The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space.HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3×10 Hepatitis C virus is an RNA virus infecting more than 170 million people throughout the world. Like the majority of other RNA viruses, it is characterised by a high level of genetic variability that is particularly marked at the level of the E1 and E2 genes encoding viral envelope glycoproteins. The 3’ end of the E2 gene (hypervariable region 1: HVR1) shows frequent mutations that have the nature of a quasispecies (QS), defined as a population of closely related but different viral genetic sequences co-existing in the host and evolving as a single unit Some authors have suggested that selection pressure due to the host's immune response plays a fundamental role in determining the significant differences in complexity observed in the QS of patients with different disease outcomes Over the last 15 years, the development of highly active antiretroviral therapy (HAART) has made it possible to restore immune responses in a large percentage of subjects affected by severe immunodeficiency due to HIV One of the limitations of these studies is that they were mainly based on a “phenetic” approach , whereas more sophisticated phylogenetic methods have recently been developed that make it possible to estimate the rate of evolution of viral genes and reconstruct their trees on a real timescale using molecular clock models based on sequences obtained at different times (heterochronous sequences) The application of the coalescent theory In a preliminary study of 32 patients with HIV/HCV coinfection enrolled in a HAART protocol, we found that, although most had very low plasma HCV RNA levels before treatment, HCV viral load remained unchanged for six months or more in the majority of immunological non-responders but increased in a large proportion of the patients whose CD4+ cell counts significantly increased more than 1.5-fold during treatment. In order to investigate the forces acting on the evolution of HCV QS during HAART and clarify the effect of a restored immune response on viral diversification, we studied the within-host dynamics of the QS populations in a subgroup of these showing an immunological response to HAART or not. QS heterogeneity was characterised before and after at least one year of HAART, and its molecular evolution and population dynamics was studied phylogenetically and compared with those of two HIV-negative and drug naïve subjects.The authors were exempted from prior approval by the IRB of Luigi Sacco Hospital, Milan, Italy. The analyses were conducted on stored serum samples collected anonymously and identified through an internal code. Individuals' clinical and demographic data, were recorded anonymously in a database. The informed oral consent obtained from our patients was registered in each chart, which at that time was in paper format. The study was conducted in accordance with the 1964 Declaration of Helsinki and the ethical standards of the Italian Ministry of Health.The study patients were retrospectively selected from a cohort of 32 patients with HIV/HCV coinfection entered in a HAART protocol whose main demographic, immunological, virological and clinical data were stored in a database see . The stoA preliminary analysis of these 32 patients showed that all of them had a low plasma HCV RNA load at the time of enrolment (mean limiting dilution titre: log 1.59±1.4 PUs-see 5′-GGDCAYCGMATGGCNTGGGA-3′ (positions 1284-1303) and 5′-GGNGSRTARTGCCAGCARTANGG-3′ (positions 1813-1791), and inner primers 5′-GCTTGGGATATGATGATGAACTGGTC-3′ (positions 1296-1321) and 5′GGTGTGGAGGGAGTCATTGCAGTT3′ (positions 1646-1623). The sequences encompassing the E1/E2 were inserted in the plasmid vector pGEM  and transfected into competent Escherichia coli JM109 cells. After overnight incubation at 37°C in agar medium, the insertion was checked by means of PCR using HCV envelope inner primers on white colonies. A total of 260 HCV-positive clones from each sample were then bi-directionally analysed by means of automated sequencing  in the presence of the specific inner primers described above, and the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems).Total RNA was extracted from 200 µl of plasma using a commercially available kit based on affinity column purification . HCV RNA was detected by means of reverse transcription (RT) nested-PCR for the amplification of the 5′ non-coding region (NCR) sequences, and its plasma levels were measured by means of limiting dilution PCR, as described elsewhere Each sample was phylogenetically analysed using a 261-nucleotide sequence included in the E1/E2 genes and encompassing HVR1 (nucleotides 1491-1571). The sequences have been submitted to GenBank (accession numbers to be given).1:AF142392; 2:D31972); HCV2e/f (D49757); HCV3a (D14311); and HCV4a (Y11604) For the purposes of subtyping, the patients' sequences were aligned with nine HCV reference sequences retrieved from the GenBank database (accession numbers in brackets): HCV1a (M62321); HCV1b (D10074); HCV2a (D00944); HCV2b (D10988); HCV2c  were aligned using the CLUSTALW program S) and non-synonymous (dN) distances were estimated for the HVR1 sequences under the Nei and Gojobori model Distance matrices were calculated using Molecular Evolutionary Genetics Analysis (MEGA) vs. 4 software An unrooted phylogenetic tree was obtained for every patient using a Bayesian approach implemented in the MrBayes program http://beast.bio.ed.ac.uk/).Dated trees, evolutionary rates and population growth models were co-estimated using a Bayesian Markov Chain Monte Carlo (MCMC) method implemented in the BEAST package 0 was evaluated as 2lnBF<2: no evidence; 2–5: weak evidence; 6–10: strong evidence; and >10: very strong evidence. A negative 2LnBF indicates evidence in favour of H0. Only values of ≥10 were considered significant. When the posterior distribution of the growth rate contained a zero value in the 95% highest posterior density (95% HPD), we did not reject the constant population in favour of the exponential growth model.Evolutionary rates were estimated using both a strict and relaxed molecular clock. As coalescent priors, we compared three parametric demographic models of population growth  and a non-parametric, piecewise-constant Bayesian skyline plot (BSP). The best fitting models were selected by means of a Bayes factor  implemented in BEAST http://tree.bio.ed.ac.uk/software/tracer/). Only ESS's of >250 were accepted. Uncertainty in the estimates was indicated by 95% HPD intervals. The trees were summarised in a maximum clade credibility tree using the Tree Annotator program included in BEAST by choosing the tree with the maximum product of posterior probabilities after a 10% burn-in; this tree was then visualised using the program FigTree 1.2 (available at http://tree.bio.ed.ac.uk/software/figtree/).The chains were run for at least 20–30 million generations, and sampled every 2000–3000 steps. Convergence was assessed on the basis of the effective sampling size (ESS) after a 10% burn-in using Tracer software version 1.3 (N/dS rate (ω) was estimated using the Maximum Likelihood (ML) approach implemented in the HyPhy software The dN ≠dS; and random effect likelihood (REL), a variant of the Nielsen-Yang approach S and dN to vary independently site-by-site. The three methods have been described in more detail elsewhere http://www.datamonkey.org/) Site-specific positive and negative selections were estimated using three different algorithms: single likelihood ancestor counting (SLAC), derived from the Suzuki-Gojobori approach The presence of recombination events was investigated using GARD The immunological responders (group A) were followed up for a mean of 26±11.1 months; the immunological non-responders (group B) for a mean of 23±7.5, and the group C controls for a mean of 65±24 months. There were no significant differences in mean peripheral blood cell counts between group A and B at baseline; during follow-up, there were statistically significant increases in the mean CD4+ cell counts  in group A but not in group B patients . The curThe phylogenetic analysis was based on a 261-nucleotide sequence included in the E1/E2 genes and encompassing HVR1. A total of 260 sequences were analysed: a mean of 11.8 (10-20) clones per patient and per timepoint.S) and non-synonymous (dN) substitution rates. At T0, there were no significant differences in any of these parameters in the whole E1/E2 sequences or HVR1 alone between group A and B , whereas the subjects in group C tended to have higher mean mutant frequencies and mean genetic distances than the HIV1-positive patients in both sequences, although the differences were not significant .S and dN showed a statistically significant (p = 0.04-by Wilcoxon's test) increase in non-synonymous and an almost significant (p = 0.07) increase in synonymous substitution rates in group A, but no changes (p≥0.1) in group B or group C , but were non-significantly different in group B  and group C  patients. These findings were confirmed when the analysis was limited to HVR1. In particular, the calculation of d group C . No corrAnalysis of the Bayesian maximum credibility trees showed that pre- and post-HAART QS segregated into separate well-supported (posterior probability>0.8) monophyletic groups in seven of the eight patients with HIV-1/HCV coinfection; in one group B subject (#7), several clones isolated at different times appeared intermixed, not segregating into significant clades. The patients for whom tree samples were available showed subsequent replacements of the main viral QS by new variants at all timepoints. In group C, the T0 and T1 clones were included in separate clades in one subject (#9) and were partially intermixed in the other (#10). Trees in −3 sub/site/month); the estimate was significantly lower than in all of the other coinfected subjects in one group B subject (#7 again): 0.17×10−3 sub/site/month (95%HPD 0.03-0.3×10−3) (st+2nd (mainly associated with non-synonymous mutations) and those at codon position 3rd (frequently synonymous). However, the median evolutionary rate in the two HIV-negative subjects with chronic HCV infection was 10 times lower than that in the HAART-treated subjects (0.29×10−3 sub/site/month), and the relative evolutionary rate at codon positions 1st+2nd was between 1.4 and 5.5 times lower than that at codon position 3rd  . There wtion 3rd .The tMRCA estimates of the root for each dated tree showed that it was no more than 12 months before T0 in all but two of the HAART-treated subjects, but preceded T0 by more than 36 months in one group A patient (#3), one group B patient (#7), and both group C patients.Comparison of the different coalescent models  showed that the BSP fitted the data better than the other models in nine out of ten patients ; the expAnalysis of the BSPs  showed a−1, corresponding to a duplication time of between 11.5 and 3.6 months.It was possible to estimate exponential growth rates in three group A patients using parametric models, which showed values of between 0.060 and 0.191 monthsComparison of the different evolutionary models allowing site-specific dN/dS rates (Niels-Yang and REL) showed that positive selection pressure was significant in three group A, one group B, and neither of the group C subjects . Using dSites under significant negative selection were found by REL and FEL in three group A, all group B and both group C subjects. Twenty-four of the 87 codons in E1/E2 (27.6%) were under purifying selection pressure, eight of which (33.3%) were included in HVR1. Twelve of the 24 sites also showed significant negative selection in internal branches .The most frequently positively selected codons were 384, 397 and 401 (which were under significant positive selection pressure in three out of four subjects), followed by sites 396 and 399, which were positive in two out of four subjects. The most represented E1/E2 codons under negative selection pressure were present in only two out of eight subjects ; all of the other sites were only detected in one patient at a time .Early studies of the impact of HAART on liver disease in patients with HIV/HCV coinfection generally suggested its beneficial effect on the progression of fibrosis Genetic distance analysis (phenetic approach) of the QS obtained before the patients started HAART and after more than one year of treatment showed that mutant frequency and mean genetic distance both significantly increased in immunogical responders, and there was a significant increase in both synonymous and non-synonymous substitution rates at the level of HVR1. On the contrary, no significant changes in QS heterogeneity parameters were observed in the group C controls.N/dS ratios after prolonged treatment A number of authors have previously studied the evolution of HCV QS in coinfection subjects during HAART. Although their findings are partially conflicting, the majority agree on two observations: a relatively low level of HCV heterogeneity before HAART, but an increase in QS complexity and dOur own analysis of dated trees showed that the clones isolated at different times in each patient were always clearly monophyletic, except in the case of one patient in group B (#7) and one in group C (#9) whose sequences isolated at T0 and T1 showed a degree of intermixing. In the majority of the HAART-treated patients, the estimated tMRCA of the tree root  Analysis of the internal node tMRCAs showed that the most heterogeneous QS population in the immunological responders emerged during a period of time in which there was a documented increase in CD4+ cell counts. This suggests that, during HAART (and particularly at the time of an increase in CD4+ cell counts), a more heterogeneous and rapidly evolving QS population arises within the host that diverges from and predominates over the few pre-existing variants.