Advances in modern medicine have led to people living longer and healthierlives. Frailty is an emerging concept in medicine yet to be explored as arisk factor in cardiac surgery. When it comes to CABG surgery, randomizedcontrolled clinical trials have primarily focused on low-risk , elevated-risk (GOPCABE) or high-risk patients (BBS), but not onfrail patients. Therefore, we believe that off-pump CABG could be animportant technique in patients with limited functional capacity to respondto surgical stress. In this study, the authors introduce the new national,multicenter, randomized, controlled trial "FRAGILE", to be developed in themain cardiac surgery centers of Brazil, to clarify the potential benefit ofoff-pump CABG in frail patients.versus on-pump CABG in pre-frail and frail patients.Primary outcomes will be all-cause mortality, acute myocardial infarction,cardiac arrest with successful resuscitation, low cardiac outputsyndrome/cardiogenic shock, stroke, and coronary reintervention. Secondaryoutcomes will be major adverse cardiac and cerebrovascular events, operativetime, mechanical ventilation time, hyperdynamic shock, new onset of atrialfibrillation, renal replacement therapy, reoperation for bleeding,pneumonia, length of stay in intensive care unit, length of stay inhospital, number of units of blood transfused, graft patency, rate ofcomplete revascularization, neurobehavioral outcomes after cardiac surgery,quality of life after cardiac surgery and costs.FRAGILE is a two-arm, parallel-group, multicentre, individually randomized(1:1) controlled trial which will enroll 630 patients with blinded outcomeassessment , which aimsto compare adverse cardiac and cerebrovascular events after off-pumpFRAGILE trial will determine whether off-pump CABG is superior toconventional on-pump CABG in the surgical treatment of pre-frail and frailpatients.th2014; last updated on March 21st 2016.ClinicalTrials.gov, ID: NCT02338947. Registered on August 29 In institutions with experience in off-pump CABG, the rates ofmajor adverse events and of complete revascularization and graft patency have beensimilar to those with on-pump CABG. These positive results have been called into question byreports of inferior graft patency and higher rates of repeat target-vesselrevascularization associated with off-pump CABG,5.One of the most controversial areas of cardiac surgery has been whether off-pumpcoronary artery bypass grafting (OPCAB) surgery is superior to conventional on-pumpcoronary artery bypass grafting (CABG) surgery. There is an ongoing debate about thebenefits and disadvantages of OPCAB. Initial trials have shown that off-pump CABG isfeasible in selected low-risk patients and offers results similar to those of CABGperformed with the conventional on-pump technique (on-pump CABG) showed that, among low-risk patients, the rate of death ormajor adverse events at 30 days after surgery was similar with off-pump and on-pumpCABG, but off-pump CABG was associated with a higher rate of incompleterevascularization at 1 year.The Randomized On/Off Bypass (ROOBY) trial.Short-term mortality and morbidity after off-pump and on-pump CABG were similar in arecent trial involving 4752 patients with a mixed operative-risk profile (the CABGOff- or On-Pump Revascularization Study [CORONARY]) study focusedexclusively on patients 75 years of age or older. However, this trial would notelucidate the potential benefit of off-pump CABG in high-risk patients because thisspecific group of German patients were moderate risk patients.The German Off-Pump Coronary Artery Bypass Grafting in Elderly Patients(GOPCABE), performed on 341 high-risk patients  undergoing on-pumpCABG or off-pump CABG, reported no significant differences in the composite ofadverse cardiac and cerebrovascular events or in any of the following outcomes:all-cause mortality, acute myocardial infarction, cardiac arrest, low cardiacoutput/cardiogenic shock, stroke, and coronary reintervention.The results of the Best Bypass Surgery (BBS) trialHowever, in our opinion, the definition of high-risk patient should be interpretedcarefully, since the EuroSCORE identifies patients based on 18 independentvariables, many of which were not considered in the study. Risk factors such asprevious cardiac surgery, critical preoperative state, emergency operation, and poorleft ventricular dysfunction were excluded. Furthermore, active endocarditis,pulmonary hypertension, other than isolated CABG, surgery on the thoracic aorta, andpostinfarction septal rupture were not considered due to the nature of the study.Chronic pulmonary disease and neurological dysfunction were not defined according toEuroSCORE, and there is no information on patients with unstable angina.. So, after a certain cut-off EuroSCORE > 4.5 or 2000Bernstein-Parsonnet score >17.75, off-pump CABG significantly reduces deathrates. Indeed, numerous large retrospective studies and meta-analyses have shownsignificant short-term improvements after OPCAB and comparable long-term outcomes. Arecent risk-adjusted analysis of the national Society of Thoracic Surgeons (STS)database assessing 876,081 patients demonstrated a significant reduction in deathand stroke  after OPCAB, seen in both low- andhigh-volume centers. Afterthat, it is important to reconsider the best approach for patients with highersurgical risk for CABG.In the real world, with more than 1500 patients, we showed lower mortality amongpatients who underwent off-pump CABG.Clearly, a base is being built with strong scientific evidence that this is the groupthat experiences the most benefit from off-pump CABG. This evidence allows not usingcardiopulmonary bypass to be the main surgical approach, and completerevascularization and greater use of arterial grafts in patients with high surgicalrisk as the second plan. However, the available RCTs were underpowered to detectsignificant differences between the groups and suffered from high selection andexclusion biases. Even more important is the fact that the available RCTs so farhave primarily focused only on low-risk , elevated-risk (GOPCABE)or highrisk patients (BBS), but not on frail patients in whom the benefits of OPCABshould be well defined. Moreover, the conversion rates  may suggest that some of the participating surgeonswere inexperienced because expert centers report conversion rates between 2% and4%, whichsignificantly affects long-term outcomes.Actually, randomized controlled trials (RCTs) have failed to demonstrate asignificant mortality benefit for OPCABWe believe that avoiding cardiopulmonary bypass should be viewed primarily as a steptoward avoiding aortic manipulation. Despite the long-term benefits of surgery, somepatients may choose percutaneous coronary intervention (PCI) for the treatment ofcomplex multivessel disease to avoid the morbidity associated with CABG, of whichstroke is the most feared. Expert OPCAB surgeons can offer equivalent durability ofgraft patency as in CABG, with a lower rate of stroke if aortic manipulation isavoided.. Thus,for example, owing to this rather conflicting evidence, either PCI or CABG surgerycould be offered to patients with chronic kidney disease, depending on thecomplexity of coronary disease and comorbidities. If PCI is recommended, appropriatemeasures should be employed to prevent contrast-induced worsening of renal function.If CABG surgery is the preferred revascularization strategy, the off-pump techniquemight reduce the risk of acute kidney injury.Authors make mistakes in pointing out that on-pump CABG should be able to beperformed under all circumstances, on all patients, at all institutions, regardlessof their cardiac volume,17. Afilalo etal. determined thatpatients with slow preoperative gait speed  had a 2 to 3fold increased risk of mortality and major morbidity for any given level ofSTS-Predicted Risk of Mortality or Major Morbidity (PROM) compared with normalspeed. Gait speed added to STS-PROM marginally increased model performance from 0.70(0.60-0.80) to 0.74 (0.64-0.84). Forty-three percent of patients died or sustained amajor complication assessed as high STS-PROM risk (≥15%) together with slowgait speed, compared with 21.7% low STS risk with slow gait and 18.9% high STS riskwith normal gait. Afilalo et al. subsequently evaluated the prognostic value of variousfrailty, disability and cardiac risk scores to identify the optimal combination topredict adverse outcome. Patients with slow gait speed and ≥3 impairments onthe Nagi disability scale predicted in-hospital morbidity and mortality above thatof the Parsonnet cardiac risk score . Lee et al. performed a retrospective review of a large cardiac registry,comparing outcomes between non-frail and frail individuals . Frailty was an independent risk factor for in-hospital mortality and mortality at 2 years .The benefits of CABG withoutcardiopulmonary bypass in pre-frail and frail patients are still undetermined.On the other hand, cardiac scores, including EuroSCORE and STS, have been developedto predict the risk of adverse outcomes following surgery. Frailty, an independentpredictor of mortality and complications, is not included in these risk algorithms.Emerging evidence suggests that frailty is a better marker of biological age andmore important than chronological ageIn conclusion, frailty is defined as a geriatric syndrome of impaired resiliency tostressors (such as cardiac surgery) that has been delineated recently in thecardiovascular literature. However, the benefits of CABG without cardiopulmonarybypass in these patients are still undetermined. We believe OPCAB remains animportant technique for the improvement of coronary surgery. The biggest questionright now is whether pre-frail and frail patients will benefit more from off-pump oron-pump CABG.versuson-pump CABG in frail patients.The aim of this paper was to describe the FRAGILE trial protocol which intends toclarify the potential benefit of off-pump CABG in pre-frail and frail patients; wewill conduct a national multicenter RCT comparing off-pump The FRAGILE study will be a national multicenter RCT to be conducted in 10Brazilian institutions. The study is already approved by a certified ethicscommittee. The study sponsor must be the Brazilian Society of CardiovascularSurgery and Zerbini Foundation. Funding will be provided by an unrestrictedgrant from São Paulo Research Foundation, which otherwise will not haveany role in the conduct of the study nor in the analysis nor in the reporting ofdata. There will be confidentiality agreement regarding data use. This RCT willbe monitored by an independent data and safety monitoring board. All the authorswill be provided revisions and comments. All the authors will be testifying forthe accuracy and completeness of the report, as well as for the fidelity of thereport to the study protocol.,not eligible for angioplasty treatment by the heart team approach and thosesuitable to undergo either off-pump or on-pump CABG.The inclusion criteria will be: participants aged ≥65 years withindication of myocardial revascularization with ≥2 criteria of frailty byFried Frailty Criteria.There is a dose-response relationship, in relation to the number of fragilitycriteria and patient outcomes. The categorization of the fragility variable wasperformed after the studies showed that the risk increases gradually over 3categories , with risk of similar events within each category.Thus, patients who had 2 or 3 criteria were considered as pre-frail, andpatients with 4 or 5 criteria were considered as frailThe exclusion criteria will be: patients