Observational studies in high-income and resource-limited settings indicate that HIV-infected adults with low vitamin D levels may be at increased risk of mortality, HIV disease progression, and incidence of pulmonary tuberculosis (TB). As a result, vitamin D3  supplementation conducted among 4000 HIV-infected adults with low vitamin D levels [25-hydroxyvitamin D (25(OH)D) <30 ng/mL] initiating ART in Dar es Salaam, Tanzania. The two primary aims of the trial are to determine the effect of a vitamin D3 supplementation regimen on incidence of (1) mortality and (2) pulmonary TB as compared to a matching placebo regimen. The primary safety outcome of the study is incident hypercalcemia. The investigational vitamin D3 regimen consists of oral supplements containing 50,000 IU vitamin D3 taken under direct observation at randomization and once a week for 3 weeks (four doses) followed by daily oral supplements containing 2000 IU vitamin D3 taken at home from the fourth week until trial discharge at 1 year post ART initiation. Trial participants are followed up at weekly clinic visits during the first month of ART and at monthly clinic visits thereafter until trial discharge at 1 year post ART initiation. Secondary aims of the trial are to examine the effect of the vitamin D3 regimen on CD4 T cell reconstitution, incidence of non-TB comorbidities, body mass index (BMI), depression and anxiety, physical activity, bone health, and immunologic biomarkers.The Trial of Vitamins-4 (ToV4) is an individually randomized, double-blind, placebo-controlled trial of vitamin D3 supplementation on incidence of pulmonary TB and mortality among HIV-infected Tanzanian adults initiating ART. The trial will also give insight to whether vitamin D3 supplementation trials for the prevention of pulmonary TB should be pursued in HIV-uninfected populations.The ToV4 will provide causal evidence on the effect of vitamin DNCT01798680. Registered on 21 February 2013.ClinicalTrials.gov, The online version of this article (doi:10.1186/s13063-017-1819-5) contains supplementary material, which is available to authorized users. Access to antiretroviral therapy (ART) coverage has rapidly increased in sub-Saharan Africa during the last decade with over 12 million human immunodeficiency virus (HIV)-infected individuals on ART in 2015 . The 1-yVitamin D is a potent immunomodulator with effects on both adaptive and innate immune responses which may play a prominent role in control of intracellular pathogens by enhancing cell-mediated immunity, production of antimicrobial peptides, and phagocytic activity of macrophages –7. AccorMycobacterium tuberculosis (Mtb)  as mean difference in concentrations (h) post-randomization immunologic biomarkers  as mean difference in concentrations and (i) risk of hypercalcemia .The overall goal of ToV4 is to investigate a vitamin DIndividuals are eligible for the trial randomization if they meet all the following inclusion criteria: (a) adult men or women aged ≥18 years old, (b) HIV-infected, (c) initiating ART at the time of randomization, (d) have low vitamin D levels with a 25(OH)D concentration <30 ng/mL at ART initiation (e) intending to stay in Dar es Salaam for at least 1 year after enrollment, and (f) provide written informed consent. Exclusion criteria include: (a) pregnancy (women of childbearing age are required to have a negative pregnancy test within a month prior to randomization) and (b) enrolled in any other clinical trial. During the trial the ART initiation criteria for the Dar es Salaam HIV treatment program changed from CD4 T cell count <350 cells/μL or WHO HIV stage 3 or 4 disease to CD4 T cell count <500 cells/μL or WHO HIV stage 3 or 4 disease in January 2016.3 trial regimen consists of oral supplements containing 50,000 IU vitamin D3 taken at randomization and once a week for 3 weeks at clinic visits under direct observation (four doses) followed by daily oral supplements containing 2000 IU vitamin D3 taken in the home starting at the fourth week until trial discharge at 1 year post ART initiation. The matching placebo regimen consists of oral placebo supplements taken at randomization and once a week for 3 weeks at clinic visits under direct observation (four doses) followed by daily oral placebo supplements taken in the home starting at the fourth week until trial discharge at 1 year post ART initiation.The investigational vitamin D3, rather than vitamin D2 , since vitamin D3 is more effective in increasing and sustaining levels of circulating 25(OH)D  . Any par3 regimen on bone health, we will measure PTH and ALP at baseline, 1, 6, and 12 months for a random sample of 800 individuals (20%). We will also assess the effect of vitamin D3 on host immune response through measurement of IL-2, IL-12, IFN-γ, and the antimicrobial peptide cathelicidin in 800 (20%) randomly selected individuals at baseline, 1, 6, and 12 months.All participants have CD4 T cell counts assessed at or before the ToV4 screening/ART eligibility visit and then every 6 months after ART initiation. Hemoglobin is also measured at the ToV4 screening/ART eligibility visit. In order to examine the effect of the vitamin DAny trial requires a high degree of compliance with experimental regimens as non-compliance can bias results and decrease the statistical power of a study. The best way to ensure full compliance is to observe each participant ingesting treatment. In ToV4 research nurses directly monitor subjects taking the weekly trial supplements , but this type of monitoring is not practical for the subsequent daily supplements taken in the home (week 4 to month 12). In order to promote adherence during the daily regimen period, all participants are encouraged to use the daily supplements at the same time every day, to put the bottle in a visible place, and identify an adherence assistant who would remind her/him to take the supplement. Compliance with the daily ToV4 supplements will be assessed three ways: (a) direct questioning about use of the supplements in the previous 24 hours and 2 weeks, (b) pill count from returned daily regimen bottles, and (c) biochemical assessment of 25(OH)D in a subset of participants longitudinally at randomization, 1, 6, and 12 months.All study participants are provided with HIV care and treatment that adhere to Tanzanian national guidelines. During the trial the ART initiation criteria within the Dar es Salaam HIV treatment program changed from CD4 T cell count <350 cells/μL or WHO HIV stage 3 or 4 disease to CD4 T cell count <500 cells/μL or WHO HIV stage 3 or 4 disease in January 2016. Per Tanzanian guidelines, zidovudine, lamivudine, and efavirenz are the choice first-line regimen with tenofovir, nevirapine, and stavudine as back-up drugs. The standard visit schedule for patients on ART is clinic visits at 2 weeks and 1 month post-initiation with monthly thereafter. Patients who are stable after 6 months of ART are eligible receive a 2-month supply of ART with a subsequent scheduled clinic visit at corresponding 2-month intervals. CD4 T cell counts are assessed at the ART eligibility visit and every 6 months after ART initiation. During the trial HIV viral load testing became available as standard of care with assessment every 6 months after ART initiation. Prophylaxis for opportunistic infections is provided in line with the Tanzanian HIV treatment guidelines. Participants receive co-trimoxazole prophylaxis if CD4 cell count is <200 cells/μL. All participants diagnosed with TB are treated according to the WHO-approved guidelines of the National Tuberculosis and Leprosy Program (NTLP) in Tanzania. All TB patients receive directly observed therapy (DOT) with a 6-month short course regimen consisting of a 2-month intensive phase of daily rifampicin/isoniazid/pyrazinamide/ethambutol (RHZE) combination tablets, followed by a 4-month continuation phase of daily rifampicin and isoniazid (RH) combination tablets. Sputa from smear-positive TB patients is collected and examined at 0, 2 and 5 months and at any other time if recurrence of TB is suspected. If the direct sputum smear is negative at the end of 2 months, the continuation phase is started and if a smear is still positive at 3 months the patient is checked for multi-drug resistance. Treatment failures and relapses are treated with an 8-month re-treatment regimen containing rifampicin throughout, with the intensive period with RHZE and streptomycin injection continuing for 3 months. Psychological and nutritional support is also provided as standard of care.An intent-to-treat analysis will be used as the primary analytic strategy for all analyses. The primary efficacy trial endpoints for this trial are (1) death and (2) incident pulmonary TB. The primary safety endpoint is hypercalcemia . We will use the use the log-rank test to test the differences in the incidence rates of death and pulmonary TB between the treatment arms, and use the Fisher’s exact test to test differences in proportion of participants with incident hypercalcemia.2) and >10% weight loss from baseline, (e) post-randomization depression and anxiety scores as assessed by the Hopkins Symptom Checklist (HSCL), (f) post-randomization physical activity as measured by METs, (g) post-randomization bone health biomarker levels (PTH and ALP) and (h) post-randomization immunologic biomarker levels . We will use linear mixed-effects models with a random intercept, a compound symmetric covariance structure, and robust standard errors to assess mean differences between baseline and post-randomization levels of CD4 T cell count, BMI, METs, 25(OH)D, PTH, ALP, IL-2, IL-12, IFN-γ, and cathelicidin by randomization group. Linear mixed-effects models will lead to robust inference for the fixed effects even if the covariance structure is misspecified. Linear mixed-effects models will also account for within-subject correlation, and accommodate varying number and timing of measurements per participant over time. We will use a generalized linear mixed model with random intercept, a compound symmetric covariance structure, and robust standard errors to assess differences in the incidence of comorbidities and complications associated with HIV and/or ART which are repeatable binomial events. The log-rank test will be used to assess differences in the incidence rates of wasting and >10% weight loss between the treatment arms.The secondary endpoints for the study include: (a) post-randomization CD4 T cell count, (b) incidence of comorbidities and complications associated with HIV and/or ART, (c) post-randomization BMI, (d) incidence of wasting D concentration, sex, age, BMI, CD4 T cell count, hemoglobin concentration, WHO HIV disease stage, pulmonary TB, receipt of isoniazid preventive therapy, ART regimen, and trial regimen compliance. To assess the statistical significance of each interaction, we will use the likelihood ratio test for risk-ratio homogeneity in the mortality, pulmonary TB, and hypercalcemia analyses and the score test in the linear mixed-effects and generalized linear mixed models for longitudinal secondary outcomes.3 supplementation may be an effective ART adjunct intervention since vitamin D deficiency is common among HIV-infected individuals, ART can further reduce vitamin D levels by altering vitamin D metabolism, multiple observational cohort studies have determined low vitamin D levels are associated with increased risk of mortality, pulmonary TB, and disease progression among HIV-infected individuals, and vitamin D3 supplements are known to be effective and safe in improving vitamin D status [3 supplementation on mortality and incidence of pulmonary TB among HIV-infected adults initiating ART in Dar es Salaam, Tanzania. A search of clinicaltrials.gov determined the only other clinical trial of vitamin D3 among HIV-infected individuals with mortality or morbidity outcomes listed as primary or secondary outcomes is an ongoing study called the “Trial of Vitamin D in HIV Progression, Birth Outcomes, and Child Health” (NCT02305927). This ongoing trial is also conducted by our research group and is a randomized, double-blind, placebo-controlled trial of vitamin D3 supplementation among HIV-infected pregnant women in Dar es Salaam, Tanzania. Accordingly, the ToV4 will likely be the first randomized trial to determine the effect of vitamin D3 supplementation on mortality and morbidity outcomes among HIV-infected adult men and non-pregnant women.Antiretroviral therapy coverage is rapidly