−3 sub/site/month (corresponding to 2.2 and 2.7×10−2 sub/site/year), significantly higher than that calculated in group C . Only group B patient #7 showed a low intra-host evolutionary rate similar to that in the group C controls. Previous studies have reported various estimates of the evolutionary rate of E1/E2 and HVR1 in HCV-infected patients −3 to 6.6×10−3 sub/site/month, a range that includes the values obtained in our group C subjects, but not the higher values observed in our HAART-treated subjects.Interestingly, we estimated very rapid within-host evolutionary rates during HAART, regardless of the immunological response to therapy: the median values in group A and B were respectively 1.9 and 2.3×10rd (which less frequently causes amino acid changes) in the group C controls was significantly higher than that estimated at codon positions 1st+2nd , whereas there were no differences between the relative evolutionary rates estimated at the different codon positions in any of the HAART-treated patients. This is in line with the increase in both non-synonymous and synonymous substitution rates found in our phenetic analysis. It is well known that synonymous substitutions are selectively neutral in the majority of cases, and we can hypothesise their increased rate was due to more rapid HCV replication and shorter generation times after the start of HAART. This hypothesis is supported by the phylodynamic analysis showing that the HCV QS populations exponentially grew during follow-up in the immunological responders, but remained constant over time in the immunological non-responders and controls. In particular, the growth in the QS population correlated with the increase in CD4 cell counts in all of the responders.Moreover, the substitution rate at codon position 3Other stochastic factors, such as a bottleneck effect due to significant changes in the size of a viral population can indifferently influence the rates of synonymous and non-synonymous substitutions. In our preliminary study, we found that the HCV viral loads were very low at baseline but increased rapidly in a significant proportion of the immunological responders. Taken together, the above data indicate that an increase in the replication activity of HCV causes an expansion of the QS population in immunological responders during HAART.One possible reason for this increase in viral replication is positive selection pressure due to the restored immune response in HAART-treated patients. The 27 amino acid N-terminal of glycoprotein E2 forming the basic ectodomain HVR1 probably is not part of a folded domain and includes epitopes that are targeted by neutralising antibodies E1/E2 glycoproteins play key roles in the life cycle of HCV The main limitations of our study are the small number of patients included and the relatively low mean number of clones (11.8) analysed for each sample. Nevertheless it has been previously shown that the statistical power of genealogy is optimized by sampling even a modest number of variants from a larger population In brief, our data suggest that HCV QS undergo exceptionally rapid evolution in HAART-treated patients with HIV/HCV coinfection, and that this is associated with an increase in QS heterogeneity in immunological responders. This seems to be due to an increase in viral replication activity during HAART, as it is also suggested by the findings of our preliminary study showing a significant increase in viral load among immunological responders. Interestingly, in the same subjects, the QS population underwent expansion at the same time as CD4+ cell counts increased, which suggests that the increase in the HCV replication is due to an adaptive response to the recovery of host immucompetence. Nevertheless, we found significant positive selection pressure in only half of our HAART-treated patients.Another possible cause of the enhancement of viral replication  is the supply of new uninfected cells susceptible to HCV infection causing an enlargement in viral replication space.Figure S1Bayesian phylogenetic trees of the HCV E1/E2 sequences at different times during follow-up. Trees obtained from subjects not included in (PPT)Click here for additional data file.Figure S2Intra-host population dynamics of the HCV QS during follow-up. Bayesian skyline plots of the effective HCV QS population size (y axis) over time  of the subjects not included in (PPT)Click here for additional data file.Table S1Best demographic models selected by Bayes Factor test.(DOC)Click here for additional data file.Table S2Amino acid substitutions at sites under positive selection pressure.(DOC)Click here for additional data file.


Administration of rifampicin along with nevirapine reduces the plasma concentration of nevirapine in human immunodeficiency virus positive individuals with concomitant tuberculosis (HIV-TB patients). Nevirapine is a much cheaper drug than its alternative efavirenz, and might be beneficial in resource constrained settings.A randomised open label trial was conducted at All India Institute of Medical Sciences, New Delhi, India. During the regimen of an antiretroviral therapy (ART), naive HIV-TB patients were randomly assigned to receive either nevirapine or efavirenz based ART with concomitant rifampicin based anti-tubercular therapy (ATT). Participants were followed for 24 months after starting ART. The end points were virological, immunological and clinical responses, and progression of HIV disease marked by failure of ART.Of the 135 HIV-TB patients, who were receiving rifampicin based ATT, 68 were selected randomly to receive efavirenz based ART and 67 to receive nevirapine based ART. The virological failure rates in the overall population, and the nevirapine and efavirenz groups were 14.1% (19/135); 14.9% (10/67) and 13.2% (9/68), respectively (p = 0.94). No significant difference was found between the groups in the rate of clinical, immunological or virological failures. The overall mortality was 17% with no significant difference between the two groups. Except for the lead in period on day 14, the mean nevirapine concentration remained above 3 mg/L. No association was found between plasma levels of nevirapine and incidence of unfavourable outcomes in this group.Outcome of ART in HIV-TB patients on rifampicin based ATT showed no significant difference, irrespective of whether efavirenz or nevirapine was used. Therefore, nevirapine based ART could be an alternative in the resource limited settings in patients with HIV and tuberculosis co-infection.01805258.NCT No.  According to the UNAIDS 2010 report on the global AIDS epidemic, there are 33.3 million people living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in the world ,5. HowevThe concomitant use of ART and ATT is associated with a reduction in the mortality in patients co-infected with HIV and tuberculosis. Nevertheless, treatment in this setting is complex because of high pill burden leading to poor adherence, drug-drug interactions and hepatotoxicity caused by both nevirapine and rifampicin. There is also a rising concern that rifampicin, a potent inducer of cytochrome P450 enzyme, reduces the plasma concentrations of nevirapine leading to virological failure and occurrence of resistance mutations -8. HowevWhile multiple studies have shown that both nevirapine and efavirenz based regimens have equal efficacy in ART naive patients without TB, but there is not much information in the literature in setting of HIV-TB co-infection ,12. The 3 and with normal renal and hepatic function were included. The other inclusion criteria were age >18 years and absence of concomitant diabetes mellitus. Patients positive in hepatitis B and C serologies were excluded to avoid confusion between drug induced and viral hepatitis. Besides these, patients on anti-epileptic drugs, immunosuppressant and other drugs that induce liver microsomal enzyme systems were also excluded. All female patients were screened with a urine pregnancy test and were excluded if pregnant. HIV infection was documented by licensed ELISA test kit (As per NACO guidelines) .Data were recorded on a pre-designed data sheet and managed on an MS-Excel spreadsheet. All entries were checked twice for any possible recording error. Mean, frequency and median were calculated for all quantitative variables along with their respective standard deviations and Interquartile ranges. Initially, considering this a pilot study, sample size calculation was not planned and data were gathered as per the sample of convenience. All analyses were done by Intention to treat analysis principle. All continuous variables having normal distribution were analysed using Student’s Of the total 135 HIV-TB patients enrolled, 67 were randomised into nevirapine (NVP) arm and 68 into efavirenz (EFV) arm of the study , in both the groups, occurred at the early stage of the treatment, when the patients were receiving both ATT and ART. Outcome of TB treatments in both the groups was comparable (p = 0 · 26).Table The combined incidence of ART failure was 28 · 4% (19/67) in the nevirapine arm and 30 · 9% (21/68) in the efavirenz arm (p = 0 · 75) with an overall ART failure of 29 · 6% (40/135). Rates of clinical, immunological and virological types of failures were compared separately, which reflected no significant difference between the groups for any category  of TB treatment in both the groups. During the 2 year follow-up, none of the patients who successfully completed their treatment relapsed. This rate of successful treatment is considered favourable and consistent with the data from older studies showing cure rates between 59 · 4% to 97 · 1% . Though ATT might be sub-optimal in the first two months of the treatment in HIV-TB co-infections, we used the standard thrice a week DOTS regimens in accordance with the standard national guidelines available to us at the start of the trial . It is pThe overall rates of adverse drug events were low in both the treatment groups. Change in ART regimen was needed in few cases for adverse effects of one of the NRTI constituents, zidovudine or stavudine. No change in regimen was required with respect to the NNRTI component of ART. This is in contrast to the studies by Manosuthi et al. and Van Leth et al. which demonstrated a higher rate of hepatotoxicity in patients receiving nevirapine requiring change of regimen ,11. ThisThe important factor in our study was that this was a randomised control study while most of other studies comparing the efficacy of nevirapine and efavirenz in HIV/TB patients were observational studies. In addition, this study comprised a long follow-up period of 2 years which allowed us to study the outcome of TB in terms of failure and relapse. The longer follow-up period also enabled us to measure directly, the effect of the two regimens on mortality rather than just measuring virological suppression. Generalisability of the outcome measures is another strength, given the high disease severity in terms of degree of immunosuppression and severity of tuberculosis at baseline of our study population, which is typical for TB and HIV co-infection cohorts in resource constrained settings. Besides this, it also included correlation with nevirapine levels up to 6 months, and close monthly follow-up.The study also had few limitations. Given the number of patients who completed the study follow-up at the end of the trial, the power of the study was less than 50%, and therefore was underpowered to detect a difference of less than 20% between the two groups. Many of the participants in the trial received stavudine, which has been recently phased out from the ART program in India. Another limitation was that the types of TB in the two groups differed significantly, and adjusting the types of TB did not alter the treatment outcomes significantly.In conclusion, the efficacy and safety of nevirapine based ART seemed to be comparable to that of efavirenz containing regimens. Owing to its lower cost and easy availability, nevirapine based ART could be an alternative in the resource limited settings in patients with HIV and tuberculosis co-infection.Authors declare that they have no competing interest.SS provided inputs to the study design, helped in data analysis and interpretation, wrote the manuscript, and did final editing. RP, RCS, SD, SK, ME, SKS, SR, BBR and SV reviewed literature, and helped in interpreting data and writing the manuscript. TV conducted laboratory tests for nevirapine levels. HA and JCS conducted laboratory tests for CD4 cell count and plasma viral load. RMP performed data analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/482/prepub