with indication of another procedure inaddition to CABG; patients who underwent emergency operation ; patients who underwent previous cardiac surgery, evenwith other approaches than median sternotomy; patients who do not have free,prior and informed consent to participate in this study.The baseline characteristics of potentially eligible but excluded patients willbe recorded in a screening log. All patients will be provided written informedconsent.Instituto de Cardiologia do Distrito FederalInstituto do Coração da Faculdade de Medicina daUniversidade de São PauloPronto-Socorro Cardiológico de Pernambuco (PROCAPE)Instituto Dante Pazzanese de CardiologiaBeneficência Portuguesa de São PauloInstituto Nacional de CardiologiaPontifícia Universidade Católica do ParanáHospital das Clínicas Samuel Libânio de PousoAlegreHospital Alberto Urquiza Wanderley de João PessoaTotal Cor HospitalEligible patients will be randomly assigned to off-pump CABG or on-pump CABG.Randomization will be performed after the baseline data, including informationabout the target vessels have been entered into a central, Internet-based,password-protected database with the use of a template.Treatment assignments will be performed in a blinded manner according to ablocked randomization scheme with a block size of ten, stratified according tothe participating center. Off-pump CABG must be routinely performed at allparticipating centers before the trial is initiated. Participating centersshould nominate individual study surgeons for each surgical technique.Study surgeons will be required to be established experts in the performance ofeither off-pump or on-pump CABG. The average number of CABG surgeries performedbefore the study will be between 50-100 off-pump surgeries for the off-pump CABGsurgeons and 250 on-pump surgeries for the on-pump CABG surgeons. It will not bemandatory for the same surgeon who operates the off-pump CABG cases to alsooperate the on-pump CABG cases, but always respecting the established protocolsfor each surgical technique.Surgical access to the heart will be gained through a median sternotomy in all ofthe patients. In order to reduce the risk of bleeding and transfusions anabsorbable hemostat is used to sternal bone marrow and recovery of red bloodcells in all patients. Off-pump surgery will be performed with the use of heartstabilizers. Patients will be heparinized with 250 IU/kg intravenously toachieve activated clotting time >200s. The proximal anastomosis will beperformed according to protocol to be discussed with our advisor. The distalanastomosis will be constructed with the help of mechanical stabilizers andcardiac positioner. Intracoronary shunts will be used routinely. On-pump surgerywill be performed in normothermia, with the use of aortic cross-clamping andcold cardioplegic arrest. Patients will be heparinized with 500 IU/kg to achievean activated clotting time >480 s. Heparin will be neutralized with 1 mgprotamine sulfate per 5000 IU given. During the trial, there will be no changesin the 2 surgical techniques that followed a pre-established protocol..All variables from the study will be stored online through a REDCap Web Platform(http://www.project-redcap.org/).The REDCap is a secure web application forbuilding and managing online surveys and databases. REDCap can be used tocollect virtually any type of data, it is specifically geared to support datacapture for research studies. This will be done through national healthcarecard, address and telephone numbers. On the basis of the registers, it will becollected copies of hospital records and death certificates during the follow-upperiod. The neurocognitive tests will be planned in accordance with thestatement of consensus of assessment of neurobehavioral outcomes after cardiacsurgery. Neurocognitive and quality of life tests will be carriedout at baseline and at 6 months postoperatively. No follow-up visits will beplanned; however, patients will be routinely seen by the referring cardiologist1 and 6 months after the operation. Hospital records and death certificates willbe blinded for the allocated treatment and forwarded to 2 randomly selectedmembers of the adjudication committee, who will assess whether each of thepre-specified outcomes had occurred. In case of disagreement, the 2 assessmentstogether with a copy of the record of the event will be sent to a third member,who will have to select the most likely assessment.We will assess health-related quality of life using the World Health Organizationquality of life questionnaireIt will comprise mortality rates and complications  occurring within 30 days of surgery. Hospitalmortality is defined as death occurring within 30 days after surgery. Myocardialinfarction is defined as the appearance of a new Q wave on the electrocardiogramwith an increase of CKMB> 100 IU/L and/or> 10% of the total CK leveland/or new wall movement abnormalities, with the exception of the septum,documented on the echocardiogram. Neurological complications are defined asstroke  or transient ischemic attack . Renal insufficiency is defined as anincrease in plasma creatinine> 2 associated with a urinary output <0.5 mLkg/h/in 12 hours. Acute respiratory distress syndrome (ARDS) is defined as thepresence of tachypnea (respiratory rate> 30 breaths/min), bilateral pulmonaryinfiltrate on chest radiography, severe hypoxemia , the need for positive pressureend-expiratory flow> 5 cmH2O, no evidence of left ventricular failure (wedgepulmonary capillary pressure <18 mmHg), and no pathological features toexplain these findings. Reoperation for bleeding is defined as the need to chestreopening in the presence of> 500 mL of blood by the chest tube within thefirst hour, >400 mL within the second hour, >300 mL within the third hour,or total bleeding >1000 mL within the fourth hour.Major adverse cardiac and cerebrovascular events after OPCAB and CABGin pre-frail and frail patients (1 year)Major adverse cardiac and cerebrovascular events after OPCAB and CABGin pre-frail and frail patients (2 years)Operative time (180 days)Mechanical ventilation time (180 days)Hyperdynamic shock (180 days)New onset of atrial fibrillation (180 days)Need for pacing >24 hours (180 days)Renal replacement therapy (180 days)Reoperation for bleeding (180 days)Pneumonia (180 days)Length of stay in intensive care unit (180 days)Length of stay in hospital (180 days)Transfusion requirement (180 days)Graft patency assessed by coronary computed tomography (1 year)Correlation between clinical and angiographic scores to prognosis (1year)Recurrence of angina (1 year)Neurobehavioral outcomes after cardiac surgery (180 days)Quality of life after cardiac surgery (180 days)Cost (180 days). Consequently,at least 630 patients will be enrolled.The sample size of the FRAGILE trial was based on the ability to detect a 40%reduction in the primary outcome in the off-pump group compared with the on-pumpgroup, assuming an event proportion after on-pump CABG of 20.8% and accepting arisk of type I and II error of 5% and 20%, respectively, and a crossover rate of5%i.e., based on treatment allocation). Therefore, we willreport treatment-received analyses according to the intervention actuallyreceived. Baseline characteristics and operative characteristics will becompared with the use of chi-square test, t-test, or Kruskal-Wallis test, asappropriate. Dichotomous data will be presented as numbers and percentages.Continuous data will be presented as mean and standard deviation or median andinterquartile range. The continuity corrected chi-square test will be used forcomparison of the 30-day endpoint. Mantel-Haenszel chi-square tests will be usedto adjust for study-center effects. Treatment effects at 30 days will beexpressed as OR and 95% CI. For the 12-month endpoint, Kaplan-Meier curves willbe constructed, and the study groups will be compared with use of the log-ranktest. HR and 95% CI derived from the Cox proportional hazards model will beprovided for the composite outcome and individual components. All statisticalanalyses will be performed with the use of the R-3.1.2 language for handling andstorage of data and performing of statistical calculations.All of the data will be analyzed according to intention-to-treat,12-14.Frailty is defined as a geriatric syndrome of impaired resiliency to stressors thathas been delineated recently in the cardiovascular literature.The results of BBS randomized trial are in contrast to these findings. Overall 30-daymortality in this trial was 4.4%, which was less than the predicted mortality rateof 7% according to the EuroSCORE but in agreement with the predicted 3.1% accordingto the STS risk score. This study shows that the risk patients were overestimated bythe EuroSCORE and this sample will be composed of low and moderate risk patients bythe STS risk score-18.Therefore, this trial does notsupport the assumption that off-pump CABG cannot improve the early outcome inhigh-risk patients.In the GOPCABE trial, there was no significant difference between off-pump CABG andon-pump CABG performed in elderly patients with respect to the composite endpoint ofdeath, stroke, myocardial infarction, repeat revascularization, or new renalreplacement therapy after surgery. With a mean age of 78 years and a predictedin-hospital mortality of 3.8%, the study cohort does not represent a population witha high operative risk. The results of previous studies are consistent with regard tohigh-risk patients,the GOPCABE study did not show a significant difference in survival or major adverseevents at 1 year after surgery. This absence of difference may be a consequence ofthe requirement for substantially greater experience with off-pump CABG in theGOPCABE trial than in the ROOBY trial.In contrast to the ROOBY trial.With the growth of minimally invasive and transcatheter cardiac interventions, theexpansion of risk prediction beyond traditional risk factors and risk scores hasbecome a high priority. Clinicians may use this integrative approach of combiningrisk scores including frailty and disability to better characterize elderly patientsreferred for cardiac interventions and identify those who are vulnerable and atincreased risk. A multidisciplinary team involving cardiac surgery, cardiology,geriatric medicine, physiotherapy, and occupational therapy may be well suited toaddress these diverse elements which contribute to postoperative risk. We hypothesizedthat the frailty status would contribute to the risk in patients undergoing on-pumpCABG and that off-pump CABG could be advantageous for frail patients.The CORONARY trial falls short of delivering a knockout victory for the off-pumptechnique. There were no significant differences in the rate of the compositeprimary outcome of death, stroke, non-fatal myocardial infarction or new renalfailure at 30 days between off-pump and on-pump CABG. However, off-pump CABG wasassociated with fewer transfusions, reoperations for bleeding, acute kidney injury,and respiratory infections or failure. Thus, the off-pump technique may be best incertain subgroups of patients and we must choose the procedure that is best forthemSome limitations of our trial are to be noted. Firstly, the assessment of graftpatency will not be performed by coronary catheterization (test which indicatedsurgery). In contrast, coronary computed tomography angiogram will be performedin all patients for this purpose. Secondly, the events will not be adjudicatedby a blinded adjudication committee. All data will be provided by the localinvestigators according to the protocol definitions.


