Emergency medicine residency programs have evaluated the use of Multiple Mini Interviews (MMIs) for applicants. The authors developed an MMI-style method called the Fast Interview Track (FIT) to predict an applicant's ‘fit’ within an individual residency program.Applicants meet with up to five residents and are asked one question by each. Residents score the applicant using a Likert scale from 1 to 5 on two questions: ‘How well does the applicant think on his/her feet?’ and ‘How well do you think the applicant will fit in here?’. To assess how well these questions predicted a resident's ‘fit’, current residents scored fellow residents on these same questions. These scores were compared with the residents’ interview FIT scores. A postmatch survey of applicants who did not match at this program solicited applicants’ attitudes toward the FIT sessions.p=0.03]) and question 2 (rho=0.5753 [p=0.01]). Among seniors, Spearman's rho was statistically significant for question 2, though not statistically significant for question 1. The chi-square measure of high scores (4–5) versus low scores (1–3) found a statistically significant association between interview and current scores for interns and juniors. Of the 29 responses to the postmatch survey, 16 (55%) felt FIT sessions provided a good sense of the program's personality and only 6 (21%) disagreed. Nine (31%) felt FIT sessions positively impacted our program's ranking and 11 (38%) were ‘Neutral’. Only two (7%) reported that FIT sessions negatively impacted their ranking of our program.Among the junior class, the correlation between interview and current scores was significant for question 1 (rho=0.5192 [FIT provided program leadership with a sense of an applicant's ‘fit’ within this program. Interview day scores correlated with scores received during residency. Most applicants report a positive experience with FIT sessions. FIT provides a useful tool to recruit applicants who fit with the residency program. Research on the use of the Multiple Mini Interview (MMI) for residency interviews has grown significantly over the last year. In September 2014, Phillips and Garmel drew attention to the use of the MMI in emergency medicine (EM) interviews and the applicant's perspective of the process .The MMI was developed as 10 stations similar to objective structured clinical examinations (OSCE) with the intention of measuring non-cognitive skills that may not be adequately assessed during a personal interview . It has Within the specialty of EM, the MMI has been a growing area of interest. Studies recently published on EM applicants’ perception of the MMI have not been favorable. Hopson et al. conducted an eight-station EM-focused MMI focused with 71 EM interns from three different residency programs . They foAnother study by Soares et al. compared the MMI with traditional interviews for applicants to one EM residency program. On an anonymous postinterview questionnaire, 89% reported that they found the traditional interview more enjoyable than the MMI process, though only 14% of applicants in this study reported that the MMI would have a negative impact on their ranking of the program .The interview day has been shown to be the most influential factor on an applicant's residency selection that is within the residency program's control , 12. BecIn order to optimize the interview day experience at our program, we developed an interview process called the Fast Interview Track (FIT). It is a less structured style of the MMI and has come to be nicknamed ‘resident speed dating’. The goal of this experience is twofold. First, it provides more time to each applicant to talk with our residents one-on-one to display the personality of our program; exposing candidates to a larger number of our current residents. Second, it provides more ‘data’ for the residency leadership when formulating our rank list and gives current residents input into our overall rank decision. We hypothesize that the FIT scores predict how well the applicant will fit in with our program. We compared the FIT scores our residents received when they were actual residents in the program with their scores on their residency interview day to examine if we are improving our ability to select applicants that fit in well.We have used the FIT format in our standard interview day since the 2011 interview season. The applicants meet with up to five of our current residents and are asked one different question at each station. These questions are selected from a bank of questions approved by the residency leadership. We developed a mix of questions, some highlighting individual candidate's personal strengths, some making the candidates to think something through to highlight their thought process, and others challenging candidates to discuss a personally perceived negative characteristic trait. On interview day, program leaders choose the five questions to be used and distribute them to the residents, mixing the question types so that all resident–candidate interactions are not biased toward one type of question .How well does the applicant think on his or her feet?How well do you think the applicant will fit in here?An applicant meets with each resident individually for 5 min in a ‘speed-dating’ format. The resident then scores the applicant on two questions:Scores are based on a Likert scale anchored by 1=poor and 5=best. These scores are then incorporated into the applicant's overall interview score, which the program leadership then uses to determine our rank list. Per our standard interview scoring process, we average the scores the applicant receives on each of the two questions; then take the sum with a maximum score of 10. The first year we instituted the FIT, corresponding to the current senior residents, we used the mean of the two scores; therefore, the maximum score was five for the first year of its use. We performed this study at a 3-year academic emergency medicine program with 45 categorical EM residents and 8 combined EM/pediatrics residents. This study was reviewed by the local institutional review board and deemed exempt. During the time the FIT sessions were utilized, 44 of the 45 categorical residents and 8 of the 9 combined EM/Pediatric residents participated in the FIT sessions during their interview day. We surveyed all current residents via SurveyMonkey and asked each resident to score their fellow residents in the two classes other than their own on the same two questions used in the FIT session. This survey was conducted in January, after the interns had been in the program for 6 months. The scores from these surveys were compared with the FIT scores that each current resident received on their actual interview day.t tests, as previous studies have shown that for five-point Likert items, the t test and its non-parametric counterpart the Mann–Whitney–Wilcoxon generally have similar power. Lastly, chi-square tests of association were conducted for the combined questions during interview and follow-up assessments in order to assess an overall association between interview and current scores. All statistical tests were two sided at a significance level of 0.05. No adjustments were made for multiple comparisons. All analyses were performed using Stata version 14.0 .The primary outcomes for this study were Spearman rank correlation coefficients between interview scores and follow-up scores to measure the strength of association between the two ranked variables. Given the non-linear relationship between variables, the Spearman rank correlation was chosen over the Pearson correlation coefficient. In addition, mean interview and current scores for both questions were assessed by paired Did the FIT sessions give you a good sense of the personality and feel of our program and residents? (Yes or No)How did the FIT sessions impact your ranking of our program? We also conducted a postmatch survey of our top ranked applicants that did not match at our program, which is our standard practice each year. Among other questions, we asked the following questions specifically pertaining to the FIT sessions:Of the 52 residents surveyed, 44 were categorical EM residents and 8 were EM/pediatrics residents. There was an even split between males and females that participated with 26 female and 26 male .Among all three groups, the mean score for question 1 was lower on the current assessment compared to the interview day assessment. This result was statistically significant among juniors but not among interns and seniors. The difference was largest among juniors, with a mean interview score of 4.12 (SD=0.51) compared with a mean current score of 3.88 (SD=0.51). However, the mean score for question 2 was higher during the current assessment compared to interview day assessment. There was no statistical significance among any groups for question2. The largest difference was again among the juniors. Among seniors, there was a data limitation for question 2.p=0.03), and for question 2, rho was equal to 0.5753 (p=0.01). Thus, among juniors, a strong and significant positive correlation was found. We rejected the null hypothesis of no association between interview and current scores and concluded that interview and current scores have a positive relationship . Among seniors, Spearman's rho was also fairly strong and statistically significant for question 2, though not statistically significant for question 1.Correlations between baseline and follow-up scores are shown in Among interns, Spearman's rho was weak, indicating that there was no tendency for the current score to be either higher or lower when the interview day score was higher .χ2=9.05, p=0.003; Juniors: χ2=5.60, p=0.02) between interview and current scores, indicating our findings are unlikely to occur if there was no association between interview and current scores. Among juniors, this further strengthens the findings from the Spearman correlation analyses. Among interns, the results are mixed but could indicate that if the sample size were larger, results from the Spearman correlation analyses could have been stronger. For seniors, the chi-square results did not reach significance, though this result did have data limitations described in the discussion section. For interns, our calculation of odds ratios showed that for those with high interview scores, the odds of a current high score were 11.5 times larger than the odds for those with a low interview score  These results were similar for juniors  but not for seniors  (.5–26.0) .We sent postmatch surveys to 49 highly ranked applicants that did not match into our program. We asked two questions about the FIT interview on this survey. We received a total of 29 responses (59%).Regarding the question ‘Did the FIT sessions give you a good sense of the personality and feel of our program and residents?’, 16 (55%) responded ‘Yes’ and 6 (21%) responded ‘No’. Seven (24%) respondents skipped this question. For the question ‘How did the FIT sessions impact your ranking of our program?’, 9 (31%) responded ‘Positively’, 11 (38%) responded ‘Neutral’, and 2 (7%) reported that the FIT sessions negatively impacted their ranking of our program. Seven respondents again skipped this question .We ask an open-ended question in our survey that states ‘How did our interview day compare to the top 5 programs on your rank list? How would you suggest improving it?’. Comments related to the FIT sessions are noted in Optimizing the interview day experience, and specifically the use of the MMI, is a topic of interest in emergency medicine. This is evidenced by the growing number of studies on the use of MMI for resident selection within our specialty and the social media discussion held on ALiEM . OpinionWe have attempted to create an interview experience that benefits all stakeholders. Joshi et al. identify three goals of the interview day: 1) predict which candidates will be successful in emergency medicine; 2) portray the program's personality and strengths accurately; and 3) optimally match the applicant and program  score or clerkship grades. We also did not compare the FIT scores with performance measures during residency training such as In-Training exam scores, evaluations on clinical rotations, or milestones assessing professionalism. This is something that we plan to investigate in the future.The data analysis of the senior class was limited by the fact that we did not have the individual FIT scores for each of the two questions. We had only the average of the two as this is what was recorded for the assessment of applicants that year. The interns had only been a part of our program for 6 months when current residents evaluated how well they fit in. This may not have been enough time to adequately assess this.The postmatch survey responses are from a small sample of applicants. We sent out 49 surveys, and 29 were completed and returned. Only 22 people responded to the questions specifically addressing the FIT sessions. This number represents about 20% of the total applicants we interviewed.The FIT sessions provide the program leadership and residents with a sense of how an applicant will fit in with our residency program. Most applicants have a positive experience in the FIT sessions. Any device or method that helps align the personalities and goals of residency programs and applicants is of benefit to all stakeholders.One question that may be raised with this process is, Does utilizing peer evaluation to ensure a good ‘fit’ in a program discourage diversity? We have not found this to be the case. Our approach to applicant selection and candidate ranking is holistic, without rigid selection or rejection criteria. The FIT interview score is a portion of the overall evaluation of an applicant, currently constituting only 10% of the overall score we use to arrive at our initial ranking. We consider the applicant's entire portfolio when determining where a candidate ends up on our final match list. We believe that the 10% contribution of FIT to the candidate's score adds value in our overall assessment and a different perspective of the candidate that was not present with application review and traditional interviews alone. In addition, the process allows the candidates to interact with more of our current residents and these residents to be engaged, contributing to our selection process. With proper training of residents on the interview process and utilizing a holistic approach, we feel the FIT interviews can enhance camaraderie and cohesiveness without detrimentally affecting the diversity of a program.The FIT session is a method that has proven beneficial for us and may be beneficial to other programs as well.


