Anaemia is one of the most frequent haematological complications in HIV-infected persons. Understandingfactors associated with recovery from anaemia during ART is vital in improving clinical outcomes since anaemia is a strong predictor of mortality.Cohort study of 12,441 HIV-infected adults initiating ART between 2004-2010 in Johannesburg, South Africa. A further 2,489 patients with prevalent anaemia at ART initiation were examined to determine the incidence and predictors of recovery from anaemia. Cox proportional hazards models were fitted to investigate predictors of recovery from anaemia.Of the 2,489 patients with prevalent anaemia, most patients  recovered from anaemia. Median time to anaemia recoverywas 3.9 months (IQR: 3.22-6.20) and incidence rate was 180 per 100person years (95% CI: 172-187). In univariateanalysis, sex, CD4 count, BMI, WHO stage, employment status, smoking status and presence of tuberculosis at initiation ofART were significant predictors of recovery from anaemia. However in multivariateanalysis, predictors of recovery from anaemia were: male sex-HR: 1.43 (95% CI: 1.29-1.59) p< 0.001, advanced WHO stage III/IV - HR: 1.17 (95% CI: 1.07-1.29) p = 0.001). There was no significant association with CD4 count in multivariate analysis.A large proportion of HIV infected patients with anaemia at baseline recover early during the course of ART. Females and those with less advanced WHO stage seem to be at higher risk of poor recovery from anaemia. Understanding the predictors for poor recovery from anaemia would allow closer follow-up and more targeted interventions thus reducing excess anaemia and mortality burden. Anaemia is one of the most frequent haematological complications seen in people with human immuno-deficiency virus (HIV) and acquired immuno-deficiency syndrome (AIDS) , 2. AmonIt has been demonstrated that haemoglobin levels provide prognostic information independent of that provided by the CD4 lymphocyte count and HIV viral load and the p3, HIV viral load, MCV, presence of opportunistic infections, zidovudine use and WHO clinical staging, there is scanty information in the literature on predictors of recovery from anaemia, more-soreinlimited-resource settings. In limited-resource settings, understanding the factors influencing recovery from anaemia is even more crucial since this setting bears a disproportionate burden of anaemia due to prevalent co-morbidities like malnutrition, tuberculosis, malaria and other parasitic infections.Better understanding of the predictors of recovery from anaemia will allow closer follow-up and more targeted interventions among patients initiating ART with anaemia thus help to improve morbidity and mortalityoutcomes. Whilst numerous studies have identifiedpredictors of persistent anaemia –18 and tIn this study, we set to examine the incidence of recovery from anaemia among patients initiating ART in a large HIV-infected clinical cohort in Johannesburg, South Africa. Also, we set to determine predictors associated with recovery from anaemia during the follow-up period.The Themba Lethu HIV Clinic in Johannesburg, South Africa is one of the largest HIV care and treatment clinics in South Africa with over 21,000 patients initiated onto ART since 2004. Details of this cohort have been published previously .We conducted a prospective cohort analysis of HIV infected ART naïve patients >18 years of age, who initiated standard first line ART at Themba Lethu Clinic (TLC) between April 2004 and June 2010 with CD4 counts =350 cells/ml. Pregnant women, women in the post-partum period (within 6 weeks post-delivery) and patients on agents which enhance haematopoiesis  were excluded from the study. At the time, standard public-sector ART in South Africa consisted of stavudine (d4T) or zidovudine (AZT) with lamivudine (3TC) and either efavirenz (EFV) or nevirapine (NVP) [Anaemia was defined according to the WHO toxicity grading systems for anaemia as haemoglobin less than 10 g/dl . In addithJune 2012.For the analysis, person-time accrued from treatment initiation until the earliest of: (1) recovery from anaemia; (2) death; (3) loss to follow-up (defined as 4 months late for last scheduled clinic visit); (4) transfer; or (5) close of the dataset on 30All data analyses were conducted in STATA version 12 . Using the Log-rank test for sample size and power estimation, assuming an alpha of 5% and assuming that more than 70% of the patients would recover from anaemia and an effect size hazard ratio of 1.5, our sample size of 2,500 was highly powered (power > 90%). We computed period incidence rates and cumulative probabilities of recovery from anaemia at initiation. Cox proportional hazard models were fitted to investigate associations between baseline characteristics and recovery from anaemia. Variables that had a p-value < 0.25 on univariate analysis were considered as potential candidates for inclusion into the multivariatemodels. We then employed the stepwise forwardselection method to arrive at the final multivariate model. The following explanatory variables available at baseline were considered for inclusion into multivariate models; gender, age, employment status, education status, smoking status, alcohol consumption status, body mass index (BMI), WHO clinical staging, tuberculosis status and baseline CD4 count. CD4 count was forced into models as a priori. The proportionality assumption was checked ) over time for each covariate) and was not violated. Overall goodness of fit of the final model was assessed using Cox-Snell residuals and there was good fit. In order to understand the extent to which loss to follow up or death may have biased our estimates, we furtherextended our analysis to compare the baseline characteristics of patients who were lost to follow up or died versus those who completed follow-up for the duration of the study. The Pearson's chi-square test and the u-Mann-Whitney tests were used to compare categorical and continuous variables respectively in this extended analysis.Permission to conduct the study was granted by the Human Research Ethics Committee of the University Of Witwatersrand and use of Themba Lethu Clinic data was approved by the superintendent of Helen Joseph Hospital.Between April 2004 and June 2010, 12,441 HIV positive subjects initiating ART at the TLC had baseline haemoglobin measurements. Among these, 26.5%  had haemoglobin level < 10g/dl. We excluded 45 patients less than 18years of age and 765 patients who either did not return for follow-up or had no subsequent haemoglobin measurements. This left 2,489 patients with anaemia for analysis. The mean age of patients was 36.7 years (s.d. 8.6), mean haemoglobin was 8.7g/dl (s.d. 1.1) and the majority of patients were female, 73.5% . Most patients 89.5%  were initiated on stavudine/lamivudine/efavirenz regimen while < 2% were initiated ona zidovudine containing regimen. This cohort was immunosuppressed at initiation of ART, mean CD4 count was 83 cells/ml (s.d. 69) and 53.1%  had advanced WHO stage (WHO stage III/IV). See After a median of 3.8 months (IQR: 3.2-6.2) and 1,235person-years of follow-up, the majority of patients 89.4%  recovered from anaemia. One hundred and forty four (5.8%) patients were lost to follow-up and 120 (4.8%) died. The overall incidence rate of recovery from anaemia was 180 per 100 person years (95% CI: 172.9 -187.9). The period with the highest recovery rate was 0 - 3 months from initiation (1.82) and the period with the least recovery rate was > 6 months. During the 0 - 3 months from ART initiation, males had a recovery incidence rate of 237 per 100 person years (py), and females had 167 per 100 py, In univariate analysis, males, patients with advanced WHO clinical stage (III/IV), employed, on d4T/3TC/NVPand those on TB treatment had a higher chance of recovery from anaemia than females, patients with WHO clinical stage I/II, unemployed, on d4T/3TC/EFV and without a TB diagnosis . Higher CD4 count at ART initiation and higher BMI were associated with reduced chance of recovery from anaemia. See We observed a very high proportion of patients who recovered from anaemia after initiating ART (89.4%). The majority of the patients (95%) recovered from anaemia during the first 3 months on ART. Sex and WHO clinical stage at initiation of ART were predictors of recovery from anaemia. Anaemia is a strong independent risk factor for survival , 10, 12.Males had a higher chance of recovery from anaemia than females. This is in parallel with findings of previous studies that females are more likely to acquire anaemia , 22, posCounter intuitively, participants who were more clinically ill  were 17% more likely to recover from anaemia compared to participants who were less clinically ill , This study, demonstrated a 27% prevalence of anaemia and it showed that more than 90% of subjects recover from anaemia within 3 months after antiretroviral therapy enrolment. Predictors of recovery from anaemia are; gender and WHO clinical staging. Since anaemia is an established strong independent risk factor of mortality in HIV-infected patients, patients less likely to recover from anaemia during ART should be identified and they should receive prompt interventions to ameliorate this. Also risk stratification among patients with the same level of anaemia is important i.e. anaemia in females should signal a worse likelihood of improvement than in males. Enrolees should be taught about balanced diets as the majority of them in the cohort were underweight at initiation.A major strength of our study is the large sample size and number of events to draw from. Additionally, we make use of routine clinical data. However, the inferences need to be interpreted with caution in light of the following study limitations. This was a retrospective study using secondary data and as such, only pre-collected information was available for analysis. Our inability to assess other important confounders which are known to have an influence on anaemia  may have limited this analysis. We also only looked at baseline variables so as to ascertain predictors of recovery from anaemia. Taking into account the longitudinal nature of some variables i.e. CD4 count and viral load measurements may have strengthened our findings. Also, because of the possibility of informative censoring (i.e. patients LTFU may have been the ones less likely to recover from anaemia) we may have overestimated our outcome. The TLC cohort is one of the largest urban cohorts in South Africa and is highly likely to be representative of the urban HIV infected persons but it may not be generalizable to rural HIV-infected persons who are more likely to have greater burden of anaemia.