Supplemental Digital Content is available in the text We aimed to estimate overall and cause-specific excess mortality of HIV-positive patients compared with the general population, and to assess the effect of risk factors.We included patients aged >19 years, recruited from January 1, 2004 to May 31, 2014 in Cohort of the Spanish Network on HIV/AIDS Research. We used generalized linear models with Poisson error structure to model excess mortality rates.In 10,340 patients, 368 deaths occurred. Excess mortality was 0.82 deaths per 100 person-years for all-cause mortality, 0.11 for liver, 0.08 for non-AIDS-defining malignancies (NADMs), 0.08 for non-AIDS infections, and 0.02 for cardiovascular-related causes. Lower CD4 count and higher HIV viral load, lower education, being male, and over 50 years were predictors of overall excess mortality. Short-term (first year follow-up) overall excess hazard ratio (eHR) for subjects with AIDS at entry was 3.71  and 1.37  for hepatitis C virus (HCV)-coinfected; medium/long-term eHR for AIDS at entry was 0.90  and 3.83  for HCV coinfection. Liver excess mortality was associated with low CD4 counts and HCV coinfection. Patients aged ≥50 years and HCV-coinfected showed higher NADM excess mortality, and HCV-coinfected patients showed increased non-AIDS infections excess mortality.Overall, liver, NADM, non-AIDS infections, and cardiovascular excesses of mortality associated with being HIV-positive were found, and HCV coinfection and immunodeficiency played significant roles. Differential short and medium/long-term effects of AIDS at entry and HCV coinfection were found for overall excess mortality. Whereas most available data on excess mortality refer to all-cause mortality, less data are available on the cause-specific excess mortality associated with being HIV-positive. This is important given that non-AIDS-defining malignancies (NADMs), cardiovascular disease, and liver-related deaths have become more frequent as a consequence of more prolonged survival of an increasingly HIV-positive population.,10Since the introduction of combination antiretroviral therapy (cART), the life expectancy of HIV-positive individuals starting cART and attaining restoration of CD4 approaches that of the general population.Therefore, we aimed to evaluate the overall and cause-specific excess mortality observed in HIV-positive subjects followed up in the cohort of the Spanish Network on HIV/AIDS Research (CoRIS) from 2004 to 2014, compared with the expected mortality in the general population in Spain, and to identify prognostic factors of excess mortality.22.1 Ethics approval was obtained from all hospitals Ethics Committees, and every patient provided written informed consent to participate in the cohort.The CoRIS cohort or CoRIS is an open, multicentre, prospective cohort of HIV-positive adults recruited while cART-naïve from 40 centers from 13 of the 17 Autonomous Communities of Spain.For this analysis, we included patients older than 19 years recruited from January 1, 2004 to May 31, 2014 (administrative censoring date). Individuals were followed up from study entry to death or last study contact, whichever arose first.2.23 count at entry ; HIV viral load (VL) at entry copies/mL ; HCV serological status at entry (positive or negative antibodies); cART initiation in the first 6 months after cohort entry; vital status; cause and date of death. Follow-up time was divided in 2 intervals: first year after the inclusion in the cohort to account for short-term mortality, and from the second year to the 10th year to account for medium/long-term mortality.We considered the following variables: age at entry ; sex ; HIV transmission category ; level of education ; geographical region of origin ; AIDS-defining condition at entry, CD4 cells/mm2.3www.ine.es), stratified by sex and age at 5-year intervals. A constant death rate within each 5-year age interval was assumed.Mortality rates in the general population, from 2004 to 2013, were obtained from the National Institute of Statistics , which has been previously applied to CoRIS.Deaths were classified using revised CoDe in CoRIS and 10th revision of the International Classification of Diseases (ICD-10) was applied in the general population and grouped as: liver disease (including HCV and HBV-related liver cancers), NADM, non-AIDS infections, and cardiovascular disease  for continuous variables for all and deceased patients.Excess mortality rate associated with being HIV-positive is defined as the difference between the death rate observed in the cohort and the expected death rate in the general population. The expected number of deaths was calculated by applying the mortality rates of the general population to the person-years (py) distribution of the HIV cohort matched by age, sex, and calendar year at risk. We used multivariable generalized linear models with Poisson error structure to estimate both the excess mortality rates (eMRs) in HIV-positive patients compared with the general population and the excess hazard ratio (eHR) for potential prognostic factors. This latter estimation should be interpreted as a common HR, but in terms of excess mortality.A competing risk analog of the generalized linear model with Poisson error structure was applied to estimate cause-specific excess mortality in HIV-positive patients. We fitted separated models for liver, NADM, non-AIDS infections, and cardiovascular deaths; individuals who developed a competing event before the event of interest were censored on the occurrence date of the competing event.P values <0.1 in the crude analysis, CD4 count at entry, and follow-up interval . We checked the sensibility of these assumption using 1-year intervals of follow-up . Further, to investigate whether the effects of the prognostic factors on overall and cause-specific excess mortality differed by follow-up interval, interaction terms between follow-up intervals and prognostic factors were tested. Due to the small number of events and to avoid overfitting, we did not consider the variable “AIDS at entry” for NADM, liver, and non-AIDS infections multivariable models. We also checked that the number of observed deaths was above the expected deaths in each subgroup.In the multivariable models, we included all the prognostic factors with  We assessed the imputation procedure and its convergence. Finally, we performed exhaustive sensitivity analysis comparing results obtained using imputed values and using only complete cases or excluding individuals with imputed outcomes.“Multiple Imputation by Chained Equations” was used to deal with missing data. We developed a multiple imputation model for each variable with missing values. Resulting models included the other incomplete variables ; the complete variables ; the outcome ; and 1 additional variable “receiving cART in the first 6 months after cohort entry). The results from the 12 imputed datasets were combined using Rubin rules.All statistical analyses were performed using STATA .33.1The total number of patients older than 19 years included in CoRIS from January 1, 2004 to May 31, 2014 was 10,340. The median follow-up was 3.20 years , and during 36,984.18 py of follow-up, 368 deaths were observed. The distribution of causes of death in CoRIS and the general population is presented in Supplementary Table 1.We found missing data on level of education , HIV transmission category , country of origin , CD4 count , VL , HCV coinfection , and cause of death . All missing values were imputed.3 (IQR 200–586), median HIV VL was 46,504 copies/mL  and 1019 (9.9%) patients with AIDS at entry. Overall, 13.4% had a positive HCV test result at entry  years. Median CD4 count was 386 cell/mm3.2The overall eMR observed in the cohort was 0.82 deaths per 100 py of follow-up , that is, the death rate was 0.82 deaths per 100 py higher in CoRIS than in the Spanish population of the same age and sex for all causes of death. Overall eMRs by potential prognostic factors are shown in Table P = 0.007). There was borderline evidence that women had lower risk of excess mortality than men . The short and medium/long-term effects of having AIDS (P < 0.001) and being HCV-positive (P < 0.001) at study entry on the excess mortality were significantly different; having AIDS at entry was a strong predictor of short-term excess mortality , whereas this effect disappeared  during the rest of follow-up. On the contrary, being HCV-positive at study entry predicted higher long-term excess mortality , whereas there was no evidence of a statistically significant effect during the first year of follow-up .In the multivariable model Fig. , CD4 cou3.3Non-AIDS-defining malignancies, liver, non-AIDS infections, and cardiovascular deaths distribution and eMRs by potential