Inflammation triggered by HRV infection  was assessed at 24 h with transcriptomic analysis and multiplex ELISA. In vitro CD14+ monocyte migration was evaluated with modified Boyden chamber. Results showed that HRV-induced monocyte migration was substantially increased in the co-culture model with asthmatic BSM, compared with control BSM. Furthermore, the well-known monocyte migration chemokine, CCL2, was not involved in this increased migration. However, we demonstrated that CCL5 was further increased in the asthmatic BSM co-culture and that anti-CCL5 blocking antibody significantly decreased monocyte migration induced by HRV-infected BE. Taken together, our findings highlight a new role of BSM cells in HRV-induced inflammation and provide new insights in mucosal immunology which may open new opportunities for prevention and/or treatment of asthma exacerbation.Asthma exacerbations, a major concern in therapeutic strategies, are most commonly triggered by viral respiratory infections, particularly with human rhinovirus (HRV). Infection of bronchial epithelial (BE) cells by HRV triggers inflammation, notably monocyte recruitment. The increase of bronchial smooth muscle (BSM) mass in asthma, a hallmark of bronchial remodeling, is associated with the annual rate of exacerbations. The aim of the present study was to assess whether or not BSM could increase monocyte migration induced by HRV-infected BE. We used an advanced  Asthma is a chronic respiratory disease characterized by chronic inflammation, bronchial hyperresponsiveness and bronchial remodeling. Asthma exacerbations still represent a major concern in therapeutic strategies since they are characterized by an increase in symptoms and a decrease in lung function that is sufficient to require a change in treatment . Asthma HRV also presented the ability to enter and replicate in monocytes when they are co-cultured with BE cells . MonocytAn important feature of bronchial remodeling was the increase of bronchial smooth muscle (BSM) mass, which has been correlated with decreased lung function , 14. IntTherefore, the goal of the present study was to assess whether or not BSM could increase monocyte migration induced by HRV-infected BE. The present study showed that HRV-induced monocyte migration was substantially increased in the co-culture of BE with asthmatic BSM, compared to that with control BSM. Furthermore, we determined that the major monocyte chemoattractant CCL2 was not involved but instead this increased migration was CCL5-dependent. Taken together, our findings highlight a new role of BSM cells in HRV-induced inflammation and provide new insight in mucosal immunology which may open new opportunities for the prevention and/or treatment of asthma exacerbation.Patients with asthma were recruited from the “COBRA” cohort  in the Clinical Investigation Center of Bordeaux  according to GINA . Non-astPrimary BSM cell culture was established, as described previously , 20. BSMBE cells were obtained from surgical specimen of control subjects (see subjects' characteristics The co-culture was established by adding the ALI-BE insert to the BSM well for 1 week. Medium used for co-culture was a mix of that used for BSM and BE (50% of DMEM supplemented with 10% FBS and 50% of Pneumacult-ALI without hydrocortisone). The BE co-cultured with BSM were infected, as mentioned above for BE alone. The absence of HRV particles within BSM cells was confirmed by digital PCR (data not shown). Supernatant were collected from three independent experiments and stored at −80°C for further analysis. In total, 18 controls and 12 asthmatic BSM cells were co-cultured with at least three different BE cells.HRV-16 was used in this study since it represents the “major group” (which utilize the cell surface receptor intercellular adhesion molecule 1) and is used in a large number of experimental studies on human primary cells. The HRV-16 was propagated in HeLa cells with 2% serum, as previously described . HeLa cePrimary monocytes were obtained from blood of asthmatic patients (see patients' characteristics Primary monocyte migration was performed using transwell chambers (Thermo Fisher Scientific) with monocytes added to the upper chamber and culture supernatant to the lower chamber of 3.0 μm pore-diameter inserts, pre-coated with PLL(20)-g[3.5]-PEG(2)  to prevent monocyte adhesion. The number of migrated cells was assessed after 4 h of migration by cell counting. The effect of chemokines on monocyte migration was assessed using neutralizing antibodies anti-CCL2 at 5 μg/ml  or anti-CCL5 at 1 μg/ml (R&D) antibodies, Armenian hamster IgG isotype control at 5 μg/ml (Biolegend) or mouse IgG1 isotype control at 1 μg/ml . For each experiment, results were first normalized with control medium (DMEM/ALI medium). Then, all the data were further normalized by the mean of the control condition (co-culture without HRV) and multiplied by 100 to display the results as percentage of migration.Monocyte migration was also assessed using micro-optical coherence tomography (μ-OCT) . BrieflyBE cells were lysed by adding 200 μl of lysis buffer RLT according to manufacturer's instruction , 24 h after HRV infection. mRNA extracts were stored at −80°C and send to PARS-I  to be assessed with nCounter® FLEX using the inflammation panel . Analysis was performed with nSolver™ Analysis software.Protein expression of CCL2 and IL-6 was assessed by ELISA in supernatants (100 μl undiluted for controls and diluted 1/10 for HRV-16) following the manufacturer's instruction . A custom Bio-Plex Assay  was performed to assess chemokine expression in co-culture supernatants (50 μl undiluted) according to the manufacturer's instruction. Special plate reader  and software (Bio-Plex manager) were used. The assay running was based on the same principle as a classic ELISA, except that all the washing steps were made with a wash station (Bio-Rad), equipped with a magnetic field that kept the microbeads to the bottom of the well while performing washing steps.p < 0.05.All statistical tests were performed on Graphpad Prism 6 software . Results were displayed as mean ± SEM values of repeated independent experiments. Statistical tests used were ordinary one-way ANOVA with Newman-Keuls or Bonferonni's multiple comparisons test and Wilcoxon tests. Results were considered as statistically significant when Since BE is the first line of defense against respiratory viruses, we first sought to assess monocyte migration in response to supernatant of HRV-infected BE cells alone, cultured in ALI. As anticipated, a significant increase of monocyte migration (62%) was observed with HRV-infected BE supernatant . Since HWe further assessed whether monocyte may cross the ALI-BE barrier. To that extent, we designed an inverted model with BE cells seeded on the inverted side of the insert of the transwell and we used an advanced system of OCT-imaging (μOCT), to perform live-imaging of inflammatory cell migration for 4 h . While nSurprisingly, HRV infection of BE co-cultured with control BSM did not increase monocyte migration . By contThe protein level of the chemokines of interest was then measured using multiplex ELISA assay . Since aTo finally assess the role of CCL5 on monocyte migration, we performed additional migration experiments. First, adding recombinant CCL5 to non-infected-BE cell medium increased monocyte migration, which was abolished using an anti-CCL5 neutralizing antibody . Second,Taken together, these results showed that the BSM from asthmatic patients increased the effects of HRV infection of the BE in terms of both pro-inflammatory response and monocyte migration in a CCL5-dependent manner.In the present study, we focused our attention on the effects of HRV infected-BE on monocytes migration. Indeed, monocytes are recruited in the bronchial wall of asthmatic patients after infection , 26. MorThese results demonstrated the interest of using an advanced model of co-culture, since the crosstalk between BSM and BE cells altered the “classical” pattern of monocyte migration. Indeed, BSM can produce chemotactic proteins and therefore attract many inflammatory cells. In the context of asthma, BSM may attract mast cells , as wellin vitro (in vitro, no effect of HRV-16 infection of BE on CCL2 production could be found in the literature. For instance, Keininger et al. did not observe any difference in CCL2 expression after HRV-16 infection, unlike infection with HRV-1B (In this study, we showed that CCL2 remained the main monocyte chemoattractant upon HRV infection when BE was cultured alone in ALI. CCL2 was considered as the main monocyte chemoattractant and was produced by many cell types, including BSM . Constitin vitro . BE cellin vitro . Howeverh HRV-1B . These ch HRV-1B . Similarh HRV-1B , whereash HRV-1B .hi CD16+ were increased in severe asthmatic patients (in vitro findings indicated that CCL5 may play a role in monocyte recruitment in rhinovirus-induced asthma exacerbation. This has to be confirmed with further in vivo or ex vivo studies.Several limitations can be discussed in this study. Firstly, BE cells were only obtained from control patients. Since the goal of the present study was to assess the role of BSM on monocyte migration induced by HRV-infected BE, we thus compared asthmatic and control BSM in a paired fashion. Moreover, when we previously evaluated the effects of BE on BSM cells, results were similar when asthmatic and control BE cells were used . Furtherpatients  which maIn conclusion, this study highlighted a new role of BSM in asthma which altered BE response against HRV infection. These results were in agreement with the double association of, on the one hand, the increased exacerbation rate in asthmatic patients with increased BSM mass , and, onAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the National Ethics Committee. The patients/participants provided their written informed consent to participate in this study.BA and HL designed the research, performed the experiments, collected, analyzed and interpreted the data, and wrote the manuscript. PE, AC, and EM performed the experiments, analyzed the data, and revised the final manuscript. MT and PG provided the human samples from the Clinical Investigation Center of Bordeaux and revised the final manuscript. TT and PB designed the research, supervised the study, analyzed the data, and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is a dearth of comparative effectiveness research examining the implementation of different strategies for active tuberculosis (TB) case finding, particularly in rural settings, which represent 60% of the population of sub-Saharan Africa.p = 0. 