The question of Highly Active Antiretroviral Treatment (HAART) simplification is important due to long term toxicity and cost of HAART.Pro-viral HIV DNA associated with Peripheral Blood Mononuclear Cells (PBMC) is a marker of the HIV reservoir which is predictive of the evolution of HIV infection. It remains the only virological quantitative marker detectable during suppressive HAART. A pro-viral DNA > 2.7log before treatment interruption is predictive of a decline of CD4 T lymphocytes after interruption.3, mean plasma HIV RNA zenith =4,44 log). Patients were on triple nucleoside analogue therapy for 7 years with plasma HIV RNA< 40 copies/ml ; mean CD4 cells = 600/mm3. Mean pro-viral DNA was 2,43 log copies/million PBMC. Treatment was switched to abacavir+lamivudine in 5 patients and emtricitabine+tenofovir in 6.We measured PBMC's pro-viral DNA in patients treated with triple nucleoside analogue therapy containing zidovudine who had developed severe lipodystrophy and/or metabolic abnormalities. A switch to a nucleoside analogue bitherapy by stopping zidovudine was proposed for patients whose pro-viral DNA was < 2.7 log. We selected 11 patients  HIV-1 DNA for the measurement of the HIV reservoir is predictive of disease progression in seroconverters whatever the mode of result expression is. J Clin Virol 42: 399-404


Herpes zoster (HZ) is common among HIV-infected individuals, but the impacts of highly active antiretroviral therapy (HAART) and HAART adherence on HZ risk have not been well studied.The effects of HAART and HAART adherence on HZ incidence were evaluated by comparing HIV-infected women on HAART (HAART use group) with the HIV-infected women remaining HAART naïve (HAART naïve group) in the Women’s Interagency HIV Study (WIHS). A 1:1 matching with propensity score for predicting HAART initiation was conducted to balance background covariates at index visit, including HIV disease stage. Kaplan-Meier method was used to compare the risk of HZ development between the matched pairs. Cox proportional hazard models were used to assess the effects of HAART and HAART adherence on HZ incidence.Through propensity score matching, 389 pairs of participants were identified and they contributed 3,909 person years after matching. The background covariates were similar between the matched pairs at the index visit. The participants had a mean age around 39 years old, and about 61% of them were Black and 22% were Latina. No significant difference in HZ risk was observed between the HAART use group and the HAART naïve group during the first year of follow-up in any analyses. In the univariate analysis, the HAART use group had marginally lower HZ risk (Hazard Ratio (HR): 0.72; 95% Confidence Interval (CI): 0.48-1.1) over the entire follow-up period. However, women with a HAART adherence level of ≥95% had significantly lower HZ risk  compared to the HAART naïve women. The association remained significant after adjusting for quality of life score and acyclovir use, but it attenuated and was no longer statistically significant after adjusting for an intermediate variable, either CD4+ T cell counts or HIV viral load.Among adult women, we observed a significant preventive effect of long-term HAART use on HZ incidence when a HAART adherence level of ≥95% was attained, and this effect was mediated through reduction of HIV viral load and improvement of CD4+ T cell counts. Highly active antiretroviral therapy (HAART) has been demonstrated to be very effective in decreasing the incidence of opportunistic infections, acquired immunodeficiency syndrome (AIDS) and death among HIV-infected patients. However, previous studies showed that the incidence of herpes zoster (HZ) is less likely than those of other opportunistic infections to be reduced by HAART as it still occurs at a relatively high rate in the HAART era -3.According to studies conducted so far, the effect of HAART on HZ incidence may differ depending on how long HAART has been used. Martinez et al. first reported a high HZ incidence in patients with AIDS soon after initiation of therapy with protease inhibitors . Later, The long-term effect of HAART on HZ incidence has also been inconsistent across studies. One study reported that the HZ incidence did not change between the pre-HAART era and the HAART era, and that being on HAART was associated with a high risk of HZ . FurtherThe objective of our study was to examine the effects of HAART and HAART adherence on HZ incidence by comparing HIV-infected women on HAART as a group (HAART use group) or stratified by HAART adherence level, with the matched HIV-infected HAART naive women (HAART naïve group) using longitudinal data from the Women’s Interagency HIV Study (WIHS).The WIHS was initiated in 1993/1994 and expanded in 2001/2002 to examine the natural and treated history of HIV disease among women. The study design of the WIHS is detailed elsewhere ,24. BrieUnlike in randomized clinical trials, use of therapy in observational studies is not based upon random assignment. Thus, unbalanced distributions of background covariates may bias estimated treatment effects. To account for this, a propensity score matching method was used in our analysis. This method approaches more closely a randomized clinical trial by balancing background covariates via matching propensity score, which is the probability of HAART initiation in our study. The propensity score was calculated for each subject at each of her visits before starting HAART using a logistic regression model, with potential confounders for HAART initiation as predictors, including age, race, education, AIDS status, CD4+ and CD8+ T cell counts, HIV viral load and quality of life score. Each HAART user was matched at the last visit before her HAART initiation to a visit of a HAART naïve participant with similar propensity score (±0.1%). For any HAART naïve participant already selected as a control, the rest of her visits were removed from the matching pool to ensure 1:1 matching ratio. Participants who had HZ history on or before the matched visits were also removed from the analysis. The matched visits are the index visits after which all follow-up occurs in this analysis.We used a longitudinal cohort design to assess the impact of HAART use and its adherence on HZ incidence. In the WIHS, questions about HZ occurrence were asked from visit 1 (October 1994) to visit 30 (April 2009). At WIHS study enrollment, participants were asked “Has a health care provider ever told you that you had shingles (herpes zoster)?” At follow up visits, participants were asked “Since your last visit, has a health care provider told you that you had shingles (herpes zoster)?” The study outcome was time to first HZ event from the index visit and participants were censored at the last study visit, death, or loss to follow up. As a participant usually had difficulty in recalling exact date of a HZ event, we used a visit date as the HZ event date when HZ was first reported at that visit, in our calculation of time to first HZ event. The exposure variables were HAART use group (yes/no) and HAART adherence level. The definition of HAART in the WIHS was guided by the DHHS/Kaiser Panel guidelines . The HAADescriptive statistics such as mean and proportions were used to assess distributions of background covariates before and after propensity score matching. Kaplan-Meier method was used to describe HZ free curves during study follow up. Cox proportional hazards models were used to estimate associations of risk factors with time to first HZ event from the index visit, as reflected by hazard ratios (HR) and their corresponding 95% confidence intervals (CI). The underlying proportional hazard assumption in the Cox models was assessed for each covariate, and we found the assumption was satisfied, with the smallest P value being 0.2959. Values of time varying variables at the last study visit were excluded from analyses to avoid temporal ambiguity. For example, acyclovir use that was reported at the visit at which HZ was first reported was not counted as acyclovir use prior to HZ since the drug may have been prescribed to treat HZ. Univariate associations were first evaluated and only variables significant in univariate analysis were included in multivariate models. For short-term HAART effect, the study was assumed to end one year after the index visit. For long-term HAART effect, the participants were followed from the index visits to either their first HZ event or censored events such as death, loss to follow up or at the last visit (visit 30). To assess the effect of HAART use on HZ incidence, we assumed that a woman who initiated HAART will continue on HAART, which is similar to an intent-to-treatment analysis in a clinical trial. To assess the effect of HAART adherence on HZ incidence, time varying HAART adherence level was used as exposure variable, which is similar to the as-treated analysis in a clinical trial. To examine whether HAART exerts its effect via immune recovery, we purposely added into the multivariate models an intermediate variable between HAART use and HZ incidence, along with an exposure variable and potential confounders. All statistical analyses were carried out using SAS 9.2 .2,813 HIV-infected WIHS women were followed from the WIHS enrollment until visit 30 (2009) and contributed 22,658 person years. Figure Characteristics of all HAART using women (2053) and all HAART naïve women (760) at the WIHS enrollment, and characteristics of the subsets of these women who were matched by propensity score at the index visit are presented in Table P = 0.113).During the follow up, the 389 matched pairs contributed 3,909 person years and reported 109 HZ events. The HAART use group had a median follow-up time of 6.9 years (interquartile range (IQR: 25th percentile to 75th percentile): 3.5-11 years) and an average HZ incidence of 25 : 20–32) per 1000 person years. The HAART naïve group had a median follow-up time of 1.5 years (IQR: 0.7-3.8 years) and an average HZ incidence of 35 (95% CI: 26–48) per 1000 person years Figure During the first year of follow up, the HAART use group and the HAART naïve group reported 13 ) and 12 ) HZ events respectively. In the univariate analysis, compared to the HAART naïve group, neither the HAART use group  nor women with a HAART adherence level of ≥95%  had significantly different risk of HZ incidence.10 increase; HR: 1.55; 95% CI: 1.30-1.84) was significantly related to higher HZ incidence. After adjusting for QOL score and acyclovir use, a HAART adherence level of ≥95% remained significantly associated with lower HZ incidence. However, after adding an intermediate variable - either CD4+ T cell count or HIV viral load, along with QOL score and acyclovir use, the association of a HAART adherence level of ≥95% with HZ incidence attenuated and was no longer statistically significant.Effects of long-term HAART use and HAART adherence on HZ incidence are described in Table Though the mechanisms by which HAART exerts its effect on HZ incidence are not yet clear, accumulating evidence suggests that HAART might have different effects on HZ incidence depending on how long HAART has been used. Many studies demonstrated that HZ occurs at a high level shortly after initiation of HAART -15, whicSince HAART became available in 1996, a steady decrease in HZ incidence over time has been observed among HIV-infected women in the WIHS coincident with their increasing access to HAART. Like many other studies -21,28, oIn this study, we found that acyclovir use was surprisingly associated with higher HZ incidence in univariate analysis. Possible explanation for this lies in that prior acyclovir use might be an indication of other herpes infections that were closely related to HZ incidence due to common cause - immunosuppression by HIV infection, rather than be used to prevent occurrence of HZ ,31. In aOur study had some advantages over previous studies. First, we used a propensity score matching method to balance background covariates. In this way, the indication bias for HAART initiation was dramatically decreased. Second, previous studies focused on HAART use but did not take into account HAART adherence level. Our study showed the protective effect of HAART use was most clear when a ≥95% HAART adherence level was attained, suggesting that HAART adherence level should always be considered in future similar studies. However, some limitations of our study should also be mentioned. First, self-reported HZ was not documented by a health professional; however, a previous study has ascribed high validity to self-reports of HZ , suggestWe have shown that individuals highly adherent to HAART regimes had a significantly lower incidence of HZ than similar individuals who were not as adherent to HAART, underscoring the importance of high HAART adherence level in the clinical management of HIV-infected individuals, in addition to its role in decreasing HIV disease progression and HIV transmission.HAART: Highly active antiretroviral therapy; HZ: Herpes zoster; HR: Hazard ratio; CI: Confidence interval; IQR: Inter-quartile range; HIV: Human immunodeficiency virus; AIDS: Acquired immune deficiency syndrome; WIHS: Women’s Interagency HIV Study.Dr. Glesby had consultancy relationship with Gilead Sciences and Pfizer. All other authors declare that they have no competing interests.: CL was responsible for study design, data interpretation and manuscript writing. CW participated in study design and statistical analysis. MJG, GD, AF, HM, TM, and RK participated in drafting and improving manuscript. MY participated in study design, coordination and drafting the manuscript. All authors read and approved the final manuscript.