An increasing number of treatment-experienced perinatally HIV-infected adolescents (PHA) are being transitioned from paediatric centres to adult HIV-care . Most ofwww.hiv-druginteractions.org) [Descriptive pilot cross-sectional study (March to June 2014). PHA under ARVs at the time of the study were assessed for concomitant medication. CSDDIs were screened and categorized using the University of Liverpool Drug Interactions Program  age: 20 years (18–22). CDC-stage C was observed in 27 (79%); 50% had ≥1 comorbidities including 3 with HCV co-infection. Drug abuse was observed in 6 (13%). The median of prior ARV regimens was 3 (3–5). Current ARV regimen included: PI: 87%, NNRTI: 27%, INSTI: 20%, enfuvirtide: 7% and CCR5 inhibitor: 4%. Median CD4 T-cell count: 568 cells/mL (279–771). Viral load <50 copies/mL: 80%. Sixty percent (27/45) had ≥1 co-medications (median 1). The most frequent co-medications were NSAIDs (40%), hormonal therapy (19%) and antimicrobials (19%). Use of herbal supplements was observed in 10 (22%). Overall, 23 (51%) had ≥ 1 CSDDIs: 19/27 (70%) with co-medication (orange flag=18 and red flag=1); and 2/10 (20%) with herbal supplements. ARV–ARV interactions were observed in 4/45 (9%): unboosted atazanavir+tenofovir (n=2), unboosted atazanavir+efavirenz (n=1) and lopinavir/ritonavir+efavirenz (n=1) . Considering patients with CSDDIs, 6 (26%) had a CSDDI that could reduce ARV levels.In this pilot study, a high prevalence of comorbidities, co-medications and CSDDIs was observed in PHA. A considerable proportion of patients had CSDDIs with a potential to cause sub-therapeutic ARV levels, what could be a concern in patients harbouring drug-resistance viruses. Therefore, clinicians should be aware of comorbid conditions pharmacologic management in order to avoid CSDDIs with ARVs agents.


Ongoing HIV-1 replication in lymphoid cells is one explanation of the persistence of HIV-1 reservoirs despite highly active antiretroviral therapy (cART). We tested the potential of cART intensification by Maraviroc plus Raltegravir to decrease proviral HIV-1 DNA levels in lymphoid cells during a randomized trial.We randomly assigned for 48 weeks 22 patients to continue their current first line regimen of Truvada® plus Kaletra® or intensify it with Maraviroc and Raltegravir. The primary objective was to obtain a 50% decrease in proviral HIV-1 DNA levels in lymphoid cells with intensification. Blood samples were drawn at W-2, W0, W2, W4, W12, W24 and W48. Plasma viremia, cellular proviral DNA and cellular RNA, 2-LTR circles and lymphocytes subsets were assayed using validated methods. Patients in the intensified group underwent a gut biopsy at baseline and W48 to measure proviral DNA levels. Statistical analysis used parametric and non-parametric tests.+ T cells activation was observed at 24 and 48 weeks, as well as in PBMCs HIV-1 RNA levels.Ten patients in each arm completed the trial. The 2 populations were comparable at baseline. No change in the reservoir size was observed in the intensified arm compared to the control arm measured in peripheral blood mononuclear cells (PBMCs). No change in the reservoir size was observed in gut proviral DNA in the intensified arm. In this group, no increase in 2-LTR circles was observed as early as 2 weeks after intensification and no change was found in residual plasma RNA levels measured by the single copy assay. However, a decrease in CD8We conclude that the intensification of a Protease Inhibitor regimen with Maraviroc and Raltegravir does not impact the blood proviral DNA reservoir of HIV but can decrease the cell-associated HIV RNA, the CD8 activation and has a possible impact on rectal proviral HIV DNA in some patients.NCT00935480ClinicalTrials.gov identifier number  With the advent of combined antiretroviral therapy (cART), HIV-1 replication can become undetectable in the plasma for years. However, HIV-1 persists in lymphoid reservoirs where residual low levels of viral replication can be found[Twenty-two adult patients on first line therapy with Truvada® (1 pill/day) and Kaletra® 400/100 (2 pills twice daily) were randomly assigned to continued cART of add Maraviroc (150 mg twice a day) and Raltegravir (400 mg twice a day) to their regimen.Inclusion criteria included to be on first line therapy with this regimen, having no contraindication to receive Maraviroc and Raltegravir, have a plasma viral load <20 copies/ml for at least 12 weeks, no contraindication for gut biopsies.+, CD8+ and CD8+CD38+ T cell subsets were measured by flow cytometry  using commercially available monoclonal antibodies .CD4HIV-1 RNA levels were measured in plasma by using the Amplicor Monitor® assay . Patients with levels <20 copies/ml where tested with the single copy assay (SCA) as previously reported.HIV-1 DNA levels were measured by using the Generic HIV® assay  according to manufacturer’s instructions.2-LTR quantification used a previously reported technique.Drug levels were measured by LC-MS.CCR5 tropism was determined on PBMC HIV DNA by sequencing according to the ANRS protocol[Lymphoid tissue was obtained by sigmoid colon biopsies at 30 cm from the anus and processed as previously reported.Only patients included in the intensified arm of the study were proposed to get gut biopsies at baseline and W48.Patients were analyzed in blood at W-2, W0, W2, W4, W12, W24 and W48.The randomization between the 2 groups was done by using a computer-based method. It was performed by our 2 technicians for clinical trials who also stored the trial data locked in their office.The primary objective was to detect at least a 50% decrease in PBMCs proviral DNA levels in the intensified group and a calculation of at least 8 patients needed in each arm was done.The secondary objectives were to measure the evolution of lymphocytes subsets, plasma viral load and PBMCs HIV-1 RNA levels in the 2 groups.Statistical analysis was done with the SPSS v20® software  and used descriptive statistics as well as parametric and non-parametric tests.The Ethics Committee and the French Agency for Drugs Security (ANSM) approved the protocol. All patients gave their written informed consent before inclusion and the randomization was performed between W-2 and W0.The protocol number on ClinicalTrials.gov is NCT00935480.We screened a total of 30 patients and 10 were included in each arm . Baseline patients’ characteristics are shown in Table Eleven patients were randomized to the continuing arm and 11 to the intensified arm.In the continuing arm, 1 patient declined to follow the protocol beyond screening and baseline as he hoped to be in the intensified arm. In the intensified arm, 1 patient stopped antiretroviral therapy after week o and was excluded from the study.Consequently, 20 patients were analyzed until week 48.Both groups were comparable in terms of age, sex, duration of infection, CD4 cell count, HIV viremia, (SCA) HIV DNA levels, CCR5 tropism and markers of activation at baseline Table .Over the 48 weeks of follow-up, antiretrovirals were well tolerated in both groups and no adverse event was observed.Adherence was correct in each group, assessed by residual through levels of lopinavir (group 1), lopinavir, Maraviroc and raltegravir (group 2). In each case, levels were in the needed range (data not shown).Regarding the SCA, one patient in each group had detectable viremia at levels <10 copies/ml. These 2 cases did not show detectable viremia at any time point during the trial. On the contrary one patient negative at baseline in the control arm was found at 8 copies/ml at week 12 and 2 patients in the intensified arm were at 3 and 15 copies/ml respectively at weeks 12 and 24. These patients did not show any resistance selection at the time cART was initiated (data not shown).6 PBMC at week 2.Regarding 2-LTR, no patient was positive at baseline. No positive detection was found during the trial for all cases but one in the intensified group who was measured at 5.84 2-LTR units/10+ T cell counts did not change over time in the 2 arms  was observed in the intensified arm following a decrease of total CD8+ T cells. This decrease in activation markers might be due to the effect of intensification on ongoing viral replication. Interestingly, we also observed a decrease of PBMCs HIV-1 RNA levels in the intensified arm after 24 weeks that can also reflect the reduction of ongoing viral replication, as this marker has been demonstrated to reflect this replication[By adding 2 new antiretroviral drugs with different mechanisms of action in first line Protease Inhibitor treated patients, we tried to impact the stability of the reservoir. The originality of this approach was the choice of drugs and the long period of intensification. However, no difference was found between intensified cases and the control group up to 48 weeks regarding the primary end point of the trial, which was a more than 50% decrease in proviral DNA levels in circulating lymphoid cells. Nevertheless we observed diverse patterns of proviral DNA evolution in gut cells but due to the small sample size we were unable to correlate these evolutions to any characteristic of the patient. No increase in 2-LTR levels, a marker of ongoing HIV-1 replication was demonstrated as early as 2 weeks after intensification. No change in CD4lication.Other trials have addressed the issue of cART intensification and showed conflicting results. In the study of Buzon et al., an increase in 2-LTR was demonstrated by adding Raltegravir. In the To our knowledge, only two studies used Maraviroc and Raltegravir in combination, 15 and Maraviroc has been shown to be an antiretroviral drug, but also to increase the expression of latent HIV. It is iOur study has some limitations. Firstly, as patients were effectively treated, CCR5 tropism could only be tested on archived virus. Secondly, the sample size was small due to the fact that having to perform gut biopsies discouraged patients. Thirdly, we were able only to measure the HIV-1 reservoir by PCR techniques and not by culture of replication competent virus. HoweverFurther studies are needed with larger samples to confirm that ongoing viral replication is present in some patients despite effective therapy and can be reduced by drug intensification. This is a major question to resolve before moving to therapeutic trials aimed at inducing a HIV functional cure, 20.In the population studied, 48 weeks cART intensification with Maraviroc and Raltegravir failed to decrease the proviral HIV reservoir in circulating lymphocytes but was able to decrease both lymphocytes activation and ongoing viral replication. A possible effect on proviral DNA in rectal cells was observed in some patients but difficult to interpret due to the low sample size.