73). In the contact-tracing arm, 1,677 contacts of 788 new TB index patients were screened, yielding 12 new patients with TB. Prespecified subgroup analyses resulted in similar results, with estimated treatment initiation ratios of 0.96  and 1.23  among historically smaller and historically larger clinics, respectively. This ratio was 1.02  and 1.08  in the Vhembe and Waterberg districts, respectively. The estimated treatment initiation ratio was unchanged in sensitivity analyses excluding 24 records whose TB registration numbers could not be verified  and excluding transfers-in . Study limitations include the possibility of imbalance on cluster size owing to changes in catchment population over time and the inability to distinguish the independent effects of the two contact investigation strategies.We conducted a pragmatic, cluster-randomized comparative effectiveness trial of two TB case finding strategies (facility-based screening and contact tracing) in 56 public primary care clinics in two largely rural districts of Limpopo Province, South Africa. In the facility-based screening arm, sputum Xpert MTB/RIF was performed on all patients presenting (for any reason) with TB symptoms to 28 study clinics, and no contact tracing was performed. In the contact-tracing arm, contacts of patients with active TB were identified , screened for TB symptoms, and offered Xpert MTB/RIF testing. The primary outcome was the number of newly identified patients with TB started on treatment. The analysis used multivariable Poisson regression adjusted for historical clinic-level TB case volumes and district. The trial was registered with ClinicalTrials.gov (NCT02808507). From July 18, 2017, to January 17, 2019, a total of 3,755 individuals started TB treatment across 56 study clinics in the 18-month period. Clinic characteristics and clinic-level averages of patient characteristics were similar across the two arms: 40/56 (71%) clinics were in a rural location, 2,136/3,655 (58%) patients were male, and 2,243 (61%) were HIV positive. The treatment initiation ratio comparing the yield of TB patients started on treatment in the facility-based arm compared to that from the contact-tracing arm was 1.04  is recommended by the World Health Organization as an approach to find patients with previously unidentified TB, particularly in high-burden settings.Few studies have compared the yield of feasible strategies to implement active TB case finding in rural settings.We conducted a cluster-randomized controlled trial examining the effectiveness of two active case finding strategies for TB in two largely rural districts of South Africa: facility-based case finding  and contact tracing (testing symptomatic contacts of newly diagnosed TB patients using Xpert MTB/RIF).p = 0.70) comparing facility-based screening to contact tracing.A total of 1,929 TB patients started TB treatment in 28 clinics randomized to facility-based screening, and 1,726 started treatment in 28 size-matched clinics randomized to contact tracing; this corresponds to a treatment initiation ratio of 1.06 .This finding is driven by lower-than-expected yield from contact tracing, suggesting that although close contacts of patients newly diagnosed with TB patients do have higher-than-average TB risk, higher-sensitivity testing approaches may be necessary to identify TB among this group and increase TB treatment initiation rates at the clinic level.Potential limitations of this study include the possibility of changing clinic populations over time, the use of two different contact investigation strategies, and limited generalizability outside of rural sub-Saharan Africa. Tuberculosis (TB) is the world’s leading infectious disease killer, causing 10 million new cases and 1.3 million deaths in 2017 , 5 but, In sub-Saharan Africa, 60% of the population still lives in rural areas . DespiteWe conducted an open-label, parallel cluster-randomized controlled trial in two districts in Limpopo Province, South Africa, comparing two active TB case finding strategies: facility-based screening and contact tracing. Clusters were eligible public-sector primary care clinics within the study area. Clinics were randomized 1:1 to receive assignment to either arm, stratified by district and three levels of historical TB patient volumes (from the year prior to conducting the intervention). Within the contact-tracing arm, clinics were further randomized 1:1 to receive either household-based or incentive-based contact tracing. The intervention period lasted for 18 months, from July 2016 to January 2018. Analyses comparing the two methods of contact tracing are not included here, as a protocol-specified second crossover study comparing these two arms is still underway, and the principal investigators remain blinded to this comparison.2) population density. The province has the lowest per capita annual income in South Africa  ), less than the 25% difference considered to be clinically important.The primary analysis, adjusting for balancing factors of district and prior clinic volumes, revealed a treatment initiation ratio of 1.04  among historically smaller clinics, 1.23  among historically larger clinics, 1.02  in the Vhembe district, and 1.08  in the Waterberg district (p = 0.78) and excluding transfers-in .Prespecified subgroup analyses by historical annual volumes and by district showed similar results, with estimated treatment initiation ratios of 0.96 . Among the 1,677 contacts, 681 (41%) were symptomatic, among whom 488 (72%) provided a sputum sample for Xpert testing. An additional 193 sputa were collected from nonsymptomatic contacts for a total of 681 contacts tested using Xpert. Twelve contacts (1.8%) tested positive for TB using Xpert. In the facility-based screening arm, among the 1,929 patients with TB started on treatment, 1,427 (74%) were screened with Xpert, and 1,060 (74%) of those screened tested positive.This pragmatic cluster-randomized trial of 3,755 individuals initiating TB treatment in 56 predominantly rural South African clinics found no difference in treatment initiation rate at the clinic level, comparing the current standard of care (facility-based screening) against feasibly delivered contact tracing based on symptom screening and Xpert MTB/RIF testing. This finding was primarily driven by a lower-than-expected yield of contact tracing, which found only 12 patients across 28 clinics during an 18-month time period. These findings suggest that although close contacts are a high-risk group  and should continue to be prioritized for case finding, the prevalence of TB among contacts may not be higher than that of individuals presenting to healthcare facilities. Diagnostic algorithms with greater sensitivity than symptom-driven Xpert MTB/RIF testing may be necessary for contact tracing to increase TB treatment initiation rates at the clinic level.The results of this study have important programmatic implications. On one hand, this trial supports the findings of numerous studies that have found a high prevalence of TB among close contacts of index patients , 3, 16. The contact investigation arm in this study represents a mixture of two strategies: traditional household-based contact tracing (in which study teams visited the households of newly diagnosed TB patients) and incentive-based tracing . It is therefore possible that the observed lack of difference between the facility-based screening arm and contact-tracing arm reflects a substantial effect due to one contact-tracing strategy, counterbalanced by a negative effect from the other strategy. It is also true that the number of contacts screened per index patient in this study (2.1) was relatively low; this reflects, in part, pragmatic decisions to investigate all newly diagnosed TB patients , to perform a single round of contact investigation (up to three visits to the household), and to use a small number of teams to conduct contact investigation across a large number of geographically dispersed clinics. Given the low yield of contact tracing in the study overall coupled with the improbability of a strong negative effect from contact tracing relative to the standard of care, however, this explanation for our results is improbable.This study has several limitations. First, we chose the number of TB patients started on treatment per cluster (clinic) as the primary outcome, rather than TB incidence per se. This was for reasons of feasibility—there are no reliable estimates of a population denominator for each clinic catchment area from which to estimate incidence. It is therefore possible that our estimates of effect are biased as a result of changing denominators at the clinic level from the historical period to the study period or differences between lab records  and clinic treatment records (used during the study period). It is worth noting, however, that study contact-tracing activities were responsible for fewer than 1% of all patients initiating treatment in that arm—making it very unlikely that this explanation alone accounts for our null findings. Second, as discussed above, the contact-tracing arm comprises a mixture of two strategies, and we treated this arm as a single intervention arm for this analysis. Third, the study was single blinded, in which investigators but not the clinics themselves were blinded to intervention assignment, and the 56 study clinics were purposively chosen based on historical TB patient volume from among all possible clinics within the two districts. Finally, this study was conducted in South Africa, an upper-middle-income country with good laboratory and transportation infrastructure, and results may not be generalizable to other settings with more constrained resources.In conclusion, we provide randomized evidence that contact tracing, implemented pragmatically using an algorithm of symptom screening followed by Xpert MTB/RIF testing, did not result in a difference in the number of TB treatment initiations compared to facility-based screening, the current standard of care. Although contact tracing identifies a high-risk population that should be prioritized for additional case detection by national TB programs, more sensitive diagnostic algorithms are necessary to increase TB treatment initiation at the population level.S1 TextThis file contains the study protocol.(PDF)Click here for additional data file.S2 TextThis file contains the questionnaires administered to all participants: TB index patients and their close contacts. TB, tuberculosis.(PDF)Click here for additional data file.S3 TextThis file contains the CONSORT checklist indicating where in the manuscript each CONSORT element is located.(DOCX)Click here for additional data file.S1 DataThis file contains the data necessary to reproduce the findings contained within this manuscript.(XLS)Click here for additional data file.