Accumulating evidence suggests that O-GlcNAc transferase, an enzyme responsible for O-GlcNAc post-translational modification acts as a nutrient sensor that links glucose and the hexosamine biosynthetic pathway to the regulation of transcriptional factors involved in energy homeostasis. In liver, glucose signaling is mediated by carbohydrate response element-binding protein (ChREBP), which stimulates glycolytic and lipogenic gene expression through its binding on a specific ChoRE DNA sequence. Modulation of ChREBP by O-GlcNAcylation increases its DNA binding affinity and its activity. ChREBP transcriptional activity also depends on the presence of several other co-factors and transcriptional factors. Among them, the nuclear Farnesoid X Receptor (FXR), a key transcription factor of bile acid metabolism involved in the gut–liver axis homeostasis was recently shown to directly interact with ChREBP, acting as a repressor on the ChoRE of glycolytic genes. Interestingly, similarly to ChREBP, FXR is O-GlcNAcylated in response to glucose. This review discusses the importance of ChREBP and FXR modifications through O-GlcNAcylation in liver and how glucose can modify their mutual affinity and transcriptional activity. The positive effects of glucose on gene expression are mediated by the transcription factor carbohydrate-responsive element-binding protein (ChREBP). ChREBP, which belongs to the Mondo family of bHLH/Zip transcription factors, is a large protein (864 a.a) that contains several regulatory domains including a nuclear localization signal  near the N-terminus, polyproline domains, a bHLH/LZ domain (amino acids 660–737), and a leucine zipper-like (Zip-like) domain (amino acids 807–847) (N-acetyl-glucosamine (UDP-GlcNAc), an obligatory substrate for β-N-acetylglucosaminyltransferase (OGT), a key enzyme allowing O-GlcNAcylation of proteins  by the glucokinase enzyme (GK) leading, in turn, to the activation of glycolytic and lipogenic enzymes including 807–847) , 3. ChREproteins  is a regproteins . Interesproteins , 11. Thil-PK, FAS, and ACC not only in the liver  were reported to be hypoglycemic in response to a short time (6 h) fasting. This phenotype can be, in part, explained by an alteration of the expression of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. Interestingly, the response to longer fasting (24–48 h) was not affected in the absence of FXR, suggesting a defective adaptative response in FXR−/− mice. Surprisingly, activation of FXR by GW4064, a specific synthetic ligand, did not increase PEPCK expression in primary mouse hepatocytes , onto its response elements (FXRE) to regulate its target genes. While largely implicated in the transcriptional control of genes controlling bile acid metabolism, FXR also recently emerged as a novel modulator of glucose homeostasis , 29. FXRatocytes . Howeveratocytes  suggesti−/− mice respond more rapidly to high carbohydrate feeding with an accelerated induction of glycolytic and lipogenic genes without, however, any difference in ChREBP mRNA levels  domain (amino acids 1–127) and via the N-terminal part of its ligand-binding domain (amino acids 215–300)  .l-PK, ACC, and FAS. This inhibition was prevented in FXR−/− hepatocytes. Importantly, using a ChoRE luciferase promoter construct, the authors reported that FXR transfection and/or activation prevented the stimulation of ChoRE-driven target genes. Gel shift analysis revealed that FXR was indeed able to bind to the L3 site (contained within the ChoRE) but not to the L4 site of l-PK promoter  assays, Caron et al.  within the ChREBP protein . This NRO-GlcNAc hydrolase (OGA), which hydrolyzes the monosaccharide. Sakiyama and co-workers (l-norleucine), a drug that decreases O-GlcNAc, the stimulatory effect of high glucose on ChREBP activity is prevented (OG). In mouse hepatocytes, overexpression of OGA led to an inhibition of ChREBP-target genes associated with a decrease of lipid droplets under high glucose concentrations. In vivo, OGT overexpression in mouse liver was associated with an increase of ChREBPOG, correlated with the induction of l-PK expression and with ChREBP recruitment to the l-PK promoter. OGT overexpression also increased ChREBP protein content without modifying ChREBP mRNA levels suggesting that the protein may be stabilized by O-GlcNAcylation (ub) was reduced when ChREBP is O-GlcNAcylated  O-GlcNAc transferase (OGT), which adds a monosaccharide to serine/threonine residues of target proteins; (ii) the -workers  first shrevented . Our labrevented . ChREBP cylation . InteresAcylated  suggestiBerrabah et al. revealed that FXR is modified by O-GlcNAcylation through its interaction with OGT, which catalyzed this reaction in response to high glucose level. O-GlcNAcylation leads to an increase of FXR protein stability, transcriptional activity, and chromatin binding through SMRT inactivation. O-GlcNAcylation of FXR occurs on serine 62 (Ser62) within the AF-1 domain. In agreement, mutation of Ser62 decreased FXR O-GlcNAcylation that correlated with an inhibition of its transcriptional activity. In vivo, nutritional experiments reveals that FXR is O-GlcNAcylated under fed conditions, which correlates with an induction of its target genes  and a decrease in hepatic bile acid content. Interestingly, a recent study reported that FXR can also be modified by SUMOylation . The improved phenotype in OGA-treated db/db mice was also associated with a significant decrease in O-GlcNAcylation of the transcriptional co-activator CRTC2 , diabetic nephropathy, and/or retinopathy . Indeed,or CRTC2  involvedor CRTC2 . Recentlstigated . Treatmehropathy . The potcontrols . The fincontrols  further controls , but alscontrols . In fastcontrols . Howeverm Figure , a domaim Figure . FurtherThe 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 Associate Editor Tarik Issad declares that, despite being affiliated to the same institution as the authors Fadila Benhamed, Gaelle Filhoulaud and Catherine Postic, the review process was handled objectively and no conflict of interest exists.