expanding globally and treatment programs are in need of interventions to prolong and improve the quality of life for HIV-infected individuals in resource-limited settings, especially during the initial months of treatment , 3. VitaD status , 34, 37.3 supplementation but also allow for generalizability of the findings. In the trial we only enroll HIV-infected adults initiating ART with 25(OH)D levels <30 ng/mL and exclude individuals with ≥30 ng/mL from randomization procedures. The 25(OH)D inclusion criteria was based on our observational cohort study of HIV-infected adults initiating ART in Tanzania which suggested there is likely little to no benefit to obtaining 25(OH)D levels >30 ng/mL in terms of mortality and pulmonary TB in our study population [3 supplementation based on their local prevalence of 25(OH)D <30 ng/mL. We also tried to maintain the generalizability of our findings by defining the study population as adults initiating ART broadly and did not restrict the study population by CD4 T cell and WHO disease stage criteria. HIV treatment programs in Tanzania and sub-Saharan Africa are moving from using a <350 to a <500 CD4 T cells/μL threshold for ART initiation based on WHO guidelines [3 supplementation in the treatment program. The ToV4 DSMB reviews event rates every 6 months and will recommend any necessary changes to the trial sample size to maintain statistical power. Overall, the ToV4 study population was carefully designed to include individuals who may experience the greatest benefits of vitamin D3 supplementation, but will also preserve the generalizability and potential ability to translate the findings into practice in Dar es Salaam and similar HIV treatment programs.The study population of the ToV4 was purposely defined by inclusion and exclusion criteria to select a population that may have significant benefit from vitamin Dpulation , 16. Theidelines . As we e3 should be considered as an ART adjunct intervention to reduce pulmonary TB and mortality among HIV-infected adults initiating treatment. The trial will also provide evidence for the effect of vitamin D3 supplementation on a wide range of secondary outcomes including: CD4 T cell reconstitution, non-pulmonary TB comorbidities and complications associated with HIV and/or ART, bone health, immunologic biomarkers, weight gain, depression and anxiety, physical activity, and incidence of hypercalcemia. The results of the trial are likely generalizable to HIV-infected adults with low vitamin D who are initiating ART in similar resource-limited settings and the trial findings will also inform whether larger vitamin D3 supplementation trials for the prevention of TB in HIV-uninfected populations should be pursued.Overall, the ToV4 will provide causal evidence if vitamin DTrial enrollment started on 24 February 2014 and recruitment is ongoing as of 26 January 2017.


The effects of antiretroviral therapy on risk of severe bacterial infections in people with high CD4 cell counts have not been well described. In this study, we aimed to quantify the effects of immediate versus deferred ART on the risk of severe bacterial infection in people with high CD4 cell counts in a preplanned analysis of the START trial.ClinicalTrials.gov, number NCT00867048.The START trial was a randomised controlled trial in ART-naive HIV-positive patients with CD4 cell count of more than 500 cells per μL assigned to immediate ART or deferral until their CD4 cell counts were lower than 350 cells per μL. We used Cox proportional hazards regression to model time to severe bacterial infection, which was defined as a composite endpoint of bacterial pneumonia (confirmed by the endpoint review committee), pulmonary or extrapulmonary tuberculosis, or any bacterial infectious disorder of grade 4 severity, that required unscheduled hospital admissions, or caused death. This study is registered with Patients were recruited from April 15, 2009, to Dec 23, 2013. The data cutoff for follow-up was May 26, 2015. Of 4685 HIV-positive people enrolled, 120 had severe bacterial infections . Immediate ART was associated with a reduced risk of severe bacterial infection compared with deferred ART . In the immediate-initiation group, average neutrophil count over follow-up was 321 cells per μL higher, and average CD4 cell count 194 cells per μL higher than the deferred-initiation group (p<0·0001). In univariable analysis, higher time-updated CD4 cell count  was associated with reduced risk of severe bacterial infection. Time-updated neutrophil count was not associated with severe bacterial infection. After adjustment for time-updated factors in multivariable analysis, particularly the CD4 cell count, the HR for immediate-initiation group moved closer to 1 . These results were consistent when subgroups of the severe bacterial infection composite were analysed separately.Immediate ART reduces the risk of several severe bacterial infections in HIV-positive people with high CD4 cell count. This is partly explained by ART-induced increases in CD4 cell count, but not by increases in neutrophil count.National Institute of Allergy and Infectious Diseases National Institutes of Health, Agence Nationale de Recherches sur le SIDA et les Hépatites Virales, Bundesministerium für Bildung und Forschung, European AIDS Treatment Network, Australian National Health and Medical Research Council, UK National Institute for Health Research and Medical Research Council, Danish National Research Foundation. The Strategic Timing of AntiRetroviral Treatment (START) trial, in people with CD4 counts of more than 500 cells per μL and who were naive to antiretroviral therapy (ART), revealed a 57% reduction in risk of AIDS and non-AIDS morbidity and mortality in participants randomly assigned to immediate ART initiation compared with those allocated to deferred initiation.10In the START study, most serious AIDS-related events, serious non-AIDS-related events, or deaths occurred in patients with CD4 counts above 500 cells per μL and the treatment effect was only partly mediated by changes in CD4 cell count during follow-up.Evidence before this studyWe searched the Web of Knowledge for search terms “randomi*” AND “trial” AND “antiretroviral” AND “bacter*” AND “infection*”, AND “HIV*” on April 1, 2016, for articles published in English only. We found no substantive randomised trial evidence published about the effect of antiretroviral therapy (ART) on risk of severe bacterial infections in people with HIV and high CD4 cell count (>500 cells per μL) before this study.Added value of this studyThe START trial was a trial of immediate versus deferred ART in 4685 participants with HIV and high CD4 cell count who were followed up for a median of 2·8 years. Our results show that ART has a substantial beneficial effect on bacterial infections, which is largely mediated by improvements in CD4 cell count in people on ART. This benefit was found also for non-tuberculosis bacterial infections. Although there were improvements in neutrophil count in the immediate ART group compared with the deferred ART arm, time-updated neutrophil count did not predict the risk of bacterial infections.Implications of all the available evidenceART has a protective effect in reducing the risk of a broad spectrum of severe bacterial infections in people with HIV, even in people with high CD4 cell count. This finding further emphasises the benefits of ART even in people with high CD4 cell count.In this Article, we report the effects of immediate ART on all severe bacterial infections. Our aim was to investigate whether the reduction in severe bacterial infection observed in the immediate-initiation group could be explained by possible increases in neutrophil counts, and the increases in CD4 cell counts that were observed in this group  that required treatment with ART. Enrolment began on April 15, 2009, and ended on Dec 23, 2013. 4685 HIV-positive people were enrolled  at 215 clinical sites caring for people living with HIV in 35 countries  and summarising the mean of all individuals.We plotted the change in neutrophil count, total lymphocyte count, haemoglobin concentration, platelet count, albumin concentration, and CD4 cell count over time at annual timepoints, on the basis of values taken at annual visits. We fitted a longitudinal mixed model with PROC MIXED in SAS (version 9.4) to assess the difference in the mean change in laboratory markers from study entry between the treatment groups. We fitted a separate mixed model for each of the laboratory markers with change since study entry as the dependent variable. We adjusted models for the baseline value of the laboratory marker and the visit number. A variance components covariance structure was specified to model the potential correlation between repeated laboratory measures collected at different visits for the same person.We used Cox proportional hazards regression to model the time to severe bacterial infection. We estimated univariable and multivariable hazard ratios (HRs) with 95% CIs to assess the association between severe bacterial infection and several covariates.2, 18·5–24·9 kg/m2, 25·0–34·9 kg/m2, ≥35 kg/m2), and smoking status. Baseline continuous covariates investigated were age and time since HIV diagnosis. Time-updated continuous covariates investigated include CD4 cell count, HIV RNA viral load (log10), neutrophil count, total lymphocyte count, haemoglobin concentration, platelet count, and albumin concentration. We stratified all models by a six-category variable describing the geographic region of residence . We assessed whether the effect of treatment group was consistent across several predefined subgroups that were defined according to characteristics at study entry, by including terms for interactions between treatment and subgroup variables in expanded Cox models. We also assessed the proportional hazards assumption for the treatment effect by fitting the interaction with log time.Baseline categorical covariates investigated include treatment group , sex, mode of HIV acquisition  and race or ethnicity . Time-updated categorical covariates investigated include body-mass index . All tests of significance were two-sided and used p<0·05 as the threshold of statistical significance. The START trial was registered with The funder had a role in study design and interpretation of data and the writing of the report through the membership of KLK  in the Writing Committee. The funder had no role in the decision to submit the paper for publication. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.4685 HIV-positive START participants enrolled between April 15, 2009, and Dec 23, 2013, were included in the analyses. Most participants were male, of white ethnicity, with same-sex mode of HIV acquisition, living in the region of Europe and Israel, and were non-smokers .120 people had at least one severe bacterial infection ; and albumin concentration 4·4 g/dL (4·1–4·6). During follow-up, the change in biomarker values since study entry was significantly higher in the immediate-initiation group than in the deferred-initiation group, for all biomarkers investigated ; neutrophil count 2830 cells per μL (2149–3700); total lymphocyte count 2200 cells per μL (1830–2659); haemoglobin concentration 14·5 g/dL (13·4–15·3); platelet count 231 × 10stigated .In univariable analysis, immediate initiation of ART was associated with a reduced risk of severe bacterial infection , 48 developed bacterial pneumonia , and 26 developed tuberculosis  was no longer significantly below 1 .Univariable and multivariable HRs for a selection of factors that were included in the Cox model are presented in 2 was associated with an increased risk of severe bacterial infection when compared with a reference group of a BMI between 18·5 and 24·9 kg/m2 . Higher time-updated CD4 cell count was associated with a reduced risk of severe bacterial infection . The time-updated CD4 cell count remained strongly associated with a reduced risk of severe bacterial infection  when time-updated values were delayed by 6 months. Adjustment for time-updated CD4 cell count in the multivariable model was responsible for the attenuation of the HR for treatment group. Time-updated neutrophil count was not associated with risk of severe bacterial infection ; which was also the case when neutrophil count was fitted as a categorical variable. Other factors that were included in the multivariable model, but were not associated with severe bacterial