Despite the major role of Gag in establishing resistance of HIV-1 to protease inhibitors (PIs), very limited data are available on the total contribution of Gag residues to resistance to PIs. To identify in detail Gag residues and structural interfaces associated with the development of HIV-1 resistance to PIs, we traced viral evolution under the pressure of PIs using Gag-protease single genome sequencing and coevolution analysis of protein sequences in 4 patients treated with PIs over a 9-year period. We identified a total of 38 Gag residues correlated with the protease, 32 of which were outside Gag cleavage sites. These residues were distributed in 23 Gag-protease groups of coevolution, with the viral matrix and the capsid represented in 87% and 52% of the groups. In addition, we uncovered the distribution of Gag correlated residues in specific protein surfaces of the inner face of the viral matrix and at the Cyclophilin A binding loop of the capsid. In summary, our findings suggest a tight interdependency between Gag structural proteins and the protease during the development of resistance of HIV-1 to PIs. PIs have high intrinsic antiviral activity and are among the most potent antiretroviral drugs (ART) available in clinical practice to date. In fact, only simplification strategies with boosted PIs have proven to be as efficacious as triple ART in maintaining continuous virological suppression3.The introduction of protease inhibitors (PIs) as part of the highly active antiretroviral therapy (HAART) have led to a dramatic reduction in morbidity and mortality rates in HIV-1–infected patients4. Moreover, mutations in the Gag polyprotein at protease cleavage sites have generally been shown to contribute to resistance to PIs by restoring the interaction with the cleavage sites and compensating for defects in viral replicative capacity6.PIs target the active site of the HIV-1 protease (PR). Protease activity is essential for the generation of full infectious viral particles through the cleavage of Gag and Gag-pol polyproteins. Despite the high genetic barrier of the PI, the emergence of mutations at the protease active site leads to drug resistance. Mutations in HIV-1 causing resistance to PIs reduce the affinity of the drug for the active site. These mutations are generally followed by a stepwise accumulation of additional mutations in protease that partially rescue its activity7. Various studies have demonstrated the direct contribution of Gag mutations to drug susceptibility. Thus, mutations at Gag cleavage site positions A431V, K436E and I437V/T conferred resistance to PIs in the absence of drug resistance mutations at the active site of the protease9. Moreover, central residues of the Gag matrix  have been directly associated with reduced susceptibility to PIs and increased viral replicative capacity11. These “alternative” PI resistance pathways have been shown to include mutations in the cytoplasmic tail of gp41 that can alter interactions between gp41 and Gag, thus affecting viral entry13. The previous studies evidence the importance of HIV-1 Gag in the mechanisms of susceptibility to PIs and support the association of Gag and protease as a functional unit. Furthermore, these studies demonstrate that, despite many years since the introduction of PIs, the determinants of virological failure have not been fully characterized.However, the above-mentioned “traditional” PI resistance pathways have been challenged by studies that evidence virological failure of PI-treated patients in the absence of PI resistance mutationsTo gain new insights into the identification of novel determinants in Gag associated with resistance to PIs, we traced virus evolution by combining Gag-protease bulk and single genome sequencing with coevolution analysis of protein sequences in 4 patients treated with PIs over a 9-year period. Using this approach, we determined hotspots of protease coevolution under pressure from PIs in Gag structural proteins, mainly in the matrix, but also in the capsid. Moreover, 3-dimensional information on coevolving sites in the matrix and the capsid shed light on the structural and functional constraints governing Gag coevolution under pressure from PIs.4. Indeed, CSM associated with drug resistance mutations in the protease have been previously associated with PI exposure in vivo and in some cases  with PI resistance4. Mutational evolution of the virus in all patients rendered virus multiresistant to all PIs over time, with the exception of darunavir .We first investigated mutational changes in HIV-1 during the development of resistance to PIs and their distribution across Gag and protease regions. We sequenced the HIV-1 Gag-protease coding region from longitudinal plasma samples in 4 patients (PT1 to PT4) over a 9-year period of antiretroviral treatment containing PIs Fig. . Sequenc4. The presence of S373P before introduction of IDV could be associated with the polymorphic nature of this residue in treatment-naïve individuals14. Therefore, S373P could be selected in the absence of PIs, thus conferring a transitory advantage for the selection of protease drug resistance mutations, as suggested by the association of S373P to weaker virological responses to SQV/r15. The stepwise accumulation of I376V (p2/NC), V128I (p17/p24) and P453A (p1/p6) in PT2 supports the emergence of complex mutational patterns in the protease, including I54A, V82F, I84V and L90M. In contrast, for PT3, I437V was present, and the selection of K436R (NC/p1) in Gag CSM was concomitant with the emergence of the I84V mutation during treatment with LPV/r. No mutations were observed at the Gag cleavage sites in PT4  and A431V (NC/p1), together with the selection of the resistance mutations I54V and V82A in the protease during treatment with RTV Fig. . MoreovePT4 Fig. .16. Overall, these data indicate that both Gag CSM and non-CSM are required for the development of resistance to PIs by HIV-1.Next, we aimed to identify the regions in Gag with preferential accumulation of mutations derived from exposure to PIs. We performed a comparative analysis of Gag sequence evolution by comparison of bulk sequences before and after the introduction of PIs in each patient. We observed how amino acid variation concentrated in p2 (28.57%), followed by NC 14.55%), p17 (14.39%) and p6 (13.46%). We found very limited variation in p24 (2.61%)  using CAPS. The sequences were equally distributed across patients and across the times from initiation of therapy with PIs . We used SGA to avoid PCR resampling and recombination events, as previously reported20, and to accurately assess inter-protein mutational linkage, which is difficult to address in independent short reads22.We performed Coevolution Analyses for Protein Sequences (CAPS) to accurately identify correlated Gag residues involved in the evolution of the protease and resistance to PIs. This method has previously been applied in numerous case studies similar to ours4, which we used as quality benchmark of our analytical tools. Moreover, our analyses established significant correlations with a total of 38 residues across Gag domains. Of those residues, 19 (50%) were located in the p17 viral matrix, 6 (16%) were located in the p24 viral capsid and 13 (34%) were distributed across p2/NC/p1/p6 proteins  rules out the potential linkage between site-preferential coevolution and site variability, thus suggesting a direct effect of pressure from PIs on Gag-protease coevolution.By using CAPS on our sequence dataset, we were able to capture previous correlations of Gag with the protease at cleavage site positions V128 (p17/p24), S373 and S374 (p2/NC), Q450, S451 and P453 (p1/p6)  binding loop or nearby. These residues are essential for binding with cellular CypA and the early events of HIV-1 replication24. Moreover, residues 280 and 331, which bridge the NTD and the CTD of p24, share properties of structural flexibility in the protein. Additionally, for G2 sites of coevolution between M46 and Gag include residues 66, 69, 111 and 124 in p17 and 280 in p24 , helix 3 , the α3/4 loop , the interconnection domain 4/5 loop  and helix 5  Fig. . Further 10 Fig. , which lSimilarly, at the quaternary protein structural level, the mapping of coevolving Gag residues revealed an organized spatial distribution Fig. . Thus, cThe immunodominant nature of Gag in the generation of HIV-1–specific CD8+ T-cell responses argues for a potential contribution of cellular immune pressure to the evolution of the virus during treatment with PIs and detectable viral load. We measured cellular immune responses in PBMC using ELISpot with a panel of overlapping peptides (OLP) covering the Gag and protease regions to evaluate HIV-1 Gag evolutionary convergence between HIV-1–specific CD8+ T-cell immune pressure and drug pressure during the 9 years of treatment with PIs.In general, immune responses in the protease were almost absent, weak and focused on a few peptides in Gag , with no major changes in breadth or magnitude during the follow-up period Table . Additio12. A direct reflection of this phenomenon is that most of the patients who experience virological failure with PIs nowadays do so in the absence of drug resistance mutations at the active site of the viral protease8. The complex mutational dynamics of HIV-1 under treatment with PIs is not fully understood. Therefore, a better understanding of the HIV-1 evolutionary pathways is crucial when attempting to identify novel determinants of virological failure during treatment with PIs. This information could be useful to build molecular models and improve drug design to ensure long-term treatment efficacy.The potency of PIs, which results from their multi-step inhibitory mechanism, leads to an unusual complexity of the mutational pathways to HIV-1 resistance26. However, most of these studies rely on unique patient-derived viral genetic backgrounds and cross-sectional analyses of bulk sequences that cannot capture the complex dynamics of Gag-protease evolution under pressure from PIs. To overcome previous study limitations, we analysed longitudinal Gag-protease sequences over 9 years of PI-containing treatment in 4 patients. Moreover, we obtained Gag-protease sequences by SGA in order to avoid PCR artifacts and to accurately assess Gag-protease inter-mutational linkage by coevolution analysis. To our knowledge, no other experimental design has offered as much detail of Gag-protease mutational dynamics of HIV-1 evolution to identify Gag hotspots potentially involved in the development of resistance to PIs. Moreover, our data help to define Gag coevolution in the setting of pressure from PIs, thus excluding natural variation, common ancestors and the selective pressure from the immune system of this otherwise highly immunogenic region28.Over the last few years, various studies have revealed the major contribution of Gag to changes in susceptibility to PIs in the presence or absence of mutations in HIV-1 protease29. In addition, out of the Gag correlated residues in the matrix, residues 55 and 58 located at helix 3 forms the globular core of the matrix through antiparallel contacts with helix 4, which is key for the structural stability of the protein. Mutations in the globular core of the matrix have been previously shown to reduce HIV-1 susceptibility to lopinavir11. Thus supporting