Although chronic sympathetic activation provides inotropic and chronotropic support to the failing heart, such activation may also have deleterious effects, including the direct cardiotoxic effects of catecholamines, activation of the renin-angiotensin-aldosterone system and an increase in myocardial oxygen demand. These observations indicate that β-blockade might be beneficial in the treatment of heart failure resulting from dilated cardiomyopathy or ischaemic heart disease. Carvedilol is a non-selective β-blocker acting on β1-, β2-, and α1-adrenoceptors. It possesses potent anti-oxidant and anti-apoptotic properties, along with neuroprotective, vasculoprotective, cardioprotective effects, and it has reduced overall mortality in patients with heart failure in controlled clinical trials. Its role in treating cardiomyopathy requires focus. The fact that anthracyclines are cardiotoxic seriously narrows their therapeutic index in cancer therapy. The cardiotoxic risk increases with the cumulative dose and may lead to congestive heart failure and dilated cardiomyopathy in adults and in children. This review focuses on recent research regarding the beneficial effects of carvedilol in the treatment of dilated cardiomyopathy and to revisit the available evidence on the cardioprotection of carvedilol when associated with anthracycline and to explain the mechanisms underlying the benefits of their co-administration. As the population ages, the incidence of cardiomyopathy is expected to increase greatly. It is a disease of the heart muscle. In most cases, cardiomyopathy causes the heart muscle to become weak, reduce the efficient functioning of the heart muscle, and diminish the ability of the heart to meet the needs of the body. When the heart can no longer pump enough blood to meet the needs of the body, heart failure (HF) is said to be present ,2.There are three major types of cardiomyopathy – dilated cardiomyopathy (DCM) , hypertrAnthracyclines are some of the most effective drugs currently available in the treatment of neoplastic diseases. However, anthracyclines have profound consequences on the structure and function of the heart causing with time a cardiomyopathy that leads to intractable congestive heart failure . The car2.Although the ultimate cause of DCM is unknown, major factors could include diabetes mellitus, coronary artery disease, inflammation of the heart muscle (myocarditis) and alcohol. DCM had been known to result from genetic causes such as mutations in several genes ,12 or no3.The therapeutic approach to DCM still remains nonspecific and symptomatic, since no specific etiology is identified. The recent introduction of angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blocker (ARB) and β-blockers greatly improved the treatment of DCM . Digital4.It is well known that the sympathetic nervous system is activated during the development of CHF, and its prolonged activation results in ventricular remodeling and progression of cardiac dysfunction . Over ex5.1-adrenoceptor blockade, the latter resulting in vasodilation. An extraordinary high degree of cardioprotection beyond that typically seen with beta adrenoceptor blockade suggested that additional properties of the drug might be responsible for this protection. Carvedilol and several of its metabolites are potent antioxidants and it has been shown to exert potent antioxidant effects in multiple cell model systems [Carvedilol is a third-generation vasodilating β blocker that has recently been shown to reduce morbidity and mortality in patients with CHF. This reduction may occur in part through β and α systems . In a la systems .Analysis of animal studies reported from our laboratory showed favorable effects of carvedilol in a rat model of DCM induced by autoimmune myocarditis ,29. In tA number of controlled clinical studies showed that carvedilol -33 was b6.The molecular pathogenesis of anthracycline cardiotoxicity remains highly controversial, although the oxidative stress-based hypothesis involving intramyocardial production of reactive oxygen species (ROS) has gained the widest acceptance . AnthracConcomitant use of dexrazoxane, a cardioprotective ion chelating agent, can reduce the risk of this drug-induced cardiomyopathy ,44. Dexret al. [2+ loading capacity and inhibition of respiratory complexes in cardiac mitochondria, and protects against doxorubicin associated lipid peroxidation of cardiac membranes [Oliveira et al.  demonstrembranes .et al. showed that carvedilol increases the cytotoxicity of doxorubicin in tumor cells by inhibiting multiple drug-resistant proteins. This demonstrates that carvedilol does not reduce the effectiveness of anti neoplastics, which is obviously important if it is to be administered together with doxorubicin [Similarly, we have reported that DNR rats showed cardiac toxicity as evidenced by worsening cardiac functions, which were evaluated by hemodynamic and echocardiographic studies, malondialdehyde level and the total level of glutathione peroxidase activity in heart tissue. These changes were reversed by treatment with carvedilol, which resulted in significant improvement in the cardiac function. Furthermore, carvedilol down-regulated matrix metalloproteinase (MMP)-2 expression, attenuated the increased protein expression of NADPH oxidase subunits and reduced myocardial apoptosis as well as improved the histopathological changes in heart induced by DNR . A studyorubicin .et al. In 6-months follow-up study, fifty patients were treated with anthracyclines, 25 of whom also received carvedilol. Co-administration of 12.5 mg carvedilol daily during chemotherapy maintained LV diastolic and systolic function [et al. reported that five cases of severe CHF due to anthracycline-induced cardiomyopathy effectively treated with carvedilol. Their LV functions as well as cardiac symptoms were persistently improved after treatment with carvedilol, suggesting that carvedilol may be an effective therapeutic strategy for anthracycline-induced cardiomyopathy as demonstrated in other forms of CHF [The first clinical trial on the prophylactic use of anthracycline-induced cardiomyopathy was performed by Kalay function . Accordis of CHF .7.Inflammatory stresses are cardinal in the pathogenesis of many cardiovascular diseases including diabetic cardiomyopathy, hypertension, atherosclerosis, myocarditis, HF and drug-induced cardiotoxicity. Many treatments for above mentioned disease target the use of anti-inflammation drug which thought to reduce the severity of the disease. Carvedilol has been reported to exert multiple activities, not only anti-oxidant and anti-apoptosis properties, but also anti-inflammation activity . In the et al. have reported that long-term treatment with carvedilol induced an improvement in symptoms of HF paralleled by an increase of IL-10 levels and a reduction of IL-18 levels, resulting in a 9-times reduction of IL-18/IL-10 ratio and reaching values similar to healthy subjects [in vitro and carvedilol dose-dependently inhibited the elevated TNF-α-mediated apoptotic process [The cytokines hypothesis for HF holds that HF progresses, at least in part, as a result of the toxic effects exerted by endogenous cytokine cascades on the heart and the peripheral circulation ,52. Intesubjects . This me process . In DCM, process . Carvedi process . A repor process .Carvedilol has been reported also to reduce the inflammatory-mediated atherogenic process . In the + T-lymphocytes and an up-regulation of CD8+ T-lymphocytes were produced by carvedilol treatment. The significant correlation between IL-1β and MMP-8 mRNA abundance and CD4+ T-lymphocytes, as well as the correlation between IL-1β mRNA abundance and hemodynamic parameters emphasize the importance of this cytokine with respect to the MMP/tissue inhibitor of metalloproteinases ratio and hemodynamic function [via the β2-receptor. β2-receptors are expressed on the Th1 T lymphocytes and antigen-presenting cells [A broader spectrum of carvedilol beneficial effect has also been confirmed in coxsackievirus-induced and autoimmune-induced myocarditis. The balance between the protective and deleterious immune mechanisms may eventually determine the course of viral myocarditis . Cytokinfunction . Therefofunction . In expefunction . This stfunction . Carveding cells ,67.Thus, it can be concluded that anti-inflammation effect of carvedilol contributes to the clinical efficacy of carvedilol in a broad spectrum of disease and may also confer a range of cardioprotective benefits. However, studies that elucidate by which carvedilol directly regulates the pro-inflammatory chemokines and inflammatory cytokines will be fully required.8.Carvedilol is contraindicated in patients with bronchial asthma or related bronchospastic conditions, decompensated NYHA functional class IV heart failure requiring intravenous inotropic therapy, severe liver impairment, second- or third-degree atrioventricular block, sick sinus syndrome (unless a permanent pacemaker is in place), cardiogenic shock, severe bradycardia or known hypersensitivity to the drug ,68.9.The number people with cardiomyopathy increases greatly with respect to time and the therapy for it still remain to be unresolved. Several classes of drugs such as β-blockers, ARB, ACE inhibitors, and diuretics are used in the management of cardiomyopathy. Carvedilol appears to have multiple modes of action, sympathetic blocking, anti-inflammatory, anti-apoptosis, and anti-oxidant activity. It also prevents the cardiac remodeling process proving its role in the treatment of cardiomyopathy .


SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome‐wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val602 variant of the non‐synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229  has a two‐fold lower affinity compared with the SLE‐associated Met602 variant for the small adaptor protein SAP. Comparison of the two variants in T‐cell lines revealed the Val602 variant to be significantly more highly expressed than CD229 Met602. Activation was diminished in cells expressing CD229 Val602 compared with CD229 Met602 as measured by up‐regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine‐tuning in the immune system with significant functional consequences.Signalling lymphocyte activation molecule ( FCSfetal calf serumHAhaemagglutininITSMimmunoreceptor tyrosine‐based switch motifJC20Jurkat clone 20MCSmultiple cloning sitePBMCperipheral blood mononuclear cellSAPsignalling lymphocyte activation molecule–associated proteinSH2 domainSrc homology 2 domainshRNAsmall hairpin RNASLAMsignalling lymphocyte activation moleculeSLEsystemic lupus erythematosusSNPsingle nucleotide polymorphismSignalling lymphocyte activation molecule (SLAM) family receptors are expressed on haematopoietic cells and include CD150 , CD48 SLAMF2), CD229 , CD244 , CD84 (SLAMF5), CD352 , CD319 , BLAME (SLAMF8) and SLAMF9 ., CD229 .The cytoplasmic tail of CD229 contains two ITSMs and an interaction between CD229 and SAP has been shown.Sle1 locus in mice.602 (TVYAQV) to Met602 (TMYAQV) switch in the first ITSM of CD229.CD229 and other SLAM family members have been linked with susceptibility to systemic lupus erythematosus (SLE), a complex autoimmune disease.m sodium pyruvate), respectively, supplemented with 2 mm glutamine, 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. Puromycin (1 μg/ml) was added to maintain protein and small hairpin (sh)RNA expression in stable cell lines. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll density gradient centrifugation and frozen at −80° in FCS containing 10% DMSO.Media were purchased from Sigma‐Aldrich . Jurkat Clone 20 (JC20) and HEK‐293T cells were grown in RPMI‐1640 and Dulbecco's modified Eagle's medium , anti‐CD3ε clone Leu‐4 , anti‐CD229 clone HLy9.1.25 , anti‐CD69 , anti‐CD3ε clone UCHT1  and anti‐CD28 clone CD28.2 (Biolegend).Antibodies used in this study were: anti‐T‐cell receptor 6 PBMCs using the PureLink® Genomic DNA Kit . DNA of exon 8 of CD229 encoding rs509749 was amplified by PCR using the primers 5′‐ACTCTGTGCTCTCCCAAGGA‐3′ and 5′‐TAGTGGCCCAAGAATGAGAGCTG‐3′ sequenced using primer 5′‐TGCACAAGCCCATCAGTGG‐3′ and analysed with geneious 6·1·5 .Genomic DNA was purified from 4 × 10R lentiviral vector was created by modifying the plasmid pU6‐shRNA‐EF1‐GOI‐IRES‐PuroR.EcoRI‐cut pU6‐shRNA‐EF1‐GOI‐IRES‐PuroR creating a multiple cloning site (MCS) downstream of the U6 promoter containing the restriction sites 5′□3′AscI, PacI, PmeI and EcoRI. The shRNAs generated by slow annealing of the 5′‐TAAGCGCGCTTTGTAGGATTCGTTTCAAGAGAACGAATCCTACAAAGCGCGCTTTTTTG‐3′ sequence with 5′‐AATTCAAAAAAGCGCGCTTTGTAGGATTCGTTCTCTTGAAACGAATCCTACAAAGCGCGCTTAAT‐3′ and of the 5′‐CGCGCCGTAGCGTTTCCTCCTCGAAATTTTCAAGAGAAATTTCGAGGAGGAAACGCTATTTTTTG‐3′ sequence with 5′‐AATTCAAAAAATAGCGTTTCCTCCTCGAAATTTCTCTTGAAAATTTCGAGGAGGAAACGCTACGG‐3′ creating the scrambled shRNA (shCTR) and the 3′‐untranslated region targeting CD229 shRNA , respectively, were cloned 3′ of the U6 promoter using the PacI and EcoRI (shCTR) or AscI and EcoRI (shCD229) sites. The CD229 sequence (NM_002348) with the EcoRI restriction sites (GAATTC) destroyed by site directed mutagenesis (GAATTT) was inserted into pU6‐MCS‐EF1‐GOI‐IRES‐PuroR via SpeI and SmaI. HA constructs were generated by cloning the secretion signal peptide from pHLsec followed by a haemagglutinin (HA)‐tag R using forward 5′‐TAGACTAGTATGGGCATCCTTCCCAGC‐3′ and reverse 5′‐TAGCCCGGGTTGATCAGGTTTAAACATAGCGTAGTCTGGGACGTCG‐3′ oligos creating a PmeI–XmaI insertion site. The DNA fragment of CD229 encoding the short extracellular linker, the transmembrane and cytoplasmic domain (residues Ser434 to Thr655) was inserted in‐frame via PmeI and XmaI to generate pU6‐MCS‐EF1‐HACD229434‐656‐IRES‐PuroR.The bicistronic pU6‐MCS‐EF1‐GOI‐IRES‐Puro® : DNA in Dulbecco's modified Eagle's medium and 1% FCS for 16 hr at 37°. The medium was replaced with RPMI‐1640 and 10% FCS and supernatant containing viral particles was collected after 48 hr and filtered (0·45 μm). JC20 cells (0·7 × 106) were resuspended in 2 ml virus‐containing supernatant, supplemented with 5 μg/ml polybrene and centrifuged in a 12‐well flat bottom plate at 30° at 1350 g for 1·5 hr. Supernatant was replaced after 16 hr by RPMI‐1640, 10% FCS and puromycin (1 μg/ml).Lentiviral particles were produced by transfecting HEK‐293T cells at 60% confluence with a 4 : 2 : 1 ratio of the lentiviral expression: psPAX2 packaging: pMD2.G envelope vectors at a 2·5 : 1 ratio of FuGENE4) or PBMCs (0·2 × 105) were plated in each well of 96‐well flat‐bottomed plates pre‐coated with the indicated concentration of anti‐CD3ε and stimulated for 6 hr or 16 hr, respectively. Surface molecules were stained with the appropriate antibodies directly before and after stimulation and expression levels were analysed using the BD FACSCalibur™ and Beckman Coulter CyAn™ ADP Analyzer. Cells were analysed based on live, single cells according to forward light scatter/side scatter characteristics unless stated otherwise. Data were evaluated using flowjo, MS excel and graphpad prism 6 software.Jurkat T cells ), mouse anti‐phosphotyrosine , goat anti‐HA  or anti‐β‐actin  followed by the appropriate secondary antibody or fluorophore‐conjugated streptavidin . Quantification of protein expression was performed using LI‐COR odyssey sa software version 1.0.Western blotting was performed as previously described.γRIIb‐ITIM (ADKVGAENTITpYSLLMHPDA) were from Peptide Protein Research Ltd, Fareham, Hampshire, UK.m HEPES, pH 7·4, 150 mm KCl, 10 mm EDTA, 0·005% volume/volume Tween 20. The signal in a reference cell coated with streptavidin was subtracted. Equilibrium binding dissociation constants were calculated using biaevaluation and graphpad prism 6 software by fitting the equilibrium data using a one‐site binding curve.Synthetic peptides all biotinylated at the N‐terminus for human CD229M (GENTMpYAQVFN), CD229V (GENTVpYAQVFN), CD244‐ITSM1 (EFLTIpYEDVKD), CD244‐ITSM2 (GGSTIpYSMIQS) and FcP‐values were calculated as indicated in the figure legends using graphpad prism 6 software. For comparison of HA expression levels, HA‐CD229 Met602 (HAM) and HA‐CD229 Val602 (HAV) data sets after stimulation with 0·5 μg/ml anti‐CD3ε from five experiments were merged and classified according to their expression levels. Cells below 25% of total HA‐expression were defined as HAlo, above 75% as HAhigh and intermediate cells as HAint. Medians of CD69‐staining intensities were used for performing paired t‐test analysis.602 or Met602 ITSMs phosphorylated on the tyrosine residues  compared with the risk allele, Met602 of CD229 (KD = 1·5 μm) .We compared binding of SAP to tyrosine phosphorylated peptides of the same length representing the CD229 ITSM containing the rs509749 SNP (TV/MpYAQ) and the more distal ITSM (TIpYCS). SAP bound to the second non‐polymorphic sequence (TIpYCS) with up to an order of magnitude greater strength , shCD229, CD229 Met602 or CD229 Val602  was more highly expressed than HA‐CD229 Met602 (HAM)  Fig. a,b confiAM) Fig. b.602 or HA‐CD229 Met602 were stimulated with anti‐CD3ε and analysed for up‐regulated CD69 expression by flow cytometry  and CD229 Val602 (KD = 3·4 μm) for SAP was measured. There was no evidence of a switch in specificity as suggested previously.The ITSMs in the variants of rs509749 in CD229 differ at the Tyr‐1 position. The amino acid at the Tyr‐1 position is not crucial but influences the three‐pronged binding mechanism of SH2 domains to ITSMs.602 was more highly expressed than the SLE‐associated CD229 Met602 in Jurkat T cells. Several SLE‐associated SNPs have been shown to change epigenetic imprints by modulating gene transcription.602 compared with CD229 Met602. Analysis of flow cytometric data indicated that CD229 Val602 was more readily internalized after CD3 monoclonal antibody‐induced activation compared with CD229 Met602, suggesting that slower internalization of CD229 Val602 is unlikely to explain the difference in expression (unpublished data). Significant differences in CD229 expression levels were not observed in PBMCs from healthy donors. There was up‐regulation of SLAM family receptors, CD229 and of CD352  in T cells from patients with SLE compared with healthy controls.The recombinant variant, CD229 Val602 variant. The lower affinity of SAP for the CD229 Val602 variant as well as the imbalance between adaptor and receptor might account for diminished activity of cells transduced with the SLE susceptible CD229 variant in comparison with CD229 Met602.An imbalance between stimulus strength, expression levels of receptor and signalling molecules can change the outcome of cellular activity, as has been shown for the SLAM receptor CD244 (2B4).In conclusion, amplification of differences at the molecular and cellular levels between the CD229 variants might be sufficient to explain how rs509749 was identified as being associated with susceptibility to SLE specifically in a family‐based study.SM designed and analysed functional experiments and wrote the manuscript. LIG and TJW performed and analysed the surface plasmon experiments. MHB directed the research and wrote the manuscript.There are no conflicts of interest.


