Leishmania-endemic areas. Although antiretroviral therapy has changed the clinical course of HIV infection and its associated illnesses, the prevention of VL relapses remains a challenge for the care of HIV and Leishmania co-infected patients. This work is a systematic review of previous studies that have described predictors of VL relapse in HIV-infected patients.Visceral leishmaniasis (VL) is a common complication in AIDS patients living in We searched the electronic databases of MEDLINE, LILACS, and the Cochrane Central Register of Controlled Trials. Studies were selected if they included HIV-infected individuals with a VL diagnosis and patient follow-up after the leishmaniasis treatment with an analysis of the clearly defined outcome of prediction of relapse.Eighteen out 178 studies satisfied the specified inclusion criteria. Most patients were males between 30 and 40 years of age, and HIV transmission was primarily via intravenous drug use. Previous VL episodes were identified as risk factors for relapse in 3 studies. Two studies found that baseline CD4+ T cell count above 100 cells/mL was associated with a decreased relapse rate. The observation of an increase in CD4+ T cells at patient follow-up was associated with protection from relapse in 5 of 7 studies. Meta-analysis of all studies assessing secondary prophylaxis showed significant reduction of VL relapse rate following prophylaxis. None of the five observational studies evaluating the impact of highly active antiretroviral therapy use found a reduction in the risk of VL relapse upon patient follow-up.Some predictors of VL relapse could be identified: a) the absence of an increase in CD4+ cells at follow-up; b) lack of secondary prophylaxis; and c) previous history of VL relapse. CD4+ counts below 100 cells/mL at the time of primary VL diagnosis may also be a predictive factor for VL relapse. L. donovani complex found in different geographical regions. These parasites have substantially different clinical, drug susceptibility and epidemiological characteristics. According to data from the World Health Organization, the areas where HIV-Leishmania co-infection is distributed are extensive. HIV infection increases the risk of developing VL, reduces the likelihood of a therapeutic response, and greatly increases the probability of relapse. A better understanding of the factors promoting relapses is essential; therefore we performed a systematic review of articles involving all articles assessing the predictors of VL relapse in HIV-infected individuals older than 14 years of age. Out of 178 relevant articles, 18 met the inclusion criteria and in total, data from 1017 patients were analyzed. We identified previous episodes of VL relapse, CD4+ lymphocyte count fewer than 100 cells/mL at VL diagnosis, and the absence of an increase in CD4+ counts at follow-up as major factors associated with VL relapse. Knowledge of relapse predictors can help to identify patients with different degrees of risk, facilitate and direct prophylaxis choices, and aid in patient counseling.Visceral leishmaniasis (VL) is the most serious form of an insect-transmitted parasitic disease prevalent in 70 countries. The disease is caused by species of the  Leishmania co-infection is a reduction in therapeutic response and high rate of relapse, which is the clinical deterioration after clinical improvement, observed in 25–61% of patients Visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) co-infection has emerged as a serious disease pattern Leishmania co-infections are increasingly being reported in countries that are also facing parasite resistance to antimonial drugs The poor therapeutic outcome, the high rate of relapse, the poliparasitic nature of VL in HIV-infected persons, as well as the atypical manifestations of the disease and the impaired access to health-care resources make HIV-infected individuals prone to enlarge the number of human reservoirs Leishmania infections are likely to lead to a greater degree of overlap and greater risk of co-infection and they justify increased alertness. From a global epidemiologic viewpoint, two parallel trends are alarming: the ruralization of the HIV pandemic and the urbanization and spread of VL Recent changes in the epidemiological patterns of HIV and Before the widespread use of antiretroviral therapy, such co-infection was common in Europe This review was conducted on all papers published before July, 31, 2010. To ensure scientific rigour, the Preferred Reporting of Systematic Reviews and Meta-Analysis (PRISMA) guidelines There were no restrictions on the publication language, date of publication, use of secondary prophylaxis, or duration of follow-up in the study. We excluded studies evaluating fewer than ten cases and studies evaluating mixed populations of HIV-infected and uninfected subjects unless separated results for HIV patients were identified. The selected articles were read in full to confirm eligibility.Data were extracted directly from the full-length articles into structured tables containing all of the descriptive variables and relevant outcomes. The following information was extracted: country and period of enrollment; sample size; clinical characteristics of the included patients; study design; the number of excluded patients if specified; statistical analyses utilized; duration of follow-up and number of subjects lost to follow-up; outcome of interest; prognostic variables assessed in each study and quality of the regression model Our selection process is illustrated in A large proportion of the patients in these studies (87.7%) were male and most were