prognostic factors are shown in Table The eMR observed from 2004 to 2014 in CoRIS was 0.08 deaths  for NADM, 0.10 deaths  for liver, 0.08 deaths  for non-AIDS infections, and 0.02 deaths  per 100 py of follow-up for cardiovascular causes.There was strong evidence that age and HCV coinfection at entry were independently associated with a higher NADM excess mortality. Patients aged 50 years old, or older at entry, had 5.15 times higher risk  compared with those aged below 50 years at entry, and the adjusted eHR for those HCV-coinfected subjects was 3.85  . HCV-coinfected subjects had 9.69 times  higher risk of liver excess mortality than HCV-negative ones. Besides, there was strong evidence that long-term adjusted eHR was lower compared with short-term eHR .A CD4 count at entry greater than 350 cells/mmHaving a positive HCV test at entry was also associated with higher non-AIDS infection-related excess mortality , and there was strong evidence that long-term adjusted eHR was lower compared with short-term eHR .No significant predictor for the crude cardiovascular excess mortality was found, hence multivariable generalized linear model was not performed.We failed to find any evidence that the effect of prognostic factors on cause-specific excess mortality had changed over time, although results might be interpreted with caution because numbers are small.4The HIV-positive subjects in Spain have experienced, from 2004 to 2014, an overall excess mortality, and also an excess mortality for NADM, liver, non-AIDS infections, and cardiovascular disease compared with the general population of the same age and sex. These mortality excesses have happened largely at the expense of higher rates in the first year after cohort inclusion, and are attributable to late HIV presentation, which could be greatly attenuated by scaling up HIV testing and early diagnoses.–17 The higher excess rates compared with CoRIS reported by Aldaz et al can be explained by their higher proportion of IDUs. The concerted action on seroconversion to AIDS and death in Europe (CASCADE) collaboration reported an excess mortality of 0.6 deaths per 100 py during 2004 to 2006. CASCADE collects data from seroconverters, hence the discrepancies with our results can be explained by different study populations. Not surprisingly, short-term mortality is less likely to happen in seroconverters because of the better clinical and immunological conditions at cohort entry; indeed CASCADE's overall excess mortality rate is similar to the 0.50 per 100 py found in CoRIS after excluding short-term mortality. Subjects with AIDS at cohort enrolment—subjects with late HIV presentation—had an increase of excess mortality during the first year of follow-up. The implications of a delayed HIV diagnosis have been previously documented. AIDS diagnosis has been related with poorer responses to cART.The excess mortality in HIV-positive patients has been reported both in Spain and elsewhere.,21,22 Our finding about the differential effect of HCV coinfection on short and long-term excess mortality raises again the question about the role of HCV coinfection in mortality associated with HIV infection, even adjusting for background mortality. Excess mortality captures both direct and indirect effects of HIV infection on mortality. Long-term excess mortality in HCV-coinfected patients may be explained by the direct impact of HCV on HIV disease progression for which data are inconsistent. Some reports have found no evidence that HCV infection accelerates CD4 decline and AIDS progression, nor that it compromises CD4 count recovery after initiation of cART.,24 Other studies have reported that HCV coinfection is associated with a higher risk of progression to AIDS and poorer CD4 recovery even after several years from cART initiation. Berenguer et al published that the eradication of HCV in HIV/HCV-coinfected patients is associated with decreases in HIV progression and lower risks of both liver and nonliver-related mortality. Long-term overall excess mortality in HCV-positive patients can be also explained by indirect mortality associated to higher exposure to drugs, alcohol, and tobacco, compared with the general population.Previous reports in CoRIS had also noticed the importance of HCV/HIV coinfection on overall and cause-specific mortality in the Cohorts of the Spanish AIDS Research Network. Besides, the toxicity of long-term antiviral treatment may contribute to liver damage and liver excess mortality. However, there is also indirect effect since hepatotoxic substances such as alcohol and illegal drugs are abused by HIV-positive subjects more often than the general population.Liver excess mortality associated with being HIV-positive can be mediated by the direct impact HIV has on the natural history of HCV. NADM excess mortality can be both directly and indirectly associated with HIV. Some studies have suggested that immunosuppression might be associated with moderated excesses of NADM and more rapid progression of some types of cancer. Besides, clear associations between immunosuppression and NADM with known viral etiology have been reported.,34 NADM excess mortality can be also attributable to the previously alleged elevated frequency of cancer risk factors in HIV-positive patients such us smoking and alcohol abuse,,35 and coinfection with oncogenic virus such as hepatitis D virus, HCV, HBV, and human papilloma virus.We observed significantly higher NADM mortality among HIV-positive patients compared with the general population, which is consistent with previous reports.,33 Patients older than 50 years have shown a poorer immunological response to cART which might also contribute to the higher NADM excess mortality observed in older patients.Hepatitis C virus coinfection and aging have been previously reported as risk factors for excess NADM rates. A second hypothesis, known as “Salmon bias,” proposes that foreign-born persons return to their country of origin when they become severe ill. Therefore, our analysis should have used general population mortality rates matched by sex, age, and also region of origin, but unfortunately, cause-specific mortality rates by country of origin are not accessible in Spain.Competing explanations for lower mortality rates in migrant populations fall into 2 broad categories. The first posits the self-selection of healthier migrants driven by labor market conditions in what is known as the “healthy immigrant effect.” Selection bias could have been introduced by the use of the general population as a proxy for background population as this population contains also HIV-related deaths. Nevertheless, as HIV-related mortality represents a small proportion of all-cause mortality in the general population of Spain, we consider correct it to use the general population mortality rates to calculate the mortality rates in a non-HIV-infected population. Misclassification of missing data due to imputation could also be discussed. However, sensitivity analysis restricted to complete cases did not reveal changes in the direction of the association between excess mortality and risk factors. Finally, the low number of deaths, when aiming to look at cause-specific mortality, may have introduced random error in some of our estimates and comparisons.There are some study limitations that merit discussion. As in other studies, some covariates  were not collected and consequently their impact on the results could not be analyzed. Another aspect that could be argued is the convenience of using different coding algorithms in the numerator and denominator of excess mortality estimations. However, it has been shown that revised CoDe classification is the best way to classify cause of death in HIV-positive cohort studies; it has been shown that applying ICD-10 system would underestimate liver mortality associated with being HIV-positive.To conclude, our results have shown overall, NADM, liver, infection, and cardiovascular excess mortality associated with being HIV-positive despite improvements in HIV disease management. As far as we know, no previous analysis has demonstrated in this very clear way the role of having an AIDS diagnosis at entry in early excess mortality and HCV coinfection in long-term excess mortality. Our results have clear implications for health policy in that promoting earlier HIV diagnosis and linkage to care will likely decrease the excess of mortality detected in the first year. It is to be expected that the scale-up of the treatment with new direct acting antivirals for HCV will impact long-term mortality too. Finally, increased efforts promoting healthier lifestyles regarding diet, smoking, and exercise are urgently needed in the current context to aim for the healthy aging of the persons living with HIV.