infection, were age (p=0·44), sex , race/ethnicity (p=0·22), time-updated smoking status , mode of HIV infection (p=0·88), time since HIV diagnosis (p=0·89), hepatitis B (p=0·67) and hepatitis C status (p=0·43), time-updated HIV RNA viral load , time-updated total lymphocyte count (p=0·10), time-updated haemoglobin concentration (p=0·20), time-updated platelet count (p=0·70), and time-updated albumin concentration (p=0·11). The tests for interaction between the treatment group and several predefined subgroups that were determined by characteristics upon study entry were non-significant.In multivariable analysis, a BMI above 35 kg/mGiven that the beneficial effect of immediate ART on risk of severe bacterial infection seems to be partly mediated by induced rises in CD4 cell count, we considered whether the effect would be lower in people with baseline CD4 cell counts above the median of 651 cells per μL than people with baseline CD4 cell counts below the median, but the univariable effect of immediate ART was similar to the overall effect .We did four sensitivity analyses to explore how sensitive the results of our study were to variations in the definition of severe bacterial infection. In one analysis, we excluded tuberculosis from the definition of severe bacterial infection . In a seImmediate ART initiation reduced the risk of severe bacterial infections in asymptomatic HIV-positive people with high CD4 cell counts by 61%. The benefit of immediate ART was attenuated in statistical models after adjustment for time-updated CD4 cell count, suggesting that the effect of immediate ART is partly mediated by the increase in CD4 cell count in the immediate ART group and hence this increase in itself or some linked immunological pathway is an important mechanism by which ART reduces risk of severe bacterial infections. By contrast, we found no evidence that the effect was mediated by the increases seen in neutrophil count with early ART, as we initially hypothesised.Our findings are consistent with those from previous studies, which show the efficacy of early ART in reducing the risk of bacterial diseases.In accordance with other studies,The relationship between BMI and risk of bacterial pneumonia was investigated in a 2013 meta-analysis,Our findings provide further evidence of a clinical benefit of early initiation of ART. Biomarkers in untreated HIV infection tend to decline even in people with high CD4 cell counts . Control31Medical Dictionary for Regulatory Activities (version 18.0). Additionally, collation of longitudinal follow-up data from 35 countries with standardised data collection methods provided a large, ethnically, geographically, and socially diverse cohort of HIV-positive people. Furthermore, rich data collection allowed for adjustment of several participant and HIV-related covariates that enabled us to assess potential association of these covariates with severe bacterial infection.The main strengths of our study are the comparison of randomised groups, the use of pre-established standardised methods for data collection, and the size and diversity of the study cohort. All serious events and deaths were reviewed by an endpoint review committee, who were blind to the treatment group, using pre-established INSIGHT criteria.One limitation of this study is that only the bacterial pneumonia and tuberculosis components of our endpoint were reported to and reviewed by the endpoints review committee. Other bacterial infections were identified only if they were classified as grade 4 events and required unscheduled hospital admission or led to death. Extrapolation of our findings to less severe bacterial infections, for which data were absent, should be done with caution. Furthermore, hospital admission might be more likely in some settings than others for a given infection. Indeed, the incidence of severe bacterial infections was notably highest in Australia whereas we might have expected higher rates in Africa, South America, and Asia. Furthermore, our analyses involved few events (n=120), which limits the study power. Additionally, although CD4 cell count and HIV RNA were measured more frequently, all other laboratory markers were measured at randomisation and annually thereafter. Therefore, for severe bacterial infection events that occurred in the first year after randomisation , there would be no follow-up measurement of these laboratory markers. This might have resulted in overestimation of the importance of CD4 cell count in explaining the treatment effect, relative to the other laboratory markers.In summary, the results of our study show the protective effect of immediate ART in reducing the risk of a broad spectrum of severe bacterial infections in HIV-positive people. The effect of immediate ART appears to be mediated in part by ART-induced increases in CD4 cell count, but not by the ART-induced increases in neutrophil count.


Management of persistently non-adherent youth living with HIV (YLHIV) with virologic failure (VF) on combination antiretroviral therapy (cART) remains challenging. One strategy has been using 3TC/ FTC monotherapy (3TC/FTC), which in the presence of the M184V resistance mutation, does not suppress viral replication nor select for additional drug resistance mutations, and reduces viral fitness with limited side effects. P1094 compared the immunologic outcome of continuing failing cART vs. switching to 3TC/FTC as a “bridging strategy” to subsequent suppressive cART for non-adherent YLHIV with pre-existing M184V resistance.+ T cell count ≥100 cells/mm3 and VF (HIV-1 plasma RNA ≥400 copies/mL (2.6 log10 HIV-1 RNA) were enrolled and randomized to continue failing cART vs. switch to 3TC/FTC. The primary endpoint (time to ≥30% CD4+ T cell decline or development of CDC class C events) at 28-weeks were assessed by Kaplan-Meier (K-M) curves in an intent-to-treat analysis.Participants with documented nonadherence, M184V mutation, CD4+ T cell count, and viral load were 15 years (Inter-quartile range (IQR) 14–20), 472 cells/mm3 (IQR 384–651), and 4.0 log10HIV-1 RNA copies/ml (IQR 3.2–4.5), respectively. Five participants, all in the 3TC/FTC arm, reached the primary endpoint for absolute CD4+ T cell decline . The Kaplan-Meier estimate of probability of primary endpoint on 3TC/FTC at 28 weeks was 0.41 (standard error 0.14). There were no CDC class C events or deaths and no statistically significant difference in frequencies of adverse events between the arms.Thirty-three perinatally acquired YLHIV participants (16 continuing cART and 17 3TC/FTC) enrolled in the study. The median age, entry CD4Non-adherent participants randomized to 3TC/FTC were more likely than those maintained on failing cART to experience a confirmed decline in CD4+ count of ≥30%. Although this study suffers from limitations of small sample size and premature discontinuation, the randomized comparison to continuing failing cART indicates that 3TC/FTC provides inferior protection from immunologic deterioration for non-adherent youth with M184V resistance. Better alternatives to 3TC/FTC such as ART with higher barriers to resistance and novel adherence and treatment strategies for nonadherent youth are urgently needed.NCT01338025Clinical Trials.gov  Durable virologic suppression using combination antiretroviral therapy (cART) is most reliably achieved for patients with excellent adherence, with small amounts of nonadherence conferring risk of emergent resistance and viral load breakthrough , 2. Howe10 c/mL[Antiretroviral management in the setting of poor adherence in children and youth living with HIV is increasingly challenging for providers. A balance must be struck between the need to continue cART to maintain immunologic integrity and the desire to simplify the therapeutic regimen, foster optimal adherence, and minimize development of resistance, while awaiting for youth to mature and develop better adherence. To improve adherence, multiple strategies have been tested , yet no gold standard has emerged , 10. How10 c/mL, and 5) 10 c/mL. Once thThis strategy has been shown to be superior to discontinuing all antiretroviral therapy in adults. Observa3, and VF (defined as HIV-1 plasma RNA ≥400 copies/mL on at least two occasions including screening at least 2 months after initiating the regimen and within 6 months of study entry). When slow enrolment was observed, a site survey was conducted and in response, the following changes were made to the eligibility criteria (March 7 2012): absolute CD4+ T cell count was lowered from 250 to 100 cells/mm3 and the time demonstrating failure from 6 months to 2 months.International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) P1094 was a multi-center , open-label, Phase IV randomized, controlled, comparative trial to evaluate continuation of non-suppressive cART (Arm A) vs. 3TC/FTC monotherapy (Arm B) for 28 weeks (in both arms) (NCT01338025). All of t3 versus ≥400 cells/mm3). Participants were enrolled online and randomized at time of enrollment via computer algorithm at the Data Management Center.Main exclusion criteria included hepatitis B infection, active malignancy within the prior two years, active opportunistic or other infection; ≥ 1 CDC class C event within 12 months of entry, or viral load >250,000 copies/mL at screening. Participants were enrolled between May 2011 and January 2013 and randomized 1:1 to the two treatment groups using a dynamic permuted block system with institutional balancing , stratified by CD4+ T cell count  received either 3TC or FTC (the choice of 3TC or FTC was left to the provider) and discontinued other ART. Sites were expected to provide and document adherence support measures (per their standard of care) during the study. The target sample size of 344 was chosen to provide 80% power to detect a difference of 0.15 between study arms in proportions of participants with immunologic deterioration using a log-rank test with a 2-sided Type I error rate of 0.05. Due to poor accrual, the final sample size was 32 eligible participants. The antiretroviral agents were not provided by the study. The study was approved by the Institutional review boards (IRBs) of all participating clinical research sites (CRS)  monitored the study conduct throughout the study.Study measurements included lymphocyte subsets at each study visit , HIV RNA testing , and safety monitoring labs including hematology . Plasma and peripheral blood mononuclear cells (PBMCs) for inflammatory and immune activation markers were collected at 0, 12, and 28 weeks. Self-reported adherence was also assessed at each study visit by collection of 3-day recall and evaluation of interventions employed to enhance adherence. Batched resistance testing was planned as part of the protocol, however, this was not performed when the study ended early.