the contribution of correlated sites in the matrix to HIV-1 evolution under pressure of PIs and potential development of resistance.Our results demonstrate that more than 50% of correlated Gag-protease residues lie in the viral matrix outside cleavage sites and are present in more than 85% of the coevolving groups. The most frequent correlated Gag residues in the matrix clustered around helix 3 (E55 and R58), the α3/4 loop  and helix 5  in the p17 crystal structure. These data are consistent with the identification of frequent Gag-protease correlations within the first 200 residues in Gag31. Therefore, modifications in matrix stability could affect virus fusogenicity. Of the coevolving p17 residues identified in our study, three out of them  have been shown to contribute to matrix stabilization and incorporation of Env into immature viral particles32. Thus, we hypothesized that the viral matrix may affect resistance of HIV-1 to PIs disturbing particle maturation and virus fusogenicity. Our observations support recent findings that describe the impact of Env in resistance of HIV-1 to PIs12.At higher protein structural levels, correlated residues in the matrix defined protein sectors on the inner face of the trimer. These surfaces, which were distributed across the tips and the centre of the trimer, identified potential novel inter-protein interfaces. In line with our findings, previous studies associated these surfaces with interactions between the C-terminal domain of the gp41 Env protein and the matrix. Indeed, Env proteins are incorporated into HIV-1 particles in a matrix trimerization-dependent manner16. Moreover, three of these positions  have previously been shown to compensate for fitness defects of p24 escape mutants driven by HIV-1–specific CD8+ T-cell responses34. In addition, mutations in the CypA at position 219 have been shown to increase HIV-1 replication of PI-resistant variants35. Thus, pointing towards evolutionary constraints in the capsid to modulate viral replicative capacity.Together with correlated residues in the matrix, our study identifies a group of correlated Gag-protease residues in the capsid. These residues comprise only 6 positions of the longest Gag protein. Residues V215, M228, T248, N252, T280 and K331 were distributed across the NTD and CTD of the viral capsid. Positions V215, M228, T248, N252 and T280 clustered either in the CypA binding loop or nearby and organized in exposed surface areas of the capsid hexamer. The limited number of correlated residues in the capsid and their distribution restricted to exposed surface areas may be associated with the genetic fragility of the protein to mutational changes Our study provides novel insights into the contribution of Gag to protease evolution during the development of resistance of HIV-1 to PIs in two ways. We identify previously unknown residues and clusters of residues in Gag that are associated with the selection of PI resistance mutations in the protease  and switches of PIs over time. Therefore, we cannot completely exclude the influence by RTI backbone treatment on subsequent viral evolution. However, recent data suggests that there are minor genetic interactions between the RT and the Protease coding regionsIn summary, our study improves our understanding of Gag-protease dynamics of coevolution associated with HIV-1 constraints under pressure from PIs. In addition, our data underline the contribution of Gag structural proteins and identify novel residues and specific protein sectors that are potentially involved in the development of resistance of HIV-1 to PIs. Coevolution analysis of HIV-1 proteins when combined with those of additional functional and structural studies, may serve as a roadmap to guide the development of novel small molecules to increase treatment efficacy against HIV-1.3; 69,000  HIV-RNA copies/ml; PT2, 389  CD4+ T cells/mm3; 457  HIV-RNA copies/ml; PT3, 635 (177–757) CD4+ T cells/mm3; 5,500  HIV-RNA copies/ml; PT4, 471 (118–630) CD4+ T cells/mm3; 37,500  HIV-RNA copies/ml. Longitudinal plasma and peripheral blood mononuclear cell (PBMC) samples were available for all 4 patients  in whom resistance to PIs had evolved over a median of 4 years after initiation of treatment with PIs. The clinical parameters (median [IQR] for each of the patients) were as follows: PT1, 122 (39–158) CD4+ T cells/mmnts Fig. .All methods and experimental protocols were approved by the Ethics Committee of Hospital Germans Trias i Pujol. The patients provided their written informed consent to participate in the study. The study was conducted according to the principles expressed in the Declaration of Helsinki.g for 90 minutes at 4 °C by ultracentrifugation. Viral RNA was amplified by RT-PCR  with the following primer sets: MMOP3 5-AATCTCTAGCAGTGGCGCCCGAAC-3 (623-647HXB2) and MMOP5 5′-TAACCCTGCAGGATGTGGTATTCC-3′ (2849-2826HXB2). The amplification conditions were as follows: 30 minutes at 50 °C, 2 minutes at 94 °C, 15 seconds at 94 °C, 30 seconds at 53 °C, and 3 minutes at 68 °C for 40 cycles. A second polymerization step was carried out to amplify the Gag-protease coding region using a nested PCR  with the specific primers BssHII 5′-TTGCTGAAGCGCGCACGGCAAG-3′ (703-724HXB2) and ClaIR 5′-GGTACAGTATCGATAGGACTAATGGG-3′ (2575-2550HXB2). The PCR conditions were as follows: 30 seconds at 94 °C, 30 seconds at 94 °C, 30 seconds at 50/52 °C and 2 minutes 68 °C for 40 cycles. PCR reactions were purified using Exo-sap to remove primers dimers and reagents at 37 °C for 5 minutes and inactivated at 80 °C for 15 minutes.Total viral RNA was extracted from longitudinal plasma samples with the QIAamp viral RNA mini kit (QIAGEN). Plasma samples with viral loads below 10,000 copies/ml, were concentrated at 23,000 HXB2), as previously described20. The cDNA was used immediately for PCR or stored frozen at −80 °C. The cDNA was serially diluted in water with herring sperm DNA solution at 1,000 ng/ml to increase reproducibility at the dilutions used for PCR amplification and underwent sets of PCR reactions with the primers as described in the previous section. The SGA cut-off value for positive wells was 30%, as described elsewhere20. Positive PCR reactions were purified by Exo-sap to remove primer-dimers and reagents at 37 °C for 5 minutes and inactivated at 80 °C for 15 minutes.Extracted total viral RNA was reverse-transcribed to single-strand cDNA using Superscript III Reverse Transcriptase  with the primer MMOP5 5′-TAACCCTGCAGGATGTGGTATTCC-3′ . Longitudinal SGA sequences were examined for amino acid variation over time using the highlighter program available at “https://www.hiv.lanl.gov”.All sequences were obtained using Sanger reactions with the Macrogen service (Netherlands) and analyzed with Sequencher (Genecodes V). Sequences were aligned to HXB2 using the Bioedit package. Phylogenetic trees of bulk and SGA nucleotide sequences were constructed using the neighbour-joining phylogenetic method with 1,000 bootstrap replicates (MEGA version 5)39. The model was implemented using the program Coevolution Analysis for Protein Sequences (CAPS)40. Briefly, CAPS compares the correlated variance of the evolutionary rates of pairs of amino acid sites, in a protein alignment adjusted for the time since the divergence of the 2 sequences they belong to. This method compares the transition probability scores between pairs of sequences at 2 particular sites, using the blocks substitution matrix (BLOSUM)41. The BLOSUM matrix applied in CAPS for each protein alignment applied depend of average sequence identity as previously described39. The significance of the CAPS correlation values was assessed by randomization of pairs of sites in the alignment, calculation of their correlation values, and comparison of the real values with the distribution of 10,000 randomly sampled values. Statistical significance was set at < 0.01. In order to correct for multiple tests for non-independence of data, CAPS implemented the step-down permutation procedure in both methods and corrected the probabilities. Gag-protease coevolution groups were constructed as previously indicated40. In brief, we analysed significantly correlated pairs (p < 0.01) to identify clusters of amino acids sharing significant correlations among all the pairs and included those in a coevolving group .A total of 171 Gag-protease SGA sequences were aligned to the HXB2 reference sequence. SGA sequences were equally distributed across patients  and follow-up points (before treatment with PIs and at the last time point available after introduction of PIs). We analysed total and patient-specific Gag-protease coevolution using a parametric model to identify coevolution events in protein-coding genesEntropy per site was calculated in SGA sequences using the entropy tool (Entropy-One) from the Los Alamos database.http://www.rcsb.org/pdb/home/home.do. The structures represent the B clade virus and were modified to show Gag correlated sites with protease using the PyMOL Molecular Graphics System, Version 1.8 (Schrödinger).Protein structures were obtained from RCSPDB—p24 (PDB 1E6J); p24 hexamer (PDB 3P05); p17 trimer (PDB 1HIW) and, protease (PDB 4dqf)—which are available at 42. Immune responses were measured using the interferon-γ (IFN-γ) ELISpot assay43. Wells were considered positive if they contained at least 50 spot-forming cells per 106 PBMCs or 3 times above the background level (2 × mean + 3 × STD). Assays were performed according to availability of cells. It was not possible to test all peptides for all patients and all time points.High-resolution HLA class I typing for alleles A, B and Cw was performed using sequence-based typing methods. HIV-1–specific immune responses were assessed over time Table  in cryopSupplementary info