Syndemics of illicit drug use and HIV remain as significant public health issues around the world. There has been increasing concern regarding the rapidly growing market of new psychoactive substances, particularly in Asia. In response, the Japanese government has increasingly banned such substances in recent years. We sought to identify the prevalence and correlates of use of quasi-legal psychoactive substances among people living with HIV/AIDS (PLHIV) in Japan.Data were derived from a nationwide survey of PLHIV conducted at nine leading HIV/AIDS care hospitals between July and December 2013. The prevalence and correlates of the use of quasi-legal psychoactive substances  among male participants were examined using multivariate survey logistic regression.Among 963 study participants, the majority (95.3%) were male. The most commonly used drug among men was quasi-legal psychoactive substances (55.3% ever and 12.8% in the previous year). In multivariate analysis, the lifetime use of tryptamine-type derivatives   and methamphetamine/amphetamine  were independently associated with recent quasi-legal psychoactive substance use.In our sample of male PLHIV in Japan, quasi-legal psychoactive substances were the most commonly used drugs. Individuals who had ever used tryptamine-type derivatives or methamphetamine/amphetamine were more likely to report recent quasi-legal psychoactive substance use, suggesting a potential shift in drug use patterns from regulated to unregulated substances among this population. These findings indicate a need for further research to examine implications for HIV care. Many countries in the world are contending with syndemics of illicit drug use and HIV/AIDS , 2. In AIn Japan, the prevalence of illicit drug use in the general population has historically been low at <3% (lifetime use) . The preIn response to the explosive spread of quasi-legal synthetic psychoactive substances , the JapFoxy ), have been categorized as “Narcotics” under the Narcotics and Psychotropics Control Law and thereby have been prohibited from possessing and consuming [Despite the tighter control of supply, the possession and consumption of Designated Substances have remained largely unregulated. By March 2013, only 13 of the 106 Designated Substances, including mephedrone and tryptamine-type derivatives  in Japan . Given tData for this study were derived from a nationwide cross-sectional survey of PLHIV conducted at nine leading HIV/AIDS referral hospitals in Japan between July and December 2013. In brief, beginning in 2003, this is a quinquennial survey that aims to investigate the health and lifestyles among PLHIV in Japan. The nine hospitals included in the survey represent tertiary care hospitals and collectively cover the eight major geographical regions in Japan. In each hospital, HIV/AIDS medical professionals were asked to recruit their patients living with HIV/AIDS in a serial manner as patients visit their hospitals. They explained the study objectives and procedures to potential participants, and obtained informed consent from them. The recruitment was continued until approximately 40% of their patients provided informed consent. While we did not count the number of patients who refused to participate in the survey, informal reports from hospitals indicated that such cases were rare. Consented patients were asked to self-complete an anonymous questionnaire and return it to an independent researcher using a self-addressed, stamped envelope provided with the questionnaire. The questionnaire elicited a range of information, including socio-demographic characteristics, HIV-related clinical histories, general health status, and substance use histories. All consented individuals received a 500 Japanese Yen (approximately US$5.00) gift certificate. The study was approved by the research ethics boards at the Saitama Prefectural University as well as each participating hospital.In total, 1,100 (61.6%) of 1,786 consented individuals completed and returned the questionnaire, including 51 (4.7%) females. For a gender-based descriptive analysis of drug use patterns, the sample was restricted to 963 (87.5%) individuals  who had complete data. For an analysis examining correlates of past year quasi-legal psychoactive substance use among men, the sample was further restricted to 895 male participants with complete data.rush, poppers). The term “quasi-legal drugs” was used because it commonly referred to the aforementioned set of quasi-legal psychoactive substances of our interest in Japan, and also because people usually do not know the exact substance contained in the product.For the present analysis, the primary outcome of interest was reporting quasi-legal psychoactive substance use in the previous year, defined as answering “Yes” to a question: “In the past year, have you used quasi-legal drugs in the form of herbal products, aroma liquid, powder, oil, bath salts, or amyl nitrites including ‘rush’ and ‘poppers’?” As such, quasi-legal psychoactive substances encompassed a range of substances that had been largely regulated as Designated Substances but had not been prohibited from possessing or consuming under the Narcotics and Psychotropics Control Law by December 2013. Specifically, these substances would include synthetic cannabinoids, synthetic cathinones, phenethylamine and thiophene derivatives, and amyl nitrites , ever used cannabis, ever used methamphetamine/amphetamine, ever used MDMA, and ever used heroin/cocaine .Then, we selected a range of explanatory variables that we hypothesized were potentially associated with the outcome. Socio-demographic characteristics included: age; education (< secondary education vs. ≥ secondary education); currently living alone (yes vs. no) and annual income . HIV-related measures included: antiretroviral therapy (ART) use (currently on ART vs. never on ART); ever had AIDS (yes vs. no); perceived route of HIV exposure ; and engagement in HIV-related peer support groups in the previous year (yes vs. no). Psychological distress was measured using the Kessler psychological distress scale (K6) . This vaFirst, socio-demographic and HV-related clinical characteristics were compared between males and females, using the Wilcoxon Rank Sum Test (for continuous variables) and the Pearson’s Chi-squared test . Since the prevalence of drug use was extremely low among women (11.1% ever and 0% in the previous year), descriptive statistics were used to compare drug use patterns between males and females.a priori-defined statistical protocol whereby all variables that were significantly associated with past year quasi-legal psychoactive substance use at the p < 0.05 level in the bivariate logistic regression analyses. In a sub-analysis, we examined the proportion of those who had never used any illicit drugs  among male participants who used quasi-legal psychoactive substance in the previous year. We also examined the proportion of those who used drugs for sexual activities in the previous year among the same aforementioned sub-sample. All p-values were two-sided. All statistical analyses were performed using SAS software version 9.3 .For bivariate and multivariate analyses of factors predicting past year quasi-legal psychoactive substance use among male participants, we used the survey logistic regression in the SAS software to account for possible clustering effects at the hospital level. A multivariate model was fit using an Foxy and methamphetamine/amphetamine. Of note, the use of 5-MeO-DPT/Foxy in the previous year substantially dropped to 0.4%. Among women, the most commonly used drug was cannabis, followed by quasi-legal psychoactive substances and methamphetamine/amphetamine.In total, 963 participants were eligible for the present study, including 45 (4.7%) females. Table Table Foxy; and ever used methamphetamine/amphetamine.Table In the sub-analysis, among the 115 male participants who used quasi-legal psychoactive substances in the previous year, 38 (33.0%) reported having never used other illicit drugs. Sixty-nine (60.0%) individuals reported having used drugs for sexual activities in the previous year.Foxy or methamphetamine/amphetamine, younger age, living alone, and male-to-male sexual behaviour as a perceived route of HIV exposure were independently and positively associated with recent quasi-legal psychoactive substance use among male PLHIV.In our sample of PLHIV in Japan, illicit drug use was much more common among males than females. The most commonly used drugs among men were quasi-legal psychoactive substances. Over half of male participants reported having ever used quasi-legal psychoactive substances, and approximately 12% reported having used these substances in the previous year. The majority (97%) of those who used quasi-legal psychoactive substances in the previous year were presumably MSM. Approximately one-third of them had no history of using other illicit drugs. In the multivariate analysis, the lifetime use of 5-MeO-DPT/Foxy were more likely to have used quasi-legal psychoactive substances in the previous year. While the cross-sectional nature of the study limits the assessment of temporal relationships, available data and literature suggest possible shifts in the patterns of drug use in this setting, from the use of fully banned to yet-to-be-fully-prohibited substances. As presented in Table Foxy were second-ranked among the drugs that have ever been used in the lifetime (28%). However, past year use substantially dropped to <0.5%. Given that these tryptamine-type derivatives became fully prohibited under the Narcotics and Psychotropics Control Law in 2005, our findings suggest that “substance displacement” might have occurred in this setting [We found that male PLHIV who had ever used 5-MeO-DPT/ setting  whereby  setting .Likewise, the independent association between lifetime use of methamphetamine/amphetamine and recent use of quasi-legal psychoactive substances could also be interpreted as switching from the former to the latter. However, a recent qualitative study reported that quasi-legal psychoactive substance use might subsequently lead to the use of methamphetamine/amphetamine among some PLHIV in Japan . In lighWe also found that those who were younger and those who were likely MSM were more likely to report recent use of quasi-legal psychoactive substances. This is consistent with a previous study conducted in Tokyo . AccordiInternationally, identifying effective regulations of new psychoactive substances is a major topic of debate , 23. In Our study has several limitations. First, as the study sample was not randomly selected, our findings may not be generalizable to PLHIV in Japan or elsewhere. Second, the self-reported data may be affected by reporting biases, including recall bias and socially desirable responding. However, we note that the anonymous nature of the questionnaire may have helped reduce some of the biases. Third, as with all observational studies, the associations between the explanatory variables and outcome assessed may be under the influence of unmeasured confounding factors. However, we sought to address this bias with multivariate adjustment of the key demographic and behavioural predictors of quasi-legal psychoactive substance use.Foxy. Individuals who had ever used these tryptamine derivatives or methamphetamine/amphetamine were more likely to report recent quasi-legal psychoactive substance use. These findings suggest a potential shift in drug use patterns from regulated to unregulated substances among this population. Given that a new regulation instituted in April 2014 fully banned presumably most of the quasi-legal psychoactive substances considered in this study, further research is warranted to examine changing drug patterns and implications for HIV care in this setting.In sum, illicit drug use was much more common among male patients in HIV care in Japan, compared to their female counterparts. Among male participants, quasi-legal psychoactive substances were the most commonly used drugs. We also found a substantial decline in recent use of 5-MeO-DPT/


Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis.Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD® 4-G (15 mg/mL) or HYADD® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints.Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis  at 56 days. Mice treated with repeated HYADDWhile we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP. Osteoarthritis (OA) is a leading cause of disability worldwide. Post-traumatic OA (PTOA) in particular is a frequent cause of disability following trauma to the weight-bearing joints. It is estimated that 12% of the nearly 21 million Americans with symptomatic OA have a post-traumatic etiology [Murine models of knee injury are critical tools to study alterations associated with PTOA in the joint. Several invasive injury models such as injecting collagenase into the joint , using a® 4-G  hydrogel is a novel HA amide derivative in which an aliphatic amine is bound to HA at the carboxylic group of the glucuronic acid in only 2% of the carboxylic groups, therefore 98% remains unmodified HA. This small chemical modification on the molecule increases the viscoelastic properties of HA and thereby increasing beneficial effects in human OA as well as in surgical models of PTOA in large animals [Hyaluronan  is a high molecular-weight, non-sulfated, unbranched glycosaminoglycan  that pla animals , 14. Inc animals .2. Most of these factors are implicated in the regulation of articular chondrocyte anabolic activity. In particular, platelet-derived growth factor (PDGF) stimulates chondrocyte proliferation and proteoglycan synthesis (reviewed in [Platelet-rich plasma (PRP) is another option for the intra-articular treatment of OA, considering the high content of growth factors in the PRP preparations. The growth factors, stored in the granules of the platelets are released after platelet activation, which can occur before injection by adding thrombin or CaCliewed in ) and traiewed in  and stimA systematic review of the efficacy of PRP has concluded that multiple sequential intra-articular PRP injections might have beneficial effects in the treatment of mild to moderate knee OA in adult patients  in compaTo date, there is no study that has tested the potential (therapeutic) effects of HA or PRP in the injured mouse knee joint after mechanical loading. Here we have analyzed joint tissues to determine whether treatment with HA or growth factor-rich preparations administered at the time of injury or later could reduce the immediate effects of loading and/or the long-term effects of joint injury.ad libitum. In total, we evaluated 150 knees from 86 mice (5–14 knees per group). Some of the knees were excluded from the analysis due to the lack of an ACL rupture  or due to lack of extraction of reliable data–for example poor tissue preparation, problems with embedding, poor orientation of sections and improper scanning. The number of mice in each group, time points and treatment options are depicted in Study procedures were approved by Washington University Institutional Animal Care and Use Committee. Mice (C57BL/6J) were procured from the Jackson Laboratory  and housed under standard sanitary and husbandry conditions as described previously . Briefly® 4-G hydrogel is a non-cross-linked HA (500–730 kDa) of fermentative origin. Fidia Farmaceutici  provided HA-derived HYADD® 4-G hydrogel in sealed syringes with two different concentrations: 8 mg/mL and 15 mg/mL. For the sake of simplicity, we used the term HA-derivative for this product. HYADD® 4-G (8 mg/mL) is commercially available with the brand name HYMOVIS® (http://hymovis.com).HA-derived HYADDWe prepared PRP from mouse blood for therapeutic use alone or combined with the HA-derivative. PRP was prepared as described previously . BrieflyMouse knees underwent 6N or 9N of compressive loading as detailed previously , 25. BriWhile mice were under anesthesia, they were injected with the indicated treatment(s) . Intra-a2 chamber at indicated time points  for analysis of 3-dimensional bone structure for trabecular bone volume fraction (BV/TV) , 28. SubKnee joints were decalcified using Immunocal  and paraffin embedded. Five-micron thick sections were cut in sagittal plane throughout the joint with every third section stained with Safranin-O. As there was no appreciable structural loss of non-calcified cartilage, we scored articular cartilage for proteoglycan depletion (loss) using a supplementary OARSI  scoring system  as described by Glasson and co-workers . SynoviaChondrocyte apoptosis was detected by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay . In order to compare the number of apoptotic cells, we examined the numbers of TUNEL-positive cells in non-calcified cartilage as it is commonly thought that apoptosis naturally occurs in calcified cartilage . Images To assess the expression pattern of extracellular matrix proteins such as aggrecan, and cartilage oligomeric matrix protein (COMP), and CD44 , we used antibody-specific immunostaining as described elsewhere , 25. SelData collection and analysis were performed in a blinded fashion. As noted above , we usedThe cartilage injury site was located at the posterior aspect of the lateral femoral condyle and was characterized by the loss of Safranin-O staining without disrupting nuclear structure. We observed complete loss of Safranin-O staining indicative of proteoglycan depletion in the non-calcified cartilage extending up to 50% of the articular surface . The carNon-immune controls are shown in the As reported by us , 25 and We observed that non-loaded knees had normal synovium as before . Both 6NWe observed some differences in bone volume fraction (BV/TV) between non-loaded and loaded knees, but the only significant difference was noted between non-loaded knees and 9N loaded knees at 56 days . No apprIn this study, we used known short-term parameters of the compression model to determine whether there were effects of HA-derivative and/or PRP. In addition, we extended the time after injury to 8 weeks (56 days) to determine long-term consequences of the single injury and whether there were long-term effects of the treatments.This study confirmed our previous findings in the compression model, which was largely characterized by the focal loss of proteoglycan at the site of cartilage injury on the posterior aspect of femoral condyle, chondrocyte apoptosis in this area, changes in the pattern of aggrecan expression, synovitis and ectopic calcification at early time points , 25. We Several new studies have shown that acute chondrocyte death caused by in vitro impact injuries results from apoptosis , 34. LisOur findings differ with other studies that have reported a beneficial effect of repeated injections of both HA and PRP in the rabbit joints after impact to patella and femoral condyle , 37. In HA interacts with its surface receptors, CD44, on chondrocytes . CD44-HAThe role of ectopic ossification and osteophytes in OA development is not completely clear, although it is thought that they mechanically stabilize an osteoarthritic joint, thereby preventing structural progression . Felson Christiansen and colleagues  have repNumerous PRP studies have been performed in laboratory and clinical research in last two decades. Of these, some studies have reported that PRP increases chondrocyte viability and proliferation , and enhAlthough PRP has been used for over 40 years for treatment of acute and chronic injuries of bone and cartilage , only a This study has some limitations. There are no studies to directly compare the effect of PRP or HA with our study, so we used the literature from other study designs such as the in vitro effects of HA in IL-1β-treated human chondrocytes, in vitro equine impaction-induced apoptosis and in vivo loading induced apoptosis in rabbit joints after impact to patella and femoral condyle. It is likely that HA has effect on other functional parameters that are beyond the scope of present study. Therefore, our findings do not represent direct comparison with the other studies. Our study also lacks appropriate positive control, as there are currently no disease modifying OA drugs or the use of some treatments has been evaluated for functional outcomes such pain and gait pattern, which we did not undertake in our study. Furthermore, the analyses were performed on the femur side as this model results in changes that occur on the femur. It would have been ideal if our study could provide more specific information on focal subchondral or trabecular bone analyses given the focal nature of the joint injury, however, this was not possible, as we could not detect any focal changes in the bone on micro-CT. Lastly, the outcomes of our study were centered on joint structure, whereas clinical findings on the effectiveness of HA and PRP are often focused on patient-reported outcome measures (symptoms)  or bioma® 4-G) at two different concentrations (8 mg/mL and 15 mg/mL) at two different loadings (6N and 9N) and at two different time points (5 days and 56 days) on these parameters: cartilage proteoglycan loss, chondrocyte apoptosis, expression pattern of aggrecan, COMP, and CD44, synovitis, ectopic calcification, subchondral bone plate thickness. Bone volume trabecular fraction was found to be significantly different between loaded (9N group) and non-loaded joints. The repeated injections of HA-derivative at 15 mg/mL and HA-derivative at 8 mg/mL+PRP, could reduce but not significantly improve the bone changes. Additionally, the injections of only PRP conferred no therapeutic efficacy, and the results were not significantly different from PBS. Other potential treatment options need to be investigated to reduce the deleterious immediate and long-term effects of traumatic injury to the knee joint.In this study, contrary to our hypothesis, there was no protective effect of HA-derivative Click here for additional data file.S2 FigA. Images with split channels from B. Images with split channels from (TIF)Click here for additional data file.


Evidence exists pointing out how non-adherence to treatment remains a major hurdle to efficient tuberculosis control in developing countries. Many tuberculosis (Tb) patients do not complete their six-month course of anti-tuberculosis medications and are not aware of the importance of sputum re-examinations, thereby putting themselves at risk of developing multidrug-resistant and extensively drug-resistant forms of tuberculosis and relapse. However, there is a dearth of publications about non-adherence towards anti-Tb medication in these settings. We assessed the prevalence of and associated factors for anti-Tb treatment non-adherence in public health care facilities of South Ethiopia.This was a cross-sectional survey using both quantitative and qualitative methods. The quantitative study was conducted among 261 Tb patients from 17 health centers and one general hospital. The qualitative aspect included an in-depth interview of 14 key informants. For quantitative data, the analysis of descriptive statistics, bivariate and multiple logistic regression was carried out, while thematic framework analysis was applied for the qualitative data.The prevalence of non-adherence towards anti-Tb treatment was 24.5%. Multiple logistic regression analysis demonstrated that poor knowledge towards tuberculosis and its treatment , cost of medication other than Tb , having of health information at every visit  and distance of DOTS center from individual home  showed statistically significant association with non-adherence towards anti- tuberculosis treatment. Qualitative study also revealed that distance, lack of awareness about importance of treatment completion and cost of transportation were the major barriers for adherence.A quarter of Tb patients interrupted their treatment due to knowledge, availability and accessibility of DOTS service. We recommend creating awareness about anti-Tb treatment, and decentralization of drug pick-ups to the lowest level of health institutions. Evidence exists that tuberculosis (Tb) is a major public health problem throughout the world, with an estimated 9.6 million annual incident cases, of which 1.2 million (12%) were co-infected with HIV and 1.5 million died globally in 2014 , 1.4 milStandard Tb treatment requires patients to take a complex combination of drugs for 2 months in intensive phase and 4 months in continuation phase for new patients . In the Even with over 80 million dollar USD funding from international agencies, and with counterpart funding from the Ethiopian government annually, Tb control remains modest . EthiopiIn Ethiopia, different studies assessed the rates and determinants of anti-Tb non-adherence. For example, a study conducted North Ethiopia reported a prevalence rate of 11.5% . AnotherThis was an institution based cross-sectional survey that mixed quantitative and qualitative methods. We followed the guidelines on programmatic management of tuberculosis in Ethiopia for diagnosis and classification of Tb cases . The zonThe study population included pulmonary Tb patients who registered 6 months before data collection time on anti-Tb treatment regime. Patients on MDR treatment regimen and patients too ill to be interviewed were excluded from the study. The sample size was calculated via OpenEpi 2.3 software using a single population proportion calculation formula using the following assumptions: 20% proportion of non adherence towards ant-Tb treatment , 95% conAnti-Tb treatment adherence status was the response variable dichotomized as adherent and non adherent. A patient belonging to either intensive or continuation phase under new or retreatment regimen who missed ≥10% of the