Thirty nine percent (n = 29) of patients had no resistance mutations in protease or reverse transcriptase suggesting that medication non-adherence was a major factor contributing to failure. Major lopinavir resistance mutations were infrequent , indicating that drug resistance is not the main barrier to future viral suppression.Limited data exist on HIV-1 drug resistance patterns in South Africa following second-line protease-inhibitor containing regimen failure. This study examined drug resistance patterns emerging in 75 HIV-1 infected adults experiencing virologic failure on a second-line regimen containing 2 NRTI and lopinavir/ritonavir. Ninety six percent of patients ( The South African antiretroviral roll out programme consists of a non-nucleoside reverse transcriptase inhibitor (NNRTI) based first-line regimen and a ritonavir-boosted protease inhibitor (PI) containing second-line regimen. Standard genotype analyses of first-line failure samples from South Africa have shown that the majority of patients remain susceptible to the second-line regimen of zidovudine (AZT), didanosine (ddI), and lopinavir/ritonavir (LPV/r) –3. With Patients with HIV-1 subtype B who experience virologic failure on an initial regimen containing LPV/r infrequently have major protease (PR) mutations detected . ResistaPlasma samples from 75 patients on a failing second-line regimen (LPV/r and 2 NRTI) were sent for HIV-1 drug-resistance testing from clinics at two large state hospitals in Johannesburg, South Africa. Virologic failure was defined as having confirmed (two consecutive measurements) of plasma HIV-1 RNA greater than 5000 copies/mL. Because pretherapy samples were not available from these patients, PR sequences were compared with those from 226 LPV/r naïve patients on failing first-line ART from the same two clinics ) of the patients in this study on failing second-line therapy (LPV/r and 2 NRTIs) did not have detectable resistance to NRTI, NNRTI, or PI. This suggests that medication nonadherence contributed to some of the virologic failure observed. In support of this, a recent study by Pulido et al.  showed tng LPV/r . In patients who did have evidence of drug resistance (46 of 75 [61%]), major LPV/r mutations were infrequent (5 of 46 [11%]). Overall, only 7% (5 of 75) of the patients in this study had major LPV/r resistance mutations. The lack of accumulation of major mutations in PR is similar to that seen in other subtype C-infected patients . Thus, dMinor LPV mutations were found in 89% (67 of 75) of patients in this study, but comparison with 226 sequences from LPV/r-naïve patients revealed that all are likely to be naturally occurring subtype C polymorphisms. Several PR polymorphisms in subtype C that could affect PI susceptibility have been noted by others . Twenty seven percent of patients (20 of 75) had both NRTI and NNRTI mutations. The high percentage of K103N and V106M is likely the result of first-line failure of a NNRTI-containing regimen. M184V and TAMs were likely selected by NRTI in either the first- or second-line regimens. However, only 15% of patients had multiple TAMs or other multi-NRTI resistance mutations (Q151M) indicating that NRTI could be used again in the majority of patients. The mutation profiles observed indicates that several third-line options are available after second-line failure. The large majority of patients (93%) would have virus that is likely to be susceptible to other boosted PI. The 4 patients with M46I would be expected to have a decreased susceptibility to indinavir, nelfinavir, fosamprenavir, and atazanavir, and the 2 patients with L76V would likely have a reduced susceptibility to indinavir, fosamprenavir, and darunavir. The sole patient with V82A would have reduced susceptibility to indinavir. The virus with I54S would be interesting to study further as the exact effect of this mutation on PI resistance is not well understood. In addition, the second-generation NNTRI etravirine could be effective in the majority of the patients (73 of 75 [97%]), although NNRTI mutations, including etravirine resistance mutations, may have declined to undetectable levels without NNRTI exposure in the second-line regimen. Additional studies would be necessary to exclude the existence of minor populations of etravirine-resistant variants. Major LPV resistance mutations were infrequent among patients on a failing second-line regimen containing LPV/r and 2 NRTI in the South African roll-out programme. Most ritonavir-boosted PIs would be a good option for subsequent therapy, possibly in combination with etravirine and NRTIs. Alternatively, the integrase inhibitor raltegravir could be used in combination with a boosted PI although comparative data are needed. The absence of HIV-1 drug resistance in approximately half of the patients suggests that better tolerated regimens and improved adherence could achieve virus suppression without the use of new classes of antiretrovirals.


The use of highly active antiretroviral therapy (HAART) has significantly reduced the incidence and progression of HIV-associated cryptococcosis. However, an early complication of HAART is the immune reconstitution inflammatory syndrome (IRIS), which may affect the CNS. The authors report a patient successfully treated for cryptococcosis and HIV who presented a late manifestation of IRIS. Neuroimaging aspects and management of CNS-IRIS in this patient are discussed in this paper. Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality . The use2O), and fungal culture was positive for Cryptococcus neoformans. She was treated with serial LP, amphotericin B plus 5′flucytosine for 14 days and then started on oral fluconazole 800 mg/day. CSF cultures were negative for C. neoformans after 14 days of amphotericin B and 5′flucytosine treatment. She was discharged for followup at the outpatient clinic and started on HAART .A 26-year-old HIV-positive female was brought in to the emergency department with a history of tonic-clonic seizures 2 days earlier and bilateral lower limb weakness. Although the patient was alert and oriented, she was unable to walk. The patient had been diagnosed HIV-positive three months earlier and started taking antiretroviral medications since then, when she was admitted to hospital with a history of fever and headache. Her lymphocyte CD4+ count was then 57 cells/mm³, and HIV viral load was 89,290 copies/mL. Serum cryptococcal latex antigen test (CLAT) was positive . Lumbar puncture (LP) was performed  was made and prednisone (80 mg/day) started. The patient recovered fully from her neurological symptoms after 10 days of treatment with prednisone. Brain MRI was repeated two weeks after admission and showed marked decrease in the volume of cerebral edema , virologic outcomes, and follow-up CD4 counts are inconsistently reported. In case reports, individual data are usually reported, giving a median baseline CD4 count of 28 cells/mm3 and VL of 5.6 log10 copies/mL, and CD4 count at event of 162 cells/mm3 and VL of 2.4 log10 copies/mL. While recognition of CNS-IRIS may be difficult in patients with a recent history of cryptococcosis taking antifungal agents and HAART, the onset of new or progressive clinical symptoms, the appearance of cerebral interstitial edema, the onset of new lesions, mass effect, and restricted diffusion in infections not typically characterized by these findings should raise the suspicion of cryptococcal CNS-IRIS [Cryptococcal immune reconstitution inflammatory syndrome (IRIS) may present as a clinical deterioration or new presentation of cryptococcal disease following initiation of HAART and is believed to be caused by recovery of cryptococcus-specific immune responses . The timPhysicians living in areas with high incidence of cryptococcosis and HIV with increasing use of highly active antiretroviral therapy should be able to recognize the clinical and neuroimaging features and select the appropriate management of cryptococcal CNS-associated IRIS.