A regimen with rilpivirine (RPV), abacavir (ABC) and lamivudine (3TC) is simple and may allow the sparing of tenofovir and protease inhibitors. However, data on use of this combination as a strategy of switch are limited. Aims of the study were to assess the long-term efficacy and safety of this regimen.Retrospective study on HIV-1 infected patients followed at the Infectious Disease Department of the San Raffaele Scientific Institute, HBsAg-negative, HLA B5701-negative, with no documented resistance to RPV, ABC and 3TC, with HIV-RNA<50 copies/mL who started RPV plus ABC/3TC from March 2013 to September 2015.The primary outcome was durability [no treatment failure (TF)]. Secondary objectives were to evaluate changes in immunological, metabolic and other safety parameters.TF was defined as the occurrence of virological failure  or discontinuation of any drug in the regimen for any reason.Patients’ follow-up accrued from the date of RPV plus ABC/3TC initiation to the date of TF (VF or discontinuation of any drug in the regimen) or to the date of last available visit.Time to TF was evaluated by use of the Kaplan-Meier curves. Mixed linear models were applied to evaluate changes in immunological, metabolic and other safety parameters.In this analysis, 100 patients starting RPV plus ABC/3TC were included. By 12, 24 and 36 months after switching to RPV plus ABC/3TC, the proportions of individuals without TF were 88% [95% confidence interval (CI): 79%-93%], 82% (95% CI:73%-89%) and 78% (95% CI:68%-86%), respectively. Time to TF was not significantly influenced by CD4+ nadir  or pre-ART viral load  or the type of previous antiretroviral regimen .Over a median follow-up of 2.9 years (IQR: 1.9–3.5), 26 subjects discontinued the treatment .In this retrospective study, long-term use of RPV plus ABC/3TC regimen is effective and safe. Efficacy of this regimen was not found to be affected by low CD4+ nadir or high pre-ART viral load. The efficacy and safety of an antiretroviral (ART) regimen based on rilpivirine/tenofovir/ emtricitabine (RPV/TDF/FTC) has been demonstrated in previous clinical trials –5.RPV is currently licensed for use in combination with other antiretroviral agents, as a single agent or a single-tablet regimen with TDF and FTC, in antiretroviral-naïve and experienced patients, HIV-1-infected adults with <100.000 HIV-1 RNA copies/ml –7.However, there is a need of additional data in regard to the use of RPV with other drugs, since in the trials above mentioned, few patients received RPV with the combination of abacavir/lamivudine (ABC/3TC), and it is not known if virological efficacy associated with a regimen based on RPV plus ABC/3TC might differ according to CD4+ nadir or pre-ART viral load. Indeed, these data could be useful in the current clinical management of patients, especially taking into account the well-known kidney and bone toxicity due to TDF ,9.The efficacy of a RPV plus ABC/3TC regimen in treatment-experienced patients was investigated in some previous retrospective studies showing 48-week proportions of virological efficacy ranging from 82% to 91% –12. MoreTherefore, the main purpose of this study was to assess the long-term efficacy of the switch to RPV plus ABC/3TC in virologically suppressed patients. The secondary objectives were to assess the safety of this regimen and whether the main study outcome is influenced by nadir CD4+ and pre-ART viral load.This is a retrospective, monocentric study on HIV-1 infected patients followed at the Infectious Disease Department of the San Raffaele Scientific Institute, HBsAg-negative, HLA B5701-negative, with HIV-RNA<50 copies/mL who started RPV plus ABC/3TC from March 2013 to September 2015. Patients with either (i) previously documented resistance  to RPV, ABC or 3TC, or (ii) history of virological failure to rilpivirine, abacavir or lamivudine, were excluded from the study. The study protocol was approved by the Ethic Committee of San Raffaele hospital and all the enrolled patients provided written informed consent.Efficacy was assessed in terms of durability and the primary outcome was the absence of treatment failure. The secondary outcomes were the virological failure and the associated resistance profile, changes in immunological and metabolic parameters and the safety profile associated with the study regimen.Treatment failure (TF) was defined as the occurrence of virological failure  or discontinuation of any drug in the regimen for any reason.Patients’ follow-up accrued from the date of RPV plus ABC/3TC initiation  to the date of TF (VF or discontinuation for any cause of any drug in the regimen) or to the date of last available visit.An a priori sample size evaluation estimated that 100 patients allowed to assess a 95% confidence interval not wider than 0.08 (i.e. 8%) around a treatment efficacy proportion ≥80% at 12- or 24-month follow-up.Time to TF was evaluated by use of the Kaplan-Meier curves; TF was analysed in strata of nadir CD4+ (≤200 vs >200 cells/μl), pre ART viral load (<100000 vs ≥100000 copies/mL) and type of previous antiretroviral regimen .Predictors of TF were evaluated by a multivariate Cox regression model; the model included age, gender, HCV-Ab, nadir CD4+, viral load pre-ART, years of ART, years with undetectable VL at the start of the RPV plus ABC/3TC, backbone included in the previous regimen, previous antiretroviral regimen, baseline haemoglobin, baseline CD4+ and estimated glomerular factor rate (eGFR).Mixed linear models were applied to evaluate changes in immunological, metabolic and other safety parameters.Among 120 patients who started RPV plus ABC/3TC, 6 patients denied consent to the use of data in the analysis; 14 of the remaining 114 patients were excluded from this analysis as they started the study regimen with HIV RNA> 50 copies/ ml.The median age was 48.7 (42.6–53.7) years, 80% were male, 79% Ab-anti HCV negative. The median years since HIV diagnosis was 11.0 (6.0–17.1), with a median nadir CD4+ 271 cells/μL (188–402). The median time on ART was 7.5 (4.4–14.1) years; 41 patients switched from a regimen based on PI+2NRTIs, 40 from 1 NNRTI+2NRTIs regimen and 19 from other treatments. In the majority of patients the backbone regimen was based on ABC/3TC (57%). The main reason for switching was the presence of toxicity (73%); among the 29 patients previously treated with a TDF-including regimen, 25 (86%) switched for renal toxicity. Patients’ characteristics are shown in By 12, 24 and 36 months after switching to RPV plus ABC/3TC, the proportions of individuals without TF were 88% (95% CI:79%-93%), 82% (95% CI:73%-89%) and 78% (95% CI:68%-86%) respectively .The time to TF was not significantly influenced by CD4+ cells nadir , viral load pre ART  or the type of previous antiretroviral regimen .Over a median follow-up of 2.9 years (IQR: 1.9–3.5), 26 subjects discontinued the RPV plus ABC/3TC treatment; the most common reasons of discontinuation were toxicity (n = 10)  and interactions with other drugs [use proton-pump inhibitors (n = 7)]. Reasons for discontinuation are shown in the The only patient experiencing VF reached 21681 copies/mL with 1180 CD4 cells/μL six months after the introduction of RPV plus ABC/3TC; the resistance mutations detected at virological failure were: L100IL, K103N, E138AE. Two patients discontinued for viral blips: the first patient had a blip (89 copies/ml) 5 months after the introduction of the study regimen, followed by HIVRNA<50 copies/ml and then by a second blip (93 copies/ml) 10 months later; the second one had one blip (79 copies/ml) 6 months after the switch, followed by a two-year period with HIVRNA<50 copies/ml, and a second blip (70 copies/ml). The physicians didn’t ascribed the occurrence of viral blips to a lack of adherence.A multivariate analysis assessing the predictive factors of treatment failure showed that none of the considered covariates was statistically associated with treatment failure.Helicobacter pylori infection and another was known being affected by gastroesophageal reflux disease (GERD).Nine drug-related adverse events (AE) emerged during the study in 9 patients: 2 events of grade 1 , 6 events of grade 2  and 1 event of grade 3 . The most common AE was epigastralgia (5/9 events); among subjects with epigastralgia (n = 5), 1 patient was treated for Only one patient reported a serious adverse event  which resolved in the 6 months following the suspension of the study regimen.Trend of immunological, metabolic and other safety parameters is reported in In this study we found a 88%, 82% and 78% efficacy at 12, 24 and 36 months after switching to RPV plus ABC/3TC with no significant differences in relation to CD4+ cells nadir or viral load pre ART or the type of previous antiretroviral regimen and thus supporting the use of this regimen even in patients with low CD4+ cells nadir or high viral load pre ART and regardless of the type of previous antiretroviral regimen.Previous studies mainly reported 48-week efficacy results with the exception of the study by Marshall et al. . The 48-Recently, Palacios and colleagues reported about the efficacy and safety of switching to ABC/3TC plus RPV in virologically suppressed HIV infected patients on HAART . At 48 wIn the SIMRIKI study, at week 48, HIV RNA<50 copies/mL was maintained by 91% of patients by intention to treat analysis .Our data also confirm the safety and good tolerability of the study regimen, already seen in registrative studies, i.e. ECHO and THRIVE –3 or in A significant improvement of CD4+ values with an increment of 32 cells/μL , haemoglobin, platelets, serum calcium and alkaline phosphatase was observed during the treatment with RPV plus ABC/3TC as well as a small and significant, although not clinically relevant, worsening of the renal function. The increase in CD4+ cell counts is likely the effect of the maintained virological suppression, while changes in bilirubin and in alkaline phosphatase are likely the effect of, respectively, atazanavir and tenofovir withdrawal. The reduction eGFR was expected, as it is known that RPV increases serum creatinine by inhibiting the OCT2 tubular transporter, which results in reduced tubular secretion of creatinine . We do nBased on these data, the RPV plus ABC/3TC regimen could be considered among treatment options for switch regimens due to its efficacy (in our study 1% of VF) also in patients with high pre-ART viral load or low nadir CD4+ cell count in addition to its safety and good tolerability. Over a median follow-up of approximately 3 years, only 10 patients changed the RPV plus ABC/3TC regimen for toxicity and only 7 patients for drug-drug interaction with proton-pump inhibitors. In fact, the RPV plus ABC/3TC regimen is characterised by a favourable drug-drug interaction profile. All these findings suggest that this regimen could be considered a good treatment option for patients with multimorbidity and the consequent problem of polypharmacy.On the other hand, the limitations of this regimen include: the presence of ABC/3TC which precludes its use in patients with HLA B5701, its low genetic barrier and also the fact that it is not a single tablet regimen. In addition, caution in the use of the investigated regimen is also needed in patients with a high cardiovascular risk because of the presence of ABC/3TC.The novelty of our findings relies on the long-term evaluation of the study regimen efficacy and safety and on the assessment of the influence of CD4+ cells nadir or viral load pre ART on these outcomes.This study has some limitations that deserve discussion. The main one is the retrospective observational study design; clinicians may have prescribed this type of regimen at individuals with specific clinical characteristics at starting which may have somewhat limited the generalizability of the study results; however, this study included both subjects with low CD4 nadir (≤200 cells/μl) or high pre-ART viral load (≥100000 copies/mL) not always included in randomized trials that allowed the calculation of stratified analyses on these factors. Another important limitation is the lack of a control arm which can make it difficult to compare the effects observed under RPV plus ABC/3TC regimen with those of other ART regimens. The other limitations include the small number of patients in the strata of CD4+ nadir and pre-ART viral load and the lack of information on the anti-inflammatory properties of the considered drugs (no data on inflammation markers).In summary, data from long-term observation of this retrospective study, confirm the efficacy, safety and the good tolerability of the RPV plus ABC/3TC regimen. The efficacy of this regimen was not found to be affected by low CD4+ nadir or high pre-ART viral load.S1 Dataset(PDF)Click here for additional data file.S2 Dataset(PDF)Click here for additional data file.S3 Dataset(PDF)Click here for additional data file.S4 Dataset(PDF)Click here for additional data file.