total prescribed dose was considered as non-adherent. For sputum follow up, patient who missed one and more sputum test will be considered as non-adherent. The covariates included: sex, age, religion, ethnicity, level of education, occupation, marital status, having treatment supporter, type of transportation, attitude of care providers, alcohol consumption, cigarette smoking, having patient supervisor, availability of health information at every visit, side effect of medication, current treatment phase, pill burden, knowledge about Tb and its treatment, distance from DOT center, traveling time and Cost of medication other than anti-Tb.History of smoking and history of alcohol consumption has been assessed as during lifetime. We asked participants to report their lifetime experiences of cigarette smoking and alcohol drinking. We recorded their response as smoker, non-smoker and ex-smoker for cigarette smoking; drinker or non- drinker for alcohol drinking. For statistical analysis, smoking responses was further aggregated to yes or no. Knowledge towards Tb and its treatment was assessed using knowledge related questions and was classified as poor, fair and good. Scores ≥75%, 74-60% and <60%, respectively, were labeled as good, fair and poor knowledge.Face-to-face interviews were conducted by six registered nurses and supervised by three health officers to solicit socio-demographic and economic factors, treatment related factors, behavioral factors and patient knowledge towards tuberculosis and its treatment. The interviews were conducted in a quiet room at the Tb clinic where patients came for follow up checkup. Study subjects who lost from follow-up were interviewed at home. Qualitative data were collated through notes and audio-recordings during interview. We used in-depth interview guide to explore barriers related with current Tb control programs and possible reasons for treatment non-adherence.P < 0.25 was considered).Quantitative data were analyzed using descriptive and inferential statistical techniques. Summary statistics including percentages and odds ratios were computed. We used binary logistic regression to analyze factors that were associated with adherence to anti-Tb medications. The analyses of bivariate logistic regression assessment were conducted to check the existence of crude association and select the candidate variables  and none was found. Multiple logistic regression analysis was used to declare the independently associated predictors. Lemeshow  and was Lemeshow .Qualitative data were analysed using thematic framework analysis –22. We cA total of 261 (96.3%) Tb patients were considered eligible: 36 patients from hospital and 225 from health centers. Data of 10 study subjects were not included in the analysis due to changing of place of residence, death and incompleteness. Table The prevalence of anti-Tb non-adherence was 24.5%. Among the total non-adherents, 36 were males, 36 were pulmonary Tb positive, and 32 were under intensive phase when analyzed by sex, Tb classification and treatment phase respectively.Table Table Tb patients who had poor knowledge about Tb and its treatment were 5 times  higher at risk towards anti-Tb non-adherence than those who had good knowledge. The relative probability of anti-Tb non-adherence among patients travelled more than 10 km to pick treatments was higher than  those travelled less than 10 km. The association of anti-Tb non-adherence among patients who didn’t get health information at every visit was 3 times  higher than among those who got. Cost of medication other than Tb was found a risk factor  to anti-Tb non-adherence.Findings of the qualitative study also supported the quantitative results. The respondents stated that there was an improvement of the program. However, they reported that the service is not decentralized, lack of drugs in stock was still observed and shortage of laboratory reagents for AFB was becoming a routine challenge. Distance, lack of awareness about importance of treatment completion and cost of transportation were the explored barriers for adherence. Decentralization of drug pick-ups and delivering health information at every visit were the suggested solutions. Table Adherence to TB treatment is fundamental for successful TB control and eradication. Nonetheless, the long-term course of the treatment might expose patients for non-adherence . In the In the current study, knowledge, distance, health information at every visit, pill burden and cost of medication other than anti-Tb were the predictors for anti-Tb treatment non-adherence. Patients who had poor knowledge towards Tb and its treatment were at risk of being non-adherent. This was suggested by the qualitative findings of the current study. The result was corroborated by findings of previous studies –29. ThisCost of medication other than anti-Tb remains another serious challenge in Tb treatment non-adherence. This was not dissimilar with the findings of previous studies , 32. ThiThe study had some limitations, which were worth noting. The institutional based nature of the study might not infer for other Tb patients who didn’t visit the institution. Similarly, the nature of cross-sectional study design does not indicate temporal relationship or causality. Self-report of adherence to medications could also be affected by recall bias. Moreover, selection bias could also have been introduced because patients who are under regular follow-up by the university clinic are likely to be receiving better care and support than those in the lower level clinics. Health workers collected data through direct interview of patients and this may subject to social desirability bias.In conclusion, this current study assessed the degree of adherence in pulmonary Tb patients receiving DOTS therapy and barriers contributing to adherence. In comparison to previous studies, our study revealed relatively high non-adherence rate among new pulmonary Tb patients. Knowledge, distance, not decentralizing of DOTs service, health information at every visit and cost of medication other than anti-Tb were the barriers for anti-Tb treatment adherence. Policies are recommended to be developed to strengthen the treatment management of TB patients. Where possible, decentralization of DOTs service to lower level of health facility structure should be prioritized.


A toxic gain of function occurs in the absence of FUS protein aggregation, involving disturbance of ribosomes and mitochondria at the endoplasmic reticulum.Devoy  FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease—with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.Mutations in  FUS (fused-in-sarcoma) account for ∼5% of familial and ∼1% sporadic ALS  is characterized by progressive degeneration of motor neurons in the brain and spinal cord, leading to muscle atrophy, paralysis and death , creating a novel frame-shifted C-terminus. We introduced the identical point mutation, g.13845A>G, into the splice acceptor site of mouse Fus exon 14   and gait analysis. Compared to wild-type littermates, at 3 months of age heterozygous FUSDelta14 mice did not show motor impairment on either test, ruling out a developmental phenotype A and B. P = 0.033, A Kaplan-Meier survival analysis to 22 months of age showed a modest but significant reduction in survival of FUSDelta14 mice compared to wild-type littermates, from 19 months of age [LogRank(Mantel-Cox) survival P = 0.0022; P = 0.028, We investigated functional motor neurons innervating the extensor digitorum hindlimb muscles, by physiological analysis of motor units. At 18 months of age motor units were significantly reduced (15%) in FUSDelta14 extensor digitorum compared to littermates . Interestingly, our mutant-specific antibody showed that regardless of having no nuclear localization signal, ∼25% FUSDelta14 protein lies in the nucleus of FUSDelta14 motor neurons  progressive degeneration of motor neurons with a dominant mode of inheritance. Crucially, we express humanized mutant FUS from the endogenous mouse Fus nuclear localization signal-deletion allele, which have late-onset motor neuron loss at 22 months of age, that importantly, is rescued by selective expression of wild-type Fus in motor neurons. Importantly, heterozygous knock-out mice in the same study did not lose motor neurons, providing the strongest evidence to date that it is a gain-of-function through mislocalization, not loss of function that leads to motor neuron death. In contrast, our FUSDelta14 mice develop motor neuron loss considerably earlier by 12 months of age. The reason for this difference is not clear, but as FUSDelta14 mice have a normal motor system in young adulthood (3 months of age) and motor neuron loss is clearly underway by 12 months, this gives us an excellent window in which to investigate early pathomechanisms. This is an important issue for understanding ALS, as we do not know when the disease begins and have limited biomarkers for disease progression. Already, our work has shown that while pathological aggregation of FUS protein in not required for the initiation of disease and motor neuron death, there is clear evidence of disturbed proteostasis.Recently, In FUS-ALS motor neuron loss is primarily driven by a gain-of-function mechanism  in contrast to knockdown/knockout studies (in vivo physiological model, and to investigate mutant-specific interactions with our novel antibody. This system is a powerful tool and is giving new insight into early stages of ALS.Our data suggest the FUS-ALS toxic gain-of-function occurs in cytoplasm and/or nucleus, in the absence of aggregation. The FUSDelta14 mouse-antibody allows us to map molecular changes longitudinally, from early development to humane end stage, in an Supplementary FiguresClick here for additional data file.Supplementary Tables S1 to S3Click here for additional data file.Supplementary Tables S4 to S10Click here for additional data file.Supplementary Materials and MethodsClick here for additional data file.


Healthy People 2020 objective is to reduce the proportion of youths exposed to onscreen tobacco marketing in movies and television (Tobacco Use Objective 18.3)  , 2010–20TUTD counts occurrences of tobacco incidents, defined as the use or implied use of a tobacco product  by an actor, in U.S. top-grossing movies each year. Trained monitors count all tobacco incidents in those movies that are among the 10 top-grossing movies in any calendar week of the year. Previous reports have used this criterion because U.S. movies ranked in the 10 top-grossing movies for at least 1 week have accounted for 96% of U.S. ticket sales  ratings . Findings were also compared with data from reports from 1991–2010  had tobacco incidents, compared with 62 (45%) of 137 in 2010; among top-grossing R-rated movies, 35 (67%) of 52 had tobacco incidents in 2016, compared with 35 (71%) of 49 in 2010 . Among yAlthough the percentage of top-grossing movies with tobacco incidence decreased during 2010–2016, the total number of tobacco incidents in top-grossing movies increased by 72%, from 1,824 to 3,145 . The totThe findings in this report indicate that although there were previously reported declines in the number of youth-rated movies with tobacco incidents observed during 2005–2010   delivered by a particular movie to children and adolescents could not be determined. Second, the sample did not include all movies. However, the samples of top grossing movies were used because they are expected to account for approximately 95% of theater tobacco-use impressions (If current trends continue, 5.6 million youths who are alive today are projected to die from tobacco-related diseases (The Surgeon General has concluded that there is a causal relationship between depictions of smoking in the movies and the initiation of smoking among young persons. The more frequently youths see smoking on screen, the more likely they are to start smoking; youths who are heavily exposed to onscreen smoking imagery are approximately two to three times more likely to begin smoking than are youths who are less exposed.Previously reported declines in number of top-grossing movies with tobacco use has continued; however, the decline in the total number of tobacco incidents has not progressed since 2010. From 2010 to 2016, the total number of tobacco incidents in top-grossing movies increased, with a 43% increase occurring among movies rated PG-13.Although there were fewer youth-rated films with tobacco incidents in 2016 than in 2010, total depictions of tobacco use has remained stable, concentrating such exposure in fewer films. Reducing tobacco incidents that appear in youth-related movies would prevent the initiation of tobacco use among young persons. An R rating for movies with tobacco use could potentially reduce the number of teen smokers by 18% and prevent their premature deaths from tobacco-related diseases.