The certain difficulties in a choice of HAART regime arise at presence at the patient with HIV-infection and tuberculosis. Enfuvirtide (ENF) not metabolized by enzymes of system cytochrome Ð450. It does not cooperate with NNRTI, PI and rifamycins and can be added to HAART regime if the patient to accept antimycobacterial drugs.Comparison of efficiency and safety of HAART regimes with and without ENF in patients with HIV-infection and tuberculosis.This research was not randomized and was lead in conditions of real clinical practice. 81 pts with HIV-infection and tuberculosis were divided on 2 groups: 1 gr. - 26 pt. with the standard regimes of HAART (2 NRTI + PI/r or NNRTI) and ENF; 2 gr. - 55 pts with the standard regimes of HAART. We determined clinical symptoms, CD4+ lymphocyte's count, viral load, haematological and biochemical parameters before, 4, 12 and 24 weeks therapy.Within 24 weeks HAART the survival rate of patients of 1 gr. has made 96,2%, and 2nd - 89,1%. After 24 weeks HAART the share of pts with VL <500 copies/ml at pts 1 gr. was 81,8% and 2 gr. - 65%  and 76% - 50% . After 24 weeks HAART the median of a gain of quantity CD4+ lymphocytes at pts of 1 gr. has made +110 cells, and 2 gr. - 69 cells . Safety of both regimes of HAART was quite good. Only 1 pt after 12 weeks of treatment has refused therapy with ENF because of local reactions.Inclusion in regimes of HAART of ENF promoted increase in a share of pts with not determined HIV RNA level and to more essential growth of CD4+lymphocyte's count. Addition to standard regime of HAART of ENF essentially did not influence on the frequency of the clinical or laboratory adverse events caused by treatment.


Identification of the determinants of access to investigational drugs is important to promote equity and scientific validity in clinical research. We aimed to analyze factors associated with the use of experimental antiretrovirals in Italy.We studied participants in the Italian Cohort of Antiretroviral-Naive Patients (ICoNA). All patients 18 years or older who had started cART (≥ 3 drugs including at least two NRTI) after their enrolment and during 1997-2007 were included in this analysis. We performed a random effect logistic regression analysis to take into account clustering observations within clinical units. The outcome variable was the use of an experimental antiretroviral, defined as an antiretroviral started before commercial availability, in any episode of therapy initiation/change. Use of an experimental antiretroviral obtained through a clinical trial or an expanded access program (EAP) was also analyzed separately.A total of 9,441 episodes of therapy initiation/change were analyzed in 3,752 patients. 392 episodes (360 patients) involved an experimental antiretroviral. In multivariable analysis, factors associated with the overall use of experimental antiretrovirals were: number of experienced drugs (≥ 8 drugs versus "naive": adjusted odds ratio [AOR] = 3.71) or failed antiretrovirals(3-4 drugs and ≥ 5 drugs versus 0-2 drugs: AOR = 1.42 and 2.38 respectively); calendar year (AOR = 0.80 per year) and plasma HIV-RNA copies/ml at therapy change (≥ 4 log versus < 2 log: AOR = 1.55). The probability of taking an experimental antiretroviral through a trial was significantly lower for patients suffering from liver co-morbidity(AOR = 0.65) and for those who experienced 3-4 drugs (vs naive) (AOR = 0.55), while it increased for multi-treated patients(AOR = 2.60). The probability to start an experimental antiretroviral trough an EAP progressively increased with the increasing number of experienced and of failed drugs and also increased for patients with liver co-morbidity . and for male homosexuals . Variability of the random effect associated to clinical units was statistically significant (p < 0.001) although no association was found with specific characteristics of clinical unit examined.Among patients with HIV infection in Italy, access to experimental antiretrovirals seems to be influenced mainly by exhaustion of treatment options and not by socio-demographic factors. Removing the barriers to accessing experimental drugs is an important goal from both a scientific and ethical point of view. In fact, generalizable therapeutic research requires a study population that is representative of the population that will eventually be in need of the drug being studied ,2. MoreoThe issue of accessing experimental drugs has been debated for persons with HIV infection since the first decade of the epidemic. When the first antiretrovirals entered clinical trials, in response to the need of patients for whom no effective treatment was available, the US issued a new regulation that allowed experimental drugs to be distributed outside clinical trials in the context of so-called Expanded access programs (EAP) . SubsequSince zidovudine was first approved for clinical use, the possibility of treating HIV infection has dramatically expanded and more than 20 drugs are now available in industrialized countries . NonetheKnowledge of patterns of access to experimental drugs by persons with HIV is still incomplete. A number of studies have analyzed the barriers that prevent access to clinical trials of antiretroviral drugs. Cross-sectional surveys on HIV patients conducted in North America found that ethnic minorities, women and patients with public or no health care insurance were underrepresented in clinical trials -14. In EThe aim of our study was to analyze the access to experimental antiretrovirals and the variation over time in the context of a national health service providing universal access to antiretroviral treatment. To this end we analyzed data recorded between 1997 and 2007 in a multicentre cohort study in Italy conducted on patients with HIV infection who were antiretroviral naïve at the time of enrolment .This analysis was conducted within the Italian Cohort of Antiretroviral-Naive Patients (ICoNA) study, an observational cohort of HIV-infected individuals who were antiretroviral naïve at enrolment . This coAll individuals signed an informed consent prior to enrolment and the study was approved by the Ethics Committee of each participating institution that are listed in an appendix.All patients 18 years or older who had started cART (≥ 3 drugs including at least two NRTI) during 1997-2007 were included in this analysis.For each subject included in the analysis we analyzed the "episodes of therapy initiation/change", defined as any initiation of a single antiretroviral drug as part of the initial or subsequent regimen.The initiation of a co-formulated drug without change in the component medicaments of the regimen was considered as a therapy change. On the other hand, change of therapeutic regimen, consisting of discontinuation of a drug without substitution, is not included in this definition and we did not consider consecutive prescriptions of the same antiretroviral separated by discontinuation. A CD4 count and a viral load determination had to be available within 6 months before and 15 days after therapy initiation/change for the episode to be included in the analysis.We speculated that for physician and patient, there is a chance of starting an experimental antiretroviral when a new regimen is started or a new drug is introduced in a cART combination. For the purpose of this analysis, we defined an experimental antiretroviral as any antiretroviral drug started when it was not yet licensed to be legally marketed and sold in Italy Table . If partDescriptive statistical techniques were used to provide a general profile of the study population.The temporal trend in the use of experimental antiretroviral was evaluated by plotting the proportion of therapy initiation/change by calendar year.We computed the proportion of therapy initiation/change with an experimental antiretroviral through a trial and an EAP by the clinical centre and we evaluated the association between them by using Pearson's correlation coefficient.Univariable association between the distribution of episodes of therapy initiation/change according to the presence of an experimental antiretroviral and selected characteristics were tested using a Pearson's chi-square test stratified by clinical centre .Multivariable logistic models were performed to assess adjusted odds ratios (AOR) for initiation of an experimental antiretroviral. In model 1 the outcome variable was the overall use of experimental antiretroviral drugs, regardless of whether the drug was obtained through a clinical trial or an EAP. In model 2 the outcome variable was the use of an experimental antiretroviral obtained through a trial, and in model 3 the outcome variable was the use of an experimental antiretroviral obtained through an EAP.Correlation between statistical units coming from the same clinical centre was taken into account by adding a random intercept to the models. We did not take into account clustering observations within patients since this outcome was a rare event (4%), and clusters without events would have been dropped by the analysis .The covariates introduced in the models concerned the characteristics of clinical centres , demographic, epidemiological  and clinical characteristics of the subjects  at the time of therapy change.We also analyzed variables indicating exhaustion of treatment options, by considering the number of experienced antiretroviral drugs and of virologically failed antiretroviral drugs (defined as plasma HIV-RNA above 500 copies per mL after more than 4 months of continuous use of the drug) . In ordeAnalyses were performed with Stata Statistical Software: Release 10 .3,752 patients were included in our analysis Figure . The med3 (IQR: 139-429.5); HIV-viral load at baseline (log 10 copies/ml) was 4.73 (IQR: 4.07-5.23). Clinically defined AIDS was present at enrolment in 16.9% of patients, while liver comorbitidy was present in 40.8%. 45.6% of patients were cared for in a teaching hospitals.The median CD4 cell count at baseline was 279/mmA total of 9,441 episodes of therapy initiation/change were analyzed of which 392 episodes, related to 360 patients , involved an experimental antiretroviral. In 192 episodes  the experimental drug was obtained through a trial, and in 200  through an EAP.The trend in experimental antiretroviral use during the study period is shown in Figure The proportion of therapy initiation/change with an experimental antiretroviral Figure , ranges Table In multivariable logistic regression Table , the proThe probability of taking an experimental antiretroviral by participating in a clinical trial (model 2), was significantly lower for patients presenting a liver comorbidity  and for those who experienced 3-4 drugs compared to antiretroviral naive patients , while it increased for multi-treated patients . No significant association was found for the number of failed antiretrovirals and the viral load.The probability of taking an experimental antiretroviral by participating in an EAP (model 3) was significantly higher for patients infected through homosexual contacts compared to those infected though heterosexual contacts and for those who were married. The probability of taking an experimental antiretroviral trough an EAP progressively increased with the increasing number of drugs experienced, with the increasing number of drugs failed and in those with a viral load at regimen change ≥ 4 log copies/ml. Patients presenting a liver co morbidity also had a higher probability of taking an experimental antiretroviral through an EAP .In all three models, age, gender, place of birth, education, clinical stage , were not significantly associated with the probability of receiving an experimental antiretroviral. Similarly, no association