New scheduling models were needed to adjust to residents’ duty hour reforms while maintaining safe patient care. In interdisciplinary night-float rotations, four to six residents from most residency programs collaborated for after-hours cross-coverage of most adult hospitalised patients as part of a Faculty-led rotation. Residents worked sixteen 12-hour night shifts over a month.We measured residents’ perception of the patient safety climate during implementation of night-float rotations in five tertiary hospitals. We surveyed 267 residents who had completed the rotation in 2015-2016 with an online version of the Safety Attitudes Questionnaire. First year residents came from most residency programs, second- and third-year residents came from internal medicine.One-hundred-and-thirty residents completed the questionnaire. Scores did not differ across hospitals and residents’ years of training for all six safety-related climate factors: teamwork climate, job satisfaction, perceptions of management, safety climate, working conditions, and stress recognition.Simultaneous implementation in five hospitals of a Faculty-led interdisciplinary night-float rotation for most junior residents proved to be logistically feasible and showed similar and reassuring patient safety climate scores. A positive working climate ensuring residents’ wellbeing and patient safety being at the origin of duty hour reforms, residents’ perception in that regard was a priority of the quality assurance process.18 Its goal is to elicit a snapshot of the safety culture through surveys of frontline worker perceptions.15 Within the first fourteen months of implementation of our night-float rotations and using the SAQ we measured residents’ attitude regarding patient safety in five hospitals.The Safety Attitudes Questionnaire (SAQ) was benchmarked in multiple care units, including general inpatient and acute care settings, and studied in association with patient outcomes.We implemented night-float rotations simultaneously in five tertiary hospitals of Université Laval in Québec City, Canada . In each site, night-float teams gathered two to four first-year residents and one or two second- or third-year internal medicine residents. Residents worked together to ensure cross-coverage of adult patient in-house calls, including new consultations at the emergency room , on most medical and surgical hospitalisation units  and in some intensive care units (sites 2 and 4). Residents worked under the immediate supervision of senior residents and on-call physicians depending on the speciality involved in each case. Residents were assigned to 16 shifts over four consecutive weeks. They worked Monday to Thursday from 8 p.m. to 8 a.m. and were exempted from daytime activities. University personnel managed residents’ distribution in collaboration with residency programs.Night-float rotations were mandatory for first-year residents from most residency programs. Instructional methods comprised video recordings, online resources, reflexive activities and logbooks. For each period, in each site, a clinical supervisor recruited among all participating specialities was responsible for residents’ follow-up and assessment. Group meetings with supervisors took place weekly. Assessment criteria focused on collaboration, professionalism and recognition of medical and surgical emergencies.We surveyed by email, with two reminders, all 267 residents who had completed the night-float rotation from August 2015 to November 2016. First year residents came from most residency programs, second- and third-year residents from internal medicine.19 The questionnaire was translated back to English electronically and all sentences maintained their semantic properties. Completed online, all questions began with “In your last night-float rotation […].” Thirty-one items of the 36-item SAQ elicit residents’ attitude toward six safety-related climate factors (five filler items).15 Answers were given on a Likert scale of 1 to 5: 1=disagree strongly, 2=disagree slightly, 3=neutral, 4=agree slightly, 5=agree strongly. In accordance with Sexton et al.15 mean scores for each of the six factors were reported on a 100-point scale using this formula: (mean score of the items of a factor – 1)*25.With permission from the authors, we translated the short form of the SAQ in Canadian-French (SAQ-FR), confirmed by four bilingual educators.18 As the sample is fairly small and groups unequal, Pillai’s trace was preferred for its robustness.20 We used Post-hoc Bonferroni tests to investigate significant differences among groups. We performed reliability analyses accordingly to classical test theory, hence we conducted item analysis to obtain Cronbach’s alpha as internal consistency coefficients and corrected item-total correlations as discrimination indices. We used IBM SPSS statistics for Windows Version 20 .We calculated differences between residents’ years of training and between sites with multivariate analysis of variance (MANOVA) to consider simultaneously the six dimensions of the SAQ.The Research Ethics Committee of Laval University, applying rule 2.5 for quality improvement of educational projects, waived ethical approval. All participants were provided a consent form before completing the questionnaires, which remained anonymous.As presented in p = .112). As for mean differences between different sites, MANOVA revealed no significant differences between the first-year and second- and third-year groups (p = .076). When both considered in the same analysis, the effect of training level*site was not statistically significant either (p = .105), nor were the effects of training level (p = .202) and site (p = .069). However, the sample remained small; hence, statistical power was limited to observe significant differences. When performing individual ANOVAs which require smaller samples but are more risky because type I error is multiplied with each combined ANOVA,20 differences between groups were significant for Perception of management between sites , but no Bonferroni test was significant. The ANOVAs on training levels were also non-significant (p = .201 to .878). SAQ-FR showed variable internal consistency levels as presented in Scores on the SAQ-FR do not seem to vary across training levels or sites. Mahalanobis distance revealed 21 cases being multivariate outliers, which were excluded from analysis. MANOVA across residents’ years of training revealed no significant differences between the first-year and the second- and third-year groups (15 Patient safety climate scores were similar across all five sites for all safety-related climate factors. Those results resonate with survey studies where residents in a night-float system felt an improvement in patient care, reporting that “better care was provided by a rested physician in spite of being less familiar with the patient.”22Faculty-led simultaneous implementation in five hospitals of a mandatory interdisciplinary night-float rotation for most junior residents was logistically feasible and showed similar and reassuring patient safety climate scores. Provided for illustrative purposes, 23Awaiting confirmation from a qualitative study underway, we believe certain characteristics of our night-float rotation had positive effects on the working climate. For example, the unique interdisciplinary nature of this rotation may be reflected in teamwork climate scores. Also, clear rules were given to residents and hospital personnel regarding call dispatch among the team to regulate the workload. Influencing the perception of management, each team could report to and had scheduled meetings with a supervisor who provided feedback and guidance. Although supervisors were not constantly on-site, all new consultations were discussed immediately with the on-call physician or senior resident. Also, second- and third-year internal medicine residents contributed to role modelling, feedback and bedside teaching, known as key elements for the educational value of night-float assignments by residents and faculty.15 Subjected to sampling bias, our response rate of 48% should be interpreted as residents’ opinion regarding the safety climate. Response rates over 60% in each night-float teams would be needed for definitive cultural assessment.14 Night-float teams were constantly moving from one physical work unit to the other. Therefore, the safety climate was assessed among the night-float teams of residents, who share common management, clinical and operational objectives. We did not include responses from the large number of health professionals in contact with residents during their rotation. Based on previous surveys, residents’ self-assessment may overestimate the quality of care and differ from what would be observed by nurses and supervisors during night floats.24 Incompletely tested in the SAQ, night-float rotations, although they offer more stability, can be associated with the sleep and alertness disturbances associated with night work.27The average response rate of SAQ in other studies is 67%.29 By studying an entire rotation, we gained insight into the hidden curriculum of the on-call period, in particular residents’ positive perception of the learning environment at night.30 Data on patients’ outcome and satisfaction with emphasis on continuity of care are needed to triangulate our results.31 This study does not aim to compare this new scheduling model with previous schedules. However, it provides observational data on a promising model that will draw interest as 12- and 16-hour work limits are applied in other provinces and countries.10Night-float rotations provide a strategic setting to study the educational outcomes of the on-call period.


Household food insecurity in South Africa is a pervasive public health challenge. Although its link to chronic health conditions is well established, its relationship to mental illness, particularly major depression, is not well-understood. Despite KwaZulu-Natal Province being the epicenter of the drug-resistant tuberculosis (MDR-TB) epidemic, and having the largest share of poverty in South Africa, this relationship remains unexamined. This study investigated the association between major depressive episode (MDE) and household food insecurity among individuals with MDR-TB.We enrolled and interviewed 141 newly admitted microbiologically confirmed MDR-TB inpatients at a specialized TB hospital in KwaZulu-Natal Province, South Africa. Logistic regression models were fitted to assess the relationship between MDE and household food insecurity, while accounting for socio-demographic status .The prevalence of MDE and household food insecurity was 11.35% and 21.01%, respectively. MDE was significantly associated with household food insecurity . Individuals who are female , young , have low educational attainment  and receive social grants  were most at risk of household food insecurity.MDE in individuals with MDR-TB was significantly associated with household food insecurity, independent of socio-economic status. Although MDR-TB is not exclusively a disease of the poor, individuals from socio-economically disadvantaged backgrounds  were more likely to experience household food insecurity. Our study underscores the need to address the co-occurring cycles of food insecurity and untreated MDE in South Africa. Despite the political advances in South Africa since the transition to democracy, there has been little progress in addressing household food insecurity , with 380 new cases per 1000 in 2016, is the leading cause of death in South Africa , and canGiven the serious nature of the disease, exposure to MDR-TB can be a psychologically devastating and life-altering experience in the absence of adequate support mechanisms , posing According to several systematic reviews, the link between mental illness and food insecurity is well-documented globally , 6, but n = 90) without primary school level education. Ethical approval was obtained from the University of KwaZulu-Natal’s Biomedical Research Ethics Committee. Five potential study participants refused to be involved in the investigation, and written informed consent was obtained from all others. Trained study personnel obtained socio-demographic data and administered the standardized questionnaire  through a face-to-face