Many people are concerned about that the quality of preventive care for patients with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection is suboptimal. Taiwan, a hyperendemic area of chronic HBV and HCV infection, implemented a nationwide pay-for-performance (P4P) program in 2010, which aimed to improve the preventive care provided to HBV and HCV patients by increasing physicians’ adherence to guidelines through financial incentives. The objective of this study was to evaluate the early effects of the P4P program on utilization of preventive services by HBV and HCV patients.Using a quasi-experimental design with propensity score matching method, we matched the HBV and HCV patients enrolled in the P4P program with non-enrollees in 2010, resulting in 21,643 patients in each group. Generalized estimating equations was applied to examine the difference-in-difference effects of P4P program enrollment on the utilization of three guideline-recommended preventive services  examinations, and aspartate aminotransferase and alanine aminotransferase (AST/ALT) tests by HBV and HCV patients.The P4P program enrollees were significantly more likely to attend twice-annual follow-up visits, to receive recommended US examinations and AST/ALT tests, than non-enrollees.The results of our preliminary assessment indicate that financial incentives offered by the P4P program was associated with a modest improvement in adherence to guidelines for better chronic HBV and HBC management. Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death worldwide –3. ChronPreventing or deferring disease progression in chronically infected HBV and HCV patients has become a major clinical and policy priority. Although antiviral treatment is the most important factor to prevent HBV and HCV progression , 14, guiDespite the establishment of guidelines, the quality of preventive care received by chronic HBV and HCV patients remains suboptimal. Studies have suggested that patients with chronic HBV do not receive adequate routine care, particularly regular follow-up visits and surveillance or testing –23. AlthHCC is a leading cause of cancer incidence and mortality in Taiwan , and theThe National Health Insurance (NHI) is a mandatory single-payer social health insurance program implemented in Taiwan on March 1, 1995. More than 90% of all hospitals and clinics in Taiwan are contracted with the NHI to provide comprehensive medical care coverage, including ambulatory and inpatient care, dental services, rehabilitation, prescription drugs and others, to > 99% of the country’s 22.5 million residents –34. A seIn addition to regular reimbursement for healthcare services, the P4P program provides participating physicians additional case management fees if their patients complete an initial enrollment visit (New Taiwan (NT)$100/visit; U.S. Dollar (US$) 3/visit) and a maximum of two routine follow-up visits per year . US examinations and AST/ALT tests are required in the initial and follow-up visits. Additional rewards are also provided for further screening, referral, and early detection of abnormalities  and HCC  .This study had a quasi-experimental design using population-based data extracted from the NHI claims files from 2009 to 2011. The NHI claims files include the NHI enrollment files, claims data, medical personnel registry, and the hospital or clinic registry. Data related to the identification of individuals were encrypted before being released to the researchers, and personal privacy was therefore protected. This study was approved by the Institutional Review Board (IRB) of National Yang-Ming University (IRB No. YM103085E).In this study, according to the P4P program’s eligibility criteria, we defined our sample of chronic HBV or HCV patients as individuals with ≥ two HBV- or HCV-related visits to the same providers during the 6 months prior to the index date. HBV and HCV infection were defined as the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes 070.30–070.33, 070.51, 070.54, V02.61, and V02.62. The index date for each patient was defined as the date of the first recorded visit for chronic HBV or HCV in 2010. We excluded patients with HCC (ICD-9-CM code 155) or hepatic coma , patients aged < 20 years, patients with incomplete or missing data, and patients who died during the 1-year follow-up.To minimize the potential influence of selection bias, we used two strategies for sample selection. First, among all eligible patients , 39,976 HBV or HCV patients of the physicians who did not participate in the P4P program were excluded. By the end of the first year, 963 physicians had participated in the P4P program. Of all 98,446 chronic HBV/HCV patients managed by these physicians, 21,646 patients (22%) had enrolled in the P4P program. Because the enrollees might have differed from the non-enrollees, we used a propensity score matching (PSM) approach to match the enrollees and non-enrollees with similar baseline patient, physician and hospital characteristics . The finFor this HBV/HCV P4P program, the NHIA developed a set of process-related quality measures to encourage the ongoing monitoring of HBV and HCV patients through regular follow-up visits, routine US screening, and AST/ALT tests. Three dependent variables were constructed in this study accordingly: (1) ≥ two HBV- or HCV-related outpatient follow-up visits per year, (2) ≥ two abdominal US examinations per year, and (3) ≥ two AST and ALT tests per year. The P4P program offers rewards for services provided by the same designated providers. Therefore, only the services provided by the same providers were included in subsequent analyses .The primary independent variable was program enrollment. Using the NHI specific payment codes for this P4P program, the P4P program enrollees were defined as patients with payment coding from P4201C to P4205C. The remaining eligible patients were referred to as the non-enrollees.Other covariates included for analysis were the characteristics of the patients, physician and hospitals. Patient characteristics included gender, age, ethnic status, socioeconomic status (SES), liver cirrhosis, number of chronic diseases, and any other mental and physical catastrophic illness. The age categories were 20–39 years, 40–59 years, and ≥ 60 years. The ethnic group categories were aborigine or nonaborigine. SES was defined as a patient’s own insurable wage if he or she was the insured or the insurable income of the insured if he or she was a dependent or did not have a clearly defined monthly wage. SES was classified into three categories. The NHI program is financed by wage-based premiums for people with a clearly-defined monthly wage, and by fixed premiums for people without a clearly-defined monthly wage. Therefore, patients with a clearly defined monthly wage were assigned to one of the three categories, in which high SES patients were defined as patients with an insurable wage ≥ NT$40,000 , middle SES patients were defined as patients with an insurable wage between NT$20,000 and NT$39,999 , and low SES patients were defined as patients with an insurable wage < NT$20,000 (US$ 625). Patients without a clearly defined monthly wage were typically farmers, fishermen, or people with low income, and were included in the same low SES group as women with an insurable wage < NT$20,000 (US$ 625) . The numPatient, physician and hospital characteristics were compared between the program enrollees and non-enrollees using chi-square tests. For the PSM method, a multivariate logistic regression model that included patient, physician and hospital characteristics, and adherence pattern prior to the P4P program, was applied to obtain propensity scores for the probability of being in one of the two groups. We employed the caliper matching method , with one-to-one matching between the enrolled group and the comparison group based on the closest propensity score, repeating the process until the smaller group (the enrollees) had been exhausted , 37. Thiβ1) represents the difference in the outcome of interest between P4P group and non-P4P group before the program was implemented. The coefficient of Time (β2) represents change of non-P4P group in the different period. The coefficient of the interaction terms (β3) reflect the impact before and after the implementation of the P4P program between the P4P and non-P4P group.McNemar’s test was used to assess the significance of the differences between the repeated proportional outcome variables before and after the implementation of the P4P program. Then we adopted difference-in-difference (DD) methodology (a pre-post design with a control group) to compare the changes (or differences) in the outcome variables before and after the implementation of the P4P program between the enrolled group and the comparison group to derive the policy impact , 39. TheTo address the correlation between repeated observations in outcomes across time for the same patient, the multivariate logistic regression model with generalized estimating equations (GEE) method was applied to examine the DD effects of P4P enrollment on three process quality measures among the patients with chronic HBV or HCV. We also analyzed the effect of P4P enrollment on the overall provision of all three requirements. For these dichotomous outcome variables, we specified a binomial distribution with logit link and the correlation matrix was assumed to be unstructured. For each model, odds ratios (OR) and 95% confidence intervals (CI) were calculated. All analyses were performed using the statistical software package SAS Version 9.4 , and a P value < 0.05 was considered significant.P < 0.001). After the PSM process, the P4P and non-P4P groups contained 43,286 matched patients, with 21,643 patients in each group. The two groups were similar in all observable characteristics.P < .0001). Specifically, the non-P4P group was associated with significant decrease in usage of regular follow-up visits , US examination , and AST/ALT tests . On the other hand, the usage rates among patients in the P4P group either increased significantly in US examination (P = 0.017) or remained unchanged in regular follow up visits. The only exception was the decrease in usage of AST/ALT tests in the P4P group.The HBV/HCV P4P program predominantly aims to encourage the ongoing monitoring of chronic patients through regular follow-up visits, routine US screening, and AST/ALT tests. The results of this preliminary assessment indicate that whereas the non-P4P enrollees were associated with significant lower usage of all three recommended services than the P4P enrollees after the implementation of the P4P program, the magnitude of change difference between the two groups before and after the P4P program was modest (5%-23%).P4P is an increasingly popular payment method for linking provider reimbursement to quality of care –27, a seIn contrast to the previous findings in other P4P programs in Taiwan, the magnitude of change differences in utilization of recommended preventive services following the HBV/HCV-P4P program was relatively small. The results highlight two key potential reasons for the minor improvement observed. First, similar to the majority of P4P programs in several other countries, provider participation in the P4P program is voluntary and the participation rate tends to be low . In the Next, although a significantly greater number of P4P program enrollees received the guideline-recommended preventive services than did the non-enrollees, the rates of receiving all three recommended services in the P4P (25.2%) and non-P4P 21.0%) groups remained unsatisfactory, particularly for services such as US examination and AST/ALT tests. Due to poor compliance, the results probably represent the minimum improvement that can be expected from the P4P program. Our results are similar to those of previous studies, which indicated that adherence to HCC surveillance is suboptimal .0% group, 48, 49.On the other hand, whereas the usage rate of all three recommended services in the P4P group remained relatively unchanged after the HBV/HCV-P4P program implementation, the usage rate in the non-P4P group was associated with significant reductions. One plausible explanation is that the financial incentives may have led providers to preferentially follow those people where they receive the greatest payment and divert human resources and attention to some people over others. This may not be necessarily ideal for a population health intervention. Further research and health policy should pay more attention on this issue.This study has a few limitations that should be noted. First, due to the voluntary nature of program participation, selection bias may be likely. To minimize the potential influence of selection bias, we used a PSM approach to match the enrollees and non-enrollees with similar baseline patient and provider characteristics , 37. HowAccording to our literature search, our study is the first nationwide population-based study to evaluate the early effects of a HBV/HCV-P4P program on the provision of recommended preventive services to patients under a universal insurance coverage system. Our large sample and comprehensive data are associated with sufficient statistical power to demonstrate a significant association between the P4P program and the quality of management of HBV and HCV patients during the first year of the program although the magnitude of differences was modest. We used PSM to match the P4P and non-P4P groups for individual and provider characteristics, which strengthens the validity of our findings . We alsoTaiwan is a hyperendemic area of HBV/HCV infection , where l