was found with the characteristics of a clinical unit. On the other hand, the heterogeneity associated with the clinical centre, not explained by the covariates included in the model, was statistically significant in all three models table .Most of the published studies assessing access to experimental antiretroviral drugs were based on cross-sectional surveys on samples of persons with HIV ,14,22,23We found that almost 10% of patients received an experimental antiretroviral at least once. Marked variations were observed in different years of the study with an overall tendency of decreasing probability of receiving an experimental antiretroviral. Demographic and epidemiological characteristics of patients were not significant determinants of receiving experimental antiretrovirals. On the other hand, antiretroviral treatment experience was associated with access to experimental antiretrovirals both in trials and in EAP. Significant variability was associated with clinical centres, although no association was found with the specific characteristics of the clinical units that were examined in this analysis.Previous studies have shown that persons with HIV have an high access to clinical trials of antiretrovirals. For example, surveys in the US found that 10-23% of patients interviewed reported experimental antiretroviral use ,14,23. TOur study also showed that 14.7% of patients who had treatment initiation/change in 1997-1999 had access to experimental antiretrovirals while this proportion decreased thereafter. However, in 2004-2007 the proportion of patients was still 3.3%, a figure which is higher than that reported for cancer patients .Treatment history was an important determinant of accessing experimental antiretrovirals, however different patterns were observed for clinical trials and EAPs. The probability of receiving an experimental antiretroviral through a clinical trial was greater for naïve patients and when treatment was changed in patients who had experienced a higher number of drugs. This probably reflects the design of clinical trials that are mainly focused on naïve and multi-experienced patients . On the Previous studies suggested that access to experimental antiretrovirals may be lower in female patients ,12, thosWe observed a marked variability in access to experimental antiretrovirals in patients cared for in different clinical centres not captured by the covariates considered in our analysis. In a previous study, characteristics of the clinical unit providing care to patients with HIV, such as patients volume and proximity to an NIH-funded HIV trial centre, were independently associated with use of experimental drugs . In our It should be pointed out that our study was conducted on patients with HIV who were enrolled in a cohort study. Therefore, it can't be ruled out that they were more likely to be enrolled in clinical trials and EAPs than patients under care who were not in clinical studies and thus we could have overestimated the probability to access experimental antiretrovirals in Italy. On the other hand, it should be noted that the study population was recruited in a very large number of clinical centres in diverse geographic locations and with different characteristics, and that the distribution of our population by age, gender and mode of HIV acquisition is similar to that of AIDS cases reported in Italy during the study period .In the analysis, we assumed that an experimental antiretroviral was obtained through an EAP if it was started after the first ethical approval in Italy of the program, and participation in a clinical trial was not recorded in the database. However, patients may have still be enrolled in a clinical trial when the EAP was active, so we may have misclassified some patients.The study design also did not allow us to investigate the possible role of a series of patient factors such as fear of side-effects, distrust of researchers, concerns about research, and interference in everyday life which have been shown to represent significant barriers to accessing antiretroviral drug trials .In summary, our study shows that a substantial number of persons with HIV had the opportunity to access experimental antiretroviral drugs in our country and suggests that gender and socio-economic factors did not represent significant barriers to accessing these drugs. This is consistent with speculation that a national health system which provides universal access to care for persons with HIV, such as the Italian health system, may also reduce disparities in access to experimental drugs, in particular when the experimental drug represent an opportunity for patients with exhaustion of treatment options.On the other hand, we also found that the chance of a patient of accessing experimental antiretroviral drugs varied widely in different clinical units: this is a potential source of non equity, and deserves further investigation. This finding also suggest the need of developing a national initiative aimed at facilitating the access of patients from different clinical centres to research on new drugs. In this context, establishing research networks among clinical centres and increasing the patient community information and involvement in clinical research may be useful tools.E.G. has received honoraria for presentation at workshops, or travel grants from Abbott, Bristol-Myers Squibb, Boheringer Ingelheim; A.G. received honoraria for presentation at workshops, or travel grants from Abbott, Bristol-Myers Squibb, Gilead, Pfizer, GlaxoSmithKline, ViiV Health Care; M.Ar. has received honorarium for expert opinion from Bristol-Myers Squibb and an unrestricted educational grant from Pfizer; M.An. has received honoraria for presentation at workshops, advisory board or research grants from Abbott, Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Janssen-Cilag, Merck, Pfizer, ViiV HealthCare; A.d'A.M has received honoraria for presentation at workshops, advisory board or research grants form Abbott, Bristol-Myers Squibb, Janssen-Cilag, Pfizer, Gilead; A.A. has received honoraria for presentation at workshops, consultancy and advisory board, or research grants from Abbott, Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Janssen-Cilag, Merck, Pfizer, ViiV HealthCare. A.C. has received honoraria for presentation at workshops, consultancy and advisory board, or research grants from Abbott, Boehringer-Inghelheim, Bristol Myers Squibb, Gilead Sciences, Glaxo-Smith Kline, Merck, Pfizer, Roche, and Tibotec (Johnson & Johnson). P.S., C.A.,G.M., D.B., G.I. declare that they have no conflict of interest.EG designed the study and drafted the manuscript. PS participated in the design of the study and drafted the manuscript. CA performed the statistical analysis and contributed with writing the manuscript. AG; DB; MA; GM; AC; A d'AM; AA; GI contributed with national coordination, data collection, and revised critically the manuscript. All authors have read and approved the final manuscript.M. Moroni (Chair), G. Angarano, A. Antinori, G. Carosi, R. Cauda, A. d'Arminio Monforte, G. Di Perri, M. Galli, F. Ghinelli, R. Iardino, G. Ippolito, A. Lazzarin, F. Mazzotta, C.F. Perno, P.L. Viale, F Von Schlosser.A d'Arminio MonforteA. Ammassari, A. Antinori, C. Balotta, P. Bonfanti, M.R. Capobianchi, A. Castagna, F. Ceccherini-Silberstein, A. Cozzi-Lepri, A. d'Arminio Monforte, A. De Luca, C. Gervasoni, E. Girardi, S. Lo Caputo, F Maggiolo, R. Murri, C. Mussini, M. Puoti, C. TortiA Cozzi-Lepri, I Fanti, T FormentiM. Montroni, A. Giacometti, A Costantini, A. Riva (Ancona); U. Tirelli, F. Martellotta (Aviano-PN); G. Angarano, N. Ladisa, (Bari); F. Suter, F. Maggiolo (Bergamo); PL: Viale, G. Verucchi, B Piergentili, (Bologna); G. Carosi, G. Cristini, C. Torti, C. Minardi, D. Bertelli (Brescia); T. Quirino, C Abeli (Busto Arsizio); P.E. Manconi, P. Piano (Cagliari); J Vecchiet, K Falasca (Chieti); G Carnevale, S Lorenzotti (Cremona); F. Ghinelli, L. Sighinolfi (Ferrara); F. Leoncini, F. Mazzotta, M. Pozzi, S. Lo Caputo (Firenze); G. Pagano, G. Cassola, G Viscoli, A. Alessandrini, R. Piscopo, G Mazzarello (Genova); F. Soscia, L. Tacconi (Latina); A. Orani, R. Rossotto (Lecco); A. Chiodera, P. Castelli (Macerata); M Galli, A. Lazzarin, G. Rizzardini, I Schlacht, A. d'Arminio Monforte, AL Ridolfo, A Foschi, A Castagna, S Salpietro, S. Merli, S. Melzi, M.C. Moioli, P Cicconi, T Formenti (Milano); R. Esposito, C. Mussini (Modena); A Gori, F Sabbatini (Monza), N. Abrescia, A. Chirianni, M. De Marco, R. Viglietti, (Napoli); C. Ferrari, P. Pizzaferri (Parma); F Baldelli, B Belfiori (Perugia); G. Magnani, M.A. Ursitti (Reggio Emilia); M. Arlotti, P. Ortolani (Rimini); R. Cauda, M Andreoni, A. Antinori, G. Antonucci, P. Narciso, V Tozzi, V. Vullo, A. De Luca, M. Zaccarelli, L Gallo, R. Acinapura, P. De Longis, M.P. Trotta, A Miccoli, F. Carletti, (Roma); M.S. Mura, G Madeddu (Sassari); P. Caramello, G. Di Perri, G.C. Orofino, M Sciandra (Torino); E. Raise, F. Ebo (Venezia); G. Pellizzer, D. Buonfrate (Vicenza).The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/38/prepub


Recent WHO guidelines for resource-limited settings recommend tenofovir in first-line antiretroviral therapy (ART) yet there are suggestions that patients receiving nevirapine with tenofovir have worse outcomes than those receiving efavirenz. We sought to compare outcomes among those taking nevirapine vs. efavirenz with tenofovir and lamivudine.We analyzed data on ART naïve, non-pregnant patients, ≥18 years old without tuberculosis co-infection, initiating tenofovir with lamivudine and either nevirapine or efavirenz between April 1, 2010 and July 31, 2011 (when South Africa’s public-sector use of tenofovir began) at Themba Lethu Clinic in South Africa. We measured virologic suppression , virologic failure , and attrition (death/loss to follow-up) all at 12 months after ART initiation. Modified Poisson regression with robust error estimation was used to estimate risk ratios (RR) and 95% confidence intervals (CI) for predictors of each outcome.2,254 patients were prescribed efavirenz, 131 nevirapine. Patients were followed a median (range) of 12.0 (0.1–12.0) person-months. 