HIV-infected individuals have increased risk of developing obstructive lung disease (OLD). Studies from developed countries report high viral load, low CD4 counts, and anti-retroviral therapy (ART) to be associated with OLD; but these findings may not be generalizable to populations in resource-limited settings.We conducted a prospective cohort study of lung function in 730 HIV-infected black South African adults. Pre-bronchodilator spirometry was performed at enrollment and repeated annually for three years. Logistic regression models were used to identify factors associated with OLD, defined as FEV1/FVC<0.70, at enrollment. Excess annual declines in FEV1 and FVC were modelled as the product-term of follow-up time and exposures using random effects regression.3 and 2655  copies/mL respectively. Overall, 25% were receiving ART at enrollment, 16% of whom reported at least 6 months of ART receipt. OLD was found in 35 (5%) at enrollment. Increasing age , current smoking , and CRP  were significantly associated with OLD at enrollment; while increasing CD4 count , viral load  and receipt of ART  were not. The median (IQR) follow-up time was 18 (12–24) months. Participants with a history of tuberculosis (TB) had a 35 mL  and 57 mL  per year excess loss of FEV1 and FVC respectively.Median (IQR) age at enrollment was 36 (32–41) years, 85% were female and 30% ever-smoked with a median (IQR) exposure of 3 (1–6) pack-years. Median (IQR) CD4 count and viral load at enrollment were 372 (261–518) cells/mmPrevalent OLD was associated with older age, current smoking and higher CRP levels, but not CD4 counts and ART, in HIV-infected South African adults. Better understanding of the long-term effects of TB, smoking and inflammation on lung function in HIV-infected populations is urgently needed. The WHO estimates 37 million people are infected with HIV, with sub-Saharan Africa accounting for nearly 70% of the global burden . WidesprAlthough HIV is an independent risk factor for OLD, key factors associated with pulmonary impairment in HIV-infected individuals are unclear. For instance, the AIDS Linked to the Intravenous Experience (ALIVE) cohort, comprising of HIV-infected and -uninfected injection drug users (IDUs) in Baltimore, USA, found high viral load, but not CD4 cell counts or heavy smoking, to be associated with OLD and excess declines in lung function , 9. ConvWhile current studies have considerably advanced our understanding of the HIV-OLD association, HIV-infected individuals at greatest risk of pulmonary impairment are yet to be identified. Furthermore, studies investigating OLD and excess lung function decline in those infected with HIV were conducted in unique populations  from high-income settings, or in populations with high levels of smoking and/or IDU, which may confound the association between markers of HIV disease and pulmonary impairment. The objective of our study was to identify factors associated with OLD and excess lung function decline among HIV-infected adults in South Africa; where nearly 20% of the adult population is living with HIV . AddressMycobacterium tuberculosis infected and ART naïve HIV-infected adults with CD4 counts ≥ 200 cells/mm3 (NCT00057122) [We established a prospective cohort of 755 HIV-infected adults (>18 years) at the Perinatal HIV Research Unit (PHRU) in Soweto, South Africa from 2008 to 2011. Participants were enrolled from two sources: 1) a recently completed phase-3 randomized controlled trial of three novel tuberculosis (TB) preventive regimens in 0057122) , and 2) 0057122) , 17. PreEnrolled participants were followed for three years. Clinical evaluations, CD4 cell count, viral load and plasma C-reactive protein (CRP) assessments were performed at enrollment and every six months thereafter. History of smoking, TB, pneumocystis pneumonia (PCP), second-hand smoking (SHS), exposure to biomass fuels , and occupation were obtained by self-report. Spirometry without bronchodilators was performed using an ultrasound spirometer  at enrollment and every 12 months thereafter. All spirometry measurements were conducted by a qualified pulmonary function technologist according to ATS/ERS standards , and the2 or Fischer’s exact tests. Percent predicted values of FEV1, FVC and FEV1 to FVC ratio were calculated using reference equations derived from an adult population of black African ethnicity from Johannesburg, South Africa [Baseline characteristics between groups were compared using the t-test and Wilcoxon rank sum test for normally and non-normally distributed continuous data respectively. Categorical data were compared by the Xh Africa .3) and categorically at relevant thresholds . Smoking was modelled as a dichotomous (ever vs never) and categorical (never vs former or current) variable. Ever smoking was defined as having consumed at least 100 cigarettes and current smokers were defined as those who continued to smoke on a regular basis. SHS was defined as having ever lived with a regular smoker since 14 years of age. ART and TB were modelled as dichotomous (ever vs never) variables. The multivariate model was constructed based on literature review and exploratory data analysis, and included factors known to be associated with OLD , markers of HIV disease , variables that were significant at the p<0.10 level in univariate analysis  and potential confounders . We additionally performed separate analysis for OLD defined as FEV1 to FVC ratio less than the 5th percentile of expected for age, sex and height using previously described reference equations [Univariate and multivariate logistic regression models were constructed to measure associations between participant characteristics and OLD, defined as FEV1 to FVC ratio less than 0.70, at enrollment . Indepenquations .Next, we measured excess change (mL/year) in FEV1 and FVC, modelled as the product-term of independent variables and follow-up time, using random effects linear regression. Participant characteristics were modelled as baseline  and time-varying  exposures. CD4 cell counts, viral load and CRP levels were modelled as dichotomous variables at clinically relevant thresholds. Multivariate models were constructed based on literature review and exploratory data analysis, and were adjusted for baseline FEV1 or FVC as relevant. Additionally, assuming the underlying biological mechanisms of lung injury to be chronic in nature, we substituted time-updated CD4 and viral load data with those obtained from 6 months prior to a spirometry evaluation in separate analyses. Finally, we performed sensitivity analysis by excluding participants who were lost to follow-up, died or otherwise did not complete at least three follow-up spirometry evaluations.All multivariate models were tested for collinearity using the variance inflation factor (VIF), and variables with VIFs of 2 or higher were reassessed. Statistical significance was determined at p<0.05. Analyses were done in STATA V.13.0 .3 and 2655  copies/mL respectively, and 146 (22%) had an undetectable viral load (<50 copies/mL). Of the 180 (25%) participants on ART, 151 (84%) initiated therapy within 6 months prior to enrollment. The median (IQR) CRP levels were 3 (1–9) mg/L and did not differ significantly by a history of TB  or current smoking   participants did not have spirometry evaluations at enrollment and were excluded from our analysis . The med p>0.30) . HoweverA total of 730 participants were included in the cross-sectional analysis at enrollment . Mean S.Multivariate logistic regression analysis identified age , current smoking  and CRP  to be independently associated with OLD at enrollment. Additionally, we found a dose-response relationship between CRP levels, modelled categorically at clinically relevant thresholds, and OLD, with levels between 3 to 9 mg/L and >9 mg/L having an 8-fold (p = 0.05) and 11-fold (p = 0.02) increased odds of OLD compared to levels <1 mg/L respectively. We did not find a significant association of CD4 cell counts  and ART  with OLD in our cohort . Similarth percentile of expected. In addition to the 35 participants with FEV1/FVC<0.70, the percentile definition detected 22 new participants with OLD (N = 57). Consistent with our previous analysis, we did not find significant associations between CD4 cell counts, viral load or ART and OLD at enrollment. While CRP levels, modelled as a continuous variable, were not associated with OLD, we did find a marginally significant association at CRP levels above 9 mg/L   .We evaluated 1705 spirometry tests over a median (IQR) follow-up time of 18 12–24) months. Failure to complete all three years of follow-up was associated with smoking (p = 0.04), higher viral load (p = 0.02) and no ART (p = 0.01), but not OLD (p = 0.21) or prior TB p = 0.83), at enrollment . We excl–24 month, at enroFinally, we performed sensitivity analysis on 105 (17%) participants who completed all 3 years of follow-up spirometry. The average FEV1 and FVC decline in this sub-group was -21  mL/year and -24  mL/year respectively. Consistent with our full cohort analysis, ever smoking and a history of prior TB at enrollment; but not time-updated CD4 cell counts and viral load, and ART use at enrollment; were significantly associated with an excess loss of FEV1. Similarly, ART receipt during follow-up among ART naïve participants at enrollment was not associated with significant differences in lung function trajectories. Interestingly, participants with a time-updated viral load of at least 13,548 copies/mL (corresponding to the highest quartile in our cohort) had a 40 mL/year  excess loss of FEV1 compared to those with viral loads less than 13,548 copies/mL .To our knowledge, our study is among the first to evaluate OLD and lung function decline in a well characterized prospective cohort of HIV-infected adults from a resource-limited setting. We found that older age, current smoking and higher CRP levels were significantly associated with prevalent OLD. Furthermore, individuals who smoked or reported a history of TB had excess lung function loss during follow up. We found a trend toward an association of OLD and viral load in our cohort, but this was not statistically significant despite our large sample size.th percentile of expected yielding a prevalence of 8%. While our estimates are lower than those reported elsewhere [OLD (FEV1/FVC<0.70) was diagnosed in 5% of our cohort at enrollment. We identified 22 additional cases of OLD with FEV1/FVC<5lsewhere , they arlsewhere , 21, 22.lsewhere , which mlsewhere .Smoking is a well-established risk factor for excess lung function decline and airflow obstruction in HIV-uninfected individuals –29. Our We found a significant association between higher CRP levels and OLD in our cohort, with the strength of association increasing at higher clinical thresholds. However, we did not find a similar consistent association between baseline or time-updated CRP levels and excess lung function decline in the prospective analysis. Discerning