Traditional genotyping assays detect viral variants present in at least 15–25% of the entire virus population. We tested the Next generation GS Junior System (NGS) setted with a detection limit of 0.05% and evaluated the clinical relevance of low prevalent mutations.NGS was performed on the plasma of 26 infected individuals who started a TDF/FTC/RPV (15 subjects) or TDF/FTC/EFV (11 subjects) cART after a routine HIV-1 drug-resistance negative test by Viroseq HIV-1 Genotyping System. Amplicon Sequencing of HIV-1 RT and PR Plate (Roche) was performed following the manufacturer's instructions. HIV-1 variants were analyzed by a specific HIV-1 tool by AVA software v. 2.7. The updated IAS resistance mutations list (March 2013) was considered for the analysis of resistance positions. Patients were followed testing viral load and immunologic parameters.Twenty four males and two females with a mean age of 43 years were included. Twenty-one were nave for cART. At baseline, median HIV-RNA was 4.57 log copies/mL (range 2.15–6.57) and CD4 count 315 cells/mcL (range 16–648). In 18 patients, NGS did not detect any additional variant relevant for the selected cART compared to population genotyping. In the remaining eight patients resistance conferring mutations to part of the ongoing regimen were detected. Single mutations E138K (two cases) and M184V in three distinct patients and V90I+G190E; M184V+A98S; Y215F+V118I+T215I; L210S+T215I+F227L; and A62V+D67G+K70N+188H in the remaining five subjects. In all cases, the mutation prevalence was inferior to 5%. The mean daily reduction of VL was −3759 copies/mL in patients without NGS detected mutations and −1045 copies/mL in those with mutations. The median KM estimates for reaching an HIV-RNA blood level <50 copies/mL were 127 days and 161 days, respectively. One patient without baseline resistance selected for M184I+E138K+T215I (NGS) after four months of TDF/FTC/RPV therapy.NGS detected low-frequency HIV-1 variants harbouring RT drug resistance mutations that could have affected the therapy outcome. However, viral decay in an early cART phase was not affected by the presence of resistant minority variants. The low prevalence of the detected mutation, the limited effect on the combination regimen and the potency of cART components could be possible explanations of our findings. Longer follow-up and larger casuistries are needed to determine the clinical relevance of NGS in routine clinical practice and eventually define a clinically relevant mutations' prevalence.


Antiretroviral therapy (ART) reduces HIV-related mortality and morbidity substantially in children. The clinical characteristics, immunological and virological outcomes were evaluated in HIV-infected children receiving ART.Twenty-six HIV-1-infected children receiving ART in Hubei province, China, were enrolled retrospectively in this study. During the period of ART, plasma viral load, lymphocyte phenotype of CD4 and CD8 cells and clinical events were assessed.P < 0.001).The median duration of ART was 41 months (18–72.3 months). In children showing clinical improvement, high viral suppression rate below log10 (2.7) copies/ml by the third months of ART was observed. The median CD4 cell counts reached to 820.5/μl by 12 months and the median ratio of CD4/CD8 increased to 0.6 by 21 months. The counts of peripheral white blood cells and red blood cells decreased in the first 12 months, while Hb concentration, MCV and MCH increased (Despite the limited small sample size, ART is an effective strategy for inhibiting HIV replication and reconstructing the immunological response in children with AIDS. Antiretroviral therapy (ART) reduces HIV-related mortality and morbidity substantially in children, and without HIV care, the progression of HIV-infection children is particularly aggressive. The World Health Organization (WHO) guidelines recommended initiating ART in all children according to their clinical stage and CD4 cell count or percentage . HoweverThe Division of Treatment and Care (DTC) of the National Center for AIDS/STD Control of China estimated nearly 6000 children infected with HIV-1 in China at the end of 2011 -9. The DEnrolment and study setting based on the WHO clinical and laboratory criteria , 26 of 6ART eligibility was defined based on the WHO guidelines . HIV-infBlood samples were collected in the morning and processed the same day at Wuhan University HIV/AIDS Treatment Center. CD4 and CD8 cell counts were measured using the single-platform assay in a FACSCalibur flow cytometer . The CD4 percentage was calculated using the formula: CD4% = absolute CD4 count/(white blood cell (WBC) count × percentage of lymphocytes). An absorbance cytochemistry and haematology analyser  was used to measure haematological parameters including hemoglobin concentration (Hb), red blood cell (RBC), and red cell indices such as mean corpuscular volume (MCV), mean corpuscular Hb content (MCH), as well as mean corpuscular Hb concentration (MCHC). Viral load (VL) was detected by a real-time reverse transcriptase polymerase chain reaction . Therapeutic failure was defined as a low CD4 cell count according to the WHO criteria or an increase in VL to > log10 (3.7) copies/ml [This program used the Free ART database to monitor current and past patients in this Program. Information was collected at the time of treatment initiation, each follow-up visit, regimen change, and treatment termination.P-values <0.05 considered significantly.Analysis of data was performed by on-treatment analysis of the observed data. Z-scores (standard deviation scores) are calculated as (variable-mean)/standard deviation. Statistical analysis was performed using SPSS 19.0 software . All tests were two-tailed with This study was approved by the Institutional Review Board of the National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention. All parents of patients signed informed consent for participation.General characteristics are shown in Table The baseline median CD4 count was 115.7 cells/μl, and this count increased markedly to 268.5 cells/μl in the third month and reached its highest level of 810.4 cells/μl at the first year of ART  copies/ml. In 23 children, the VL decreased to less than log10 (2.7) copies/ml within 3 months of ART and VL was maintained at this level until the preparation of this study . All children demonstrated a change to megaloblast-like red blood cell , and the lymphocyte count decreased to 1.8 ± 0.6 × 109/L (P = 0.02). Of the patients with severe anemia, none required a change of regimen because of symptomatic anemia or blood transfusions. Other haematological parameters evaluation included macrocytosis and thrombocytopenia . In contrast, Mean of red blood cell count decreased from 4.0 ± 0.4 × 10Our study shows that ART (AZT/3TC/NVP) should be effective in achieving an adequate response in HIV-infected children. Most HIV-children showed improved clinical, immunological and virological outcomes. Such therapy results are similar to those reported for adults  and a smThe immunological outcomes from receiving ART showed evidence of significant immunological reconstitution in most of the cohort. After 12 months of ART, most HIV-infected children with available CD4 cell percentage data attained near-normal immune status (e.g. CD4 cell percentage of 34%). The CD4 cell counts increased to the highest level in the first year, and children with the most profound immunosuppression at the onset had the greatest increases in CD4 cell count (from 17 to 510 cells/μl). However, these children still had the lowest absolute CD4 cell counts, and a significantly higher proportion experienced an opportunistic infection. This suggests that, although immune function improves significantly in these children irrespective of the baseline CD4 cell count and percentage, the greater the baseline immunodeficiency, the more likely the patient will remain at risk of opportunistic infections during the first year of receiving ART .The survival rate is determined mainly by age-risk in the first six months of ART, whereas the patient’s sex, baseline CD4 cell percentage, baseline WHO clinical stage and initial drug regimens are not significantly associated with death in different age groups . Our datOur results are similar to those from resource-rich settings , althougHIV-infected children are significantly shorter and lighter than healthy children, and the growth differences increase with age . Our datART causing macrocytic anemia has been reported elsewhere . MacrocyIn conclusion, despite the limited small sample size, ART is an effective strategy for inhibiting HIV replication and reconstructing the immunological response in children with AIDS.ART: Antiretroviral Therapy; NRTIs: Nucleoside Reverse Transcriptase Inhibitors; NNRTIs: Non-nucleoside Reverse Transcriptase Inhibitors; VL: Viral Load.The authors declare that they have no competing interests.ZJ and ZD wrote the manuscript and coordinated the NPFAP project, collected the data. Both authors read and approved the final manuscript.