The voluntary medical male circumcision (VMMC) program in Zimbabwe aims tocircumcise 80% of males aged 13–29 by 2017. We assessed the impact ofactual VMMC scale-up to date and evaluated the impact of potentialalterations to the program to enhance program efficiency, throughprioritization of subpopulations.We implemented a recently developed analytical approach: the age-structuredmathematical (ASM) model and accompanying three-level conceptual frameworkto assess the impact of VMMC as an intervention. By September 2014, 364,185males were circumcised, an initiative that is estimated to avert 40,301 HIVinfections by 2025. Through age-group prioritization, the number of VMMCsneeded to avert one infection (effectiveness) ranged between ten(20–24 age-group) and 53 (45–49 age-group). The cost perinfection averted ranged between $811 (20–24 age-group) and $5,518(45–49 age-group). By 2025, the largest reductions in HIV incidencerate (up to 27%) were achieved by prioritizing 10–14, 15–19,or 20–24 year old. The greatest program efficiency was achieved byprioritizing 15–24, 15–29, or 15–34 year old.Prioritizing males 13–29 year old was programmatically efficient, butslightly inferior to the 15–24, 15–29, or 15–34 agegroups. Through geographic prioritization, effectiveness varied from9–12 VMMCs per infection averted across provinces. Through risk-groupprioritization, effectiveness ranged from one  to60  VMMCs per infection averted.The current VMMC program plan in Zimbabwe is targeting an efficient andimpactful age bracket (13–29 year old), but program efficiency can beimproved by prioritizing a subset of males for demand creation and serviceavailability. The greatest program efficiency can be attained byprioritizing young sexually active males and males whose sexual behaviorputs them at higher risk for acquiring HIV. Zimbabwe is one of 14 countries that are scaling up voluntary medical malecircumcision (VMMC) as part of implementation of a comprehensive package of HIVprevention and treatment services including HIV testing and counseling andantiretroviral therapy (ART) among others . With hiIn light of these results, Zimbabwe adopted VMMC as a key HIV strategy in 2009 andcreated a steering committee to undertake advocacy, protocol development, and apilot study of the VMMC program in the country , 6. Unli1) estimate the population-level epidemiological impact of theVMMC program to date, and 2) explore whether Zimbabwe has anopportunity to enhance the program’s efficiency by focusing effort onsubpopulations.Because the country is developing a costed operational plan for the VMMC program, itis critical to reevaluate the current program scale-up plan, by estimating theepidemic impact of the scale-up to date, and by determining whether makingsubpopulations a priority for intervention could improve program efficiency. Programefficiency is defined as optimizing program impact (the number of HIV infectionsaverted) and minimizing program cost. Therefore, the objectives of this study are toWe implemented a recently developed analytic approach to address these questions andinvestigate gains in VMMC program efficiency. The approach is expressed in terms ofthe age-structured mathematical (ASM) model and accompanying three-level conceptualframework . DetaileBriefly, the ASM model is a population-level deterministic compartmental modelthat consists of a set of coupled nonlinear differential equations . The modThe three-level conceptual framework takes into account epidemiologic, healtheconomics, program efficiency, and policy outcome measures, in addition toprogrammatic feasibility . The firDemographic andHealth Survey (DHS) 2005–06 [The model was parameterized using current empirical epidemiological and naturalhistory data from sub-Saharan Africa . The cou 2005–06 , a natio 2005–06 . HIVpre 2005–06 , 18. Dem 2005–06 , 21. We  2005–06 .We estimated the impact of what has been achieved by the VMMC program to dateby using the actual number of VMMCs that the program in Zimbabwe hasimplemented between 2009 and 2014 [In the modeled baseline intervention scenario (reference scenario), thescale-up of the VMMC program was initiated in 2010, with a“catch-up” phase up to 2017, and a“sustainability” phase up to 2025. The catch-up phase ends byreaching 80% VMMC coverage in the 15–49 age bracket. This coverage ismaintained in the sustainability phase by circumcising the incoming cohortsinto the 15–49 year old population. The scale-up of VMMC was assumedto be at a fixed rate.This baseline VMMC scenario assumes 80% VMMC coverage among 15–49 yearold males per the World Health Organization (WHO) and UNAIDS recommendationfor VMMC scale-up plans .The sceThe scenarios for prioritizing specific subpopulations were based on age,geographic location, and the sexual risk profile of males. For eachsubpopulation scenario, 80% VMMC coverage by 2017 was assumed.Prioritization of a male subpopulation is meant here as intensifying demandcreation and service availability for that specific subset of males.Prioritization is not meant to be an exclusion of VMMC services to anyone.These services need to be available to all males.For the age-group prioritization scenarios, we prioritized each of thefive-year age bands  andalso wider age brackets . Two different VMMC scale-up scenarios wereconsidered in prioritizing the 13–29 year old age bracket, which isZimbabwe’s current age target. In the first scenario, theprioritization was informed by current program data for the age distributionof VMMCs in Zimbabwe—the distribution of VMMCs to males younger than15 versus males older than 15 [For the geographic prioritization scenarios, the VMMC intervention wasprioritized to 15–49 year old males based on the distribution of HIVinfection across the provinces in Zimbabwe, with each province prioritizedseparately.For the sexual risk-group prioritization scenarios, the VMMC intervention wasprioritized to 15–49 year old males based on their sexual riskbehavior profile—that is, according to their specific sexual riskgroup.We conducted a multivariate uncertainty analysis to specify the range ofuncertainty in the effectiveness of VMMC with respect to variations in thestructural parameters of the model. Monte Carlo sampling from uniformprobability distributions was used for the uncertainty in the biological andbehavioral parameters of the model. We assumed an uncertainty of 20% around thepoint estimates of all parameters. Each set of new parameters was used to refitHIV prevalence time series data in Zimbabwe. We implemented 500 uncertainty runsfor each modeled intervention scenario to derive the mean value and associated95% uncertainty interval for the effectiveness of the VMMC program.By September 2014, Zimbabwe circumcised 364,185 males. Assuming the current agedistribution of VMMCs, the program is estimated to avert 40,301 HIV infectionsby 2025. If these VMMCs had been delivered exclusively within the officialtarget age group of 13–29 year old, the program would instead avert anestimated 44,022 HIV infections by 2025, nearly 10% more infections.In order to achieve 80% VMMC coverage by 2017 (the catch-up phase) among the13–29 year old males, a total of 2.17 million VMMCs are required . An addiAssuming the current age distribution of VMMCs , with 29By targeting 15–49 year old males, 2.52 million VMMCs are required inthe catch-up phase to achieve 80% VMMC coverage by 2017 . MoreoveOur findings demonstrate that the current VMMC program in Zimbabwe targets anefficient and impactful age bracket (13–29 year old) and is averting asignificant number of HIV infections. However, the epidemiological and healtheconomic benefits of the program can be maximized by intensifying demand creationand service availability for specific subpopulations, while maintaining VMMC serviceavailability to all adult males. Program efficiency and public health and economicbenefits of VMMC scale-up can be improved by giving priority to young sexuallyactive males and males whose sexual behavior puts them at higher risk of acquiringHIV.Since the start of the VMMC initiative in 2009, VMMC scale-up in Zimbabwe haswitnessed an accelerated uptake among males. However, nearly 29% of the completedVMMCs to date are within the 10–14 age group . FocusinBased on the different outcome measures within the three-level conceptual framework, the optIf the program aims to achieve the largest reduction in HIV incidence rate in theshort term (by 2017), the optimal age groups to prioritize are 15–19 and20–24 year old. If the program aims to achieve the largest reduction inincidence rate in the intermediate or long term (by 2025 or 2045), the optimal agegroups to prioritize are 10–14, 15–19, and 20–24 year old.Thus, by reaching males during or immediately prior to the age at which HIVincidence rate peaks, the VMMC program in Zimbabwe can optimize the public healthbenefits of VMMC.As part of reevaluating the VMMC program plan, the expansion path curve delineatedhow Zimbabwe’s VMMC program can be expanded efficiently to include more agegroups, as feasible by total VMMC program cost . With exThe VMMC program in Zimbabwe could also focus on improving program efficiency throughgeographic prioritization. The efficiency of the program can be enhanced byprioritizing geographic areas (e.g. provinces) whose HIV prevalence exceeds that ofthe national HIV prevalence. VMMC effectiveness can be improved by as much as 19%(by 2025) by giving priority to Matabeleland South, Matabeleland North, andBulawayo—the provinces with the highest HIV prevalence . HoweverDespite the challenges in identifying males whose sexual behavior puts them at higherrisk of acquiring HIV ,substanIn a previous study, the ASM model and the three-level conceptual framework wereapplied to Zambia to investigate program efficiency gains through subpopulationprioritization . OverallA key consideration influencing our model projections is the uncertainty surroundingfuture HIV incidence projections. It is not possible to precisely quantify the scaleof uncertainty in HIV incidence estimates. Different scale-up plans for ART andother HIV interventions can change HIV incidence projections, thereby potentiallyaffecting the outcome of Zimbabwe’s VMMC program. Nevertheless, in our studyfor Zambia , we showWe used an elaborate mathematical model to capture the complexity of HIV dynamics,but modeling predictions can depend on model structure and the accuracy of the dataused to parameterize the model. Our study did not cover all aspects of VMMC scaleup, such as those related to logistical feasibility, social dimension, and communityrelationships. The modeling results presented here are conditioned on therepresentativeness of the data input, such as time-trend data for HIV prevalence,baseline male circumcision prevalence, demographics, VMMC unit costs, and discountrates. To address the uncertainty in model input, we conducted a multivariateuncertainty analysis to assess the impact of changes in the structural andbiological parameters of the model . This anThe ASM modeling tool and the three-level conceptual framework implemented hereprovide an opportunity for more strategic programming in Zimbabwe by focusing on theefficient use of limited resources. This approach was implemented to demonstrate theoptimal public health and economic benefits of making subpopulations the focus ofVMMC services and demand creation—results that can inform national policy andprogramming. Zimbabwe may decide to intensify demand creation and serviceavailability for specific subpopulations to optimize VMMC’s epidemiologicaland health economics impact. The current VMMC program plan in Zimbabwe is alreadytargeting an efficient and impactful age bracket (13–29 year old), but notthe optimal one. Prioritizing males between the ages 15–34 years will improveVMMC program efficiency. Efficiency can also be enhanced by prioritizing geographicareas with higher HIV prevalence than the national HIV prevalence, but the gains arenot substantial. The program’s efficiency can be enhanced immensely byprioritizing males whose sexual behavior puts them at higher risk for acquiring HIV.Last but not least, any policy deliberations of the reevaluated targets should takeinto consideration programmatic feasibility on the ground.