62.2% were female and median (IQR) age was 37.7 years (31.5–44.1). Patients prescribed efavirenz had similar initiating CD4 counts (median 132 for both regimens) but were somewhat more likely to be WHO Stage III or IV (39.6% vs. 33.6%) than those prescribed nevirapine. No difference in attrition was found . Among patients with ≥1 viral load within 1 year on ART, those prescribed nevirapine were as likely to reach virologic suppression  but more likely to experience virologic failure  than those prescribed efavirenz.Our results support the notion that, among patients prescribed tenofovir and lamivudine, virologic failure is more common among those taking nevirapine than among those taking efavirenz. Longer-term follow up and larger studies will be needed to confirm this finding. In many resource-limited settings, until recently lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) were the most common nucleoside reverse transcriptase inhibitor (NRTI) choices for first-line antiretroviral treatment (ART). While the change to tenofovir-based regimens continues, it is unclear what the best non-nucleoside reverse transcriptase inhibitor (NNRTI) is to pair with tenofovir. The 2010 WHO treatment guidelines recommended either nevirapine (NVP) or efavirenz (EFV) as they found no evidence of better outcomes with either regimen, but did note the simplicity of the efavirenz regimen (as a once daily pill) and the lower cost of nevirapine. In South Africa, the shift to tenofovir-based regimens began in April 2010. Approval for analysis of the data was given by the Human Research Ethics Committee  of the University of the Witwatersrand and by the Institutional Review Board of Boston University. As per the South African Medical Research Council’s Guidelines on Ethics for Medical Research and the Declaration of Helsinki, individual patient consent was not needed. For this retrospective analysis of routine medical records, the analytic datasets were de-identified and no information was included in the dataset that could reveal a patient’s identity.3 or a WHO Stage IV condition The data for this analysis come from the Themba Lethu Clinic in Johannesburg, South Africa, 3 and men with CD4 counts >400 cells/mm3 are cautioned against initiating nevirapine due to increased risk of severe adverse events. Under the 2010 guidelines, standard public-sector ART regimens for patients initiating treatment include tenofovir with lamivudine and either nevirapine or efavirenz. Efavirenz is recommended when TB co-infection is present Patient laboratory blood tests  are taken at ART initiation and monitoring tests  are conducted at six months, twelve months, then yearly thereafter. If a viral load result is between 400 and 1000 copies/ml, a repeat viral load is usually conducted 6 months later. If, however, a viral load result is >1000 copies/ml then a repeat viral load is typically conducted 3 months later. Patient follow-up occurs roughly every other month for the first six months, then every three months if the patient is considered stable. Information on deaths are recorded through passive surveillance and through linkage with the National Vital Registration System (last done in September 2011). 3 for women and >400 cells/mm3 for men) initiated on nevirapine were excluded due to increased likelihood of adverse events.We analyzed data on treatment eligible, ART naïve, non-pregnant patients, ≥18 years old, without tuberculosis co-infection, initiating a regimen containing tenofovir, lamivudine, and either nevirapine or efavirenz between April 1, 2010 and July 31, 2011. Patients with high baseline CD4 counts (>200 cells/mmOur primary exposure was the drug regimen patients were initiated on: tenofovir-lamivudine-nevirapine (TDF-3TC-NVP) or tenofovir-lamivudine-efavirenz (TDF-3TC-EFV). Under treatment guidelines, nevirapine is distributed as a 200 mg tablet taken once daily for two weeks and twice daily thereafter. Efavirenz is prescribed as a once daily 600 mg tablet or as two 200 mg tablets taken once daily for patients weighing less than 40 kilograms. 3, 50–99 cells/mm3, 100–199 cells/mm3, ≥200 cells/mm3), defined as that CD4 count closest to the date of ART initiation from one year prior to 14 days after the treatment initiation date, WHO Stage  at ART initiation, defined by the clinician or from analysis of recorded co-infections, and anemia at ART initiation, defined using hemoglobin level (g/dL) as none , mild , moderate (8 to <11 for men and non-pregnant women), or severe (<8 for men and non-pregnant women).Our other covariates of interest were age at ART initiation, sex, baseline CD4 count  and virologic failure . Patients lost to follow up or who died were censored at their last observation. Our secondary outcome was attrition (death or loss to follow-up). Loss to follow up was defined as ≥3 months late for a scheduled clinic visit with no subsequent visit. Our last outcome was regimen switches, defined as a switch of at least one drug from the initiating regimen. Patients were followed for a maximum of twelve months after ART initiation. Person-time accrued from the date of initiation until the treatment outcome was observed. If the treatment outcome was not observed within twelve months of ART initiation, patients were followed until death, transfer, loss to follow up, or completion of one year of follow up.We analyzed data by baseline treatment regimen. We calculated frequencies of outcomes during the first year on treatment and present these as simple proportions. We also analyzed predictors of treatment outcomes by calculating crude and adjusted risk ratios using modified poisson regression with robust error estimation. 3 and male patients prescribed efavirenz with CD4 counts >400 cells/mm3 were excluded from the analytical dataset.In addition, we conducted two sensitivity analyses. The first examined whether or not the association between nevirapine use and virologic failure remained at a different definition of failure. For this analysis, we defined virologic failure as a single failing viral load >1000 copies/ml after at least 4 months on treatment. The goal of the second sensitivity analysis was to determine if the application of the nevirapine exclusion criteria of high baseline CD4 counts to the efavirenz group would impact the results. For the second analysis, female patients prescribed efavirenz with CD4 counts >200 cells/mm3 for women and >400 cells/mm3 for men), pregnant women, those with TB, and patients who initiated other regimens were removed from further analysis, leaving 2,385 subjects – 2,254 (94.5%) on TDF-3TC-EFV and 131 (5.5%) on TDF-3TC-NVP.3,390 patients initiated treatment between April 2010 and July 2011. Of those, 2,534 (74.8%) initiated TDF-3TC-EFV while 169 (5.0%) received TDF-3TC-NVP. Patients on nevirapine were more likely to be pregnant than those on efavirenz (8.3% vs. 0.2% among women) and those with TB were more likely to receive efavirenz than nevirapine (10.8% vs. 1.8%). Patients prescribed nevirapine with high baseline CD4 counts (>200 cells/mmPregnant women were younger than included patients (median: 31.0 vs. 37.7) while patients co-infected with TB were of a similar age (median: 36.6). Both pregnant women  and TB patients  had lower median baseline CD4 counts than included patients  and lower baseline hemoglobin levels . While the baseline BMI value for patients co-infected with TB was lower than included patients (median: 19.2 vs. 22.5), pregnant women had a higher median baseline BMI value than included patients (25.3).Included patients were followed for a total of 2,064 person-years, for a median (range) of 12.0 (0.1–12.0) months per person. Of all included patients, 62.2% were female and the median (IQR) age was 37.7 years (31.5–44.1). Patients prescribed efavirenz had similar initiating CD4 counts (median 132 for both) but were somewhat more likely to be WHO Stage III or IV (39.6% vs. 33.6%) at ART initiation than those prescribed nevirapine .Among the 2,385 patients, 415 (17.4%) died or were lost to follow up within one year of ART initiation in a median (IQR) of 4.7 (3.9–7.6) months. No difference in overall attrition was found between patients who received nevirapine compared to those who received efavirenz both before  and after adjusting for baseline characteristics . Among the 1,850 patients who had at least one viral load measurement between one month and one year after ART initiation, viral suppression was common. 1,593 (86.1%) achieved viral suppression and, after adjusting for baseline characteristics, those prescribed nevirapine were as likely to achieve suppression as those prescribed efavirenz . 1,769 patients had ≥1 viral load measurement after four months of follow up. Of those, 1105 (62.5%) had just one viral load measurement while 553 (31.3%) had two viral loads conducted and 111 (6.3%) had 3 or more viral load measurements. Of the 1,769 included patients, 117 (6.6%) experienced virologic failure. In an unadjusted model, patients on nevirapine were almost 3 times more likely to fail than patients prescribed efavirenz . However, after adjusting for baseline characteristics, the effect was less pronounced but those on nevirapine were still 80% more likely to experience virologic failure than those on efavirenz  ; though,Of the 2,385 patients included in the analysis, 241 (10.1%) switched at least one drug in their regimen within one year of ART initiation. 29.0% of nevirapine patients (n = 38) switched compared to 9.0% of efavirenz patients (n = 203). For patients initiated on efavirenz, the most common switch was from tenofovir to stavudine (26.6%), followed by efavirenz to lopinavir/ritonavir (20.2%). For nevirapine patients, the most common switch was from nevirapine to lopinavir/ritonavir (36.8%), followed by nevirapine to efavirenz (26.3%).Among the 1,769 patients included in the failure analysis, 360 (20.4%) had at least one failing viral load between 4 and 12 months after treatment initiation. More nevirapine patients (28.3%) than efavirenz patients (19.9%) experienced a single failing viral load resulting in an unadjusted risk ratio of 1.42 . After adjusting for baseline characteristics, an increased risk of approximately 40% for nevirapine compared to efavirenz patients remained. .Of the 2254 patients initiated on efavirenz who were included in the analytical dataset, 265 (11.8%) people with high baseline CD4 counts (263 women with CD4 >200 cells/mm3 and 2 men with CD4 >400 cells/mm3) were excluded. 1,504 patients were included in the failure analysis who had at least one viral load after 4 months on treatment. Using our original definition requiring two consecutive failing viral loads >1000 copies/ml, 112 (7.4%) patients failed, 96 (6.8%) of whom were initiated on efavirenz and 16 (16.2%) on nevirapine. Both before  and after adjustment  for baseline characteristics, patients initiated onto nevirapine were over twice as likely to fail as efavirenz patients.Concern over use of nevirapine with tenofovir was raised in 2004 when a small clinical trial noted patients on tenofovir-lamivudine-nevirapine experienced a high rate of failure after 24 weeks on therapy despite high adherence levels. Other studies have reported conflicting results. A small study of tenofovir with either lamivudine or emtricitabine and nevirapine found that at 24 weeks, all patients were virally suppressed at <400 copies/ml. We found low rates of failure (6.6%) in this cohort receiving tenofovir-lamivudine-nevirapine; in keeping with the findings presented in several previous studies. However, our study does support the notion that among patients prescribed tenofovir and lamivudine, virologic failure is more common among those taking nevirapine than among those taking efavirenz. Over twelve months of follow up, we found that while achievement of virologic suppression was comparable between regimens at over 75% for both nevirapine and efavirenz based regimens, patients initiated on nevirapine were almost twice as likely to experience virologic failure as patients initiated on efavirenz. The results of previous studies showed smaller effects than our findings and may suggest our results are overestimates.Our study has several strengths. We were able to compare nevirapine and efavirenz directly in a single study and were able to reduce confounding by indication by excluding patients who would be prescribed a regimen due to pregnancy or tuberculosis. Still our findings should be considered along with several limitations. First, we only had data on 131 patients who were prescribed nevirapine along with tenofovir and this led to imprecise estimates. There is also some evidence emtricitabine in combination with nevirapine and tenofovir may be comparable to regimens including both tenofovir and efavirenz, Treatment failure among patients receiving tenofovir-lamivudine-nevirapine has been high in several studies compared to tenofovir-lamivudine-efavirenz, including our own. Combined with the lack of strong evidence of teratogenic effects of efavirenz, the results of these studies have prompted many programs to favor efavirenz. Our results support this decision. However, many patients who have already initiated nevirapine will likely remain on this regimen and continued follow up of these patients is necessary.