Recent treatment patterns and cost data associated with HIV in the United States are limited. This study assessed first-line persistence and healthcare costs of HIV-1 in patients by treatment line and CD4 cell count.MarketScan Commercial Claims and Encounters Database (2007–2011) and Lab Database (2007–2010) were used to construct two HIV-1 cohorts: 1) newly treated HIV-1–infected patients with ≥6 months' continuous enrollment prior to first third-agent drug claim (Newly Treated Cohort) and 2) CD4 cell count test results (CD4 Measurements Cohort). All patients were ≥18 years old and without hepatitis co-infection. The Kaplan-Meier method was used to measure treatment switch rates. Generalized linear models  were used to compare healthcare costs by treatment line and CD4 cell count controlling for potential confounders.Newly treated patients  had mean age of 41, 82% were male, and 20% had experienced AIDS-defining events at baseline. Over 20% of newly treated patients switched initial treatment regimen within 2 years. Average unadjusted (and covariate-adjusted) total healthcare cost/year was $33,674  for first-line, $39,191  for second-line, and $39,882  for third-line treatment. Covariate-adjusted costs of care on second- and third-line treatments were significantly more expensive than first-line treatment . The CD4 Measurements Cohort included 803 CD4 measurements . Costs associated with CD4 measurements <100 cells/µL were 92% higher than those with >350 cells/µL (p<0.001). For higher CD4 cell counts, the majority of expenditures were for antiretrovirals .Despite modern advances in antiretroviral therapy and medical care, direct medical costs of HIV-1–infected patients increase after treatment switch and with lower CD4 counts, consistent with previous costing studies. Human immunodeficiency virus (HIV) infections remain prevalent within the United States (US), where approximately 1.26 million people are afflicted and 50,000 new infections occur annually While effective, ARV treatment does not represent a cure for HIV infection, and treatments must be taken for the duration of a patient's lifetime. Patients are typically treated with a combination of drugs, including at least two nucleoside reverse transcriptase inhibitors (NRTIs) and at least one drug from the other, more potent drug classes . Third agents are treatments from the non-NRTI, protease inhibitor, and integrase inhibitor drug classes and are responsible for the large improvement in the treatment of HIV disease by combination therapy observed in the last 2 decades. These combinations are costly; the most popular combination (efavirenz/emtricitabine/tenofovir) is published to have an average wholesale price of $2,253.88 per 30 tablets Treatment switching is often the result of intolerance or virologic failure (the inability of ARVs to suppress circulating virus to undetectable limits or rebound of virus after previous suppression). In the case of the latter, future regimens may be less effective Recent estimates of the economic burden of HIV disease and costs of treatment switching are not available. The most recent literature reporting cost data associated with HIV in the US reflects costs by CD4 cell count prior to 2006 MarketScan Commercial Claims and Encounters Database (2007–2011) and the MarketScan Lab Database (2007–2010)  were analyzed to determine total costs for HIV-1–infected patients. These data sets include claims compiled from 100 employers and 12 health plans throughout the US, representing more than 30 million covered lives. Patients with any HIV-1 diagnosis code  in any inpatient, outpatient, or service claim between January 1, 2007, and December 31, 2011, who were ≥18 years of age and had no comorbid diagnosis codes for HIV-2 (ICD-9-CM 079.53) or hepatitis C  were considered for inclusion in the analysis. From these data, two patient cohorts were constructed: 1) Newly Treated Cohort—patients initiating ARV treatment, including a third-agent drug and at least one NRTI; and 2) CD4 Measurements Cohort—patients with one or more CD4 cell count test result data.The Patients included within the Newly Treated Cohort were those with ≥6 months' continuous enrollment prior to the first claim for a third-agent ARV , ≤30 days of NRTI use prior to first third-agent claim, at least 1 month of continuous enrollment after first third-agent claim, and use of at least one NRTI during the follow-up period . The patEach CD4 measurement was a unique observation defining the CD4 Measurements Cohort, with the date of CD4 measurement serving as the index date . To alloPatient characteristics included age, geographic region, health plan type, and gender, as available. In addition, categories of comorbidities were defined, and the Charlson Comorbidity Index . Cost outcomes were standardized into monthly units within the CD4 Measurements Cohort (costs per person-month  =  sum of costs observed over CD4 measurement period x 30 divided by number of days in CD4 measurement period) and yearly units in the Newly Treated Cohort . All cost data were inflated to 2011 US dollars using the Bureau of Labor Statistics Consumer Price Index for medical care Cost analysis for the Newly Treated Cohort was limited to treatment lines ≥90 days. Without patients' viral load data, it was not possible to identify whether the reason for treatment switch was due to intolerance, virologic failure, or other. One could assume that treatment switching within 90 days would likely be due to reasons of intolerance and the subsequent regimen's persistence would not be affected by the switch. For this reason and to allow for sufficient time for cost accrual, the cost results are restricted to only treatment lines ≥90 days in length. Within the CD4 Measurements Cohort, results are presented for measurement periods with ≥1 ARV prescription to minimize the effect of missing ARV cost data due to the possibility of patients receiving treatment outside of their health plans.Analyses to address the study objectives were primarily descriptive in nature. Age data were presented as means and standard deviations along with medians and interquartile ranges. Age by categorical strata, gender, health plan type, geographic region, Charlson Comorbidity Index, and presence of AIDS-defining conditions were described using proportions. The Kaplan-Meier method was used to estimate probability of initial treatment switch accounting for the presence of censored data due to different lengths of follow-up time.Because cost data are always positive and not normally distributed, generalized linear models with a gamma distribution and log link were used to compare healthcare costs by treatment line and CD4 cell count controlling for potential confounders including Charlson comorbidity index, presence of AIDS-defining events, sex, age, region (Newly Treated Cohort only), and type of health plan. From these models, adjusted mean costs were calculated for treatment line and CD4 cell count strata using the coefficients from the generalized linear model, setting all other parameters within the model to their mean value. Unadjusted mean costs were calculated as simple means. Percentage of costs attributable to different sources  was similarly modeled to control for covariates. Due to the high number of zeroes for inpatient and ER costs, a two-part model was created. First, the probability of having a non-zero cost, adjusted for covariates, was estimated by logistic regression. Second, this probability was multiplied by the adjusted mean cost estimated by a generalized linear model of the observed non-zero costs. Sensitivity analyses were conducted for cost models by including treatment lines <90 days in length within the Newly Treated Cohort and including CD4 measurements without an ARV prescription fill in the measurement window within the CD4 Measurements Cohort.MarketScan database during the study intake period , a total of 9,931 patients met inclusion criteria for the Newly Treated Cohort  were observed to switch from first line to a second line of treatment in 132,487 months of patient follow-up. Of those, 108 (7.0%) had switched to a third line of treatment during the observation period. Kaplan-Meier estimates accounting for censored follow-up times predicted 6.1%, 9.3%, 14.4%, and 22% of the patients on first-line treatment switched treatment at 3 months, 6 months, 12 months, and 24 months, respectively .Within the Newly Treated Cohort, when considering lines of treatment ≥90 days long, unadjusted total mean costs per person-year increased with each subsequent treatment line: $33,674 for first line, $39,191 for second line, and $39,882 for third line. After adjusting for covariates, second-line was 24% (p<0.001) and third-line was 41% (p = 0.006) more expensive than first-line treatment . AdjusteWithin the CD4 Measurements Cohort, inpatient admissions became an increasing cost driver at lower CD4 cell counts . UnadjusThis study identified significantly higher healthcare costs associated with treatment switches and lower CD4 cell counts within a current managed-care claims database reflecting modern-era treatments. We also identified that 22% of newly treated patients switched third-agent treatments within 2 years, confirming that despite the increase in the potency and number of HIV treatments available, more than one-fifth of patients do not achieve long-term persistence on their initial regimens and move to second-line regimens, where the costs of treatment seem to increase.These estimates were obtained from a retrospective analysis of a database including cost years that are more recent than what is currently available in the published literature and are most generalizable to HIV-1–infected patients who are covered by commercial insurance, are under the age of 65, and who do not have comorbid hepatitis C infection. Other patients not included within these parameters may have lower or higher costs due to lack of access to medical care  or additional health care requirements to manage comorbidities . Older database analyses have shown healthcare costs to be higher for patients with lower CD4 cell levels. Two studies reported mean annual costs of patients with <50 cells/µL to be $35,000–$40,000, while patients with >350 cells/µL were estimated to have annual costs of approximately $15,000 2000  USD. HeaMarketScan database provides what is perhaps the most clinically rich, commercially available data set for costs stratified by CD4 cell count. However, it has a few limitations. The CD4 laboratory data were primarily from a small proportion of total patients who lived in the Northeast region (94.4%), which may limit generalizability of these results to the entire population if differences exist in treatment practices and associated costs across regions. The database was limited in the availability of lab results for patients outside of the Northeast region. The limited geography among patients with CD4 cell measurements and the fact that all patients in our sample had healthcare insurance and access to medical care also contributed to the small number of (patients with) measurements in the lower CD4 stratum (12 out of 803 with <100 cells/µL).The Due to complex treatment patterns within the Newly Treated Cohort, we were able to select patients with concurrent use of NRTIs alongside their third-agent treatments but were unable to ensure that these patients were consistently using at least two NRTIs, which would be consistent with clinical guidelines for appropriate HIV treatment. However, given that the US guidelines regarding multiple NRTI use within combination treatment regimens have existed for nearly 20 years, the likelihood that any patient in our sample used only one NRTI is probably low.Although costs by CD4 cell level and line of treatment were measured within the same database, these two estimates are possibly correlated. As patients progress through lines of treatment, their CD4 cells may concurrently decline, an important confounder in cost estimates. Because of the limited CD4 cell data available, there was very little patient overlap between the Newly Treated and the CD4 Measurements Cohorts (n = 29), precluding any adjustment of the treatment line costs for CD4 cell level. A data set based on a chart review or other clinical records may be necessary to properly estimate costs of treatment switching while adjusting for reasons for switch and CD4 cell levels. This is evidenced by the increasing proportion of non-ARV drug costs , which cMarketScan and may have underestimated costs for patients who receive supplemental healthcare not included within the claims database. This is a particular problem for estimating the costs of HIV in the US, where AIDS Drug Assistance Programs provide treatment to approximately one-third of infected patients A limitation typical of all retrospective claims database analyses is that cost data were limited to medical care received within plans included in To obtain an analysis as close to the “real world” as possible, we restricted the primary CD4 cell measurement analyses to be representative of expected treatment patterns and the treatment line analyses to be representative of switching for reasons of treatment failure. These results confirmed that HIV infection continues to be a costly disease even in the era of modern ARV treatments and provided a recent estimate of costs by treatment line and CD4 cell level, filling a gap in the US economic literature. Our analysis showed that during the years 2007 through 2011, US patients receiving first-line treatment had significantly lower total healthcare costs than patients receiving second-line or third-line therapies. This indicates that further investment to develop additional treatment regimens to suppress viral replication and preserve patients' CD4 cells through multiple lines of treatment are fundamental to long-term HIV disease management and reduction of the economic burden of advanced HIV disease.