N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC50s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy.Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified  Chloroquine, celgosivir, and balapiravir are the only drugs that have reached the clinical-trials stage; however, no results have been reported for these studies . Repurposing of drugs is one way of fast-tracking antiviral discovery, as it builds on preexisting knowledge of the candidate drug. In cases where the drug has been proven to be safe for human use for other conditions, this process helps to bring the drug to human use faster than a new candidate drug, which, according to industry estimates, takes 12 to 15 years from discovery to therapeutic use (1280) for anti-dengue virus compounds. LOPAC1280 covers most major pharmacologically relevant target classes, and many of them are approved as drugs for human use, thus providing an excellent platform for repurposing as antivirals. In this study, we report the screening of LOPAC1280 for dengue virus inhibitors using an imaging-based screening approach with Huh-7 cells. We identified three inhibitors from the library that specifically blocked early stages of dengue virus RNA replication. Our data further underscore the value and economic benefits of drug-repositioning efforts that could be harnessed to augment the antiviral pipeline.Dengue virus (DENV) is a mosquito-borne pathogen that imposes maximum burdens on the economy and public health in large parts of the Americas, Southeast Asia, and Africa. According to recent estimates, close to 60 million symptomatic cases occur every year, and the overall economic burden of dengue is nearly $9 billion . Vaccine2–Huh-7 cells; DENV-2, Japanese encephalitis virus (JEV), and West Nile virus (WNV) isolates; virus growth conditions; and procedures for determining titers were described previously . (See th50 (50% inhibitory concentration) experiments were performed by using 2-fold dilutions of the inhibitor starting from an 8 μM concentration. A total of 30,000 cells were plated in a 48-well plate, infected with DENV-2 at a multiplicity of infection (MOI) of 3, and incubated with 2% Dulbecco's modified Eagle's medium (DMEM) containing inhibitors. Supernatants were collected at 24 h postinfection (p.i.), and viral titers were estimated by plaque assays on BHK-21 cells. For Western blotting, cell lysates were prepared as described previously . RNA was isolated from the concentrated supernatant by using TRIzol-LS (Thermo Fisher Scientific). The aqueous phase obtained after the chloroform step was mixed with RNA binding buffer and processed for RNA isolation by using an RNA Clean and Concentrate kit (Zymo Research). A total of 400 ng of RNA was transfected into HEK293 cells by using Lipofectamine 2000 (Thermo Fisher Scientific). At 4 h posttransfection, medium containing dimethyl sulfoxide (DMSO) or inhibitors was added. At 72 h postinfection, total RNA was isolated as described above. The amount of DENV negative-strand RNA was estimated as described below.TC method.Total RNA was isolated from cells by using either TRIzol or a Quick-RNA MicroPrep kit (Zymo Research). DENV RNA levels were estimated as described previously, using TaqMan one-step real-time PCR (Applied Biosystems) , 11. Thez projection images are shown.Huh-7 cells grown on coverslips in a 24-well plate were infected and treated with 4 μM inhibitors as described above. At 24 h p.i., cells were processed for immunofluorescence to visualize double-stranded RNA (dsRNA) as described previously . For assP values were estimated by a Mann-Whitney test or one-way analysis of variance (ANOVA).GraphPad Prism software was used for all graphical representations and statistical analyses. All experiments were performed with two or three replicate samples, and results in each figure are a compilation of data from experiments performed at least two to three times. 1280 using high-throughput screening where both the reduction in the number of virus-infected cells as determined by a fluorescence imaging assay and the reduction in viral titers in the supernatant were used as endpoints. Huh-7 cells were infected with DENV-2 and treated with either 10 μM inhibitors or DMSO as a control. Cells were stained with DENV E antibody, and infected cells were visualized by using the Cell Analyzer imaging platform. We identified 30 compounds as primary hits that showed >50% inhibition of DENV infection. We confirmed the inhibitory activity of these compounds by a secondary screen using two independent assays measuring (i) viral titers in the supernatants by plaque assays and (ii) viral RNA levels at 24 h p.i. in cells treated with 10 μM inhibitors . Based on the secondary screen, we selected three inhibitors, N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate, which consistently showed >90% inhibition of both viral titers and viral RNA levels without cytotoxic effects . We next confirmed whether the inhibitors had any effect on flaviviruses closely related to DENV, such as Japanese encephalitis virus and West Nile virus. Huh-7 cells were infected with JEV and WNV, and virus titers were estimated at 24 h p.i. Surprisingly, none of the three inhibitors had any effect on either JEV or WNV titers . To further confirm that the observed inhibition of DENV infection is not specific to Huh-7 cells, we infected A549 cells with DENV-2 and treated the cells with inhibitors as described above. Viral titers from the supernatants were estimated by plaque assays at 24 h p.i. Cells were fixed and stained with DENV E antibody, and the percentage of DENV-positive cells was analyzed by fluorescence-activated cell sorter (FACS) analysis. We observed the complete absence of both virus-positive cells and viral titers in supernatants of cells treated with inhibitors compared to DMSO , suggesting that the inhibitors may be targeting a factor(s) or pathway(s) that is expressed only in cells of human origin.We screened LOPAC to DMSO  to f. Th50s of N-desmethylclozapine, fluoxetine, and salmeterol in Huh-7 cells infected with DENV-2. Cells were infected with DENV-2 and treated with increasing concentrations of each of the inhibitors, and viral titers were measured by plaque assays at 24 h p.i. The IC50s of N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate were 1 μM, 0.38 μM, and 0.67 μM, respectively  and at 48 h postinfection (for a time of addition of 24 h). The addition of inhibitors was inhibitory up to 12 h postinfection, whereas at 24 h postinfection, it had no effect . At 24 hInternalized DENV has been shown to undergo membrane fusion in the late endosomal compartment . TherefoN-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate, as dengue virus replication inhibitors in a DENV screening assay. All three inhibitors reduced dengue virus infection with IC50s in the high-nanomolar/low-micromolar range. Cells treated with these inhibitors had low levels of dsRNA and negative-strand intermediates. This finding, along with the time-of-addition and RNA transfection experiments, demonstrated that the inhibitors interfered with early stages of dengue virus RNA replication. The effect was specific to dengue virus, as the inhibitors did not have any effect on other related flaviviruses, such as JEV and WNV, or on respiratory syncytial virus, a negative-strand RNA virus, and rotavirus, a virus with a double-stranded RNA genome. Finally, we showed that treatment with all three inhibitors led to lysosome enlargement and autophagosome formation, possibly affecting endolysosomal pathways that are specifically involved in early stages of the DENV life cycle. Although a number of studies have characterized direct-acting antivirals targeting dengue virus proteins   . The adm (TNF-α) . SimilarN-desmethylclozapine, fluoxetine, and salmeterol leads to an alteration in autophagosome/lysosome biogenesis affecting early stages of the DENV life cycle. This observation also hints at differences in the pathways used by related flaviviruses at early stages of infection.Previous studies have shown that both RSV and RV induced autophagy at later stages of infection , 30. RSV33–N-Desmethylclozapine is a major metabolite of clozapine and a potent 5-hydroxytryptamine 2 (5-HT2) serotonin receptor antagonist. Clozapine is a antipsychotic drug. Salmeterol is a β2-adrenergic receptor agonist and has anti-inflammatory properties (N-desmethylclozapine and salmeterol to inhibit enteroviruses. It is likely that the inhibitors identified in our study may affect a pathway that is common to DENV and enteroviruses. Characterization of the host factors involved in early stages of DENV infection and its similarities with the enterovirus life cycle would help in identifying antiviral targets. Further work on in silico docking with known structures of dengue virus proteins and the generation of resistant viruses would provide clues with respect to the ability of these inhibitors to directly affect dengue virus replication. We believe that our study has provided new use for compounds that are approved for human use for other conditions. The safety profile and efficacy of these drugs need to be validated in pilot trials with dengue virus-infected patients. As these drugs would be used during the viremic phase of infection and for shorter periods in humans, we believe that the side effects observed with prolonged-dose regimens for other conditions may not be pertinent to viral infections. Finally, if these drugs are inhibiting dengue virus replication via cellular targets, the identification of molecular targets would help in the further development of safer and more potent antivirals against dengue virus.operties . In conjoperties . There aoperties . Fluoxetoperties . Fluoxetoperties . An evaloperties . We have