temporality of the association between CRP levels and OLD is a matter of considerable debate. While some studies have found a temporal association between CRP and accelerated lung function decline , 35, thiLarge cross-sectional studies from multiple countries have identified an independent association between a history of TB and OLD –45. AddiWhile our findings differ from those reported from high-income settings, they are similar to those reported from a recent multi-center study of HIV-infected adults with comparable demographic and clinical characteristics . ContrarAnother possible explanation for these differences may be the effect of smoking on HIV-associated lung injury. Airflow obstruction in smokers is primarily CD8 T cell mediated, and is associated with cellular infiltration and alveolitis . This meCurrent evidence on the association between ART and OLD in adults is conflicting. Oxidative stress, immune reconstitution and autoimmune reactions have been proposed as possible mechanisms for ART associated lung injury ; howeverOur study has limitations. Post-bronchodilator spirometry was not performed in our cohort and we could not test for reversibility of airflow obstruction. Second, the cross-sectional nature of our primary analysis precludes the identification of a temporal relationship between host factors and prevalent OLD. However, with the exception of CRP, cross-sectional findings are consistent with those from our longitudinal analysis. Third, a history of TB was assessed by self-report and may be subject to recall bias or misclassification. However, this cohort was recruited from a clinical trial where TB was rigorously assessed  and fromDespite these limitations our study is among the first to report on factors associated with OLD and excess lung function decline in HIV-infected individuals prospectively evaluated from a well characterized cohort in a high HIV-TB burden setting. In contrast to published work, our findings extend our understanding of HIV associated pulmonary impairment by studying a cohort of relatively well controlled HIV disease with minimal exposure to confounders such as smoking, IDU, biomass fuel use and occupation. Our findings are likely generalizable to HIV-infected individuals receiving active clinical care in resource limited settings, who account for a vast majority of the HIV burden globally. In conclusion, prevalent OLD was associated with older age, current smoking and higher CRP levels; but not CD4 count and ART; in HIV-infected South African adults. Better understanding of the long-term effects of TB, smoking and inflammation on lung function in HIV-infected populations is urgently needed.S1 Tableth percentile of expected at enrollment.57 (8%) participants had FEV1/FVC<5(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


According to World Health Organization (WHO) guidelines, which have also been adopted by the National AIDS Control Organization (NACO), India, Efavirenz-based Anti-Retroviral Therapy (ART) is better in Human-Immunodeficiency-Virus (HIV)-infected patients who are also being treated with Rifampicin-based Anti-Tuberculous Therapy (ATT). However, Efavirenz is much more expensive. We hypothesize that Nevirapine is a cheaper alternative that possesses equal efficacy as Efavirenz in HIV-Tuberculosis (TB) co-infected patients.A parallel open-label randomized clinical trial was conducted at All India Institute of Medical Sciences (AIIMS), New Delhi and National AIDS Research Institute (NARI), Pune. Those who were ART-naïve and co-infected with TB were randomized to receive either Nevirapine (Group 1)- or Efavirenz (Group 2)-based ART along with Rifampicin-based ATT. ATT was begun first in ART-naïve patients according to the NACO guidelines, with a median of 27 days between ATT and ART in both groups. The primary endpoint was a composite unfavourable outcome (death and/or ART failure) at 96 weeks, and the secondary outcome was successful TB treatment at 48 weeks.n = 144)- or Efavirenz (n = 140)-based ART after a median ATT-ART gap of 27 days. The median CD4 count was 105 cells/μl, with a median viral load of 820,200 copies/μl and no significant difference between the groups. Composite unfavourable outcomes were reported in 49 patients in the Nevirapine group and 51 patients in the Efavirenz group , 0.63,1.43, adjusted). There was no difference in successful TB treatment outcome between the groups . The results were similar, showing no difference between the groups in the two centres of the study after adjusting for disease stage.A total of 284 patients (mean age 36.7 ± 8.1 years) were randomized in a 1:1 ratio to receive either Nevirapine  Date of registration: March 2013.The online version of this article (10.1186/s12879-017-2864-0) contains supplementary material, which is available to authorized users. Tuberculosis is the second most common opportunistic infection  among newly diagnosed HIV-positive cases, with an incidence rate of approximately 10% . In 2015Nevirapine metabolism is more sensitive to induction of hepatic enzymes than that of Efavirenz; therefore, Efavirenz-based regimens are advocated as the first-line treatment in HIV-TB co-infected patients –9. The iTherefore, this study was conducted to measure outcomes in HIV-TB co-infected patients with Rifampicin-based ATT and either Nevirapine- or Efavirenz-based ART in ART-naïve patients.A randomized, parallel design, open-label clinical trial was conducted at AIIMS, New Delhi, and NARI, Pune between September 2007 and December 2013. Eligibility criteria included ART-naïve patients, co-infected with TB, who had not been started on ATT and were aged >18 years with no Diabetes Mellitus or co-infection with Hepatitis B or C. Patients taking anti-epileptic drugs, immunosuppressants or any other drugs that could induce liver microsomal enzyme systems were also excluded. All female participants were screened with a urine pregnancy test and were excluded if found to be pregnant since at the time of conducting the study, there were concerns regarding the safety of Efavirenz during pregnancy.HIV infection was confirmed using a licensed ELISA kit. All patients underwent a physical examination, complete blood count, liver and kidney function testing, chest X-ray and abdominal ultrasonography (USG), along with tests for Hepatitis B Surface Antigen (HBsAg), anti-Hepatitis C Virus (HCV) antibodies, and Venereal Disease Research Laboratory (VDRL) testing. All HIV-positive patients were screened for TB. Pulmonary tuberculosis was diagnosed by sputum microscopy for Acid-Fast Bacilli (AFB) , chest X-ray and response to empirical ATT in sputum smear-negative patients with radiological or clinical findings suggestive of TB. Extra-pulmonary TB was diagnosed using radiographic, cytopathologic, histopathologic and biochemical assessments. ART-naïve patients co-infected with TB were randomized before the start of ATT in a 1:1 ratio to receive either Nevirapine- or Efavirenz-based ART with Rifampicin-based ATT.ATT was given in the form of thrice-weekly therapy as DOTS (Directly Observed Therapy Short-course).After ATT, ART was initiated, with a median ATT-ART gap of 27 days. ART was started after ATT to decrease the chances of IRIS. In the ART regimen, Zidovudine, Lamivudine or Stavudine combined with either twice per day Nevirapine or once per day Efavirenz were given. ART doses were given according to NACO guidelines  . Viral load was similarly measured at 0, 6 and 24 months using the AMPLICOR HIV-1 Monitor test, manufactured by ROCHE Diagnostics, and Abbott’s real-time HIV-1 qualitative assay. Routine investigations (complete blood counts and kidney and liver function tests) were performed at 0, 3, 6, 12, 18 and 24 months. Patients were enrolled after due consent was taken in their vernacular language. The protocol was approved by the ethics committees of the respective institutes.Clinical failure was defined as a new or recurrent WHO Stage 4 condition after at least 24 weeks of ART. Immunological failure was defined as a decrease in CD4 count from the baseline values; either a 50% decrease from the peak CD4 count during the treatment or persistent counts below 100 cells/μl after 24 weeks of treatment. Virologic failure was defined as viral load >400 copies/μl after at least 24 weeks of ART. Combined ART failure was defined as the development of clinical, immunological or virologic failure at any time during the treatment. The composite unfavourable outcome was defined as either ART failure or death due to any cause during the 96-week follow-up period. Treatment outcomes of ATT were defined as per Revised National Tuberculosis Control Programme (RNTCP) guidelines, with successful treatment being defined as either completed treatment or cure.The primary outcome of the study was to assess the proportion of subjects after 96 weeks who had a composite unfavourable outcome. The secondary outcome was an assessment of successful treatment of TB at 48 weeks.A sample size of convenience was taken due to the paucity of both resources and time.Block randomization with variable block size was used as a method of randomization to generate random numbers for allocation of patients into one of the two study groups. Codes were kept in an opaque envelope arranged serially, which was opened after the patient was found eligible for enrolment. These envelopes were kept with a person not involved in the study. The study flow chart is provided in Fig. The means and standard deviations (SD) were calculated for data with normal distribution, while medians and ranges were calculated for quantitative variables following non-normal distribution. The effect size of primary outcome at 96 weeks was computed using the difference in two proportions, and its 95% confidence interval (CI) was determined. Time to primary outcome was analysed using the Kaplan-Meier survival analysis method, and overall survival curves between the two groups were compared using the log-rank test. All analyses were performed following the intention to treat principle. Continuous variables with normal distribution were computed using Student’s t-test. Ordinal variables and variables following non-normal distribution were analysed using the Wilcoxon rank-sum test. Analysis of covariance was used to compute the effect size , adjusting for stage of disease. The primary outcome analysis was the difference in the proportions of composite unfavourable outcomes at 96 weeks in the two groups, and the secondary outcome analysis was the comparison of successful TB treatment at 48 weeks. Statistical analysis was performed using STATA version 12.0 .Of the 284 HIV-TB co-infected ART-naïve patients enrolled, 144 were randomized to receive Nevirapine (group 1) or 140 Efavirenz (group 2). The baseline characteristics of the patients in the two groups are summarized in Table The two groups were not significantly different except in WHO staging of disease, with significantly higher proportions of Stage 3 patients in