structured interview. Information on participants’ HIV status was obtained from the National Health Laboratory Service based at the study site.We prospectively enrolled and interviewed those with microbiologically confirmed MDR-TB who were consecutively admitted to a referral-based specialized TB hospital in KZN between September 2015 and October 2016. The inclusion criteria were consenting adult individuals (defined as aged 21–59 years) with no prior history of MDR-TB treatment. This study utilized data from an investigation that monitored neurocognitive impairment in participants with MDR-TB, requiring basic literacy to participate in the assessment and excluding individuals  was the main covariate of our study and was diagnosed using the Mini International Neuropsychiatric Interview version 6.0 (MINI 6.0) , a strucThe analysis consisted of two components: first, descriptive statistics summarized the participants’ socio-demographic and clinical status. Second, logistic regression models were fitted to assess the association between MDE and household food insecurity while adjusting for socio-demographic and clinical status. We generated a final outcome measure that combines mild to severe insecurity categories to classify household food insecurity to fit the logistic regression models. STATA 15 was used for the analyses, with the model fit being assessed using the Akaike’s Information Criterion , where tn = 110; 78.01%), black , and had not completed secondary level education . The prevalence of household food insecurity was 21.01% (n = 29), with approximately a third receiving a social grant . The overwhelming majority were HIV+ , and the proportion meeting MDE diagnostic criteria was 11.35% (n = 16).The socio-demographic characteristics of the 141 MDR-TB study participants are presented in Table p = 0.06). The Akaike’s Information Criterion (AIC) of the full model (model 2) was 124.23. Although model fit value of model 1 was lower, the significance of MDE under both models remain the same. The model 2 was retained and its findings reported due to the potential importance of income in our study.The results of the regression analysis are indicated in Table The purpose of this study was to examine the association between major depressive episode and household food insecurity among individuals with MDR-TB from an area considered to be at the forefront of the outbreak in sub-Saharan Africa. Two important points emerged from our investigation. First, we found the proportion of severe food insecurity 14.49%) in individuals with MDR-TB to be markedly higher than that in the general South African population (4.8%) [4.49% in Our main finding is consistent with various investigations , 43–46, Notably, many studies about the role of depression and food insecurity have been conducted among vulnerable and marginalized groups, such as pregnant women , 33, 43,clinical rather than community sample. Our study participants were receiving intensive inpatient treatment for MDR-TB at a specialized TB hospital, which may have had a positive impact on their depressive symptomatology.The 12-month prevalence of MDE in our MDR-TB sample (11.35%) was higher than the South African general population estimate of 4.9% . HoweverThe limitations of the investigation are the cross-sectional and modest sample size associated with our study design, which precludes any causal inference about the effect of depression on food insecurity. Our study is theory-driven , but we also acknowledge the possibility of reverse causation (effect of food security on depression), a point also considered in a recent study from KZN that found a significant association between food insecurity and depression among refugee and migrants . As noteNotwithstanding the limitations, the significant association between depression and food insecurity among people with MDR-TB highlights the multifaceted health and social challenges that affected individuals who undergo in a setting considered to be at the epicenter of the MDR-TB epidemic in sub-Saharan Africa. Our current study comes at a time when such complex development challenges are further complicated by the growing threat to food security due to climate change in South Africa . This wi


Different microRNAs are dysregulated in ovarian cancer where some of them have proved to be valid biomarkers. miRNA profiling analyses have shown that the different histotypes of ovarian carcinoma display differential expression of specific miRNAs. In the present study, we used miRNA-sequencing and Real-Time qPCR to detect the expression levels of miRNAs belonging to the miRNA-192/215 family, namely miR-192, miR-194, and miR-215, in different types of ovarian neoplasia, finding that miR-192, miR-194, and miR-215 were upregulated in ovarian carcinomas of the mucinous subtype, but downregulated in other types of carcinoma and in sex cord-stromal tumors. The expression of the said miRNAs was 6-fold higher in mucinous tumors compared to the other histotypes making them candidates for a possible role as diagnostic biomarkers. They play an important regulatory role by targeting specific mRNAs for degradation or translation repression2. In so doing, they may influence the development and/or progression of some types of neoplasia as many transcripts are affected simultaneously, leading to profound alteration of signaling pathways3.MicroRNAs (miRNAs) are non-coding RNAs with diverse biological functions4, liver5, large bowel6, and ovaries7. Lately, several research groups have aimed to characterize the miRNA signature in the various types of ovarian cancer (OC). The Cancer Genome Atlas consortium launched the first cooperative effort to identify the miRNA profile in high-grade serous ovarian carcinomas8 (HGSC), after which many other studies followed focusing on miRNA expression in also the less common OC subtypes10. Several miRNAs were found to be deregulated11 such as miR-1419 and miR-19212 which are overexpressed exclusively in endometrioid and mucinous carcinomas, respectively. They might hence be diagnostic biomarkers in these tumor subtypes. Also other miRNAs have been shown to be valid prognostic and predictive biomarkers in ovarian carcinomas13. For example, overexpression of the miR-200 family17 is correlated with a better response to paclitaxel in patients with OC18, and miR-9 upregulation was associated with an improved outcome19 and sensitivity to cisplatin20. Recently, circulating miRNAs such as miR-21, miR-200c, and miR-1246 were demonstrated in the blood of patients with OC22.miRNA deregulation is a pathogenetic mechanism in cancers of the lungDespite the increasing interest in the role of miRNA in ovarian carcinogenesis, little is known about this topic in the less common malignant tumors of this site. We therefore investigated the expression status of miRNAs belonging to the miRNA-192/215 family, namely miR-192, miR-194, and miR-215, in different types of ovarian tumors, ranging from sex cord tumors to various carcinomas.−11), miR-194 log2FoldChange of 5.9 (padj = 8.86e−12), and miR-215 log2FoldChange of 5.8 (padj = 2.6e−7).Downstream analyses of miRNA-seq data gave informative results on 66 samples out of 89. The DESeq. 2 analysis found a mean of 250 miRNAs differentially expressed in each group (data not shown). We narrowed down these results taking into consideration only miRNAs showing more than 4-fold overexpression and Padj minor of 0.005. Using these guidelines/parameters, we found that the miRNAs belonging to the miR-192/215 family were consistently upregulated in mucinous carcinomas , Zinc finger E-box-binding homeobox 2 (ZEB2), and Mouse double minute 2 homolog (MDM2) genes was performed as these genes are known to be targeted and regulated by the miR-192/215 family23. The expression levels of the three mentioned genes were similar in all ovarian tumors included in our series. No significant inverse correlation was found between the expression of these genes and expression of miR-192, miR-194, and miR-215 in the mucinous carcinomas or in the other groups of ovarian tumors analyzed (data not shown).Since the number of mucinous carcinoma samples analyzed for miRNA differential expression was low (n = 3), we decided to validate the bioinformatic analyses performing Real-Time qPCR for the mentioned miRNAs in an independent cohort of 73 samples from several subgroups of ovarian tumors, i.e., different histological subtypes of carcinoma as well as sex cord-stromal tumors, among which were included samples from 19 mucinous carcinomas. The results obtained by Real-Time qPCR are shown in Fig. 25. miR-200a and miR-200c were found upregulated in serous, endometrioid, and clear cell carcinomas, whereas miR-200b and miR-141 overexpression was characteristic of the endometrioid and serous histotypes26. Moreover, endometrioid carcinomas have shown upregulation of three additional miRNAs, namely miR-21, miR-203, and miR-2059. Calura et al.12 found the clear cell histotype to be characterized by a higher expression of miR-30a whereas mucinous tumors had higher levels of miR-192 and miR-19412. Our results are in accordance with previous findings inasmuch as both miR-192 and miR-194 were found overexpressed in mucinous carcinomas but downregulated in the other histotypes. However, we found also another miRNA belonging to the same family upregulated in the majority of mucinous carcinomas, miR-215. This further emphasizes the specificity of the expression profile of this miRNA family in mucinous ovarian carcinomas and highlights the possibility that these molecules may be used as diagnostic biomarkers.Several studies have identified different miRNA signatures in the various types of OC and some of these miRNAs are successfully used as diagnostic and prognostic markers23, colorectal cancer27, and multiple myeloma28. Our analysis showed variable expression of miRNAs in the mucinous tumors; more precisely, in three carcinomas, all three miRNAs were down-regulated, two tumors showed overexpression of miR-194 and miR-215 but downregulation of miR-192, and the last three carcinomas showed no expression of the three miRNAs. We checked if the observed expression differences among the mucinous samples correlated with parameters such as tumor stage, grade, recurrence, or time of death, but found nothing  or if some epigenetic silencing mechanism is operative. Additional studies should shed more light on the importance of these pathways.23 and multiple myeloma28. When expression of this family of miRNAs is restored in these malignancies, the miRNAs act as tumor supressors repressing the oncogenes TYMS, ZEB2, and MDM229. In order to gain new insights into the pathogenesis of mucinous carcinomas in particular, and ovarian carcinomas in general, we checked the expression status of the aforementioned three genes targeted by the miR-192/215 family, expecting to find different expression patterns in mucinous carcinomas compared to the other groups of ovarian tumors analyzed. Interestingly, the mean expression of the three genes  was upregulated in all ovarian tumor subtype analyzed. These results suggest that also other mechanisms than miRNAs are active in the regulation of these genes in ovarian tumors. Recently, Zhang et al.30 showed that miR-192/215 can also act as oncomirs promoting epithelial-mesenchymal transition in gastric cancer repressing the tumor suppressor gene nonsense mediated mRNA decay associated PI3K related kinase (SMG1). These findings suggest that the miR192/215 family miRNAs may exert oncogenic functions in mucinous carcinomas.Previous studies showed the miR-192/215 family of miRNAs to be downregulated in renal cell carcinomahttp://helseforskning.etikkom.no), project numbers 2.2007.425 and 2011/2071. All methods were performed in accordance with the guidelines and regulations approved by the institutional review board . Written informed consent was obtained from the patients.The material consisted of 155 fresh frozen samples from ovarian tumors surgically removed at The Norwegian Radium Hospital from 1998 to 2008. Eighty-nine samples (53 high-grade serous carcinomas (HGSC), 17 endometrioid carcinomas, 7 low-grade serous carcinomas (LGSC), 5 clear cell carcinomas, 4 borderline tumors, and 3 mucinous carcinomas) were investigated by means of next generation sequencing . Sixty-six other samples  were used as an independent series to validate the results from NGS and bioinformatic analyses. As part of this validation, 12 mucinous carcinomas were yet again reexamined by an expert pathologist, immunostained for CK7 and CK20, and additional staining was performed for PAX8 to identify the primary versus metastatic origin of the ovarian tumor  and QIAcube (Qiagen) according to the manufacturers’ recommendations. RNA concentration and purity was measured using a Nanovue Spectrophotometer .https://www.sequencing.uio.no/). The sequencing library was created using the ScriptMiner Small RNA-seq Library Preparation Kit . RNA was sequenced using an Illumina HiSeq. 