Despite known limitations of positron emission tomography (PET) for mediastinal staging of non-small cell lung cancer (NSCLC), radiation treatment fields are generally based on PET-identified disease extent. However, no studies have examined the accuracy of FDG-PET/CT on a per-node basis in patients being considered for curative-intent radiotherapy in NSCLC.In a prospective trial, patients with NSCLC being considered for definitive thoracic radiotherapy (± systemic chemotherapy) underwent minimally invasive systematic mediastinal evaluation with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) following noninvasive staging with integrated PET-CT.P = 0.005.Thirty patients underwent EBUS-TBNA, with TBNA performed from a mean 2.5 lymph node (LN) stations per patient . Discordant findings between PET-CT and EBUS-TBNA were observed in 10 patients . PET-occult LN metastases were demonstrated by EBUS in 4 patients, whereas a lesser extent of mediastinal involvement, compared with FDG-PET, was demonstrated by EBUS in 6 patients, including 2 patients downstaged from cN3 to pN2. LNs upstaged by EBUS were significantly smaller than nodes downstaged by EBUS, 7.5 mm (range 7–9) versus 12 mm (range 6–21), A significant proportion of patients considered for definitive radiotherapy (+/-chemotherapy) undergoing systematic mediastinal evaluation with EBUS-TBNA in this study have an extent of mediastinal NSCLC involvement discordant with that indicated by PET-CT. Systematic EBUS-TBNA may aid in defining the extent of mediastinal involvement in NSCLC patients undergoing radiotherapy. Systematic EBUS-TBNA has the potential to contribute significantly to radiotherapy planning and delivery, by either identifying occult nodal metastases, or demonstrating FDG-avid LNs to be disease-free. Consequently, at completion of therapy, the pathologic extent of disease is discretely defined for surgical patients.Thoracic surgical guidelines identify mediastinal sampling as being selective (involving only selected suspicious nodes), or systematic (exploration and biopsy of a standard set of lymph node [LN] stations in each case).2 in contrast to surgical candidates, comprehensive staging of the mediastinum is not routinely performed in this patient group. This is potentially clinically significant, as, despite the imperfect negative- and positive-predictive value of PET,10 radiation treatment fields are generally constructed on the basis of PET-identified disease extent.11 Sensitivity of PET/CT is even poorer when individual nodal stations are considered separately.12 Hence, any false-positive nodal activity will result in an unnecessarily extensive field of radiation with consequent greater risk of toxicity, whereas PET-occult nodal metastases will result in the risk of geographic miss, increasing the likelihood of local disease recurrence.In contrast, although invasive pathologic confirmation of mediastinal NSCLC disease is recommended before radical intent radiotherapy (± systemic chemotherapy), there is no consensus regarding the extent to which pathologic evaluation of the mediastinum should be performed. Thus, although pathological confirmation of mediastinal LN involvement is recommended before radical radiotherapy (with or without chemotherapy),13; however, no previous studies have undertaken systematic mediastinal evaluation of patients with locally advanced NSCLC.Thus, accurate pathologic characterization of the mediastinum in patients receiving radical radiotherapy for NSCLC (±chemotherapy) has the potential to improve treatment outcomes both in terms of disease control and treatment toxicity. One prior study has demonstrated EBUS may detect PET-occult LN metastases in patients being considered for stereotactic radiotherapy for clinical Stage I NSCLCWe hypothesized that systematic mediastinal evaluation with minimally invasive EBUS-TBNA in NSCLC patients being considered for radical radiation therapy may identify disease extent discrepant of that indicated by PET-CT. This may have significant implications for radiation treatment planning and consequently treatment-related outcomes. We conducted a prospective observational study to examine this hypothesis and findings are presented here.Melbourne Health Institutional review board approval was granted for performance of this prospective observational study. All patients provided written informed consent.We performed a prospective multicenter observational cohort study in 3 tertiary centers in Melbourne, Australia.Eligible patients were those undergoing mediastinal evaluation with EBUS-TBNA for diagnosis\staging of suspected\known NSCLC wherein noninvasive imaging and\or clinical condition indicated the likely treatment modality would be external beam radiotherapy (±systemic chemotherapy) with curative intent, following discussion at a Lung Cancer Multidisciplinary Meeting. Diagnoses other than NSCLC, and the presence of medical comorbidities precluding bronchoscopy, resulted in exclusion from the cohort.th edition of the Lung Cancer Stage Classification, the TNM descriptors for which are reviewed in detail elsewhere.14Patients underwent noninvasive staging with PET-CT before bronchoscopy with pretreatment staging established according to the 7Integrated PET-CT was performed before EBUS-TBNA in all patients at 3 accredited Australian PET centers according to standard institutional protocols using one of GE discovery 690 , Discovery STE , or Biograph 64/40 .16 with a dedicated linear array bronchoscope .EBUS-TBNA was performed under conscious sedation as previously described17 LN station anatomy was identified according to endobronchial and sonographic landmarks as previously described.18 Any identified LN ≥6 mm in diameter was sampled via EBUS-TBNA, regardless of sonographic findings.19Convex probe EBUS evaluation was performed in a systematic fashion, commencing with the highest contralateral mediastinal (N3) LN, as previously described20 The aspirate was deemed adequate if lymphocytes were observed, or diagnostic if malignant cells were observed. If inadequate, TBNA was repeated once more. If adequate benign lymphocyte tissue was seen, progression to evaluation of N2 LNs was undertaken, commencing from the most superior PET-negative station, then proceeding as above to inferior N2 stations . Once PET-negative stations were evaluated, we proceeded to EBUS-TBNA of PET-positive LN stations.Rapid on-site cytologic examination (ROSE) of TBNA aspirates was performed using a rapid Romanowsky stain , as previously described.21 Pathology outcomes were based on final histocytologic reports following examination of these specimens.For each TBNA, following transfer of initial TBNA material to slides for ROSE, all subsequent materials were placed in formalin solution to allow the preparation of a cell block for histological evaluation and immunohistochemistry (IHC), as previously described.PET-CT imaging for all patients in whom EBUS and PET-CT returned discordant findings regarding the extent of mediastinal involvement underwent independent blinded review to confirm findings from the original PET report. A systematized approach to this was undertaken as follows:A single PET physician (DG) blinded to the EBUS results and PET-CT reports obtained DICOM image files for the discordant FDG-PET studies and viewed the studies using OsiriX imaging software . Positive nodes were classified as nodes demonstrating FDG uptake greater than mediastinal blood pool uptake. The specific nodal stations assessed were 2R, 2L, 4R, 4L, and 7, and the reviewing PET physician reported each nodal station individually.46 given the surgical risks of mediastinoscopy,26 wherein EBUS suggested a lesser extent of mediastinal disease than was suggested on PET, planning was undertaken on the basis of PET findings alone.The aim of this observational study was to compare diagnostic accuracy of EBUS-TBNA with PET. Where a greater extent of mediastinal involvement was demonstrated by EBUS, this information was incorporated into radiotherapy planning. Despite the high negative predictive value (NPV) of EBUS-TBNA,t test with Welch correction using GraphPad InStat . For all analyses, the level of statistical significance was set at 0.05.Categorical variables were presented as summary statistics, including simple proportions. All reported confidence intervals are 2-sided. Sensitivity, specificity, and accuracy of the 2 methods were calculated according to standard definition. Continuous data were analyzed with the unpaired Thirty-five patients undergoing EBUS-TBNA consented to inclusion in the study. All patients underwent EBUS within a maximum of 15 days of performance of PET-CT. Five patients were excluded from the study  or positive ROSE specimen at superior mediastinal LN station (n = 2). Thus, LN sampling was performed from a mean 2.5 LN stations per patient .Adequate samples were obtained from all sites examined by EBUS-TBNA. Mean long-axis size of sampled LN was 16 ± 7.8 mm . Twenty-four percent of sampled LNs were ≤10 mm.Findings regarding the extent of mediastinal disease on PET-CT and EBUS were concordant in 20 of 30 participants . T-, and N-stage of these participants are recorded in Table Discordant findings were observed in 10 of 30 