the Nevirapine group and Stage 4 patients in the Efavirenz group. The 48-week follow-up data were available for 127(88%) and 129 (92%) patients in the Nevirapine and Efavirenz groups, respectively. The 96-week follow-up data were available for 103 (71.3%) and 94 (67.1%) patients in the Nevirapine and Efavirenz groups, respectively.P = 0.79, adjusted)  Table . There wSuccessful TB treatment outcome at 48 weeks (secondary outcome) was comparable between the two groups  Table  and viraThere were no significant differences in either the composite unfavourable outcome or the TB treatment outcome between the groups in the two study centres.Ruling out opportunistic infections (OIs) and treating the same, along with early initiation of ART, are the mainstays of HIV management. TB, being the second most common OI in PLHIV , causes n = 116).Our results differ from the study performed by Swaminathan et al., which showed higher efficacy in the Efavirenz group vs the Nevirapine group at week 24 (85% vs 65%). However, in this study, participants were given once daily Nevirapine; furthermore, the study was a noninferiority trial, with fewer patients .The ATT outcomes were similar in both of the groupsAll participants were on DOTS therapy, and ART was started within a median of 27 days. With the introduction of daily Rifampicin-based ATT from 2016 under RNTCP, it remains to be seen how the interaction of ATT with ART therapy, especially Nevirapine-based therapy, will impact the clinical course. It is possible that the higher rate of failure in the Nevirapine group that was observed by Boulle et al. was because of this daily therapy.IRIS was documented in a total of 8 patients (3%) (6 in the Nevirapine group and 2 in the Efavirenz group). This finding is in contrast to the meta-analysis performed by Muller et al. , where tEfavirenz is metabolized through CYP2B6. Polymorphism in this enzyme complex has been implicated in Efavirenz-associated side effects, especially neurotoxicity. The prevalence of poor phenotypes of CYP2B6 has been reported to be as high as 20.56% in a study performed in similar population . HoweverAs discussed by De Nardo et al. , there aRandomization at the onset with a long-term follow-up of 96 weeks is the strength of our study. The TB treatment success rates in our study were consistent with the data from older studies showing cure rates between 59.4% to 97.1% .The limits of our study include a high rate of loss to follow-up (approximately 30%) and a sample size of convenience. With the introduction of daily Rifampicin-based ATT under RNTCP, DOTS with thrice-weekly ATT has been phased out; therefore, these results cannot be extended to the current treatment regimens for HIV-TB co-infection.Nevirapine and Efavirenz were equally effective in terms of overall mortality and chances of ART failure. The TB treatment outcomes were similar between the two groups.


Understanding the mechanisms of drug resistance can facilitate better management of antiretroviral therapy, helping to prevent transmission and decrease the morbidity and mortality of people living with HIV/AIDS. However, there is little data about transmitted drug resistance and acquired drug resistance for HIV/AIDS patients in Shanghai.A retrospective cohort study of HIV-infected patients who visited the Department of Infectious Disease from June 2008 to June 2015 was conducted in Shanghai, China. Logistic regression analysis was performed to analyze risk factors for drug resistance among HIV-infected people with virological failure. The related collected factors included patient age, gender, marital status, infection route, baseline CD4 count, antiretroviral therapy regimens, time between HIV diagnosis and initiating antiretroviral therapy. Factors with p<0.1 in the univariate logistic regression test were analyzed by multivariate logistic regression test.There were 575 subjects selected for this study and 369 participated in this research. For the antiretroviral therapy drugs, the rates of transmitted drug resistance and acquired drug resistance were significantly different. The non-nucleoside reverse transcriptase inhibitor (NNRTI) had the highest drug resistance rate  and protease inhibitors (PIs) had the lowest drug resistance rate . Logistic regression analysis found no factors that were related to drug resistance except marital status  and the time span between HIV diagnosis and initiating antiretroviral therapy .NNRTI had a higher DR rate compared with nucleoside reverse transcriptase inhibitor (NRTI) and PIs, consequently, LPV/r was a reasonable choice for patients with NNRTI drugs resistance in China. Only married status and a time span≤6 month between the HIV confirmed date and the time initiating antiretroviral therapy were risk factors for TDF drug resistance. Both baseline HIV-RNA load and resistance test is crucial for TDR diagnosis, and frequent monitoring of HIV-RNA load is crucial for ADR identification and intervention. Treatment adherence still plays a positive role on the outcome of ART. Combined antiretroviral therapy (cART) has significantly decreased the morbidity and mortality of people living with HIV/AIDS (PLWHA). It has Many factors can influence the presence of ADR. A low CD4 count and high HIV-RNA virus load (V-L) at baseline can contribute to a high ADR rate ,7. In adDistinct features on the control and management of the HIV epidemic in China should be a primary concern. Our team’s previous study summarized drug resistance characteristics of NRTI and NNRTI, but related data in recent years is scarce. With the development of ART drugs in China, a change had taken place in the types of drugs resistance in recent clinical settings . ART regThe study protocol was submitted and approved by the Shanghai Public Health Clinical Center Ethics Committee. The Ethics Committee authorized this study without written informed consent from participants because the study was retrospective and anonymous.This retrospective cohort study included HIV-infected patients who were patients at the Department of Infectious Disease of Shanghai Public Health Clinical Center from June 2008 to June 2015 in Shanghai, China. Subjects had a HIV-RNA virus load higher than 1000 copies/mL and volunteered for drug resistance testing. The WHO stage of participants was assessed at the first visit by clinicians. Regardless of whether or not patients started ART, all were included in this study. ART regimens conformed to the current Guideline of Diagnosis and Treatment of AIDS in China: Zidovudine (AZT) and Lamivudine (3TC) with either Efavirenz(EFV) or Neviripine(NVP); Stavudine(d4T) and 3TC with either EFV or NVP; Tenofovir(TDF) and 3TC with EFV or NVP; and other regimen (3TC)+Abacavir(ABC)+lopinavir/ritonavir (LPV/r), AZT /d4T +3TC+ didanosine(ddI)/ indinavir (IDV), AZT+IDV+EFV, d4T+ddI+NVP.Patients who were tested for drug resistance were recorded in the Hospital Laboratory Retrieval System (HLRS). This study utilized the HLRS to obtain the list of patients that had been tested. At the same time, this research reviewed patient demographic characteristics including gender, age, infection route, marital status, and HIV confirmed time, as well as disease relevant information including CD4 counts, HIV-RNA load, and ART drug resistance from the Hospital Information System(HIS). See pol gene spanning the polymerase domain of PR and RT regions. RT-PCR was mainly conducted with Recombiant RNase Inhibitor  and Reverse Transcriptase M-MLV . Nested PCR was performed with Ex Taq . HIV-1 genotypic and ART drug resistance analysis was performed using the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/).Blood samples for HIV-RNA virus load and CD4+ T-Cell count measurement were analyzed with COBAS TaqMan  and CYTOMICS-FC500, respectively, at the Shanghai Public Health Clinical Center affiliated with Fudan University. Viral RNA was extracted from 140μl plasma using QIAmp Viral RNA Mini Kit . Reverse Transcription (RT) and nested Polymerase Chain Reaction (PCR) were used to amplify an approximate 1300bp fragment of the Data analysis was conducted using IBM SPSS version 20.0 . Continuous variables were described with mean and standard deviation (SD); categorical variables were described by numbers and percentages. Categorical variables were analyzed with the chi-square test or Fisher’s exact test and continuous variables were analyzed using the t-test. Risk factors for drug resistance were analyzed using the logistic regression test. If factors were determined to be p<0.1 in the univariate logistic regression test, they were then analyzed using a multivariate logistic regression test. All hypothesis testing was two-sided, with a level of α = 0.05.Five hundred and seventy five subjects were considered for inclusion in this study. Based on the exclusion criteria, 206 patients were excluded. The 369 remaining subjects were enrolled in the study. Among these participants, 294 were treatment-naïve patients and 75 were ART-experienced patients, as shown in For ART naïve patients and ART-experienced patients, there were no differences in demographic characteristics of age, gender, marital status, or infection route between the DR and non-DR groups. CD4 counts were relatively low among participants, with 278 patients (75.3%) below 200cells/μl. In ART naïve patients, more than half the patients (161/294) had a HIV-RNA virus load higher than 4 log10 . In ART-experienced patients, no more than 30% (22/75) had HIV-RNA virus load higher than 4 log10.Among ART naïve patients, most participants (224/294) had drug resistance testing within six months of their HIV confirmation date (HIV CD). Among ART-experienced patients, more than half the participants (38/75) started ART within six months of their HIV CD, while one-third of patients (25/75) had drug resistance testing within a year of starting ART. Among 75 ART-experienced patients, the most used regimens were TDF+3TC+EFV (20/75) and AZT+3TC+EFV (17/75). We analyzed subtypes and found that there were 102 subtype B, 16 subtype C, 164 subtype CRF01_AE, and 12 CRF07_BC among the ART naïve patients. In the ART-experienced patients, there were 38 subtype B, 5 subtype C, 32 subtype CRF01_AE, and 0 subtype CRF07_BC .TDR and ADR rates were significantly different among ART drugs. Understandably, PIs (LPV/r) had the lowest DR rate: 1.7% TDR rate and 2.67% ADR rate. NNRTI (EFV and NVP) exhibited the highest DR rate: 10.88% TDR rate and 53.33% ADR rate for both EFV and NVP. AZT had the lowest TDR rate (0.68%) among first-line commonly used ART drugs, but its ADR rate was not comparably low (25.33%). As expected, NRTI had a moderate DR rate compared with NNRTI and PIs . See To determine the factors associated with drug resistance, logistic regression analysis was conducted. See The hospital provided optimal adherence support and education but a few subjects had unsatisfying adherence for different reasons, see This study identified 54 patients with TDR among 294 ART naïve patients