+ CD45RO- CD95-) and activated (CD38+ HLA-DR+) cells within total CD3+/CD4+ and CD3+/CD8+ viable lymphocytes was determined. In addition, the percentage of recent thymic emigrants (RTE) (CD31+ CD27+ CD45RO- CD95-) among CD3+/CD4+ lymphocytes was determined[Plasma and Ficoll-Hypaque density gradient isolated peripheral blood mononuclear cells (PBMCs) were cryopreserved within 8 hours of blood draw. Plasma markers of inflammation and immune activation were determined with ELISA kits, using duplicate samples on the same plate per subject, for D-Dimer , sVCAM-1 , and soluble CD4 (sCD14), interleukin-6 (IL-6), and high sensitivity C-reactive protein (hsCRP) . These markers were selected based on correlation with morbidity and mortality in studies of HIV-1 infected adults–27. Multetermined.The primary endpoint was time to time to immunologic deterioration, defined as first ≥30% decline in absolute CD4+ T cell count from baseline or development of CDC class C events. Additional analyses were done on rates of adverse events or deaths, change in viral load, changed in biomarkers of inflammation and immune activation, and adherence at the end of follow-up; as well as baseline immune characteristics on the 3TC/FTC monotherapy arm. Kaplan-Meier curves and log-rank tests were used to compare the treatment arms with respect to time to immunologic failure. The prob3 (IQR 384–651) and 4.0 log10HIV-1 RNA copies/ml (IQR 3.2–4.5), respectively. A median of 4 interventions  had been used to address nonadherence prior to study. The median duration on study intervention of 27 weeks (IQR 12–34) was not different between the two treatment arms.Enrollment occurred May 10, 2011 through January 16, 2013 and the study was discontinued in February 2013 due to slow accrual; all participants were off study by May 15, 2013. Thirty-three participants were enrolled, out of targeted enrollment of 344, of whom one was ineligible and did not have any post-entry visits . We presFive participants, all in the 3TC/FTC monotherapy arm, reached the primary endpoint for decline in CD4+ T cell count  while those on the cART arm were estimated to gain 3.82 cells/mm3 per week (p = 0.10). The median (IQR) change in CD4+ T cell count over the study period was 44  for the cART arm and -70  for the 3TC/FTC arm .To test the null hypothesis of identical rates of change in CD4+ T cell counts over time on study, repeated measures modeling was used. The model  included10HIV-1 RNA copies vs. an increase of 3.87 (IQR -3.73 to 4.48) log10HIV-1 RNA copies/ml in the 3TC/FTC arm (p = 0.08), although the difference was not statistically significant. Four (67%) of the six participants on cART with viral load data for week 24/28 had decreases in log viral load of more than 0.5, while none of the six participants on 3TC/FTC did There were no deaths. There was only one grade 3 or higher adverse event reported; one participant on the cART arm had a grade 4 total bilirubin, attributable to receiving atazanavir. There were no significant differences in self-reported adherence between the two arms at baseline or during the study period. However, compared to baseline, at the end of the study period, fewer participants in both treatment arms reported missing doses in the prior 3 days, 47% at baseline vs. 33% at the end in the cART arm and 59% vs. 18% in the 3TC/FTC arm. Participants in the cART arm had a median decline in viral load of -3.78 (IQR -4.25 to -2.25) IogBiomarkers of inflammation and immune activation were assessed for participants in the 3TC/FTC arm followed at domestic sites with available samples. Fourteen participants had biomarker data  available at both baseline and Week 24/28 (9 in the 3TC/FTC arm and 5 in the cART arm). There were no significant differences between arms in the change from baseline for any of the markers . As an eTo our knowledge, this is the first and only randomized control trial comparing 3TC/FTC monotherapy to continuing cART in any population of individuals living with HIV. In this multi-site study of YLHIV with persistent virologic failure on cART, those who were randomized to 3TC/FTC monotherapy were significantly more likely to experience ≥30% decline in absolute CD4+ T cell count over 28 weeks compared with those maintained on failing cART. The change from baseline in markers of inflammation and immune activation did not differ between the two groups. Immune correlates at baseline were not different between those who did and did not experience immunologic decline in the 3TC/FTC arm, though this was performed on a limited sample size.3 compared 3TC monotherapy to discontinuation of cART and found that the monotherapy strategy was superior to cART discontinuation [3. Others have shown that withdrawing the NRTIs as opposed to interrupting PIs or NNRTIs in a failing regimen may have the greatest impact on immune decline, a different approach than was used in our study [A prior study of adults with relatively robust baseline median CD4+ T cell counts of 570 (IQR 466–636) cells/mminuation . Specifiur study .3) who were switched in an uncontrolled, one-armed study from a failing (VL> than 400 copies/mL) cART regimen to 3TC monotherapy[3 at 24, 36, and 48 weeks was 36%, 57%, and 70%, respectively. When the investigators analyzed the participants stratified by the failing regimen at baseline (PI vs. reverse transcriptase inhibitor (RTI)-based cART (NNRTI or NRTI)), 81% of participants on PI-based cART vs. 40% of participants on RTI-based cART reached the clinical endpoint, despite similar baseline CD4+ T cell counts and other characteristics. The decline in absolute CD4+ T cell was not statistically different between the two groups, however, patients who were initially on PI-based cART had greater increases in the viral replication capacity, perhaps due to outgrowth of viral strains lacking PI resistance mutations detected on resistance testing in the absence of ART pressure. The authors concluded that 3TC monotherapy may be considered as a short-term strategy for “selected” patients failing RTI-based cART, but not PI-based regimens due to concern for increased viral fitness and greater likelihood of CD4+ T cell decline. Our study is unable to look at differences between PI vs. RTI-based cART as NNRTI-based regimens were excluded. Given the known lower barrier to resistance in NNRTI-based regimens and the possibility of rapid accumulation of resistance in the setting of continued nonadherence, we did not feel that it was ethical to randomize participants to continue NNRTI-based regimens in setting of continued nonadherence.It is possible that the use of the comparison cART group on PI-based regimens may have led to the dramatically different outcomes between the two study arms. Opravil et al studied 26 clinically stable, significantly pre-treated adults  that have demonstrated the ability to maintain immunologic integrity, however, these strategies have been employed in the setting of virologic suppression or simplification of multi-agent ART with simplification. This st3, therefore, we cannot generalize to those with lower baseline CD4+ T cell counts. However, one would anticipate, as was seen in the Castagna study[The P1094 study has several important strengths. Specifically, it was a randomized controlled study conducted through a highly experienced network, with standardized comprehensive measurements, including immunology and immune activation. Further, the participants were from multiple sites in the U.S., South America, and Thailand. The participants had a wide range of CD4+ T cell counts and were failing on contemporary antiretroviral treatment regimens. The study findings must be interpreted with some important study limitations in mind. First, the early closure due to slow accrual resulted in a small sample size, leaving the study under-powered to make certain comparisons and conclusions. Further, ARV drug exposure  were not collected to corroborate self-reported adherence. A relatively healthy population participated with median initial CD4+ T cell counts > 500 cells/mmgna study, that suThe findings of this first randomized controlled trial to evaluate 3TC/FTC monotherapy vs. continuing failing cART, do not support the use of 3TC/FTC monotherapy as a strategy to address nonadherence in patients failing cART, particularly in settings such as the U.S., where there are other options available, even in the setting of drug resistance. Clinicians will need to continue to assess the specific barriers and options for their young patients who are failing cART and struggling with adherence in order to make the best care decisions with and for their patients; the poor immunologic outcomes observed with 3TC/FTC monotherapy should be factored into those decisions. In all settings, the focus needs to be on increasing the availability of ARV regimens that have high barriers to resistance, low pill burden, and are available in suitable formulations and durably sustainable delivery mechanisms for youth. High priorities include fixed dose combination formulations for second and third line regimens, long-acting, injectable, and other novel cART delivery systems, as well as innovative ways of delivering care and enhancing adherence, particularly for youth, given their challenges with medication adherence.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Due to the limited availability of viral load testing for treatment outcome monitoring in resource limited settings, identifying predictive factors of antiretroviral treatment failure will help in selecting clients who will benefit most from the targeted use of viral load monitoring. Little is known about the predictors of treatment failure in the study area. This study was conducted to determine factors that predict first-line antiretroviral therapy failure among HIV-infected adult clients at Woldia Hospital, Northeast Ethiopia. For this study, antiretroviral therapy treatment failure was defined as the fulfillment of clinical and/or immunological criteria set by WHO.Case-control study was carried out from November to December 2014. Cases were adult clients who were on failing first line regimen and on active follow up while controls were those adult clients on a non-failing first-line regimen for 36 months and above and on active follow up. Data was entered in to Epi Info version 7 and was exported to SPSS version 20 for analysis. Binary logistic regression model was used to identify predictors of ART failure.4count was not significantly different among cases and controls (105 (IQR = 60–174)vs.131 (IQR = 72.5–189.0); p = 0.301). The median peak CD4 count in the failure group (230 (IQR = 123–387)) was significantly low compared to the non-failure group (463 (IQR = 348.5–577)) [p < 0.001]. High peak CD4count  and longer duration on ART  were protective of treatment failure. In addition stavudine based regimen , low baseline BMI , unemployment  and formal educational level  were independently significant predictors of treatment failure.A total of 59 cases and 245 controls were included in the analysis. Sixty three percent of the participants were females and the median age at ART enrollment was 33 years . The median baseline CDIn this setting low peak CD4count, shorter duration on first line ART, d4T based regimen, low baseline BMI, unemployment and formal educational level were significantly associated with increased treatment failure. Retaining patients on their initial first line regimen with appropriate follow up and improving their socioeconomic status through various livelihood initiatives should be strengthened. Globally, an estimated 36.7 million people were living with Human Immunodeficiency Virus (HIV) in 2015. Sub-Saharan Africa remains most severely affected, with nearly 25.5 million people living with HIV and accounting for 69.5% of the people living with HIV worldwide [The introduction of Anti-Retroviral Therapy (ART) was a critical turning point in the history of HIV disease and the gradual evolution of HIV infection into a chronic non-fatal condition. The primary goal of ART is to achieve and sustain maximal viral suppression , 5. The 4 cells/mm3’ in 2005 to a recommendation of ‘less than 350 CD4 cells/mm3’ in 2008. Currently, adults with WHO clinical stage III & IV irrespective of CD4 cell count or CD4 cell count less than 500 cell/mm3 or all TB/HIV co-infected and pregnant & lactating women irrespective of CD4 cell counts are eligible for ART. The basic first-line regimen contains 2 Nucleoside Reverse Transcriptase Inhibitors (NRTIs) as a backbone and 1 Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs). The choice of second-line regimen depends on the Anti-retroviral drugs (ARVs) used for first-line and that retain activity against the virus. Generally, one or more drug from a new class, usually Protease Inhibitors (PIs), is included [In Ethiopia, fee based ART was launched in 2003 and subsequently in 2005 free ART program was initiated. Guidelines for starting ART have progressively shifted away from the initial threshold of ‘less than 200 CDincluded .4 cell count to baseline (or below) or a 50% reduction from on treatment peak value or presence of persistent CD4 cell count below 100 cells/mm3. In addition, clinical failure is the occurrence of new or recurrent WHO stage 4 or some stage 3 conditions [Antiretroviral treatment (ART) failure is associated with virologic failure, immunologic failure, and/or clinical failure. Virological failure is said to be occurred when plasma viral load become above 1000 copies/ml in two consecutive measurements within a three month interval with adherence support after at least six months of using ART. Whereas, immunological failure is defined as a fall in CDnditions .According to the 2011 Ethiopian Demographic and Health Survey, the prevalence of HIV in Amhara region is 1.6%, which is slightly higher than the national prevalence. The HIV situation in the region is one of the worst in the country, especially in the urban areas,having one third of People Living with HIV/AIDS (PLWHA) and those in need of ART found. Woldia town is a transport corridor in the Eastern part of Amhara and is