patients  Detailed information regarding CT-, PET-, and EBUS-identified stage, and findings at the LN stations returning discrepant findings are presented in Table EBUS-TBNA identified malignancy in 4 LNs wherein PET-CT had not detected disease, thus identifying a greater extent of mediastinal involvement in 4 patients . Three patients were upstaged by EBUS-TBNA and in 1 further patient, extent of disease was greater than noted on PET because of more proximal involvement of LN disease not resulting in stage advancement. Median size of LN upstaged by EBUS was 7.5 mm (range 7–9).EBUS-TBNA demonstrated only benign lymphocytes in 11 mediastinal LNs (6 patients) wherein PET-CT had indicated the presence of disease. Thus, in 6 patients, EBUS identified a lesser extent of mediastinal disease than PET, including 2 patients downstaged from N3 to N2 . Median size of LN downstaged by EBUS was 12 mm (range 6–21).P = 0.005)LNs upstaged were significantly smaller than those downstaged . Our results suggest a significant proportion of this group has an extent of mediastinal nodal involvement different to that indicated by noninvasive PET-CT, with clear implications for radiotherapy planning performed on the basis of imaging alone. We identify 33% of such patients have inaccurate characterization of the mediastinum by PET-CT, with EBUS-TBNA demonstrating a greater extent of disease in 4 patients and a lesser extent of disease in 6  patients are not wrongly excluded from potentially curative resection. Such a recommendation regarding FDG-avid nodes in patients in which N2 disease has been confirmed at an alternate site is lacking. Our results indicate that in patients with FDG-avid LN metastases, other sites of FDG-avidity may be false-positive results.Previous reports suggest a very high NPV of EBUS-TBNA, varying from 0.91 to 0.99.7 or minimally invasive10 lymph node sampling. Similarly, false-positive PET-CT findings were suggested by EBUS-TBNA in 20% of our cohort, consistent with previously reported specificity of 83% to 87% for PET-CT detection of mediastinal disease wherein prevalence of disease is >20%.2Our observation of variation between EBUS- and PET-determined mediastinal disease extent is consistent with surgical literature, although this variation has not previously been demonstrated in pN2 NSCLC patients undergoing nonsurgical treatment. EBUS detected PET-occult nodal involvement in 13% of our cohort, consistent with the 5% to 16% upstaging rate among patients PET-staged cN0 following surgical2 Importantly, postsurgical studies suggest that the median size of metastatic foci in mediastinal lymph nodes involved with NSCLC is 7 mm.30 This is less than the accepted limit of detection of PET/CT31; therefore, almost certainly a proportion of patients have a mediastinal disease extent greater than that indicated by PET/CT. Such lesions may only be detected by invasive means. Multiple studies have demonstrated the ability of EBUS-TBNA to identify PET-occult N2 disease,10 with high sensitivity preserved even in evaluation of subcentimeter disease.10 Therefore, our finding of EBUS-detected PET-occult disease occurring in nodes with median size 7.5 mm is unsurprising.Published reviews have reported a sensitivity of integrated PET-CT of just 62% for detection of mediastinal lymph node metastases.32 which likely reflects the increased biologic propensity of tumour to spread to mediastinal nodes given it has already acquired the capacity to metastasize to hilar nodes. Consequently, both ACCP and ESTS guidelines on NSCLC staging recommend (Grade 1C) that invasive staging be performed before resection in those staged cN1 by PET.33Interestingly, 2 of 4 patients upstaged by EBUS were patients with cN1 disease (based on PET-CT). Postoperative pathologic upstaging may occur in over 25% of patients staged cN1,35 thai is, in a location not subjected to radiotherapy. Rates of local disease recurrence are significantly higher in lymph node stations receiving suboptimal radiation doses.36 This indicates the importance of accurately defining the extent of disease before radiotherapy for treatment of NSCLC.Accurate characterization of extent of mediastinal disease may have major ramifications for treatment outcomes. In a significant proportion of patients who demonstrate treatment failure following radiotherapy, disease is seen to recur “out-of-field,”The results of this study suggest that systematic mediastinal staging with EBUS may impact the delivery of radiotherapy by either identifying occult nodal metastases (thereby reducing the risk of geographic miss) or demonstrating FDG-avid lymph nodes to be disease-free  in a proportion of patients. The dosimetric consequences of discordance of PET/CT- versus EBUS-defined mediastinal staging need to be evaluated in this context. Our findings will be important in informing future studies on systematic mediastinal pathologic staging before radical radiotherapy (±chemotherapy).37Evidence for the potential impact on treatment outcomes for more accurate pathologic staging of the mediastinum may be inferred from studies examining outcomes in patients receiving stereotactic ablative radiotherapy (SABR) for early stage I NSCLC. Higher than expected regional LN failure rates are seen in patients receiving SABR in which staging was performed on the basis of PET alone, with the authors suggesting these outcomes indicate the potential utility of minimally invasive EBUS-TBNA for LN staging before radiation therapy.2 sonographic features,19 risk stratification models,38 or used artificial neural networks39 to identify patients at higher risk of postsurgical upstaging. Larger studies will be required to examine the applicability of such tools to cohorts similar to ours, or to identify specific clinicoradiologic features predictive of a higher rate of detection of occult disease .We have enrolled a consecutive sample of patients undergoing EBUS-TBNA mediastinal assessment. Previous studies have identified clinical factors ,27 indicating EBUS-detected disease is a reliable true-positive result. Negative EBUS-TBNA at FDG-avid sites is associated with a NPV of 0.91 to 0.99,42 and negative EBUS-TBNA in NSCLC surgical candidates predicts a very low prevalence of metastatic disease in sampled nodes, sufficient to obviate the need for confirmatory mediastinoscopy preoperatively.40 Previous studies have confirmed that pathologic staging is the criterion standard for mediastinal staging, with FDG-avidity on PET not associated with risk of recurrence in patients histologically negative mediastinal lymph nodes.43 Nevertheless, future studies may consider surgical confirmation of negative EBUS-TBNA results before excluding FDG-avid LN from radiation treatment fields.This was a prospective observational pilot study. Surgical confirmation of discrepant results was not performed. Previous meta-analyses have suggested a 0% false-positive rate with EBUS-TBNA,45 suggesting that such an approach may reduce accuracy of complete characterization of mediastinal nodes in NSCLC patients before radiation. Potential benefits of selective sampling  may be offset by reduced diagnostic performance, although this remains to be examined.We have undertaken systematic staging of the mediastinum. It is unclear whether selective lymph node targeting  would identify the same proportion of disease not accurately characterized by PET-CT. “Skip” metastases are a well-recognized phenomena,Not all stations were sampled in each patient, with a mean 2.5 LN stations sampled per patient. This is in part because of termination of the procedure following a positive ROSE result, but also is because of no lymph nodes being identified, or only LN <5 mm at a particular station. Although systematic LN sampling was not performed, a systematic EBUS mediastinal examination was performed in each patient.48 as EUS allows sampling at sites not amenable to EBUS-TBNA 18; however, this requires an additional procedure. More practically, the linear array EBUS bronchoscope may allow performance of EBUS-TBNA and transoesophageal sampling (EUS-B-FNA) by a single operator. Such an approach may further improve diagnostic accuracy of minimally invasive mediastinal assessment50 and should be considered for future studies.We have used only EBUS-TBNA in minimally invasive staging. Diagnostic accuracy in mediastinal staging may be improved when both EBUS-TBNA and EUS-FNA are performed,Our pilot study demonstrates that systematic mediastinal staging with EBUS-TBNA in nonsurgical NSCLC patients being considered for radical radiotherapy idenitifes a significant proportion of patients with an extent of mediastinal disease differing from that indicated by non-invasive PET-CT. These results suggest systematic minimally invasive staging should be considered for all patients before definitive thoracic radiotherapy to accurately assess pathologic stage of disease, and to ensure treatment fields most accurately encompass all sites of disease.Comprehensive mediastinal staging with EBUS may improve the delivery of radiotherapy by either identifying occult nodal metastases (thereby reducing the risk of geographic miss) or demonstrating FDG-avid lymph nodes to be disease-free  in a proportion of patients.