2500 instrument and the Illumina software pipeline was used to process image data into raw sequencing data. Only sequence reads marked as “passed filtering” were used in the downstream data analysis.miRNA sequencing was performed on 89 samples at the Norwegian Sequencing Center at Ullevål Hospital Oslo . The reads were aligned using the alignment program STAR32. The miRNA detection rate was assessed for each sample by correlating the number of reads mapped to reference miRNAs obtained from a freely available miRNA database (http://www.mirbase.org/) and the number of detected miRNAs. An arbitrary cutoff was set at 1.5 million mapped reads as suggested by Metpally et al.33, and all samples whose reads were below that threshold were removed from further analyses (13 out of 89). Finally, the read counts were analyzed for differential expression using the Bioconductor package DESeq. 234 comparing each tumor histotype against all the others. Only results with a padj minor of 0.005 were considered.The raw data were trimmed using Cutadapt−ΔΔCt (Livak) method35.The expression of the miRNAs and genes analyzed in this study was assessed with Real-Time qPCR on 73 samples. The PCR analyses were performed using the CFX96 Touch Real-Time PCR detection system . The reactions were carried out in quadruplicate using the TaqMan Universal Master Mix II with no UNG  following the manufacturer’s protocol. Human Universal Reference Total RNA  was used as internal reaction control, whereas two commercially available controls, MVP Total RNA Human Ovary  and Human Ovary Total RNA , were used as reference for relative expression normalization. The Real-Time data were analyzed with Bio-Rad CFX manager 3.1 (Bio-Rad). The normalized expression was calculated using the 2RNU6B gene (TM:001093) was used as reference as it is stably expressed in ovarian tumors 36. The mean Cq values of RNU6B for each sample were checked to be in the range 26–27 before performing the relative normalized expression to avoid bias in the expression analysis.Ten ng of total RNA were reverse transcribed with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s protocol. miRNA expression was assessed with Real-Time qPCR using the TaqMan MicroRNA assays (Applied Biosystems) for miR-192 (000491), miR-194 (000493), and miR-215 (000518). The ZEB2 (Hs00207691_m1), MDM2 (Hs00540450_s1), and TYMS (Hs00426586_m1). RPL4 (Hs_01939407_gH) was used as a reference gene because it shows stable expression in ovarian cells37.One µg of extracted total RNA for each tumor was reverse-transcribed in a 20 μl reaction volume using iScript Advanced cDNA Synthesis Kit according to the manifacturer’s instructions . Gene expression was assessed with Real-Time PCR using the TaqMan Gene Expression Assays (Applied Biosystems) for Supplementary Dataset 1


Purpose: Our purpose was to evaluate whether omitting high-dose clinical target volume radiation (CTV-HD) around the gross tumor volume (GTV) in patients with oropharyngeal squamous cell carcinoma (OSCC) treated with intensity-modulated radiotherapy (IMRT) was associated with increased local failure.Methods and materials: Patients diagnosed with stage I to stage IV OSCC between December 2004 and April 2017 were retrospectively reviewed. All patients were treated with radical radiotherapy using IMRT, with or without neoadjuvant or concurrent chemotherapy. In accordance with institution guidelines, CTV-HD was not used. Local failure was defined as disease persistence or reappearance at the primary tumor site. When primary failure was documented, the computed tomography/positron emission tomography (CT/PET) scan that showed primary failure was fused with the original treatment scan. Each recurrent tumor was contoured to evaluate the pattern of recurrence. Recurrences were categorized as in-field, marginal, or out-of-field if >95%, 20%-95%, or <20% of the recurrent tumor volume, respectively, was encompassed by the 95% high-dose prescription isodose line of the original treatment plan. We then determined whether omitting CTV-HD was associated with increased locoregional failure.Results: A total of 272 patients with OSCC were assessed. The median follow-up from initial treatment was 43 months (range: 3-194 months). Seven patients were lost to follow-up. The overall five-year survival rate was 87%. The three- and five-year disease-free survival rates were 86% and 83%, respectively. Forty-one patients had 53 treatment failures . Fourteen (87.5%) of the local recurrences were in-field, one (6.25%) was marginal, and one (6.25%) was out-of-field.Conclusion: Our analysis of patients with oropharyngeal cancer suggests that local failure is mostly in-field and potentially due to radioresistance, rather than a marginal miss of the tumor. It suggests that omitting CTV-HD is feasible and safe. Patients with advanced head and neck squamous cell carcinoma (HNSCC) are increasingly treated with radiotherapy, with or without chemotherapy, as the only curative nonsurgical treatment. This therapeutic strategy achieves high rates of local tumor control of over 80% for stages 1-2 and 60%–70% for stages 3-4 [The superiority of IMRT has been confirmed by randomized controlled trials, which have established IMRT as the current standard of care for the treatment of HNSCC ,3. HowevLocal radiotherapy treatment failure can be related either to the radioresistance of the tumor or the geometric miss of the target tumor treatment volume . The locStudy designA retrospective review was performed by examining patient charts, treatment plans, and diagnostic imaging studies of patients with oropharyngeal squamous cell carcinomas treated at our hospital. Patient consent was waived for reviewing patients' records and images by the institutional review board (IRB) of our hospital. Patients with histologically proven squamous cell carcinoma of the oropharynx, stage I to stage IV, using American Joint Committee on Cancer (AJCC) 7th edition, who had been treated with radical IMRT radiotherapy, with or without neoadjuvant or concurrent chemotherapy, were selected and their charts reviewed. Patients whose primary treatment modality was surgery were excluded. Patients who were treated with IMRT for recurrent disease or palliative radiotherapy, or who presented with distant metastases, were also excluded. When local treatment failure was documented, recurrence was assessed and delineated on post-recurrence computed tomography (CT) or CT/positron emission tomography (PET); then, the image was fused with the original treatment planning CT image, focusing on the area of recurrence. Failures were scored as in-field, marginal, or out-of-field if >95%, 20%-95% or <20% of the recurrent tumor volume, respectively, was encompassed by the 95% high-dose prescription isodose line of the original treatment plan. Imaging reports of recurrence and other available information, such as reports of physical examinations or fiberoptic laryngoscopy reports, were reviewed for recurrence delineation for patients with no available post-recurrence imaging.Patient populationBetween December 2004 and June 2017, 272 patients with histologically proven squamous cell carcinoma of the oropharynx, stage I to stage IV, were treated with radical radiotherapy, with or without concurrent chemotherapy, using an IMRT technique. We noted 139 patients with a tonsil primary tumor, 113 patients with a primary tumor at the base of the tongue, and 20 patients with a soft palate primary tumor. Clinical characteristics of our patient population are presented in Table TreatmentAll patients were immobilized with a thermoplastic head and neck mask and Accufix shoulder immobilization, and then they underwent computed tomography (CT) simulation scanning with 3-mm slices. Intravenous (IV) contrast was used in all patients. Diagnostic PET scan or magnetic resonance imaging (MRI) was fused with the planning CT scan from the original treatment. The attending physician defined the GTV, CTV, and organ at risk (OAR) volumes by contouring every CT slice. The GTV was defined to include the visible tumor and involved lymph nodes shown on CT, MRI, and/or PET imaging and thorough physical examination, including fiber-optic laryngoscopy. According to radiotherapy practice guidelines at our hospital, an accelerated hypofractionation schedule was used, with differential dose allocation to target volumes in a manner similar to the method described in Radiation Therapy Oncology Group (RTOG) protocols . The GTVFollow-upThe tumor response to treatment was assessed two to three months after completion. Evaluation included a clinical examination with fiberoptic laryngoscopy and CT imaging. The consideration of biopsy was based on clinical or radiological criteria. Patients were followed up with a physical examination every six to eight weeks during the first year after treatment, every three months for an additional two years, and then every six months until discharge at five years.Local recurrence analysisA total of 272 patients with OSCC were assessed. Of this group, 139 patients had a tonsil primary tumor, 113 patients had a primary tumor at the base of the tongue, and 20 patients had a soft palate primary tumor. Median follow-up was 43 months (range 3-194 months). Seven patients were lost to follow-up. The five-year overall survival was 87%. The three- and five-year disease-free survival rates were 86% and 83%, respectively  margins, if any, to be added to the gross disease for IMRT treatments. Other authors have looked at the incidence and location of local failures as an indication of adequate target coverage. Eisbruch et al. found that in 133 patients, 17 of 21 recurrences were in-field  . SchoenfPrecise target volume delineation, both for improving treatment efficacy and for reducing radiation-induced toxicity, remains an area of active investigation. Identifying and integrating post-treatment imaging findings, such as site of failure, with the corresponding point in the original treatment plan, is essential to define high-risk areas and optimize guidelines for volume delineation. At our center, it is standard practice to omit CTV-HD radiation, in an attempt to minimize the high-dose treatment volume and the risk of acute and late toxicity-related complications. By performing a co-registration between the imaging of relapses and original treatment planning images in our study, we have shown that most treatment failures occurred predominantly in the GTV. In our study population, out of all local failures, only two patients were found to have a marginal or out-of-field failure, confirming that an appropriate target volume definition and adequate target dose coverage without the addition of a CTV-HD margin is safe and may lead to a possible reduction in acute and long-term radiation therapy-related toxicity. LimitationsOne limitation of our study is its retrospective nature. In addition, at the time of recurrence, there may be significant changes in the external tumor contour of these patients due to factors, including patient weight loss and the changing shape of recurrent tumors. Also, in 30% of patients with local failure, the actual imaging was not available, and for these patients, we relied on clinical notes and radiology reports.Our analysis of patients with oropharyngeal cancer suggests that local failure is mostly in-field and potentially due to radioresistance, rather than a marginal miss of the tumor. It suggests that omitting CTV-HD is feasible and safe. Further validation of these findings is of interest in order to optimize volume definition guidelines in future head and neck cancer treatment protocols.