hot spot area for HIV infection , 8. In tMeeting universal access rapidly and maintaining patients on appropriate treatment regimen are crucial to the long-term success of ART program. Patients are required to consistently adhere to the lifelong therapy to minimize the development of drug resistance . DespiteMonitoring individuals receiving ART is important to ensure successful treatment, identify adherence problems and determine regimen switch in case of treatment failure. Viral load monitoring is a more sensitive and an early indicator of treatment failure. However, most ART programs in resource limited countries didn’t have access to viral load testing. This limited use of viral load monitoring has been identified as a key reason for the lower rate of switching to second-line regimen than expected .Although viral load monitoring is the gold standard method to diagnose ART failure, it is not accessible in resource-limited settings like Ethiopia. Hence, the diagnosis and monitoring of first-line treatment failure and the decision to initiate second-line treatment are largely based on clinical and immunologic assessment of patients. Clinico-immunologic determination of ART failure, in the absence of viral load, are associated with unnecessary switches to second-line in the absence of virologic failure and the prolongation of a failing regimen and a late switch to second-line. Late switches of first-line regimens, which contain NNRTIs, are associated with accumulation of mutation and lead to cross-resistance to other NNRTI drug that might be used as a second-line option , 12, 13.In Ethiopia, according to FMOH report in January 2013, approximately 1.5% patients currently on ART are receiving second-line treatment . The ratRecent surveys by WHO indicated that there are increased signs of both transmitted and acquired HIV Drug Resistance (HIVDR) in areas where ART program was expanded. Little is known about factors that predict treatment failure in Ethiopia, in general, and in the study area, in particular. Identifying such factors has programmatic importance, as cost effective interventions can be made at program level to maintain the efficacy of standard regimens , 18.Different studies indicated that immunologic criteria have low sensitivity to detect virologic failure as they caused a large number of patients to be misdiagnosed and continued to take a failing regimen. In addition, their specificity & predictive values are also low, leading to unnecessary switches with increased cost for the expensive second-line drugs , 21, 22.4 cell counts (<50 cells/mm3) have equivalent or greater rate of immune recovery in the first year of ART compared to those with a higher baseline CD4 cell counts [Despite the limitation in the diagnostic capacity to detect treatment failure in sub-Saharan Africa, high viral suppression rate for substantial length of time and enhanced immunologic recovery has been documented. Patients with very low baseline CDl counts , 24, 25.4 cell count change [4 cell count and body weight change were associated to immunological failure [4 cell count and low baseline viral load. The result reflected that these groups of patients might have low adherence level due to the lower risk of developing opportunistic infections [Discordant virological and immunological responses are observed during ART suggesting a complex interaction between virological response and the CDt change , 23. A 2 failure . A similfections , 27. Confections .Although different cutoff points are used to define virologic failure according to the national guidelines, one study done at Jimma, Southwest Ethiopia, reported a 5.3% virologic failure at 6 month of treatment  . Meanwhi4 cell count, rate of CD4 decline, prior exposure to ARVs and treatment interruptions as determinants of treatment failure [Several studies have identified non-adherence, low baseline CD failure , 33, 34. failure . Prior e failure , 334 cell count is another important determinant factor for antiretroviral treatment failure. It is known to increase the risk and episodes of opportunistic infections and high attrition rate [2 and baseline CD4 cell count were found to be associated with treatment failure in this study [Baseline CDion rate . In addiion rate . A case is study . The occis study , 36. Butis study , 37, 38.2 or change in body weight from the baseline, and advanced WHO clinical stage are also additional predictors of treatment failure. These factors are related to each other and their combination weakens the host immunity by predisposing it to different opportunistic infections [4 cell count, first and current antiretroviral regimens and time on ART [A cross sectional studies from Kenya and Cameroon found that duration of antiretroviral treatment was not associated with treatment failure, while a retrospective cohort analysis from Uganda reported that the level of treatment failure was twice at 24 months compared to 12 months of treatment among cohorts , 31, 38.fections , 36, 37.fections , 32, 34.fections , 31, 40.e on ART . Supporte on ART .Given the limited laboratory facilities, the diagnosis of first-line antiretroviral treatment failure and the initiation of second-line treatment are largely dependent on clinical and immunologic assessments which are less sensitive and predictive of virologic failure. This study aimed to identify factors that predict Antiretroviral Therapy (ART) failure, which will provide information for clinicians for appropriate decision and management of patients on antiretroviral treatment. In addition, this particular study highlighted important factors apart from the already known factors investigated in other study settings. Program planners and clinicians will also use the findings to develop diagnostic algorithm to prioritize patients who need targeted viral load monitoring, considering the available resources.The factors include patients related factors, virus related factors, programmatic and drug factors leading directly and indirectly to antiretroviral treatment failure . The objFacility based case-control study of HIV-infected adults on ART and on follow up was carried out at Woldia Hospital from November to December 2014. The study got ethical approval from Wollo University, College of Medicine and Health Sciences. Cases were adult patients who experienced first line regimen failure and switched to second-line regimen and on active follow up while controls were those adult patients on a non-failing first-line regimen for 36 months and above and on active follow up. Cases were required to have been on ART for at least 6 months before switching to second-line regimen.4 cell count was measured every six months and assessment of opportunistic infections and side effects done every visit. Second line regimen consists of boosted protease Inhibitor after a failing first-line regimen. Viral load testing is available at the Bahirdar regional health research laboratory, 360 kilometers Northwest of Woldia, for a limited number of patients suspected of treatment failure. The hospital uses clinical assessment and immunologic evaluation to identify ART failure.The study was conducted from November1 to December 30/ 2014 at Woldia hospital in Woldia town, 521 kilometers northeast of the capital Addis Ababa and having an estimated population of 67,760 . Woldia According to the monthly report of the hospital, as of October 2014, 9076 HIV infected peoples were ever enrolled, of whom 6272 started antiretroviral treatment. There were 3713 clients, currently, on ART, of whom 3599 were on first line and 114 were on second line regimen .HIV-infected adult patients, who were put on second-line and first-line ART based on the national guideline, were the source population for the cases and controls, respectively. The study population, for cases, was sampled from the list of patients on active follow up and taking second-line regimen. Controls were sampled from patients taking a first-line regimen for 36 months and above and on active follow up. Data relevant to this particular study was collected from eligible participants and their medical record simultaneously.Clients initiated for first-line ART at Woldia Hospital were included on the study, where as those with serious illness and with incomplete relevant data from medical record were excluded.4 cell count ≤ 100, treatment interruption and prior exposure to ARV drug. Then, the largest sample size in most literatures was found to beCD4 cell count ≤ 100, as independent variable was used. The sample size was calculated using Epi Info version 7 considering the following parameters: proportion of CD4 cell count ≤ 100 (58.9% in cases and 37.1% in controls) [Sample size was calculated for the known significantly associated independent variables: non-adherence, CDontrols) , 5% margThere were 114 adults currently on second line ART by the end of October 2014. Of these, 35 were transferred in from other health facilities. All the remaining 69 patients who fulfilled the inclusion criteria were included in the study without sampling for their relative small number. Out of whom, 10 were excluded because of the incompleteness of relevant clinical data form their medical record. By the end of October 2014, 3,306 adults were currently on first line ART. The average daily patient flow for drug refill was 55, during the month preceding the data collection. During the data collection period, there were 38 working days and making a total of 2,090 study population from which the sample was taken. The required number of controls for this study (n = 245) was sampled by systematic random sampling from the 2,090 patients who came for refill during the data collection period . At the 4 cell count, baseline body mass index, baseline hemoglobin, WHO stage, adherence to ART , functional status, occurrence of TB while on ART, initial ARV regimen, prior ARV exposure, treatment interruption (for 7 and more days and as reported by cases and controls), history of drug substitution, recurrent pneumonia and missed appointment.The dependent variable for this study is occurrence of antiretroviral treatment failure. For this study, antiretroviral therapy treatment failure was defined as the fulfillment of clinical and/or immunological criteria set by WHO. This was determined by reviewing whether the client was diagnosed with treatment failure or not on his/her medical record. The independent variables include: Socio-demographic variables , HIV sero-discordance and disclosure, body weight change, baseline CDThe data collection tool was developed from prior studies and adapted to this particular study. A structured interviewer administered questionnaire and patient medical record review was used to collect data on the dependent and independent variables. The Amharic version of the questionnaire was used to collect data from study participants and re-translated back to English version for data entry. Major components of the data collection tool were socio-demographic variables, baseline & follow up clinical and laboratory variables. The data collection tool was pre-tested on 25 participants and, based on the findings, corrections were made. The interview was conducted on the day of a scheduled clinic visit upon getting verbal consent to enroll in to the study. A unique identification number was assigned to sampled cases and controls.Data was entered using Epi Info version 7 and was exported to SPSS version 20 for analysis. Data was cleaned and edited by running simple frequencies and cross tabulation before analysis. Mean , median  and percentages were used to summarize participants characteristics in each group. A chi-square/Fischer’s exact test and independent t-test were used to compare the differences among the categorical and continuous variables between the two groups. Bivariate and multivariable logistic regression modelswereused to identify predictors of ART failure. Independent variables with p < 0.2 in the bivariate analysis were included in the multivariable analysis. Covariates that were a priori known to be significantly associated with the outcome were included into the model regardless of the level of significance in the bivariate analysis. Covariates in the final model were selected by backward step-wise selection procedure. Model of goodness of fit was done by Hosmer-Lemeshow goodness of fit test. Crude and adjusted Odds Ratio with 95% confidence intervals was computed and statistical significance was considered