young adults; the median or mean ages reported varied from 27 to 37 years . In the Leishmania-specific PCR (polymerase chain reaction) of peripheral blood or bone marrow samples. Three studies In the majority of the studies, the diagnosis of VL was established by direct demonstration of amastigotes  or by the observation of promastigote growth in samples cultured in specific media. In one study The drug used in the treatment of the primary episode of VL was reported for 89% of the treated patients. Of this total, 73.4% of cases (733 patients) were treated with pentavalent antimonial drugs, 12.4% with amphotericin B deoxycholate (124 patients), and 2.1% (21 patients) received amphotericin in lipid formulations. A minority of patients (1.2%) received pentamidine isethionate and three papers included patients treated with miltefosine Three studies explored the impact of mono or dual antiretroviral therapy at VL diagnosis Two studies CD4+ lymphocyte count at VL diagnosis and follow-up has been studied in relation to risk of relapse. Nine articles Leishmania treatments given, time from VL diagnosis to the introduction of protease inhibitor therapy, HAART compliance, the presence of an AIDS-defining disease before VL diagnosis and the presence of serum anti-Leishmania antibodies were not substantially different between relapsing and non-relapsing patients. Tuberculosis co-infection Other variables explored in relation to relapse are shown in The statistical quality and the presentation of methods and results in many studies were poor. In nine studies, the Kaplan-Meier method was used in a univariate survival analysis to analyzed VL relapse. Three prospective studies and two retrospective cohort studies employed Cox regressions for multivariate analysis of independent predictors. One study randomized patients to compare prophylaxis  and performed multivariate analysis to compare relapse rates by logistic regression, including some predictors as covariates. None of these six studies mentioned collinearity assessment  or developed a risk score for relapse based on their multivariable results. Also, none of the multivariate analyses reported a goodness-of-fit test of their models. Other studies analyzed isolated relapse predictors by univariate association tests in series of prospective or retrospective cases or in intervention studies.The present study is the first systematic review of predictors of VL relapse in HIV-infected patients. Our main conclusions are that VL relapse in HIV-infected patients receiving HAART is high and that secondary prophylaxis provides some protective effect but does not completely prevent the occurrence of relapse. We found that patients who did not relapse showed significantly higher CD4+ count at follow-up than patients with relapsing course. Our analysis also suggests that CD4+ count greater than 100 cell/mL at VL diagnosis reduces the odds of relapse. Unlike other opportunistic infections there are some reports of VL relapse in patients with a CD4+ count greater than 200 cell/mL in Ethiopia, and rarely in Europe Most cases reported showed severe reductions in T cells. It could indicate that VL affects HIV-1 patients who exhibit a significant disturbance of cellular immunity; however, VL by itself may reduce CD4+ lymphocyte counts Relapses of VL are suggested to occur mainly in individuals with poor responses to antiretroviral treatment who have no improvement in CD4+ counts Leishmania. Fernandéz-Cotarelo et al. It is known that HIV patients who are receiving HAART have fewer opportunistic infections and recent data shows that there has been a decline in the incidence of VL after the introduction of HAART Leishmania infection Previous episodes of VL were strongly associated with relapse. Also in agreement with the immune-inflammatory theory, it was hypothesized that the enhancement of the Th-2 response following one early relapse could complicate or prevent the later control of Secondary prophylaxis seemed to only partially protect against relapse. Some of studies that observed a reduction in VL relapse following the use of secondary prophylaxis had few patients on HAART, which may not reflect the current reality. Data analysis suggests that the small sample sizes and heterogeneity of regimens used make the results less robust. Nevertheless, the evaluation of these studies through meta-analysis indicates a clear benefit of secondary prophylaxis in reducing VL relapse. Based on six studies whose data were available, the average relapse rate in patients not receiving secondary prophylaxis was 67%, while in the secondary prophylaxis arm, the relapse rate was 31%. Given this difference, the estimated total sample size needed for a study with 80% power would be 70 patients. Three out of the six studies examining secondary prophylaxis were not able to demonstrate statistical significance, possibly because of small sample sizes. It is important to emphasize that despite the heterogeneity of prophylaxis regimens used; statistical results are positively homogeneous in meta-analysis.L. donovani, the predominant causative agent of VL in east Africa and south Asia, is a more virulent and anthroponotic species than L. infantum. Another plausible explanation for this difference may be the influence of other variables that can affect the host immune response such as nutritional status and the presence of other infections and co-morbidities.Thresholds for safe discontinuation of secondary prophylaxis for Spanish patients have been suggested to be CD4+ counts of 200 It has been postulated that the maintenance of an undetectable viral load