Ciona robusta, is a model organism that has been adapted to experimental studies of host/microbiome interactions. Ciona variable-region containing chitin-binding proteins (VCBPs) are innate immune effectors, composed of immunoglobulin (Ig) variable regions and a chitin-binding domain (CBD) and are expressed in high abundance in the gut. It was previously shown that VCBP-C binds bacteria and influences both phagocytosis by granular amoebocytes and biofilm formation via its Ig domains. We show here that the CBD of VCBP-C independently recognizes chitin molecules present in the cell walls, sporangia (spore-forming bodies), and spores of a diverse set of filamentous fungi isolated from the gut of Ciona. To our knowledge, this is the first description of a secreted Ig-containing immune molecule with the capacity to directly promote transkingdom interactions through simultaneous binding by independent structural domains and could have broad implications in modulating the establishment, succession, and homeostasis of gut microbiomes.The gut microbiome of animals consists of diverse microorganisms that include both prokaryotes and eukaryotes. Complex interactions occur among these inhabitants, as well as with the immune system of the host, and profoundly influence the overall health of both the host and its microbial symbionts. Despite the enormous importance for the host to regulate its gut microbiome, the extent to which animals generate immune-related molecules with the capacity to directly influence polymicrobial interactions remains unclear. The urochordate,  The gut microbiota consists of diverse communities of microorganisms, including: bacteria, archaea, viruses, fungi, and other microbial eukaryotes. Investigations utilizing diverse model systems have defined an active role of the microbiota in both normal host physiology  and diseA recent report suggests that fungi do not efficiently colonize the digestive tract, rather their presence in the gut may be linked to both diet and the oral flora . HoweverAlthough there is a dearth of knowledge on if and how fungi interact with host immune system components under normal “commensal” or healthy conditions or during times of microbial dysbiosis, much is known about how diverse innate immune receptors interact with fungal pathogens , 27. InnC. robusta, four members of the VCBP gene family  have been described -encoding gene families in two protochordate model systems –38. In Cescribed . Unique escribed . VCBP-C escribed , 38. Theescribed , 38; a pCiona tunic with 70% ethanol and washing with sterile ASW  for 3–4 h. All steps to isolate fungi were carried out under a laminar hood, in aseptic conditions, using sterile materials. Briefly, after sterilization of the rile ASW , gut wasFungal species were identified by sequencing (Sanger) across the interspersed spacer (ITS) region of the 18s rRNA gene. Briefly, using Dneasy PowerSoil kit , DNA extraction was performed on fungi grown in liquid medium for 5–7 days; fungal-specific18s rRNA amplicons were generated with standard ITS primers . SequenEach fungal isolate was grown on a YPD agar plate for about 5 days and fixed with 4% paraformaldehyde in PBS overnight at 4°C. Plates were washed three times for 10 min in PBS and small pieces of colonies were embedded and frozen on dry ice in OCT freezing media (Fisher Scientific), and stored at −80°C. Thin sections (7 μm) were collected on poly-L lysine -coated slides. Immunofluorescence staining was performed using a recombinant, customized, probe possessing a N-terminal human Fc domain fused to the VCBP-C CBD domain, and named the Fc-CBD-C probe. The specificity of this probe for chitin, and the general staining procedures, were previously validated and described in Dishaw et al . BrieflyFungal strains were grown in YPD liquid medium and orbital shaking at 20°C for 7 days, following which fungal bodies were fixed in 4% paraformaldehyde in PBS overnight at 4°C. Samples were washed several times in PBS and processed for whole mount immunofluorescence staining with the Fc-CBD-C probe as described above. Controls were run in parallel and images acquired as described in the previous section Immunofluorescence of cultured fungal isolates cryo-sectioned and labeled with Fc- CBD-C probe.Spores were isolated from fungal cultures grown for 7 days in 10 ml YPD liquid medium with orbital shaking at 20°C. Briefly, the culture was filtered using twice folded sterile gauze and centrifuged at 2200 × g for 15 min. Spores were re-suspended in 1 ml YPD medium and, either were fixed for immunofluorescence staining with the Fc-CBD-C probe or incubated with VCBP-C recombinant protein, as describe below.For the CBD-C binding, spores were fixed by adding formamide  to 400 μl of re-suspended spores, followed by incubation for 30 min with gentle agitation. Spores were pelleted at 2600 × g for 10 min, re-suspended in PBS and plated on poly-L-lysine coated circle glass coverslips (see above). The spore suspension was incubated for 15 min to allow attachment to the coverslip, then the immobilized spores were incubated overnight at 4°C with the Fc-CBD-C probe and BSA 0.5% in PBS was used as blocking solution before detection with secondary antibody.In order to characterize the biding of VCBP-C, 300 μl of spores were incubated for 2 h with gentle agitation, either with 50 μg/ml VCBP-C recombinant protein , was used to confirm some of these observations; specifically, WGA recognizes chitin fibers localized only on fungal surfaces and not in the inner cell walls , 44; henAcremonium sp. Acrostalagmus sp., Mucor sp., and Penicillium sp., were grown in liquid medium, processed for isolation of spores and incubated with recombinant VCBP-C protein. Intact VCBP-C exhibits the same binding pattern to the cell wall surface of these species ,  species  that was species , whereas species . Specifi surface ; in Peni regions . WGA lec regions  and the  regions , furtherCiona robusta is a model organism that in recent years has been adapted to studies of host-microbe interactions within the gut  when it is present in combination with costimulatory PAMPs , in locations that are not privately-owned nor protected in any way. Ciona is considered an invasive species and is not regulated or protected by environmental agencies in the United States. The collection service contracted in this study maintains current permits and licenses for collection and distribution of marine invertebrates to academic institutions. Handling of live animals was in accordance with the guidelines of our academic institutions. Animals were recovered and brought to the laboratory alive and maintained in clean water with aeration. In accordance with general animal protocols, the least number of animals required per experiment were utilized. Animal waste products were disposed of in accordance with USF Health and Safety Guidelines.The research described herein was performed on MK431096, MK424129, MK424120, MK423932, MK424124, MK424125, MK424126, MK424132, MK424131, MK424130, as reported more in detail in the The sequences reported in this manuscript have been deposited in the Genbank database with the following accession numbers: AL designed, performed, and analyzed experiments and wrote/edited the manuscript. JC and GL edited the manuscript. LD conceived the original idea and wrote/edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and objective: The trajectories of long-term and domain-specific cognitive alterations over a decade after stroke are largely unknown. This study aims to investigate the dynamic alterations of domain-specific cognitive performance among young stroke survivors over 10 years after their first stroke.Methods: A prospective cohort study was carried out on 38 young stroke survivors (aged 18–65 at stroke onset) living in the community at 10 years after their first stroke. The cognitive outcomes were assessed repeatedly at 1 week, 7 months, and 10 years after their first stroke on the sub-domains: process speed , visual attention (Bells test), visuospatial function , executive function , verbal function [Letter fluency (FAS) from D-KEFS and CD], working memory (Digit Span from WAIS), immediate memory (RCFT and CD), and delayed memory (RCFT and CD). Global cognition was evaluated with Mini mental state examination at the two later time-points.Results: We found a delayed significant improvement of working memory with total recovery 10 years after participants' stroke. Visuospatial function recovered already at 7 months and remained stable at 10-year follow-up. Process speed demonstrated a significant decrease at 10 years compared to 7 months after stroke onset, a decrease which could be compensated by enhancements of other cognitive domains. No further deterioration was found in verbal function, immediate-, and delayed memory, and executive function during 10-year follow-up. Global cognition improved by on average two points between 7 months and 10 years. Education level and fatigue showed low to moderate positive correlations with cognitive improvements.Conclusions: The concordance of cognitive improvements between domain-specific and global cognitions strongly suggest that some young stroke survivors do improve their cognitive outcome over a 10-year period following their first stroke. This finding fills a gap of knowledge with respect to the dynamic trajectory of post-stroke cognition, with important implications in clinical practice. Stroke is the second most common cause of acquired cognitive impairment and is strongly associated with increased disability, dependency, institutionalization, and mortality . ThromboCurrently, the few studies regarding the longitudinal effect of stroke on cognition have shown mixed results, demonstrating either a trend toward deterioration –5, stabiThe aim of the current study was to investigate dynamic alterations of the domain-specific and global cognitive performances among young stroke survivors over 10 years after their first stroke.This study was a single-center prospective cohort study of stroke survivors with 10-year follow-up. It was carried out at the Department of Neurological Rehabilitation, University Hospital of Northern Sweden. Ethical approval was obtained from the regional Ethical Review Board in Umeå, Sweden, D-nr 2015/144-31.All young patients (>18 years to 65 years) who had suffered a first-ever stroke between January 2004 and December 2007 with neuropsychological assessment within the first year after stroke, at Stroke Centrum, University Hospital of Northern Sweden were contacted and provided informed and written consent via letter and telephone for recruitment to the study. Exclusion criteria were severe dementia, severe aphasia, and severe comorbidity/non-community-dwelling, recurrence of stroke/TIA and other physical and/or psychiatric disease after first-ever stroke.There was a total of 425 first-ever young stroke patients in the Riksstroke registry and medical journals between January 1st, 2004 and December 31st, 2007 within the catchment area. The Riksstroke registry has a proximately cover-rate of 86%. Neuropsychological assessments (NPAs) were performed for 108 of the young stroke patients within 1 year after their stroke. The reason for the initial NPA was unknown to us but possibly due to patients' capability to be assessed and access to a psychologist at that time. NPAs were often carried out within 1 week after stroke debut. Some of the participants were assessed again at approximately seven (IQR 2–10) months after stroke. After a thorough recruitment process, 38 stroke survivors participated in the 10-year follow-up with written consent, while 49 declined due to various reasons . All parr = 0.82–94) -R and WAIS-III was replaced by WAIS-IV at 10-year follow-up due to practical reasons. Previous validation studies have shown that WAIS-IV has the same construction as WAIS-III/R , with ve0.82–94) . Swedish0.82–94) . In addi, visual attention (Bells test), visuospatial function , executive function , verbal function (Letter fluency (FAS) from D-KEFS and CD), working memory (Digit Span from WAIS), immediate memory (RCFT and CD), and delayed memory (RCFT and CD) (The following cognitive domains were examined: process speed  and CD) .One month prior to the scheduled appointment for NPA, the participants received various questionnaires to complete at home and return on the NPA occasion. Depression, anxiety, and fatigue were assessed with the Beck Depression Inventory-II (BDI-II), Beck Anxiety Inventory (BAI), and Fatigue assessment scale (FAS). Baseline data were collected from the Riksstroke registry and medical records. Participants also provided information regarding their education level, weight, height, and employment status.t-test, Fisher's exact test, or Chi-square test when appropriate.Demographic characteristics are presented as mean ± SD, number