Multi-experienced HIV-infected patients bear the burden of treatment-related toxicities over the years. This study evaluated the correlation between bioimpedance- and DXA-quantified changes of adipose tissue with duration of infection and duration of combined antiretroviral therapy (cART) in HIV-seropositive patients.A cross-sectional study, belonging to prospective grant PNCDI2 no.62077/2008, was conducted in a national reference hospital in 2011-2012. DXA whole-body adipose tissue analysis and bioimpedance analysis revealed fat and lean tissue (% and grams), android/gynoid distribution, arm+legs/trunk ratio and waist/hip ratio (WHR).There were 78 patients enrolled, including the control seronegative group. HIV-seropositive patients had equal sex distribution, median age of 33 years with mode of 20 years and body mass index of 23.6 kg/sqm . Duration of diagnosed infection, duration of cART and number of previous therapeutic regimens had medians of 69 months , 57 months  and 2 , respectively. Among all variables, WHR correlated with duration of infection , duration of cART  and duration of number of therapeutic regimens . Also fat and lean tissue, as grams, correlated with duration of cART - rho= -0.26, p=0.048 for both.In this young treatment-experienced HIV-infected population undergoing antiretroviral therapy, the waist/hip ratio correlated best with the number of previous therapeutic regimens, inversely proportional. Adipose changes, measured by bioimpedance and DXA, were rather correlated with treatment duration than with time from diagnosis. Patients experiencing the history of antiretrovirals still present adverse effects on long term, which could influence their adherence to antiretrovirals.


Since antiretroviral therapy must be taken lifelong, persistence and safety have become the goals to achieve. Protease inhibitors, in particular atazanavir (ATV) with or without ritonavir (r), represent a highly prescribed class in real life long-term treatment.We conducted a retrospective cohort study in HIV-1-positive patients who were followed at the Infectious Diseases Unit, DIBIC Luigi Sacco, University of Milan. Data regarding viral load, CD4 lymphocytes and the mean blood chemistry parameters were collected at baseline, first, third, sixth months from the beginning of therapy and then every six months. Factors related to persistence of therapy with ATV and time-dependent probability to reach a CD4 cells count >500 cells/µL were evaluated with Kaplan-Meier curve and Cox model.A total of 1030 patients were evaluated: 183 received therapy with ATV/r as naïve, 653 switched to ATV/r as a second or following line and 194 switched to unboosted ATV from previous ATV-free regimens. A total of 138 patients shifted to unboosted ATV from a previous ATV/r regimen (17 from naïve ATV/r and 121 from experienced ATV/r). The median duration of therapy was 38 months (95% CI 29–73) in ATV/r naïve patients, 36 months (95% CI 23–53) in unboosted ATV group and 35 months (95% CI 31–43) in patients switched to ATV/r. We observed no significant difference in the persistence of the three regimens (p=0.149). Female  and patients with CD4<200 cells/µL at baseline (HR=1.433 95% CI 1.086–1.892 p=0.011) were at increased risk of regimen interruption, whereas starting therapy with a backbone containing abacavir  resulted protective. In multivariate analysis no significant difference between the three regimens was observed regarding reaching a count of CD4 cells >500 cells/µL. Factors associated to a poor CD4 gain were each extra Log of viral load at baseline  and CD4<200 cells/µL at ATV start ; conversely, females  had a higher probability of CD4 recovery.Antiretroviral regimens containing atazanavir with or without ritonavir were durable and well tolerated, an elevated viral load and CD4 <200 cells/µL at baseline resulted related to regimen discontinuation and reduced CD4 recovery.


Approximately 30% of HIV-1-infected patients receiving antiretroviral therapy who achieve virologic control have unsatisfactory immune reconstitution, with CD4+ T-cell counts persistently below 350 cells/μL. These patients are at elevated risk for clinical progression to AIDS and non-AIDS events. CD4+ T-cell depletion following infection and persistent immune activation can partially explain this low CD4+ T-cell recovery. Recent data suggest a link between the tryptophan oxidation pathway, immune activation and HIV disease progression based on overstimulation of the tryptophan oxidation pathway by HIV antigens and by interferon-gamma. This overstimulation reduces levels of circulating tryptophan, resulting in inflammation which has been implicated in the development of neurocognitive dysfunction. Niacin (vitamin B3) is able to control the excess tryptophan oxidation, correcting tryptophan depletion, and therefore represents an interesting strategy to improve CD4 recovery.We aim to design a crossover proof-of-concept study to assess supplementation with an extended-release form of niacin (Niaspan FCT™) in combination with antiretroviral therapy, compared to antiretroviral therapy alone, on T-cell immune activation as defined by changes in the percentage of CD8+ CD38+ HLA-DR+ T-cells.This randomized, open-label, interventional crossover study with an immediate versus deferred use of Niaspan FCT for 24 weeks will assess its ability to reduce immune activation and thus increase CD4 recovery in 20 HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression. A substudy evaluating neurocognitive function will also be conducted.This randomized trial will provide an opportunity to evaluate the potential benefit of oral extended-release niacin, a drug that can indirectly increase tryptophan, to reduce immune activation and in turn increase CD4+ T-cell recovery. The study will also allow for the evaluation of the impact of Niaspan FCT on neurocognitive function in HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression.NCT02018965).This study was registered with ClinicalTrials.gov on 17 December 2013 (registration number: The online version of this article (doi:10.1186/1745-6215-15-390) contains supplementary material, which is available to authorized users. Approximately 30% of antiretroviral therapy (ART)-treated patients who achieve virologic control have an unsatisfactory immune reconstitution, with CD4+ T-cell counts persistently below 350 cells/μL , 2. ThesRecent data suggest a link between the tryptophan (Trp) oxidation (TrpOx) pathway , immune activation and HIV disease progression , 16. Indet al. [Trp is an essential amino acid required for the biosynthesis of proteins, serotonin and niacin, which is a vitamin B3 compound . Both HIet al. ; both diIn vitro data has shown that Trp depletion can significantly impede T-cell reconstitution by reducing both CD4+ T-cell proliferation and survival [survival , 26. ARTsurvival –29. Ongosurvival , 31.Collectively, these data strongly suggest that declining Trp levels are associated with immunosuppression, mood disturbances and cognitive impairment, thereby leading to the hypothesis that oral Trp supplementation should contribute to improved immune responses and cognitive function. However, in clinical studies, direct Trp supplementation in the presence of ongoing immune activation was associated with increased risks of neurotoxic problems, oxidative stress and possibly the development of eosinophilia-myalgia syndrome, a rare and deadly flu-like condition , 33.An indirect way to increase Trp levels while minimizing the side effects of direct Trp supplementation is to provide exogenous niacin, a key player in the TrpOx pathway. Niacin, also known as vitamin B3, nicotinic acid and vitamin PP, is able to control excess Trp oxidation by a process of reverse inhibition . It has In one small study, four ART-naïve HIV-infected individuals were treated with a supplement of 3,000 mg/day of immediate-release (IR) niacin for two months. The treatment was not associated with the usual side effects such as flushing (described as warmth), redness, itching and/or tingling, and resulted in a 40% increase in plasma Trp levels and clinical benefits . HoweverNiaspan FCT™ is an extended-release (ER), oral form of niacin that is dosed once each night when cholesterol synthesis and fatty acid mobilization are at their peak. Niaspan FCT has intermediate-release characteristics and is thus designed to minimize both liver enzyme elevation and flushing .Niacin is the most effective agent at raising high-density lipoprotein cholesterol levels, either as a monotherapy or in combination with other agents, despite the fact that it has not consistently shown improved patient outcomes . HoweverSince the beginning of the AIDS epidemic, HANDs have been commonly observed in individuals in the advanced stage of the disease . More reet al. reported that enhanced Trp degradation was associated with impaired QoL in patients with cancer [Schroecksnadel h cancer . Collecth cancer , 22.Given that ER niacin can indirectly increase Trp levels, we seek to further assess the effects of ER niacin on neurocognitive functions and QoL among HIV-infected individuals who choose to participate in a substudy of this clinical trial.This pilot crossover study will be conducted to examine the ability of ER niacin to reduce immune activation, which may in turn increase CD4 recovery in HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression. The main hypothesis to be tested is