with two sided P-value < 0.05.Quality of data was assessed before, during and after data entry. Every filled data collection tool was thoroughly checked for completeness and consistency at the data collection point by the supervisor and before entry by the principal investigator.A total of 304 participants (245 controls and 59 cases) were included in this study. The response rate for controls was 100% and 95% for cases. The median age for cases was 33 years (IQR: 27–38) and for controls was 32 years (IQR: 28–40). There were more female than male clients in the control group accounting two third, while the proportion of female and male is approximately equal among cases. Eighty percent of the cases had some educational background (elementary and above) while half of the controls didn’t write or read. Fifty six percent of the cases were unemployed compared to one third among controls .4 count for controls was higher for controls and the mean time required attaining the peak CD4 count was also longer for controls compared to cases. Additionally, the mean duration of months on first line ART was shorter for cases as compared to controls.Voluntary counseling and testing (VCT) was the most common point of entry for HIV testing. HIV sero-status disclosure and discordance rate was similar among the two cohorts. ARV treatment interruption was higher among cases than controls (16.4% vs. 1.2%). There was no difference in the history of prior ARV exposure before starting ART among cases and controls. The median peak CDThere was a difference in baseline Body Mass Index (BMI) between the two cohorts. Among cases 37 (62.7%) had a baseline BMI < 18.5, whereas 109 (44.5%) of controls had a baseline BMI < 18.5. A higher proportion of cases (27.1%) had negative or no change in weight from baseline in the first 12 months of ART compared to controls (15.1%). Regarding the initial ARV regimen type, the commonest first line regimens were Stavudine/Lamivudine/Nevirapine, 40%; Zidovudine/Lamivudine/Nevirapine, 27.7%; Stavudine/Lamivudine/Efavirenz,10.5% followed by Zidovudine/Lamivudine/Nevirapine, 7.2%. But there was no significant difference in the type of NRTIs (Stavudine vs. Zidovudine) and NNRTIs (Nevirapine vs. Efavirenz) used in the first line regimen between the cases and controls.4 count, hemoglobin and history of drug substitution were not statistically different among the two cohorts (Compared to controls a higher proportion of cases had history of incident tuberculosis after ART initiation (6.5% vs. 16.5%). In addition cases had slightly higher non-adherence and recurrent pneumonia (more than 2 episodes). Baseline WHO stage, functional status, CD cohorts .The bivariate analysis showed that age at ART enrollment was not associated with ART failure. Females were less likely to develop treatment failure compared to males. HIV infected adults who had some educational level above elementary had increased risk of developing ART failure. The risk appeared to be higher for those with tertiary level education. Unemployed patients had 2.5 times increased risk of ART failure .4 count, hemoglobin and history of drug substitution did not show any significant association with the occurrence of ART failure. Compared to the reference group, patients with baseline BMI < 18.5 kg/m2 and with negative or no change in weight from the baseline within 12 months of ART initiation had twice the risk of developing ART failure. Patients with incident tuberculosis after initiation of ART had three times increased risk of developing treatment failure.Baseline WHO stage, CD4 count and with longer time to attain peak CD4 count had decreased risk of treatment failure. The median peak CD4 count and mean duration to achieve peak CD4 count was 230 cells/μl and 16.5 months for cases and 463 cells/μl and 37.2 months for controls. In the bivariate analysis the type of initial ARV regimen used (d4T vs. AZT or NVP vs. EFV) did not show any significant association with treatment failure. The risk of treatment failure was lower among patients with longer duration on first line ART .Patients with high peak CDARV treatment interruption and occurrence of recurrent pneumonia were also significantly associated with treatment failure but the precision of estimation was weak as evidenced by the wide confidence interval .4 count and duration on first line ART were independent predictors of ARV treatment failure after controlling for other factors. Receiving stavudine based first line regimen had three fold increased risk of treatment failure compared to Zidovudine based regimen. In addition peak CD4 count achieved during the first line ART and low baseline BMI (< 18.5) were also significant predictors of treatment failure. Patients who attain high peak CD4 count were less likely to develop treatment failure . Although the time required to attain peak CD4 count was associated with treatment failure in the bivariate analysis, this effect couldn’t be demonstrated in the multivariate model.In the multivariate analysis initial ARV regimen type, unemployment, having formal education (Grade 1–12), low baseline BMI, peak CDPatients who were unemployed and with formal education had increased risk of ARV treatment failure. Furthermore duration on first line ART was another factor significantly associated with treatment failure. Patients who failed on ART had shorter duration on first line therapy. The mean duration on first line ART was 48.4 months for cases and 76.5 months for controls . History of treatment interruption and the occurrence of recurrent pneumonia were also associated with treatment failure but the power of estimation was weak as seen with the very wide confidence interval.4 count and WHO clinical stage [Variable that were not significantly associated with treatment failure in the multivariable analysis includes age, sex, weight change, incident TB, baseline CDal stage .Females account to two third of the adults on treatment and majority of the index patients has disclosed their HIV status to their partners. Similar finding was reported from other study in Ethiopia . DiffereThe median age of the study participants was comparable to a finding from Debremarkos hospital study and close to half of the participants were in the age group 25–34 years . VariousResults from this study revealed that duration of first line ART regimen was an important determinant of treatment failure. Patients, who were on their first line regimen for longer period, appear to have low risk of treatment failure. A large observational cohort study by Dragsted et al have found that a gradual and significant decline in the rate of immunologic failure over time. In fact the trend over time showed a 28% decrease with each additional year since the initial immunologic response . ContrarStavudine use as NRTI backbone was significantly associated with a threefold increased risk of treatment failure compared to Zidovudine based regimen. However, a study from Kenya showed that Zidovudine based regimen was associated with treatment failure partly explained by the early adverse effects which includes nausea and vomiting that potentially affect treatment adherence . Ethiopi4 count at ART initiation was low (mean CD4 = 132 cells/microliter). Interestingly, however, both advanced WHO clinical stage and low CD4 count were not significantly associated with treatment failure. This is similar to findings from Kenya where both WHO stage [4 count was found to be the strongest predictor of treatment failure. Low peak CD4 count was significantly associated with increased risk of failure. Singh et al in their study found that the mean peak CD4 count for the non-failure group was twice that of the failure group [4 count was equal or greater compared to those with high baseline CD4 count. However, their CD4 count remains low (< 200 cells/microliter) for longer period increasing the risk of morbidity and subsequent disease progression [Various studies have shown that low baseline CD4 count and advance disease stage were significant predictors of treatment failure explained by the frequent occurrence of opportunistic infections that leads to disease progression subsequently increased risk of failure , 32, 39.HO stage , 34 and re group . This figression .Unemployed patients had about 5 times higher risk of treatment failure compared to employed patients. The possible explanation could be those patients who were unemployed may have low income that hinders them from getting the opportunity for early and better care and support. This is in agreement with a finding by Melsew et al . The othOf the well-known factors, adherence was one of the strongest predictor of treatment failure , 34, 40.Prior ARV exposure for PMTCT purpose was reported to be very low in this cohort probably reflecting the low coverage and enrollment to PMTCT service in the early period of the HIV treatment program in Ethiopia. Studies from other African countries have found that an increased risk of treatment failure among patients with prior ARV exposure , 33. In The occurrence of recurrent pneumonia (> 2 episodes) was another predictor of treatment failure. Similar finding was reported from other study . The occSome studies have revealed that patients with low baseline BMI (< 18.5) and negative or no change in weight have weakened immunity and blunted immune response and increased risk of treatment failure , 36, 37.Regardless to the study’s strengths and the inevitable intrinsic limitations of using case control study for the study, the following limitations worth mentioning. One of the limitations of this study was the determination of treatment failure through clinical and/or immunological criteria due to the absence of viral load testing to confirm its presence. In addition, the use of adherence measurement based on health care provider’s assessment inadequately detects poor adherence even though this method used to be the usual clinical practice in the Ethiopian context. Moreover, the exclusion of patients who were initiated for ART in other health facilities may introduce bias. Not standardizing the cases and controls in terms of duration of treatment may introduce bias in predicting the outcome.4 count achieved during the first line therapy that has not been demonstrated in the Ethiopian context. In addition it was revealed that the risk of treatment failure in patients with an initial response to ART decreases with a longer duration on first line treatment.This study yielded a valuable insight to the various predictors of treatment failure. Apart from the well-known factors identified elsewhere, this study have found a novel risk factor of peak CD4 count, advanced WHO stage and occurrence of incident TB were not significant predictors of treatment failure in this study.Another interesting finding was the negative association between educational level and immunologic response. Being unemployed, stavudine based initial regimen, having recurrent pneumonia and history of antiretroviral treatment interruption for more than 7 days were also significantly associated with increased risk of treatment failure. While low baseline CD4 cell count achieved during ART should be monitored closely. In addition, programs managers should have their own efforts in improving socio-economic status of HIV infected patients through different livelihood initiatives should be strengthened. Moreover, besides the clinical care provided to patients on treatment, the integrated nutritional counseling and supplementary food support program with in the HIV treatment platform should be further strengthened. To enhance the evidence in this regard, some of the predictive factors identified in this study needs to be further explored with longitudinal studies and larger sample size to ascertain their association, by researchers. In addition, as cumulative evidences revealed from various studies adherence is the most important determinant of treatment failure, a validated measure of adherence shall be considered in future studies.With a maturing HIV treatment program in Ethiopia monitoring of patients on first line treatment to identify those who are more likely to develop treatment failure is highly crucial. Clinicians should exert their effort to maintain patients on their initial regimen for as long as possible duration with appropriate care and close follow-up. Among the different markers of treatment failure the level of peak CDS1 File(PDF)Click here for additional data file.