with/without number of cases (%) or median [25–75% interquartile ranges (IQR)] as appropriate. Baseline characteristics in patients were compared using a Student p-value was adjusted to account for multiple comparisons using Dunn's multiple comparisons test. A Spearman correlation test was used to obtain the analysis correlation coefficient. Statistical analyses were performed using GraphPad Prism software version 6.0., with a P < 0.05 being considered significant.Data from NPA are presented in the medium raw scores with 25–75% IQR because of the limited and varying number of participants at the early stage. The NPAs at the early stage were presented at two time-points . No adjustment was made on missing values. Numbers of raw scores on each assessment /time-point are clearly presented in P < 0.01).Baseline characteristics of study participants, stroke survivors who declined participation and all first-ever young stroke patients within the catchment area under the same period are presented in SD 10.6) years. All participants were living in the community. Most participants  had no or only slight disability (mRS = 0–2), the remaining three participants had moderate disability (mRS = 3). More than two thirds of participants  were overweight (BMI >25). Approximately one third of participants  had anxiety problems (BAI >8), four participants  had moderate anxiety and two participants  suffered severe anxiety (BAI >25). Eleven participants  were depressed (BDI-II >13), with three having moderate  and one severe  depression (BDI-II >28). Almost half of the participants  had received education for more than 12 years. Four participants were still working full time ; and six participants were working part time at 10-year follow-up. No significant difference with respect to basic characteristics was observed between men and women at 10-year follow-up  (P < 0.0001) . The digit span total score among participants reached a similar level as no-stroke individuals at age 55–64 years   and in D 0.0001)  compared64 years . Digit s64 years .n = 1 or 4) were tested in the memory domain at 1 week after stroke onset (Immediate memory assessed by RCFT  and CD  showed nke onset .p = 0.0002) already at 7 months after stroke onset compared to the results at 1-week post-stroke. The improvements remained at 10-year follow-up    and reac= 0.003) .Attention examined with Bells test  showed sp = 0.03) after 10 years compared to the 7-month follow-up. However, WAIS-Coding, not TMT-A, demonstrated a significant speed enhancement at sub-acute phase but returned to the former level at the chronic stage . Compared to the results at 7 months after stroke onset, with a median score of 27 with 25–75% percentile (25–29), there was a weak but significant improvement in global cognition (p = 0.02) among participants .n = 19) demonstrated a low to moderate but significant positive correlation to working memory that is forward and backward digit span  and backward digit span  , p = 0.04]. Education (n = 37) showed a low but significant positive correlation with working memory, that is forward and backward digit span  and backward digit span  , p = 0.02].Fatigue  at 10-year follow-up . NotablyThere were no correlations demonstrated between cognitive improvements and age, gender, anxiety, or depression among participants at 10-year follow-up.The current study provides a unique longitudinal cognitive evaluation of young stroke survivors using a comprehensive neuropsychological test battery repetitively from acute (1-week), subacute (7-month) to chronic (10-year) stages after stroke onset. To our knowledge, no such valuable cognitive data have been reported previously. We found significant improvements and recoveries with respect to working memory, visuospatial function, and global cognition, after initial post-stroke cognitive decline among the participants at 10 years after stroke. Visuospatial function improved already at 7 months post-stroke but working memory was enhanced mainly at the chronic phase, 10 years after stroke. The remaining cognitive domains, such as attention, immediate and delayed memory, and verbal and executive function, remained relatively stable over 10 years after initial post-stroke declines. Processing speed was the only domain in the cohort that demonstrated a significant decrease in one of three subtests at 10-year follow-up compared to 7-month follow-up. Education level and fatigue, but not stroke sub-type, age, and gender, showed low to moderate positive correlations with cognitive improvements among participants.The most striking finding in the current study was significant improvement of working memory in all Digit span assessments at 10 years after stroke. No such late cognitive recovery among stroke survivors has previously been reported according to our knowledge, although good functional outcome is common among young stroke survivors . This suOur results are however contradictory to two previous studies with relatively large samples  showing cognitive decline at 6 or 10 years after stroke , 4. The Intriguingly, Digit span forward (which considers pure short-term memory and rote learning) had improved already at sub-acute phase after stroke; but the more challenging Digit span backward and Digit span total score reflected real working memory recovered later at the chronic stage. This result is highly encouraging because it indicates that a delayed improvement of working memory does occur at least among these younger community-dwelling stroke survivors at chronic phase. This finding suggests that more complicated functions, such as working memory, may require longer time (more than a year/ years) to recover. This prolonged improvement of working memory after stroke illustrated in this study provides important implications for cognitive rehabilitation, driving, and care planning in clinical practice.Compared to working memory, visuospatial function assessed with Block design demonstrated another temporal profile. There was an early significant recovery to a similar level as their peers  already Beside long-term recoveries on working memory and visuospatial function, our results demonstrated that other cognitive domains such as verbal fluency, immediate and delayed memory, and executive function were relatively stable over 10 years. This is important knowledge for clinicians and patients because a significant amount of previous data could only point out the cognitive decline after stroke at certain time-points , 4. Our The reason for the cognitive improvements in the current study was not investigated at this stage. Because no organized cognitive rehabilitation was supplied after the initial hospitalization in this cohort, participants' struggle to be active and/or live independently in their daily lives may presumably be a crucial element for their cognitive improvements. These daily activities could possibly trigger activity-induced neuroplasticity  to furthIn contrast, process speed examined by a Symbol search showed significant decline at 10-year follow-up but not process speed measured with Coding and TMT-A. A possible explanation could be that Coding demands cognitive processes other than Symbol search, such as visual short-term memory, visuo-motor coordination, and attention . The purGlobal cognition measured by MMSE also improved by on average two points between 7-month and 10-year follow-ups in the present study. Together with previous studies showing global cognitive recovery from 3 weeks to 1 year after stroke onset , 11, 12,A small but significant positive association was found between level of education and working memory. This result is in line with early evidence that working memory capacity is correlated with learning outcomes in literacy and numeracy , 29. TheWe are surprised about the positive association between fatigue and improved cognitive function. This relation could possibly be explained by the fact that fatigue was assessed by answering questionnaires at home prior to NPA, and thus mainly reflected fatigue level in participants' daily lives. Presumably, those participants who performed well in the NPA and struggle in daily life have to use more energy to achieve better functional outcomes with fatigue as a drawback. This presumption may suggest various reasons behind the fatigue at different stages after stroke. Therefore, awareness of the importance of balance between activity and rest over time should be enhanced in both stroke survivors and medical staff.The current study has several unique strengths. We demonstrated repeatedly domain-specific cognitive outcomes by NPAs at acute, sub-acute, and chronic phases over 10 years after stroke onset, providing us with a distinctive opportunity to detect longitudinal cognitive alterations over time. Moreover, trajectory of post-stroke cognition was examined by both MMSE and NPAs in the same cohort. It provides not only more comparative information between global- and domain-specific cognition but also more comprehensive information regarding multiple cognitive domains: process speed, visual attention, as well as visuospatial, executive, and verbal functions, and in addition working, immediate, and delayed memory types. Furthermore, all assessors were blind to the previous assessments in order to reduce possible sources of bias.However, some methodological limitations need to be addressed. First, we are aware of the small number of participants and some variations of the number of participants at early time-points in the current study. The current findings need to be replicated in other studies with a larger sample size. Second, no cognitive data were collected for those who denied participating in the study. One may assume that the deniers may have worse clinical outcomes. However, demographic characteristics did not show any significant differences between participants and deniers, indicating that selection bias was unlikely during the follow-ups. Third, our results were generalized only from community-dwelling participants without aphasia. Nevertheless, rather than generalizing the findings to the entire young stroke population, the current study suggests mainly that cognitive recovery does occur during both sub-acute and chronic phases after first-ever stroke, at least among some young stroke survivors who are living in the community.This study demonstrated that significant cognitive recoveries on visuospatial function and working memory do occur at sub-acute and chronic stages after initial post-stroke cognitive decline in young community-dwelling adults after first-ever stroke. No further deterioration in overall cognition occurred among these young stroke survivors over 10 years' time after stroke. The age-related decline of processing speed at 10-year follow-up could be compensated by other cognitive improvements and stabilizations over time. This finding fills a gap of knowledge concerning the dynamic trajectory of post-stroke cognition over 10 years after stroke. The current results may supply strong clinical implications on rehabilitation and care planning in clinical practice.EE contributed to study conception, sample collection, interpretation of data, and revising the manuscript, provided final approval of the version to be published; agreed to be accountable for all aspects of the work. XH contributed to study conception, supervision of the acquisition, interpretation of data, drafting, and revised the manuscript including the figures, tables, and references and provided final approval of the version to be published and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of the study is to describe oncologists’ perceptions and opinions about patient eligibility, guidelines, and barriers for use of granulocyte colony-stimulating factor (G-CSF), overall and stratified by their affiliation with the Oncology Care Model (OCM). In May 2018, we invited and recruited practicing US oncologists from a national database for an online survey. Level of agreement was identified using a seven-point scale, ranging from strongly disagree to strongly agree. Of 200 participating oncologists, 70 were OCM-affiliated. Overall, 65% of oncologists agreed or strongly agreed that all patients at high risk of febrile neutropenia (FN) should receive prophylactic G-CSF, and half agreed or strongly agreed that benefits of G-CSF outweigh the potential adverse effects. The most common barriers to G-CSF use for patients at high risk of FN included patient refusal (37.1% of OCM-affiliated oncologists vs. 21.5% of non-OCM-affiliated oncologists), not on protocol/not supported by guidelines (32.9% vs. 23.1%), lack of reimbursement to practice (30.0% vs. 15.4%), and concerns about insurance coverage (22.9% vs. 26.9%). More OCM-affiliated oncologists reported that their practices offer and strongly encourage adherence to a specific protocol for G-CSF use (49.2%) versus non-OCM