and 52 patients with ADR among 75 ART-experienced patients. For both ART naïve and ART-experienced, participant characteristics between the DR and non-DR groups were comparable. Different HIV-1 subtypes can affect the presence of drug resistance ,12. In CZidovudine (AZT) was the first nucleoside reverse transcriptase inhibitor for HIV-1 infections , and manEFV and NVP, as NNRTIs, had the highest drug resistance rate, with 10.88% in the ART-naïve group and 53.33% in the ART-experienced group. NVP is no longer recommended as a first-line ART drug due to its severe adverse effects  and medication complexity (induction period for reducing side effects) . SimilarTo determine the risk factors of ART drug resistance among the HIV positive population with virological failure, logistic regression analysis was conducted. The results showed that initiating ART within six months from HIV determination to ART initiation was a risk factor for drug resistance of TDF and ddI (p<0.05 for both treatments). However, this situation was not associated with the severity of HIV infection, as evidenced by baseline CD4 count>200ul  or HIV-load>4log10 . Thus, it was related to non-pathological factors, e.g. psychological or social problems. Recent studies showed  that earAdherence to treatment also plays a crucial role in the presence of DR ,30. ManySeveral limitations of this research should be noted. First, this was retrospective research. The baseline HIV-RNA load was not available and the surveillance frequency of HIV-RNA load was relatively low (once a year) due to local government policy. Therefore, only a marginal percentage of patients (294 of nearly 5000) could afford HIV-RNA load and DR tests at the baseline, and infrequent HIV-RNA load resulted in delayed VF exposure . In addition, the following conditions were not observed dynamically with DR, which may further support this research.In conclusion, NNRTI had a higher DR rate compared with NRTI and PIs, and LPV/r was a satisfying substitute for NNRTI based on this study of patients in China. Only marital status and a time span ≤ 6 months between HIV CD and the initiation of antiretroviral therapy were risk factors for TDF drug resistance. For didanosine, the interval ≤6 month between HIV CD and the start of antiretroviral therapy was the only risk factor. Though early ART can increase DR risk, it is still advised through consideration of risk-benefit analysis. A baseline HIV-RNA load and resistance test is crucial for TDR diagnosis and frequent monitoring of HIV-RNA load is crucial for ADR identification after the initiation of ART. ART adherence can also play a positive role on treatment, so HIV healthcare providers should take effective measures to improve PLWHA adherence.S1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


To present the development and validation of the WebAd-Q Questionnaire, a self-report instrument to monitor adherence to antiretroviral therapy in HIV/AIDS centers in Brazil. The WebAd-Q is an electronic questionnaire that has three questions about the use of antiretrovirals in the last week. It was constructed from interviews and focus groups with 38 patients. Its validity was tested in a study with a sample of 90 adult patients on antiretroviral therapy for at least three months. We used electronic monitoring bottles, pill counting, and self-report interview to compare adherence. The WebAd-Q was answered on the sixtieth day, twice, with at least one hour of interval. The viral load of the patients was obtained from the service records. We have analyzed the agreement between the answers to the WebAd-Q, the associations, and the correlations with viral load and performance compared to other measures of adherence. Among the invited patients, 74 (82.2%) answered the WebAd-Q. No difficulties were reported to answer the questionnaire. The average answer time was 5 min 47 sec. The set of three questions of the WebAd-Q obtained agreement of 89.8%, with Kappa of 0.77 (95%CI 0.61–0.94). The non-adherence answers of the WebAd-Q were associated with detectable viral load. We obtained moderate viral load correlations with the non-adherence scale according to the WebAd-Q. For the three questions of the WebAd-Q, patients with non-adherence answers were also reported as less adherent according to the other measures of adherence. The WebAd-Q answered all the issues considered relevant in the validation of questionnaires, was well understood by patients, was associated with viral load, and obtained good agreement and good performance compared to the other measures. The feasibility analysis of its implementation still depends on a national study on its applicability. The success of antiretroviral therapy (HAART) for HIV/AIDS heavily depends on timely diagnosisIn Brazil, among the estimated 781,000 persons living with HIV (PLHIV) in 2014, 649,000 (83.1%) were diagnosed. Among the diagnosed PLHIV, 405,000 (62.4%) received antiretroviral therapy from the Brazilian Unified Health System (SUS). Approximately 88% of the persons on HAART had an undetectable viral load,The current Brazilian clinical treatment protocol,,,In this context, in addition to the essential monitoring of adherence in the individual clinical follow-up,,However, there is no consensus in the literature on a gold standard for measuring adherence to HAART. There are different methods of measurement, such as self-report, pill counting, pharmacy records, and electronic monitoring devices. Each method has advantages and disadvantages, which vary according to the context and the desired objectivesSelf-reports as questionnaires structured for interviews or self-completion are commonly used to measure adherence, both in observational studies and in the collection of information from health centers, given their operational simplicity, low cost, and possibility to address several dimensions of adherence,In order to monitor adherence, self-report questionnaires must present an adequate balance between psychometric, theoretical, and pragmatic propertiesThe theoretical properties involve the interpretation given to the measure and the adaptation of the instrument to its purpose. On the other hand, pragmatic properties are those that enable the implementation of the instrument: low cost, non-intrusive, can repeat the measure over time, self-applied, fast in the collection and organization of answersIn Brazil, the methods used to measure adherence to HAART are usually non-specific and dependent on the individual initiative of professionals. The lack of a standardized resource prevents the routine and homogeneous monitoring of the rates of adherence in health centers and hinders the evaluation of the results of activities aimed at improving adherenceThis study presents the development and validation of the web version of a self-report instrument to monitor the average rates of the adherence to HAART in health centers: the Web Adherence Questionnaire (WebAd-Q).The WebAd-Q Questionnaire was developed and tested in a study performed between 2008 and 2010. The instrument was proposed based on literature review and the previous experience of the research team in assessing the adherence of persons living with HIV and other chronic diseases. Inspired by a self-report questionnaire previously developed by the team to measure adherence to drug therapy for hypertensionThe WebAd-Q was developed to evaluate adherence at the collective level, that is, to provide average rates of adherence for management use in HIV/AIDS centers and in operational research studies.The construction of the questionnaire sought to respect the following attributes: evaluate the main components of the act and the process of taking medication; have high sensitivity for non-adherence; mitigate the socially desirable response and memory biases; and be easily understandable and feasible for the routine services of the SUS. The answer to the questionnaire is anonymous, as the purpose of the questionnaire is not to evaluate individual adherence, but rather to allow health centers to periodically obtain the rates of adherence for all patients or groups of patients.The WebAd-Q is designed to provide self-answers on a computer, accessible via the InternetFor this stage of development of the instrument, we defined an intentional sample of 38 patients, from both sexes, different social strata, education level, ethnic groups, and different therapeutic regimens, who are monitored at the STD/AIDS Reference and Training Center of the State of São Paulo (CRT-DST/AIDS-SP) and at the Clinic of Medical Specialties of São Bernardo do Campo (SAE-SBC). The operation of the answer system and the database was tested at CRT-DST/AIDS-SP.Based on the consultations made, the research team instructed the final design and supported the web designer in the design of the application. The patients showed preference for the female version of the main character. The speeches of the characters were recorded by actors, guided by the research team.The WebAd-Q begins with an introduction to the questionnaire made by the character “Coquetel”, which explains the procedures of the questionnaire and ensures the confidentiality of the participant. Then, the character “Silvia” appears, a patient who is in a health center where she went to get her medication. Silvia introduces herself, comments that sometimes she has difficulty taking her medication, and asks the respondent three questions: 1) “And you? In the last seven days, did you take any medication from your cocktail outside the time appointed by your doctor?”; 2) “In the last seven days, did you stop taking any medication from your cocktail?”; 3) “In the last seven days, did you take more or less pills from your cocktail?” .Possible answers for each question are: “No” (adherence answer), “Yes” or “I don’t know/I don’t remember” (non-adherence answers). In addition to the measures for each individual question, we constructed a scale of non-adherence of four categories, taking into account all three questions: 1) no non-adherence answer, 2) one non-adherence answer, 3) two non-adherence answers, and 4) three non-adherence answers.For the validity study, we invited 90 adult patients on HAART for at least three months at the SAE-SBC, which is a health center that monitored approximately 800 persons on HAART at the time of data collection. The calculation of the sample size had sensitivity of 80%, precision of 10%, and prevalence of adherence of 70% as parameters. Patients were invited according to a convenience sample, according to the order of arrival in the center, from August to October 2009.Patients who agreed to participate signed an informed consent and were instructed to use a special Medication Event Monitoring System (MEMS) bottle for 60 days, whose lid has a microprocessor that is activated when open, recording the date, time, and duration of the opening. Participants were asked to use the MEMS for the medication with the highest number of doses or pills of their