that ER niacin co-administration with ART will be associated with decreased T-cell activation as determined by changes in the percentage of activated CD8+ CD38+ HLA-DR+ T-cells. If so, during administration of ER niacin, participants should experience a time-dependent decrease in T-cell activation, and such a decrease may reverse after ER niacin is discontinued. We will also examine the effects of ER niacin on cognition. To this end, we will assess the neurocognitive function and QoL in participants using standardized neuropsychological (NP) tests and questionnaires.This is a randomized, open-label, interventional crossover study  number PT006) with an immediate versus deferred use of ER niacin for 24 weeks to assess the ability of oral ER niacin to reduce immune activation (as determined by the percentage of activated CD8+ CD38+ HLA-DR+ T-cells), which may in turn increase CD4+ T-cell recovery in HIV-infected individuals who achieve suboptimal immune responses despite sustained virologic suppression. Participants will continue to take their ART treatment as prescribed throughout the study. The study design is shown in Figure Participants will be recruited at the Chronic Viral Illness Service, Montreal Chest Institute of the McGill University Health Centre (MUHC), one of the largest hospital-based centers in Montreal, Quebec, Canada, and at the Clinique médicale l’Actuel, the largest private medical centre in Montreal.Prior to physical contact with any patient by the study staff, the HIV specialist at each HIV clinic will conduct chart reviews of prospective HIV-positive participants ahead of their scheduled clinic visits to determine which patients successfully treated with ART did not complete immune restoration. Based on this information, the research nurse and/or the referring HIV specialist will approach potential trial participants upon arrival at the clinics. At this time, the trial staff will inform the patient about the trial and invite him or her for eligibility screening and possible enrolment into the trial. Prior to enrolment, trial participation eligibility will be documented, and written informed consent will be obtained from eligible patients by the treating physician  visit to be considered eligible for entry into the study: 1) documented HIV infection by western blot, enzyme immunoassays or viral load assay; 2) aged 21 years or older; 3) viral load <50 copies/mL for the last three months; 4) CD4+ T-cell count ≤350 cells/μL; and 5) on stable ART ) for more than 12 months.Subjects who meet any of the following criteria will be excluded: 1) prior history of hypersensitivity reaction to niacin or any other component of the study drug; 2) prior history of flushing; 3) liver disease (including co-infection with hepatitis B or C virus) or unexplained persistent elevations of serum transaminases; 4) alanine aminotransferase (ALT) or aspartate aminotransferase (AST) or alkaline phosphatase >2.5 times the upper limit of normal (ULN); 5) active duodenal or gastric peptic ulcer; 6) active bleeding disorders; 7) history of gout; 8) active AIDS events in the last three months as determined by the treating physician; 9) unstable angina or acute phase myocardial infarction; 10) diabetic or potentially diabetic with hypercholesterolemia; 11) renal dysfunction; 12) co-enrollment in another study involving neurocognitive evaluation; or 13) pregnant or nursing or planning to become pregnant. Infection with syphilis, for which testing is done at screening, is not an exclusion criterion if the patient is treated; participants will be tested again at weeks 24 and 48.The study medication is ER niacin  500 and 1,000 mg ER film coated tablets (FCT) which will be taken at bedtime. The dose for this study is that recommended in the Niaspan FCT product monograph for the treatment of dyslipidemia . ER niacOnce a participant is deemed eligible and has provided consent, they will be randomized to one of two treatment arms in a 1:1 ratio to receive the immediate or the deferred intervention with ER niacin. Prior to the initiation of the trial, a CTN staff member not directly associated with the implementation of the trial will use an SAS module  to generate sequential participant study identification numbers which will be loaded onto a webpage randomization system. The treatment groups will be: arm 1: ER niacin supplementation for 24 weeks followed by 24 weeks of ART alone, and arm 2: 24 weeks of ART alone followed by ER niacin supplementation for 24 weeks. When a participant is deemed eligible, the study coordinator signs onto the password-protected randomization webpage which issues the allocation. A transaction file records the study identification number, allocation and date and time of randomization.Scheduled visits for arm 1 of this study will be screening (up to four weeks prior to treatment), week 0 (baseline), week 4, week 8, week 12, week 16, week 20, week 24, and week 48/final visit (or other if terminating early). Table Scheduled visits for arm 2 of this study will be screening (up to four weeks prior to study enrolment), week 0 (baseline), week 24, week 28, week 32, week 36, week 40, week 44 and week 48/final visit (or other if terminating early). Table At baseline, the following clinical information will be collected for all participants: age, education, ethnicity, handedness, communication skills , list of current psychoactive medication, dosage and date of treatment initiation, psychiatric disorders, duration of HIV infection, current ART regimen (duration in months), review of ART history, CD4+ T-cell count within the past three months, nadir CD4+ T-cell count, HIV plasma viral load within the past three months and any preexisting medical conditions, signs, or symptoms.Assessments performed at scheduled study visits will monitor safety  and will determine the dermatological effects of flushing. At the follow-up visits, some or all of the following assessments will be done: plasma HIV RNA levels, hematology, fasted biochemistry, CD4+ T-cell and CD8+ T-cell counts, plasma levels of Trp and Kyn, T-cell activation and inflammatory markers and testing for syphilis if the participant tested positive at baseline (week 0).A dietary survey to determine the pre-study dietary niacin levels for each participant will be completed at week 0 (baseline). This survey will be performed by an MUHC dietician with expertise in HIV patient management. Participants will be instructed to keep their diet constant during the study.Adherence for both ER niacin and ART will be measured using the Simple Medication Adherence Questionnaire (SMAQ) . This quQoL will be assessed with the World Health Organization quality of life instrument in patients with HIV infection brief (WHOQOL-HIV BREF) , an instMood will be assessed by the Center for Epidemiologic Studies Depression scale (CES-D), and the Profile of Mood State (POMS) questionnaire. The CES-D is a 20-item short self-report scale designed to measure depressive symptomatology in the general population . It has Changes in cognition will be assessed with the Hopkins Verbal Learning Test Revised (HVLT-R™) and the Cambridge Neuropsychological Test Automated Battery (CANTAB™). The HVLT-R is a pencil and paper test that explores verbal episodic memory, learning, recall and verbal recognition processes . The CANThe main hypothesis to be tested in this study is that ER niacin co-administration with ART will be associated with decreased immunosuppressive Trp catabolism and T-cell activation as determined by the proportion of activated CD8+ CD38+ HLA-DR+ T-cells.The primary objective of this study will be to assess the impact of ER niacin supplementation in combination with ART compared to ART alone on T-cell immune activation as defined by changes in the percentage of CD8+ CD38+ HLA-DR+ T-cells.The secondary objectives of this study will be to: 1) assess the change in total CD4+ T-cell count after ER niacin administration; 2) explore the effect of ER niacin on Th17 and Treg cell frequencies in blood; 3) explore the effect of ER niacin on cytokines and inflammatory markers such as interferon-alpha (IFN-α), IL-1, IL-6 and IL-17, interferon gamma-induced protein 10 (IP-10), D-dimers, highly-sensitive C-reactive protein (hsCRP) and LPS; 4) assess the influence of ER niacin on Trp plasma levels; 5) assess changes in cholesterol and triglyceride levels; 6) evaluate the impact of ER niacin on QoL, fatigue, depression and neurocognitive scores; and 7) explore ER niacin tolerance.The primary endpoint of this study will be changes in the percentage of CD8+ CD38+ HLA-DR+ T-cells which will be used as a marker for the change in the level of T-cell activation. Enumeration of the CD38 activation marker on total CD4+ and CD8+ T-cells, as well as on naïve, memory and effector cell subsets, will be performed by eight-color flow cytometry. This will allow for the precise identification of which cell subpopulation(s) is or are more likely to downregulate CD38 expression following ER niacin administration.The absolute numbers of activated T-cell subsets will be computed using: 1) the white blood cell (WBC) count; 2) the percentage of lymphocytes; 3) the percentage of CD4+ and CD8+ T-cells; and 4) the percentage of each CD4+ or CD8+ T-cell subset .Levels of T-cell activation will be assessed in blood samples on weeks 0, 24 and 48. This analysis will be performed using specific monoclonal antibodies (mAbs) as previously reported , 