oncologists (31.3%). Despite recommendations from national guidelines and strong evidence from randomized, controlled clinical trials, only two thirds of oncologists agree or strongly agree that all patients at high risk of FN should receive primary G-CSF prophylaxis. Decisions about G-CSF prophylaxis may be affected by factors other than risk of FN, such as patient choice, practice protocols/guidelines, lack of reimbursement, and insurance coverage.The online version of this article (10.1007/s13187-019-01638-8) contains supplementary material, which is available to authorized users. Febrile neutropenia (FN) following myelosuppressive chemotherapy is associated with significant morbidity, mortality, dose delays, and dose reductions –3. The rIn the USA, the real-world use of G-CSF among patients at high risk of FN does not reflect the recommendations of national guidelines. G-CSF prophylaxis is observed in 17 to 74% of patients receiving chemotherapy regimens associated with high risk of FN, suggesting underutilization of an important preventive measure –16. TherThe timing of prophylactic pegfilgrastim, the most commonly used G-CSF, is also an issue. National guidelines recommend that pegfilgrastim should be administered 1 to 4 days or 1 to 3 days after the last dose of chemotherapy and that pegfilgrastim administration on the same day as the last dose of chemotherapy is sometimes justified , 12. HowData are also lacking regarding the influence of oncologists’ practice setting. The use of G-CSF may differ in academic centers versus community clinics or in practices participating versus not participating in the Oncology Care Model (OCM). The OCM is a Medicare performance-based payment model that aims to provide higher-quality, more highly coordinated, more patient-centered oncology care at the same or lower cost , 21.The objective of this survey was to describe the perceptions and opinions of US-based oncologists about G-CSF use in patients receiving myelosuppressive chemotherapy. Specifically, we sought to explore oncologists’ perspectives about G-CSF prophylaxis for FN; barriers to G-CSF use across various treatment settings; patient-level risk factors for FN; and differences in G-CSF use, specifically pegfilgrastim, stratified by affiliation with the OCM.This was a national cross-sectional survey of US oncologists conducted by an independent third party, Adelphi Research. Prior to initiation of the study, the protocol was approved by a centralized, US-based independent review board, Quorum Review (QR#33224/1). After approval, oncologists who currently prescribe G-CSFs were invited and recruited from a national database of 13,800 oncologists hosted by M3 Global Research. Oncologists from Vermont and Maine were excluded because of state prohibitions on compensation for surveys, and oncologists from Massachusetts were excluded due to state requirements for double blinding.The study targeted recruitment of approximately 200 US oncologists. Targets for specific subgroups of interest included approximately 75 oncologists affiliated with an academic cancer center and approximately 50 oncologists affiliated with the OCM. Oncologists who consented completed a screening section prior to the actual survey so that their eligibility could be assessed according to the following criteria: (1) licensed and board-certified in the USA in oncology and/or hematology for at least 3 years, (2) treated at least 40 unique oncology patients with chemotherapy in the past 6 months, (3) treated at least six oncology patients with a G-CSF in the past 6 months, (4) self or family member not affiliated with a marketing, pharmaceutical, or biotechnology company, and (5) proportion of professional time devoted to patient care ≥ 40% if affiliated with an academic center or ≥ 60% if affiliated with a community center.The survey questionnaire was developed using standard survey methodology . Before The finalized version of the questionnaire consisted of 58 questions: 12 screening questions, 4 demographic questions, 6 questions on G-CSF use overall and by type, 5 questions about predictors of G-CSF use, 23 questions about types of G-CSF selected, and 8 questions about future G-CSF prescribing. The survey collected information on primary affiliation , patient-level risk factors, criteria considered to determine a patient’s FN risk level, drivers of and barriers to G-CSF use, and perceptions about G-CSFs. The survey took 30 min to complete . The level of agreement was identified in the questionnaire using a seven-point scale, where responses from 1 to 7 indicated strongly disagree, disagree, somewhat disagree, neither agree nor disagree, somewhat agree, agree, and strongly agree, respectively. To minimize missing data, the electronic questionnaire was designed in such a way that a question had to be completed before the subsequent question was made available to the participant.https://www.qresearchsoftware.com).Continuous variables were summarized as mean, standard deviation, median, and interquartile range, and categorical variables were summarized as number and percentage with binomial 95% confidence interval (CI). Outliers were identified as values beyond 3 standard deviations and excluded from the analyses. The seven-point scale for level of agreement was categorized into three groups: strongly disagree or disagree; somewhat disagree, neither agree nor disagree, or somewhat agree; and agree or strongly agree. To understand the differences between groups, survey data was further stratified by OCM and academic center affiliation. Analyses were conducted using Q Research Software  were hematologist-oncologists and 86 (43.0%) were medical oncologists.n = 130; 65.0%) and community clinics . Detailed characteristics of the participating oncologists stratified by institutional affiliation are presented in Table Overall, oncologists reported practicing for an average of 13 years, and each participating oncologist provided G-CSF support for an average of approximately 80 patients in the past 6 months. The most common practice settings reported by oncologists were group practices (36.0%) and major academic centers (25.0%). The majority of the 200 oncologists were primarily affiliated with non-OCM practices (In response to the question regarding what guidelines they follow for G-CSF use (Q.B1a), most oncologists reported awareness of the NCCN guidelines (70.5%), followed by ASCO guidelines (38%), institution-specific or practice-specific guidelines (17%), American Society of Hematology guidelines (12%), no guidelines (11%), Centers for Disease Control and Prevention guidelines (6%), and other (2%).Almost two thirds of oncologists agreed or strongly agreed with the statement that patients at high risk of FN should receive primary G-CSF prophylaxis. According to practice setting, the figures were 57.3% for academic center, 69.6% for community practice, 60.0% for OCM-affiliated, and 67.7% for non-OCM-affiliated. More OCM oncologists reported that their practices strongly encourage adherence to a specific treatment protocol for G-CSF use  compared with non-OCM oncologists . Additionally, oncologists affiliated with practices where specific treatment protocols are strongly encouraged were more likely to say they use G-CSF for primary prophylaxis for patients in the adjuvant chemotherapy setting  compared with oncologists affiliated with practices that somewhat encourage specific treatment protocols .Participants were asked to select the recommended timing for administering pegfilgrastim prefilled syringe (PFS) from four predefined timeframes. The majority of the oncologists in both the OCM 72.5%) and non-OCM (75.0%) subgroups indicated that pegfilgrastim PFS should be administered between 24 and 48 h following completion of chemotherapy administration. Almost a quarter of oncologists in both the OCM (23.5%) and non-OCM (22.8%) subgroups said that the recommended timing of pegfilgrastim PFS is within 24 h of the last day of chemotherapy administration  to 100 (most important). Based on the mean ratings, the factors considered most important in determining whether a patient is at high risk of developing FN were chemotherapy regimen, preexisting neutropenia or bone marrow involvement with tumor, and prior experience of neutropenia . Additionally, half of oncologists (50.0%) agreed or strongly agreed that the benefits of G-CSF therapy outweigh the potential adverse effects, with no difference observed according to OCM affiliation , not being on protocols set by the practice or guidelines (26.5%), and concerns about insurance coverage (25.5%). OCM oncologists perceived barriers to prescribing G-CSF as prophylaxis differently than non-OCM oncologists did, with more OCM oncologists than non-OCM oncologists reporting lack of reimbursement to the practice/organization  and patient refusal  as barriers , 67.0% of oncologists reported that the use was increased somewhat or significantly. Subsequent questions, which allowed oncologists to select multiple reasons for increased use of OBI, showed that oncologists most commonly attributed the increase to convenient dosing (66.7%) and patients’ need for an active lifestyle (45.5%). Non-OCM oncologists reported convenient dosing as an influential factor more frequently than OCM oncologists .The majority of the oncologists in this survey reported adhering to at least one national guideline for G-CSF use –12, but Furthermore, almost a quarter of the oncologists in this survey said that the recommended timing of pegfilgrastim PFS administration is within 24 h of the last dose of chemotherapy administration. This misinterpretation of recommended timing could be one of the reasons for almost 12% of chemotherapy cycles in the real-world setting being given with same-day pegfilgrastim administration .The reasons for same-day pegfilgrastim administrations are multifactorial and have been explored in a survey of oncologists who prescribed at least one same-day dose of pegfilgrastim in the past 6 months . PreviouCompared with non-OCM oncologists, OCM oncologists were more likely to have their practices offer or strongly encourage adherence to a specific protocol for G-CSF use. The OCM sites may be more likely to adhere to a specific protocol for G-CSF use because the practice performance is graded not only on outcomes such as all-cause hospitalization and emergency room visits but also on medication use . AnotherCompared with academic oncologists, community oncologists rated hepatic and renal dysfunction and recent surgery as more important determinants of high risk of FN. The perception of risk factors for FN may be different for community oncologists compared with academic oncologists, and this may explain the slightly higher use of G-CSF for primary prophylaxis reported by community oncologists in this study. This finding is similar to previous survey studies of ASCO oncologists, which detected greater use of prophylactic G-CSF by oncologists practicing in a fee-for-service setting , 27.We restricted the study to US oncologists who met the eligibility criteria, volunteered to participate in an online survey, and had used G-CSFs in their practice. Thus, the results may not be generalizable to non-US oncologists, to US oncologists who were ineligible to participate, or to oncologists who do not use G-CSFs yet treat patients who are otherwise eligible for prophylactic FN treatment. The selection of oncologists using physician panel is not truly random, and those volunteering to participate may not be representative of source population of oncologists. The survey collected information on oncologists’ perceptions that were based on recall; therefore, the results may not reflect routine practice. Substantial variations in patterns of G-CSF use have been noted between oncologists belonging to the same community practice ; howeverDespite G-CSF approval since 1991 and the availability of evidence-based guidelines, FN prophylaxis in appropriate patients remains uneven. Our survey shows that not all oncologists follow national guidelines. Noncompliance with prophylactic G-CSF use in appropriate patients can increase FN risk, leading to increased morbidity and potentially increased health care costs. Further education of oncologists on the downstream consequences of not adhering to national guidelines could help reduce the underutilization and inappropriate timing of G-CSF, consequently reducing the risk of FN. Pegfilgrastim OBI is now increasingly used for next-day pegfilgrastim injection, reducing the travel burden and likely reducing the risk of noncompliance as a result of the travel burden. Barriers to G-CSF, including concerns about lack of reimbursement and patient refusal, differ by OCM affiliation and need to be explored further and overcome to improve coverage for G-CSF prophylaxis in appropriate patients.ESM 1(DOCX 230 kb)