antiretroviral prescription, as well as open the bottle only when taking the pills. They were also instructed to return to the health center after 30 and 60 days bringing the MEMS bottle.The MEMS bottle allows us to construct two measures of adherence: MEMS dose of seven days  and MEMS time of seven days .We also counted the pills given to the patients at the beginning of the study, after 30 days, and after 60 days. The patients were instructed to return on the thirtieth and sixtieth day with the pills not taken, recorded as “returned”. We made a percentage measure on non-adherence by the number of pills returned in relation to the number of pills supplied. The individual who returned 5% or more of the supplied pills was considered non-adherent.On the thirtieth day, the participants reported their adherence to a trained interviewer using a previously tested self-report questionnaireThe results of the viral load of each patient were collected routinely in the SAE-SBC. In this center, viral load tests are requested from all patients in the follow-up every four months. For each patient, we analyzed the result of the viral load test closest to the answer to the WebAd-Q, collected before or after answering the questionnaire. As a mathematical resource to calculate the viral load log, we used the unit to express individuals with undetectable viral load.For the analysis of the social, demographic, and clinical data of the participants, we calculated the absolute and relative frequencies and the respective 95% confidence intervals (95%CI) of the qualitative variables, and the medians, means, and respective standard deviations of the continuous variables.The WebAd-Q was answered by the patients at the end of the sixtieth day of research, in a specific room for this purpose, after receiving instructions from previously trained health professionals. We asked patients to answer to the WebAd-Q twice, with a minimum interval of one hour, in order to estimate the test-retest agreement using the Kappa index. We calculated the results of the WebAd-Q and the other measures of adherence, with 95%CI.In summary, on the thirtieth day, the patients performed electronic monitoring of doses and time, counted the pills, and answered the self-report questionnaire with the interviewer. On the sixtieth day, they again performed the electronic monitoring and pill counting and responded to the WebAd-Q. At the end of the study, we obtained the measure of viral load closest to the answer to the WebAd-Q, collected routinely in the center.We tested the associations between detectable viral load and the answers to the WebAd-Q, with 95%CI (p < 0.05). We used the Spearman coefficient to analyze the correlation between the scale of non-adherence of the WebAd-Q and the viral load log, according to the viral load measure closest to the answer to the questionnaire. In addition to total correlation, we tested correlations for different groups of respondents, according to the distance between the date of the answer to the WebAd-Q and the date of viral load collection: up to seven days, from seven to 30 days, more than 30 days. We compared the answers for each question of the WebAd-Q with the other measures of adherence using the Mann-Whitney test.Of the 90 invited patients, 74 (82.2%) answered the WebAd-Q questionnaire. The average follow-up time in the study was 60.7 days [standard deviation (SD) of six days]. Among those who did not answer the questionnaire, there were two (2.2%) refusals and 14 (15.6%) drop-outs.Among the 74 participants in the study, four (5.4%) reported difficulty in using MEMS, which resulted in the loss of information for MEMS dose and MEMS time in the last seven days. Two participants (2.7%) did not complete the self-report interview. Altogether, 17 patients  had a detectable viral load (> 40 copies/mL) in the exam with the closest date.The average time to answer the WebAd-Q questionnaire was 1 min 53 sec (SD = 1 min 53 sec). There was no statistically significant difference in the time to answer the questionnaire between patients with adherence or non-adherence answers, considering all three questions (p = 0.272). No patient reported difficulties in answering on the computer.Of the total number of participants, 59 (79.7%) answered the questionnaire twice. In the test-retest, the answers for the set of three questions of the WebAd-Q obtained agreement of 89.8%, with Kappa of 0.77 (95%CI 0.61–0.94).This study analyzed the validity of a self-administered questionnaire to measure adherence to antiretroviral therapy in care centers for PLHIV, and we observed high levels of test-retest agreement and good agreement between the answers and other indicators of adherence.,The associations between the answers to the WebAd-Q and viral load suggest consistency of the measures of the questionnaire for each question individually. The scale of non-adherence obtained by the WebAd-Q had moderate correlation when compared to the clinical outcome evaluated by the viral load, which is similar to that obtained by other self-report HIV questionnairesThe scale seems to fit the purpose of the follow-up, as it provides health professionals with a numerical and synthetic measure of non-adherence for all patients, which helps in the identification of the most frequent types of non-adherence and provides subsidies for comparison, replanning, and evaluation of strategies.This study used several measures of non-adherence, and it obtained different results for this measure, from 25% in the three-day self-report interview to 69% in the seven-day electronic monitoring measure that included time. This variability is expected, since different instruments evaluate different aspects of medication intake. The electronic monitoring records the opening of bottles and the pill counting records the number of pills not taken. The self-report records the pills taken according to the information given by the patient. All these methods are subject to reliability problems: the openings of the bottle may not match the pills taken, pills may be discarded, memory on the pills taken may fail, and the answer of the patient may be influenced by the interviewer or the environmentIn a systematic review, Simoni et al.Given this diversity, the validation of the WebAd-Q sought an expanded scope, based on the comparison with both viral load and different types of concurrent measure. Since there is no gold standard established and the comparison with the clinical outcome was satisfactory, we understand that the criterion (or concurrent) validity by comparison with other methods cannot be evaluated similarly as in studies on the accuracy of diagnostic tests. Thus, if the various methods evaluate different dimensions of adherence, we have to understand the comparison as an overall indicator of consistency between the measures obtained. This study showed that the groups of patients who provided non-adherence answers to the WebAd-Q were also considered less adherent by the other methods.The questionnaire ensures the anonymity of the respondents, minimizing the desirable response bias. Although the research environment requires informed consent, the high non-adherence results obtained suggest that patients were comfortable to admit that they did not take or that they changed the dose of their medication.The study showed the good pragmatic properties of the WebAd-Q: patients reported no difficulties in answering it; the average time to answer it was 5 min 47 sec, which is short considering that the questionnaire has an introduction and acknowledgments at the end; and the database is easily fed with answers, which dismisses the use of typists and can be consulted and shared online.,The purpose of the WebAd-Q was to develop a managerial instrument for health centers, with the provision of averages for the non-adherence of a group of patients in an autonomous, standardized, anonymous, and routine way. This would not be possible with an instrument that required a long time from patients and professionals, or if it were too complex to be answered and analyzed. That is why we chose the self-reported computer questionnaire, similar to the instruments used in other studies on adherence to HAARTThe development of the instrument together with patients was fundamental in this aspect, since it ensured that respondents had friendly and interesting graphic elements and questions. These attributes were shown by several patients already in the first tests of the WebAd-Q in a large HIV/AIDS center, the CRT-DST/AIDS-SP, where a very diverse population answered the questionnaire in a “real-life” environment.This study had limits regarding sample loss during the two months of the study, especially regarding the use of electronic monitoring bottles. Some of the drop-outs may also be related to difficulties in the use of MEMS, previously reported in the literatureAlthough the rate of answers to the questionnaire remained above 82%, the decrease in sample number reduces the statistical power of the study. This may have hindered the observation of statistically significant results, particularly in relation to the comparison with other measures, which yielded some borderline results, but still enough for the criterion validity.Another limit to be considered is that, in the context of the study, the concomitant use of different methods to measure adherence, together with viral load collection in the routine of the health center, may have generated greater attention and care from the patients in relation to the medication taken, reducing possible memory biases when answering the WebAd-Q.Regarding the sociodemographic characteristics of the participants, we can observe the profile of users of a public health center in a medium-sized city in the state of São Paulo, which does not necessarily correspond to the profile of PLHIV in Brazil. For example, we can observe that the study participants presented a higher education level in relation to the average profile of Brazilians living with HIV, according to data from the last epidemiological bulletin on HIV/AIDSIn view of this limitation, the use of the WebAd-Q in the routine of the SUS was later tested in an applicability study conducted in a national sample of HIV centers from different regions of the country, which will be addressed in future publicationsThe WebAd-Q is a multidimensional questionnaire that includes taking all medications at the times and doses prescribed during the last week, with good reliability, patient friendly, and feasible for routine application in health centers. Therefore, in this validation study, the WebAd-Q showed adequate properties for the monitoring of the rates of non-adherence to HAART. The feasibility of its implementation in the routine of HIV centers will be analyzed in a national study, conducted after this study, which will seek to reflect the heterogeneity both of the persons under treatment in the country and the characteristics of the health centers that treat them.