The most common reasons for switching HIV-1 therapy in patients with virologic suppression are treatment regimen simplification and resolving tolerability issues. Single-pill regimens that include an integrase inhibitor are recommended options. A retrospective clinical audit was performed to determine the motivations for switching to dolutegravir (DTG)/abacavir (ABC)/lamivudine (3TC) at high HIV-caseload general practice clinics in Australia. The most common reasons for switching from a prior suppressive therapy to DTG/ABC/3TC were simplification of regimen, resolving toxicity/intolerance and patient preference . Kaplan–Meier analysis showed that the probability of patients remaining on DTG/ABC/3TC therapy at 12 months was 95.1%. Switching to DTG/ABC/3TC from a range of other regimens was associated with a discontinuation rate of 3.2%, with 2.5% of patients discontinuing due to adverse events and no patients discontinuing due to virologic failure. Switching to DTG/ABC/3TC was a viable treatment strategy in this cohort of Australian patients. The STRIIVING study demonstrated the non-inferiority of switching from a variety of antiretrovirals (ARVs) onto dolutegravir (DTG)/abacavir (ABC)/lamivudine (3TC) in comparison to staying on baseline (current) ART.4 The rate of AEs leading to discontinuation of DTG/ABC/3TC (4%) in this patient population was similar to that observed in DTG treatment-naive studies.49The need to switch HIV-1 therapy because of virologic failure and drug resistance has decreased with improvements in antiretroviral therapy (ART). As a result of these improvements, there is now a rationale for switching therapy in some patients with virologic suppression. Reasons to consider switching therapy in such patients include adverse events (AEs) and simplification of regimen.The present paper describes a retrospective clinical audit at high HIV-caseload primary care practices in Australia to determine why virologically suppressed patients switched therapy to DTG/ABC/3TC fixed-dose combination and the clinical outcomes following this switch.Patients identified across six primary care practices, who had received DTG/ABC/3TC alone for ≥1 day following a switch from suppressive ART (<50 HIV-1 RNA copies/ml), were included. These patients were required to have switched to DTG/ABC/3TC between 1 April 2015 and 31 March 2016, and to have been maintained on DTG/ABC/3TC alone after the switch. Patient files were reviewed by each general practice and individual cases submitted via a systematic electronic survey.The primary outcome was the percentage of patients remaining on DTG/ABC/3TC therapy with HIV-1 RNA < 50 copies/ml. Time on treatment was calculated from the date of first DTG/ABC/3TC script and censored on 1 April 2016. Kaplan–Meier survival methods were used to determine probability of DTG/ABC/3TC continuation at 12 months of treatment. All other results are descriptive.This project was approved by an Australian private not-for-profit ethics committee registered with the National Health and Medical Research Council and all patient data were de-identified.Data from 443 patients were included: 97% male and 45% ≥50 years. Of the 443 patients, two patients discontinued from DTG/ABC/3TC after 1 April 2016 and the data of one patient were received after the study closing date; the data from these three patients were included in the study. A summary of the most recent regimen prior to switching therapy is shown in The probability of patients remaining on DTG/ABC/3TC therapy at 12 months was 95.1% (95% CI 88.8–97.9) by Kaplan–Meier estimate .The most common reasons patients switched therapy to DTG/ABC/3TC were simplification, toxicity/intolerance and patient preference . After excluding prior DTG-based regimens as the most recent before the switch, toxicity/intolerance as a reason for switching therapy increased to 21%. Few (2%) patients switched therapy for reasons attributed to cost, suboptimal adherence or drug–drug interactions. The most common toxicities/intolerances leading to the discontinuation of prior therapy and switching to DTG/ABC/3TC were nervous system disorders, renal and urinary disorders, and gastrointestinal disorders. Pre-existing toxicity/intolerance events resolved in 43 of 58 (74%) patients, including nine of 13 (69%) with nervous system disorders, eight of 12 (67%) with renal and urinary disorders and ten of ten (100%) with gastrointestinal disorders. Patients with toxicity/intolerance events that did not resolve after switch onto DTG/ABC/3TC had a median time on treatment that was lower (191 days [IQR 100–239.5]) than that of the overall population (266 days [IQR 176–325]).Fourteen patients (3.2%) discontinued DTG/ABC/3TC; none of these were due to virologic failure . Disconthistory of ARV resistance number of previous regimens time on most recent regimen most recent agent class in prior regimen rationale for regimen switch DTG not used in prior regimen (3.7%) or a combination of DTG + ABC/3TC alone not used in prior regimen (3.0%)Nine of 14 patients who discontinued DTG/ABC/3TC switched therapy back to their prior regimen .This retrospective clinical audit demonstrated that the relative probability of staying on DTG/ABC/3TC therapy at 12 months after switching from another regimen was 95.1%.The most common reason for switching from a prior suppressive therapy to DTG/ABC/3TC was simplification. For those who switched therapy due to toxicity/intolerance, most pre-existing events, including over two-thirds of nervous system disorders, resolved. Of patients who ceased DTG/ABC/3TC therapy, all remained virologically suppressed on their subsequent regimen, which was often the regimen they had switched from.4 Other randomised controlled trials assessing stable switch strategies to single-pill regimens containing rilpivirine or elvitegravir have reported similar discontinuation rates due to AEs.10–14 The rates of discontinuations due to psychiatric AEs observed in this Australian cohort were consistent with the phase 3 clinical trials of DTG in treatment-naïve patients5–9 as well as many real-world evidence studies.15–19 Some real-world evidence studies have reported higher rates of discontinuations due to psychiatric AEs,20–22 possibly because of differences in study populations, or perhaps due to reporting and/or channelling bias. This tenet is supported, in part, by an analysis of the OPERA cohort study, in which patients receiving DTG were found to be more likely to have a history of psychiatric disorder at baseline than patients receiving other core agents.19The rate of discontinuations due to AEs in this study was low and comparable to that of the phase 3b stable switch study STRIIVING .This audit report was subject to the general limitations of a retrospective cohort analysis, including limits to data collection imposed by pre-specified end points in the study protocol. Consequently, data were not collected for a range of parameters including other regimens switched to during similar time frames, AEs that did not lead to discontinuation and the severity of AEs leading to discontinuation. In addition, there are limitations associated with use of DTG/ABC/3TC, including the need to test for HLA-B*5701 allele status before initiating therapy and, since DTG/ABC/3TC is a fixed dose tablet, it should not be prescribed for patients requiring dose adjustment.In this real-world retrospective audit, switching to DTG/ABC/3TC from a range of other regimens showed a low rate of discontinuation. Few patients were reported to discontinue due to AEs and no patients discontinued due to virologic failure, supporting DTG/ABC/3TC as a viable treatment strategy in this Australian patient population.