52. Bri6 cells/mL per well and will be stimulated with 5 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin  for two hours at 37°C. A total of 2 μg/ml brefeldin A  will then be added to block cytokine secretion and cells will be cultured for 18 hours at 37°C. PBMCs will be surface stained followed by fixation and permeabilization using the Cytofix/Cytoperm Permeabilization kit  for intracellular staining for IL-17A and IFN-ɣ (positive control). Treg cells will be defined by flow cytometry as being CD3+ CD4+ CD25high CD127low FoxP3high and Th17 cells as being CD3+ CD4+ IL-17A+ upon PMA and ionomycin stimulation [PBMCs will be cultured in 48-well culture plates at 0.5 to 1 × 10As IDO enzyme is expressed by monocytes and dendritic cells, we will also assess the impact of ER niacin on the activation of monocytes and dendritic cells (DCs). The expression of the activation markers CD69 and HLA-DR on CD14+ monocytes, as well as the expression of CD86 and CD83, markers of DC activation and maturation on both CD11c+ myeloid DCs and CD123+ plasmacytoid DC, will be evaluated by flow cytometry.Although inflammatory markers such as D-dimer and hsCRP are related to cardiovascular non-AIDS events during HIV infection, it has become clear that HIV induces a hypercoagulable state as a consequence of impaired endothelial function despite ART. The effect of ER niacin on the levels of IFN-α, IL-1, IL-6, IL-17, IP-10, D-dimer and hsCRP, as well as on the microbial translocation markers sCD14 and LPS, will be assessed by Enzyme-Linked Immunosorbent Assay ELISA .Plasma levels of Trp and Kyn will be measured by an automated on-line solid-phase extraction-liquid chromatographic-tandem mass spectrometric (XLC-MS/MS) method as previously described , 53.in vivo data, a convenience sample of 20 participants was chosen without formal power calculations. The data that will be obtained should be sufficient to assist in such calculations for future large studies.Because this proof-of-concept study will explore a phenomenon with little The primary comparison is the change in the percentage of CD3+ CD8+ HLA-DR+ T-cells between the period of ER niacin plus ART  and the control periods of ART alone . If the treatment effects in the two time periods look distinctly dissimilar, which may occur due to carryover effects of treatment, we will focus on the comparison between arms 1 and 2 at the end of 24 weeks.In each time period, a between-groups t-test will be used to compare the change in the percentage of CD3+ CD8+ HLA-DR+ T-cells during the ER niacin plus ART treatment with the change in the percentage of CD3+ CD8+ HLA-DR+ T-cells in the ART treatment alone. If the results of these comparisons in the two treatment periods are similar, then within each treatment order, a paired t-test will be used to compare the change in the percentage of CD3+ CD8+ HLA-DR+ T-cells during the ER niacin plus ART period with the change in the percentage of CD8+ CD38+ T-cells during the ART-alone period and the results of these two comparisons will be combined to generate a pooled estimate of the treatment effect. The same analytic approach will be used for the secondary outcomes. The primary comparison would be based on 20 pairs of observations, with each subject receiving active ER niacin compared to the control time-points  in different time-points. The ratio of the difference between the study groups to the standard deviation of the differences would have to be fairly large (0.625) to achieve a power of 80%. All statistical tests performed will be two-tailed with significance determined by reference to the 5% level, unless otherwise stated.The same analytic approach will be used for the secondary outcomes as for the primary outcome. The secondary outcomes are: the change in total CD4+ T-cell count, changes in Th17 cells and Treg cells in blood and gut mucosa samples (from substudy), changes in cytokines and in inflammatory markers such as IFN-α, IL-1, IL-6, IL-17, IP-10, D-dimers, hsCRP, sCD14 and LPS, the change in Trp plasma levels, changes in monocyte and dendritic cell immune activation, changes in cholesterol and triglycerides and changes in QoL, fatigue, depression and neurocognitive scores, using computerized and pencil and paper tests .All patient data and electronic files will be stored in the secure environment of the Chronic Viral Illness Service of the McGill University Health Centre,  and in the CTN database . All data will be anonymized for publication. Only researchers affiliated with the study will have access to participant data. Study progress and safety will be evaluated in an ongoing fashion by a CTN Data and Safety Monitoring Committee (DSMC).Biological samples will be stored at the Chronic Viral Illness Service of the McGill University Health Centre for analysis in the current trial and for future use in ancillary studies.The safety and tolerability of Niaspan FCT will be evaluated by vital signs and AE monitoring, both spontaneously reported by the participant and actively sought at each clinic visit. In addition, biological tolerability will be evaluated by hematology, biochemistry and any other clinical, laboratory or diagnostic tests conducted on the participants during the course of the study. The trial medical monitor and the trial investigator will receive and review the lab results for all participants for all assessed safety variables. The DSMC will also review safety information. The toxicity of Niaspan FCT will be assessed using the World Health Organization toxicity scale. Any AEs and laboratory abnormalities that occur during the study will be evaluated by the trial investigators and grade 3 and 4 AEs will be recorded on case report forms (CRFs). Grades 1 and 2 AEs specific to the effects of flushing will also be recorded on the CRFs. The ‘Bays-Ballantyne niacin-induced flushing and persistence of use questionnaire’ will be used during ER niacin administration to follow the participants’ perception of the side-effect of flushing . If requWritten informed consent will be obtained from all study participants. The study protocol and consent forms have been approved by the Research Ethics Board of the McGill University Health Center (reference number: 11-019GEN) and by Health Canada’s Therapeutic Products Directorate (reference number: 9427-M2303-44C). The study will be conducted in accordance with the applicable Health Canada regulations, International Conference on Harmonization guidelines on current Good Clinical Practice and the Declaration of Helsinki.ART therapy has significantly decreased HIV-associated morbidity and mortality over the last 15 years. Despite the substantial benefits of ART, immune reconstitution may not be complete for approximately 30% of patients who achieve virologic control . These pWe have designed a proof-of-concept study to assess supplementation with Niaspan FCT (an ER form of niacin) in combination with ART, compared to ART alone, on T-cell immune activation as defined by changes in the percentage of CD3+ CD8+ HLA-DR+ T-cells. A reduction in immune activation may improve CD4 recovery in HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression. Our study also includes a neurocognition substudy that examines the effect of niacin on QoL, fatigue and depression.It has been previously reported that a 10-fold difference exists between uninfected healthy controls and treated aviremic HIV-infected participants in the level of activated CD3+ CD8+ HLA-DR+ T-cells , 56. It Another objective of this study is to examine Niaspan FCT tolerance. Flushing at the initiation of niacin therapy is the most commonly reported side effect and is defined as redness, warmth, itching and/or a tingling sensation on the face, neck, chest and back . This isFor this pilot study, a convenience sample of 20 participants was chosen without formal power calculations. Should the results of this study suggest that niacin is tolerable, effective at reducing T-cell activation and/or has a positive effect on QoL, fatigue, depression and neurocognition, future studies will be performed. As a crossover study, two other comparisons will be made between the two arms: 1) the period of niacin plus ART in arm 1 will be compared to niacin plus ART in arm 2; 2) the period of ART alone in arm 1 will be compared to ART alone in arm 2. This will allow for the further evaluation of the potential continuity of the hypothesised effect of niacin following its discontinuation. The data obtained in this pilot study should be sufficient to assist in formal power calculations for such future studies.The results of the trial, regardless of outcome, will be disseminated through the traditional routes of scientific peer-reviewed publications, through international and national specialist conferences, and through the CTN. BL and MAJ will be responsible for initially drafting these manuscripts and professional writers will not be used for any of the publications. Authorship will be based on the criteria defined by the International Committee of Medical Journal Editors . We aim The trial was started in February 2012. We expect enrolment to be complete by November 2014.Participant Informed Consent.(PDF 122 KB)Additional file 1:


Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations.HIVDB.stanford.edu.The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively.The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence. Antiretroviral therapy (ART) has changed the dynamic of the HIV epidemic through suppression of HIV-1 RNA to undetectable plasma levels (<50 copies/ml). However, latent HIV infection of CD4+ T cells, established early after infection, necessitates lifelong ART . TreatmeGenotyping of the proviral reservoir may inform treatment decisions, identify occult DRM and uncover community phylogenetic relationships. Retrieving HIV resistance data from PBMC may be less expensive, but the clinical significance of DRM in proviral DNA remains controversial ,15–17. MIntegrated HIV proviral DNA in T central memory cells can be transcriptionally reactivated leading to virological failure ,2,21. SoThe resistance mutations detected by genotyping proviral DNA after virologic failure have been found to closely mirror those obtained from concomitant vRNA ,27–29 unHere, to assess the prevalence and the pertinence of DRM archived in proviral DNA, 120 subjects, 82 suppressed to <50 copies/ml and 38 with 68-171,000 copies/ml of plasma RNA, were sequenced from one or both compartments. We identified virologic failures among suppressed patients by evaluating the longitudinal relationship between archival DNA and RNA drug resistance patterns. PBMC genotyping may identify transmission linkages and add information to optimize ART among suppressed patients.One hundred and twenty HIV infected individuals were recruited from the Peninsula AIDS Research Consortium clinic at the San Mateo County AIDS Program and the San Francisco Public Health Department clinic, and enrolled in a longitudinal study of drug resistance. Participants were recruited from public health clinics during attendance for routine HIV care.Written informed consent was obtained for each participant and the study was approved by the institutional review board “Administrative Panels for the Protection of Human Subjects” from Stanford University for the protocol 7861 “Antiretroviral Drug Resistance in Blood, Oral, and Genital HIV-1: Implications for Pathogenesis and Transmission of Drug Resistance and Secondary Prevention in Community Based Treatment Programs”.Study visits occurred every 6 months over a total of 2 years. Blood specimens were obtained at each visit and transported to the laboratory within 6 hours of collection. Plasma was removed after centrifugation and PBMC were separated from whole blood by density gradient centrifugation and stored at -70°C in 90% human serum, 10% Dimethyl Sulfoxyde (DMSO). Viral load (VL) and CD4 measures were performed by the clinical laboratories at San Mateo County hospital using Roche Cobas Amplicor Monitor v1.5  and BD FACSCalibur , respectively.Virus was concentrated from 1 ml of plasma by centrifugation for 20 min at 20,000g to pellet the virus, 800μl of supernatant was discarded and RNA extracted from the 200μl residua with QIAamp viral RNA Mini kit  as per the manufacturer’s protocol. vRNA was used as template for reverse transcription using SuperScript II Reverse Transcriptase  and random hexamer primers, using cycling parameters of 10min at 25°C, 30min at 45°C and 15min at 70°C, and the RNA template degraded with RNAse H for 20 min at 37°C. DNA was extracted from pelleted PBMC using QIAamp DNA Blood Mini kit (QIAGEN Inc.).At baseline, vRNA and proviral DNA, were sequenced from viremic patients (n = 38) while suppressed patients (n = 82) provided only a proviral DNA genotype. Only the vRNA was sequenced from patients who virologically failed during follow-up (n = 11).st round PCR primers; MAW26 (5´-TTGGAAATGTGGAAAGGAAGGAC-3´) and RT21 (5´-CTGTATTTCTGCTAT TAAGTCTTTTGATGGG-3´) and 2nd round with PRO1 (5´-CAGAGCCAACAGCCC CACCA-3´) and RT20 (5´-CTGCCAGTTCTAGCTCTG CTTC-3´), with Platinum Taq DNA Polymerase (Invitrogen), 50pmoles of each primer, 2mM of Magnesium Chloride and 0.5mM of dNTPs. Forty cycles for the 1st and 35 for the 2nd round of 94°C for 15s, 55°C for 20s and 72°C for 2 min, then 10min at 72°C produced a nested PCR product, purified using QIAquick PCR Purification kit (QIAGEN Inc.), as per manufacturer’s protocol. After quantification, 30ng were sequenced using the Big Dye Terminator kit  and a set of 4 bidirectional primers by capillary electrophoresis on a 3130xl Genetic Analyzer (Life Technologies).A 1,300-bp fragment encompassing the protease (PR) and the first 300 amino acids of the reverse transcriptase (RT) gene was generated from DNA and cDNA for PCR amplification, followed by a nested PCR with 1Sequences were assembled using Geneious R6 genetic analyzer  . DRM werOne hundred and twenty subjects were enrolled: their demographic, immunologic and virologic baseline characteristics are presented in 3 among suppressed patients and 305/mm3 among viremic patients. Cumulative ART history included exposure to TDF (85%), AZT and/or d4T (57%) and 57% and 66% had received an NNRTI- or PI- based regimen respectively.At study entry 82 subjects (68%) were suppressed to <50 copies/ml and 38 (32%) had >50 copies/ml of plasma HIV-1 RNA with a median of 5,865 copies/ml. The median CD4+ cells count was 422/mmOverall, resistance profiles in both vRNA and proviral DNA were concordant in 32/38 viremic patients at baseline (84%). Twenty-four patients had wild-type genotype in both vRNA and DNA and 14 had DRM see ; 12 had Among viremic patients, 18/38 (47%) had been on treatment at baseline for a median of 4 years (ranging from 1 to 13 years). Half were wild-type and the other half harbored resistance in vRNA and proviral DNA, and 4 of them displayed DRM in vRNA that were not present in proviral DNA  in contrast with the concordant genotypes among 32 with higher virus load . Considering only viremic patients with VL >1,000 copies/ml, concordance between vRNA and proviral DNA was 97%. Finally, among 12 patients known to have been previously exposed to NNRTI, 4 had NNRTI resistance mutations in proviral DNA while among 22 exposed to PI, only 3 had PI resistance mutations.pol gene with modestly higher CD4 cell counts .Most of the 82 suppressed subjects were on treatment, 3 were reported to be off of treatment and harbored wild-type genotypes in proviral DNA see . Twenty-Of the 82 study subjects with <50 copies/ml at study entry, 11/82 (13%) subsequently had a VL >50 copies/ml at a mean of 39 weeks. Seven of the 11 had virologic failure with >1,000 copies/ml , among them 3 had DRM in proviral DNA at baseline , this group shares common characteristics, which highlight the dynamic of HIV epidemic among drug users and their sexual partners in the 1990s .In summary, our study showed that proviral DNA and vRNA drug resistance profiles of viremic subjects are concordant  and are consistent with a median of 87% describe in the literature ,41–45. DRNA genotyping is the gold-standard template for HIV drug resistance testing and requires RNA extraction, reverse-transcription followed by PCR and sequencing reactions. vRNA is labile and genotyping protocols mandate separation and storage of plasma at -70°C within 6 hours. In addition, reverse transcription is the most critical and costly step in RNA genotyping . While tDRM, predominantly NRTI and NNRTI resistance mutations, were found in PBMC proviral DNA among 26% of subjects with suppressed plasma RNA levels who enrolled in a community treatment program in California. Those with PBMC DRM had failed previous ART and were subsequently suppressed on either a boosted PI or NNRTI based regimen. Stable reservoirs of NRTI and NNRTI DRM (particularly M184V and K103N) in PBMC are common at virologic failure of a first-line NNRTI based regimen ,34,47–51The significance of low level viremia  remains controversial –61. ThesIn assigning clinical significance to DRM in PBMC, the study is limited by the potency of modern suppressive regimens with relatively small numbers with virologic failure over a mean follow-up of 46 weeks. However the frequency of DRM at entry among suppressed individuals who developed virologic failure 4/11 (36%) was similar to DRM found among those who remained suppressed, 17/68 (25%), suggesting that DRM in PBMC are not themselves a risk for virologic failure, and were only expressed in a fraction of those with later virologic failure.nd generation NNRTI and 3TC/FTC respectively and can be attributed to APOBEC3G/F editing [APOBEC3 cytidine deaminases restrict endogenous and exogenous retroviruses by inducing G to A hypermutations in GG or GA dinucleotides context. HIV-1 counteracts this antiviral activity through Vif. Kieffer et al showed that hypermutated sequences comprise >9% of archived species in resting CD4+ cells . In our  editing ,68.3) reflecting a robust immune response. However, archival DRM, even in hypermutated sequences, can shape the phenotype of the circulating viral population through recombination with replication competent virus, leading to resistant strains [Fourati et al demonstrated that the HIV-1 genome is often defective in PBMC after a long-term HAART . In our  strains . Defecti strains .Sequencing proviral DNA provided additional information about DRM, which may impact treatment decisions, particularly in the setting of low level viremia or suppression. Moreover, new information about phylogenetic relationships and the prevalence of DRM, albeit as archival mutations among suppressed individuals may identify historic transmission dynamics in a community. Here we found linkage among African American men and women in East Palo Alto, consistent with a reported peak in the IDU epidemic more than 20 years ago in this community . Althoug