Background. CD4+ T-lymphocyte monitoring is not routinely available in most resource-limited settings. We investigated predictors of time to CD4+ T-lymphocyte recovery in HIV-infected children on highly active antiretroviral (HAART) at Korle-Bu Teaching Hospital, Ghana. Methods. Time to CD4+ T-lymphocyte recovery was defined as achieving percent CD4+ T-lymphocytes of 25%. We used Cox proportional hazard models for identifying significant predictor variables. Results. Of the 233 children with complete CD4+ T-lymphocyte data, the mean age at HAART initiation was 5.5 (SD = 3.1) years. The median recovery time was 60 weeks (95% CL: 55–65). Evidence at baseline of severe suppression in CD4+ T-lymphocyte count adjusted for age, age at HAART initiation, gender, and having parents alive were statistically significant in predicting time to CD4+ T-lymphocyte recovery. Conclusions. A targeted approach based on predictors of CD4+ T-lymphocyte recovery can be a viable and cost-effective way of monitoring HAART in HIV-infected children in resource-limited settings. HIV primarily targets CD4+ T-lymphocytes, and recent studies have shown that during primary HIV infection, HIV viral replication in CD4+ T-lymphocytes in gut-associated lymphoid tissue (GALT) results in significant CD4+ T-lymphocyte depletion , 2. ChroThe therapeutic goal of highly active antiretroviral therapy (HAART) is to suppress HIV viral replication and restore immune function . The standard of care for monitoring treatment in HIV-infected children is the routine laboratory monitoring of CD4+ T-lymphocyte percentage or count and HIV viral load at least every 3-4 months . HoweverSeveral studies have demonstrated that the efficacy of HAART in HIV-infected children in resource-limited countries is comparable to that of children in resource-rich countries –19. GiveThis was a single center retrospective study at the Pediatric HIV/AIDS Care program at Korle-Bu Teaching Hospital in Accra, Ghana. The study population consisted of all HIV-infected children on HAART, since pediatric antiretroviral therapy became available in 2004. All the patients included in the analysis were on their first-line regimen of nonnucleoside analog-based HAART consisting of zidovudine (AZT) or stavudine (d4T) plus lamivudine (3TC), plus either nevirapine (NVP) or efavirenz (EFV). We analyzed longitudinal data extracted from medical records of children on treatment with at least two CD4+ T-lymphocyte determinations between June 2004 and December 2009. The study was reviewed and approved by the Ethics and Protocol Review Committees of University of Ghana Medical School and Yale School of Medicine.The main study outcome was time to CD4+ T-lymphocyte recovery, defined as achieving and maintaining a target CD4 percentage of 25% after initiation of HAART. Because of well-known large natural decline and variation in absolute CD4+ T-lymphocyte numbers in early childhood, the percentage of CD4+ T-lymphocytes is preferentially used in the management of pediatric HIV infection; the target is to achieve at least 25% of CD4+ T-lymphocytes according to age on treatment , 21.The predictor variables included gender, age at start of therapy, WHO clinical staging and WHO immune classification at baseline, mode of transmission, HIV and living status of parents, TB diagnosis and treatment, being a graduate of prevention of mother-to-child transmission (PMTCT) program, and adherence to treatment regimen. Since 2004, CD4+ T-lymphocyte counts and percentages were done at least every 6 months. CD4+ T-lymphocyte count and percentage were quantified by a dual-platform flow cytometry technology using an FACSCount system  at the clinical laboratory at Korle-Bu Teaching Hospital according to manufacturer's instructions. Samples were processed within four hours. The laboratory is certified by the South African Public Health Reference Laboratory and they participate in an external quality assurance testing program by the South African Public Health Reference Laboratory. Viral load testing is not available routinely. N and percentage. Kaplan-Meier approach was used to describe the cumulative proportion of CD4+ T-lymphocyte recovery as of particular time in the followup. We used  to plot the trajectories of recovery function over time. The median recovery time across the categories of specific patient characteristics was evaluated with the log-rank test. We used unadjusted Cox proportional hazards (CPHs) models to evaluate independent associations between each of the patient characteristics and the rate of CD4+ T-lymphocyte recovery. The magnitudes of the associations were summarized with hazard ratios (HRs) and 95% confidence intervals (95% CI). Adjusted associations were obtained from multivariate CPH. Significance was established with alpha of 0.05. All analyses were performed with SAS 9.2 .Patient characteristics were summarized with Three hundred and fifty-one HIV-infected children were started on HAART between 2004 and 2009 at the Pediatric HIV clinic at Korle-Bu Teaching Hospital. During the study period, 233 of the children on HAART had at least two CD4+ T-lymphocyte counts and were included in our analysis. The followup time ranged from 11 to 349 weeks, with the median followup of 127 weeks. There were two deaths; one died after meeting the study's primary outcome , and the other died after 54 weeks without recovery. Both patients were included in the analysis. P < .0001). The median baseline CD4+ T-lymphocyte count was 423 (IQR: 272–742), 531 (IQR: 305–774), and 174 cells/mm3 (IQR: 35–371) for children less than one year old, between one and five, and six and older, respectively.The baseline CD4+ T-lymphocyte count of children between one and five years old was significantly higher than those older than six years (P = .06) and both parents with HIV positive status (.10). One hundred and seventy-two (73.8%) of study subjects achieved immunological recovery, that is, achieved a target CD4+ T-lymphocyte percentage of 25%, during the study period. In the unadjusted analyses, the median recovery time for all ages was 60 weeks (95% CL: 55–65). Children between ages one and five years recovered faster, with median recovery time of 52 weeks (95% CL: 38–60 weeks), followed by children less than one year old , and then children six years or older . The median recovery time was shorter for children with no or moderate evidence of immune suppression at the start of HAART therapy and at least one parent alive . We alsoKaplan-Meier curves of the cumulative probability over time of CD4+ T-lymphocyte recovery stratified by age category at start of HAART, gender, WHO immune classification, and parental living status are illustrated in P = .002), higher baseline CD4+ T-lymphocyte count , and having at least one parent alive  were significantly associated with faster CD4+ T-lymphocyte recovery. We found a border line association between female gender and time to recovery . There was no statistically significant association between mode of HIV transmission, parental HIV status, WHO clinical staging, treatment adherence self-report, previous TB diagnosis and treatment, and CD4+ T-lymphocyte recovery , gender (P = .051), age at start of HAART (P = .014), and WHO immune classification at start of HAART (P < .0001). The probability of recovering faster was two times greater in children with at least one parent alive than those with both parents deceased . Females were 1.37 times more likely to achieve faster recovery than males . Children who were started on HAART between one and 5 years of age had 1.59 times greater rate of recovery than children 6 years of age or older.Because baseline CD4+ T-lymphocyte was significantly associated with age at the start of HAART therapy and since WHO immune classification at the start of HAART incorporates the effect of child's age, we retained WHO immune classification in the multivariate model instead of baseline CD4+ T-lymphocytes. In a multivariate model , statistOur findings corroborate with earlier, though few and far in between, reports on the efficacy of NNRTI-based HAART regimens in HIV-infected children in resource-limited countries. Moreover, we identified predictor variables that could be useful in developing a targeted approach to CD4+ testing and cost-effective monitoring of HAART with CD4+ T-lymphocyte counts where resources are limited. Furthermore, children with correlated variables predictive of slow CD4+ T-lymphocyte recovery could be identified and followed more frequently and carefully. Seventy-four percent of study participants achieved immunological recovery during the study followup period of mean duration of 110 (SD = 67.7)  weeks. The magnitude of CD4+ T-lymphocyte recovery is consistent with other reports on pediatric antiretroviral therapy in resource-limited countries , 22, 23.N = 3). It is worth mentioning that the very few numbers of HIV-infected children from the program attest to the merit of the PMTCT programs. One can speculate that the faster recovery rate of graduates of PMTCT program who go on HAART may be due to good followup and strong patient compliance. Although studies from sub-Saharan Africa have demonstrated the efficacy of HAART in HIV-infected children, the predictors of immunological response have not been investigated systematically . In mostOur finding that children aged 1–5 years at the start of HAART achieve CD4+ T-lymphocyte recovery significantly faster than older children confirms the results from previous pediatric studies in both resource-limited and resource-rich countries , 24, 27.Consistent with previous reports from resource-rich settings , 31–33 aP = .13). We are inclined to infer that this is really a surrogate for having one versus both parents alive, hence the stronger observed association of parental living status with the outcome in our study. Having at least one parent alive was found to predict rate of immune recovery. This relationship may be rather complex and there may be other contributing factors, considering the extended family structure and support in Africa. Interestingly, a study from Cambodia found that children who were orphaned were more likely to fail therapy, and the authors attributed this to poor adherence to treatment . Among cCD4+ T-lymphocyte count is a significant parameter in monitoring ART in HIV-infected individuals , 35–37. Our study, like other retrospective studies, has inherent limitations and also additional limitations due to operational constraints of resource-limited settings. It is a single center study, and, therefore, one has to be cautious in generalizing our findings. Moreover, data on viral loads were not available, which precluded us from examining the effect of HAART on viral load and the correlation between viral load and CD4+ T-lymphocytes. Our findings have to be validated with effect of HAART on viral load decline, since it is not uncommon to have discordance in the gain of CD4+ T-lymphocytes and decline in viral load. Furthermore, the data on adherence to therapy was by self-report and not validated, so we did not use it in the final model. However, with these caveats, our findings are consistent with previous studies, while our proposal of targeted approach to CD4+ T-lymphocyte testing is worth further investigation and validation.Our study demonstrates that a clinically meaningful and robust immune recovery in HIV-infected children on HAART is attainable in resource-limited countries and that there are patient characteristics that can predict time to immune recovery. Based on these predictors, a followup analysis can examine how well we can predict a time to CD4+ T-lymphocyte recovery for an individual child so that a targeted approach to CD4+ T-lymphocyte testing could be adopted as a viable and potentially cost-effective approach in monitoring HAART in HIV-infected children in resource-limited countries.