HAART is very effective in suppressing HIV-1 replication in patients. However, patients staying on long-term HAART still develop various HIV-associated neurological disorders, even when the viral load is low. The underlying pathogenic mechanisms are largely unknown. Emerging evidence implicated that persistent neuroinflammation plays an important role in NeuroAIDS. Although residual virus or viral proteins are commonly thought as the causal factors, we are interested in the alternative possibility that HAART critically contributes to the neuroinflammation in the central nervous system (CNS). To test this hypothesis, we have determined the effect of NRTIs on the expression of proinflammatory cytokines in the various CNS regions. Mice (C57Bl/6) were administered with AZT (Zidovudine 100 mg/kg/day), 3TC (Lamivudine 50 mg/kg/day) or D4T (Stavudine 10 mg/kg/day) for 5 days, and cortices, hippocampi and spinal cords were collected for immunoblotting. Our results showed that NRTI administration up-regulated cytokines, including IL-1β, TNF-α and IL-6 in various CNS regions. In addition, we found that NRTIs also up-regulated Wnt5a protein. Importantly, BOX5 attenuated NRTI-induced cytokine up-regulation. These results together suggest that NRTIs up-regulate proinflammatory cytokines via a Wnt5a signaling-dependent mechanism. Our findings may help understand the potential pathogenic mechanisms of HAART-associated NeuroAIDS and design effective adjuvants. About 35 million people have died of HIV-1 infection, and there are around 36 million people living with HIV2. Although there is still no cure for HIV-1 infection, the highly active antiretroviral therapy  has been proved to be a very effective therapy for inhibiting the viral replication, significantly decrease HIV-associated mortality and morbidities, and become the standard treatment for HIV patients3.Human immunodeficiency virus-1 (HIV-1) was identified as the etiologic pathogen for acquired immunodeficiency syndrome (AIDS) over three decades ago5. Convergent evidence suggests that the prevalence of HIV-associated neurological disorders (HAND) in HIV patients on HAART remains high7. HAND in post-HAART era significantly affect the quality of life of HIV patients and may directly contribute to them on-adherence to treatment. However, the potential mechanism by which HAART contributes to HAND is still poorly understood, and interventions are not available.Despite its efficiency in suppressing HIV-1 viral load to a very low level, long-term HAART is associated with various detrimental effects. Among the critical HAART side-effects are the damages in the nervous system8. Antiretroviral drugs also led to neuronal damage and death in animal models9. Neurotoxicity appears to associate with major types of antiretroviral drugs in HAART, including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitors (PI)13.NRTIs are the backbone in current HAART, and ample evidence indicates NRTI-associated neurotoxicity in both peripheral nervous system (PNS) and CNS17, is probably contributed by their mitochondrial toxicity20.Neurotoxicity is a suggested mechanism by which HAART could contribute to HAND. Progressive neuron loss was reported in HIV patients on HAART23. A consistent finding in the postmortem biopsies of HIV patients is neuroinflammation, as indicated by the presence of activated microglia and up-regulated pro-inflammatory cytokines24.HIV infection and toxic viral proteins such as gp120 and Tat are commonly thought as the cause of neuroinflammation in HIV patients. Indeed, the activity of gp120 and Tat in inducing neuroinflammation has been demonstrated in cultured glial cells27 and animal models31. However, the potential contribution of HAART drugs to the manifestation of persistent neuroinflammation has not been conclusively tested. Because HIV patients usually stay on long-term HAART, this question is clinically relevant.Chronic neuroinflammation is implicated in various neurological diseases, including HANDIn this study, we test the hypothesis that long-term administration of NRTIs to mice induces neuroinflammation. We measured the expression level of IL-1β, TNF-α and IL-6 in different CNS regions from mice that were administered with AZT (Zidovudine 100 mg/kg/day), 3TC (Lamivudine 50 mg/kg/day) or D4T (Stavudine 10 mg/kg/day) for 5 days by western blotting. Our results showed that NRTIs up-regulated the cytokines in CNS, and that Wnt5a signaling played a critical role in NRTIs-induced cytokine up-regulation.34. As HAART is the currently common treatment to suppress HIV replication in patients, we wanted to determine the potential effect of NRTIs, the essential components in HAART, on neuroinflammation in the CNS. Mice  were subcutaneously injected with AZT (100 mg/kg/day), 3TC (50 mg/kg/day) or D4T (10 mg/kg/day) for 2, 5, 10, or 14 days and CNS tissues including cortices, hippocampi and spinal cords, were collected at the end of NRTIs treatment. Western blotting analysis was performed to determine the expression levels of IL-1β, TNF-α and IL-6. Preliminary experiments indicated an increase of the cytokines was already evident at day 5 after NRTIs administration. Thus, we focused our analysis on this time point to save animals.Persistent neuroinflammation is considered to contribute to the development of HANDAs shown in Fig. 37 and is up-regulated in the spinal cord of ‘pain-positive’ HIV patients on HAART38. We determined if AZT, 3TC and D4T administration (5 days) up-regulated Wnt5a protein in the cerebral cortex 40 was administered via the mouse nasal cavity at 3 hours after each NRTI injection. The cerebral cortex, hippocampus and spinal cord were collected for Western blotting after 5 days of drugs administration. We observed that BOX5 administration significantly attenuated NRTIs-induced up-regulation of inflammatory cytokines in the CNS regions  in three CNS regions .Our results show that all three tested NRTIs induce cytokine up-regulation in general, despite that drug-specific effects are observed for different NRTIs. Although it is commonly thought that residual virus and/or viral protein are the causal factors for the commonly observed persistent neuroinflammation in the CNS, our findings indicate that we need to at least include the antiretroviral regimens containing NRTIs as potential critical etiological factor underlying the neuroinflammation in the CNS of HIV patients on HAART. Although some “old’ NRTIs (e.g. AZT and d4T) that are no longer recommended for the first-line treatment are included in the study, this insight may have a general clinically-relevant implication, and is consistent with the reported up-regulation of inflammatory cytokines by other HAART components such as NNRTI and PI in the CNS47.In this study, we have tested the hypothesis that NRTIs cause neuroinflammation in the CNS. It is well documented that NRTIs can cause neurotoxicity51. Thus, NRTI-induced proinflammatory cytokines revealed in this study may directly contribute to the development of neuroAIDS/HAND56. In support of this notion, Zheng et al. found that TNF-α is involved in neuropathic pain induced by 2′,3′-dideoxycytidine (ddC) in rats57. This new understanding may add significant insights into the pathogenesis of neuroAIDS from the perspective of antiretroviral therapy. Flower et al. reported an anti-inflammatory activity of NRTIs (e.g. AZT and d4T) in primary human retinal pigment epithelium cells or Raji/TK+ cells58. It will be interesting to examine if NRTIs have pro- and anti-inflammatory effects in different biological systems.Cytokines such as IL-1β, TNF-α and IL-6 are implicated in inflammatory responses associated with various CNS damages61, including neuroinflammation63, this study reports for the first time an important role of Wnt5a signaling in the regulation of NRTI-evoked CNS neuroinflammation.Our results further suggest a Wnt5a-mediated mechanism by which NRTIs up-regulate cytokines in the CNS. Specifically, we show that Wnt5a is also up-regulated by NRTIs Fig. , and thaTaken together, our findings suggest that HAART may contribute to neuroAIDS pathogenesis by inducing neuroinflammation in the CNS.Wnt5a signaling probably playscritical role in this pathogenic process by regulating NRTIs-induced cytokines, which could directly contribute to neuroAIDS-related neurodenegeration  were purchased from Shanghai Ling Chang Biological Technology Co., Ltd. All procedures used in the study were approved by the Zhejiang Sci-tech University Experimental Animal Ethics Committee, and all experimental procedures were carried out in accordance with relevant guidelines and regulations. Specific experimental procedures are described below.cord: The whole spines of euthanized mice were exposed by cutting along the spine skin on the back. After exposure, the spine was cut near the tail cavity. The spinal cord was flashed out with precooled PBS. Collected spinal cords were frozen in liquid nitrogen and stored at −80 °C freezer.Mice were euthanized with inhaled anesthesia ether, and heads were quickly removed and put into phosphate buffer saline (PBS) precooled on ice. The skulls of completely soaked heads were opened and the brains were removed to clean dishes. Cerebral cortices and hippocampi were carefully dissected and frozen immediately inliquid nitrogen. Frozen tissues were stored in a −80 °C refrigerator.Spinal NRTI drugs: zidovudine (AZT), lamivudine (3TC) and stavudine (D4T) were purchased from Sigma (Sigma-Aldrich). NRTIs were dissolved in PBS to a final concentration of AZT (8 mg/ ml), 3TC (4 mg/ml) and D4T (0.8 mg/ml) and stored at −20 °C.NRTIs were administered by SC injection. First, the injection site was disinfected with 75% alcohol. Skins of the back of the mouse was gently raised for slowly injecting the drug (0.25 ml/20 g) into the subcutaneous space. After injection, the injection site was pressed gently with alcohol wipes for a moment to prevent drug leakage. Injection volume:0.25 ml/20 g/day.64, at 3 hours after NRTI injection. Briefly, a mouse was placed into a fixator tube . A small hole was made in the bottom of the fixator to expose the nostrils. One person held the animal with the left hand, and gently fixed the mouse head with the right hand to properly expose the nostrils. Another person applied the drug to the nostrils with a 10 μL pipette. Left and right nostrils of mouse were separately administered with BOX5 (10 μL). Drug solution drops were applied to the nostril slowly, usually with one drop every few seconds, and the next drop was applied only after the previous one was completely absorbed. After completing drug application to one nostril, the same procedure was performed to the other nostril. The time interval was about 1 minute. The animal was released from the fixation device after the completion of the whole procedure. Injection volume:10 μL each nostril per day.Mice were administered with BOX5 (dissolved in PBS to the concentration of 0.5 μg/μL) by nasal dripping as previously describedCollected tissues were homogenized in cell lysis buffer (Beyotime Institute of Biotechnology), with protease inhibitors (Biotool).The homogenates were centrifugated for 10 min , and protein concentration in the supernatant was measured using the BCA Protein Assay Kit (BIOMIGA). Equal amounts of total protein (40–60 μg) were loaded and separated on 12% SDS-PAGE by electrophoresis . Proteins on SDS-PAGE were blotted onto PVDF membranes . The membranes were blocked with 5% skim milk dissolved in TBST  solution for 2 h, and incubated sequentially with primary antibodies in TBST against IL-6 , IL-1β , TNF-α , Wnt5a , α-tubulin  or GAPDH  for 2 h.After washes with TBST3 times for 10 minutes each and the membranes were incubated with goat anti-rabbit IgG  or goat anti-mouse IgG (1:30000 abcamab97023) secondary antibody conjugated with horseradish peroxidase (HRP) in TBST, After washes with TBST3 times for 10 minutes each and the protein bands were visualized by ECL (Advansta). Tubulin and GAPDH were used as loading controls.65. Briefly, paraffin sections were collected and deparaffinized. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. The sections were incubated in EDTA (pH 8.0) buffer at 95–100 °C for 20 min for antigen repair. The pre-treated sections were incubated with primary antibody at room temperature (25 °C) for 0.5–1 h, followed by incubation with secondary antibody for 30 min and staining with DAB (Gene Technology). The slides were lightly counterstained with hematoxylin, rinsed with distilled water, dehydrated in ethanol and mounted.Immunohistochemistry was performed as described