protects against the development of VL It is important to note that a wide range of therapeutic drugs were utilized for the treatment of VL in the studies we have reviewed. There was no notable difference in the relapse rate with regard to specific VL treatment used ; however only four studies explored this association and most of them included a limited number of patients and only two Although we have made an extensive review, our analysis includes studies with different definitions of cure and different lengths of follow-up. Cure is seldom defined parasitologically in these studies and the difference between treatment failure and relapse is arbitrary in some studies. It is possible that some episodes of relapse in the group of patients in which parasitological cure were not documented by bone marrow examination were treatment failures rather than relapses. Moreover, re-infection was not distinguished from relapse in any paper. There is a high degree of heterogeneity in the evaluated populations as shown by the wide range of reported mortality (6.5% to 83.8%), treatment failure (0 to 47.6%) and relapse rates (20% to 70%). These studies included patients with different degrees of immunosuppression, and different treatment and prophylaxis regimens. Also, there are differences in the study designs, the types of statistical methods used and the prognostic variables included in analysis. These variations may have resulted in patient selection bias or low statistical power, thus hampering a meta-analysis of all studied predictors of relapse. In spite of these limitations, we believe that the meta-analysis results of secondary prophylaxis are consistent, considering the available evidence. In addition, the quality of published reports was heterogeneous and usually poor. Despite these limitations, this review may assist clinicians in making decisions and may also help in the design of future studies.The results of this systematic review suggest there are identifiable predictive factors of VL relapse, such as previous episodes of VL relapse and lack of recovery of CD4+ lymphocyte numbers after primary visceral leishmaniasis. HAART did not produce the anticipated decrease in the incidence of VL relapses and more data is needed in order to better assess the evolution of VL in the HAART era. In contrast, secondary prophylaxis was shown to be protective against relapse. CD4+ count below 100 cells/mL at the time of VL primary diagnosis is a potential predictor of relapse.Based on these observations, a high-risk population might be identified and such patients might then be eligible for secondary prophylaxis. Strong surveillance will certainly contribute to improved data quality for decision-makers in this complex scenario. Randomized trials to compare the efficacy of different drugs and their role either in treatment or in prophylaxis are required.Table S1Parasitological control. Identification of Leishmania amastigotes by direct examination or by isolation of promastigotes in culture of tissue samples dAmB: amphotericin B deoxycholate LAmB: liposomal amphotericin LipAmB: amphotericin B lipid complex. PA: Pentavalent antimonial compounds SD: standard deviation IRQ: interquartile range : median μ: mean.(DOC)Click here for additional data file.Table S2VL: Visceral leishmaniasis Parasitological confirmation: identification of Leishmania amastigotes by direct examination or by isolation of promastigotes in culture of tissue samples #Serology confirmation: Leishmania direct agglutination positive §Biologic confirmation: identification of Leishmania amastigotes by direct examination or by isolation of promastigotes in culture of tissue samples or Leishmania-specific PCR on peripheral blood/bone marrow dAmB: amphotericin B deoxycholate LAmB: liposomal amphotericin B LipAmB: amphotericin B lipid complex PA: Pentavalent antimonial compounds Hemo: transfusion route IDU: intravenous drug user HETERO: heterosexual contacts HOMO: men who have sex with men sexual: heterosexual or homosexual contacts SD: standard deviation IRQ: interquartile range ♯ if the information was available (DOC)Click here for additional data file.Table S3Yes: positive association No: negative association VL: Visceral leishmaniasis HAART: highly active antiretroviral therapy HVC: hepatitis C virus * multivariate analysis.(DOC)Click here for additional data file.


Non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutants have been shown to emerge after interruption of suppressive NNRTI-based antiretroviral therapy (ART) using routine testing. The aim of this study was to quantify the risk of resistance by sensitive testing and correlate the detection of resistance with NNRTI concentrations after treatment interruption and virologic responses after treatment resumption.Resistance-associated mutations (RAMs) and NNRTI concentrations were studied in plasma from 132 patients who interrupted suppressive ART within SMART. RAMs were detected by Sanger sequencing, allele-specific PCR, and ultra-deep sequencing. NNRTI concentrations were measured by sensitive high-performance liquid chromatography.Four weeks after NNRTI interruption, 19/31 (61.3%) and 34/39 (87.2%) patients showed measurable nevirapine (>0.25 ng/ml) or efavirenz (>5 ng/ml) concentrations, respectively. Median eight weeks after interruption, 22/131 (16.8%) patients showed ≥1 NNRTI-RAM, including eight patients with NNRTI-RAMs detected only by sensitive testing. The adjusted odds ratio (OR) of NNRTI-RAM detection was 7.62  with nevirapine or efavirenz concentrations above vs. below the median measured in the study population. Staggered interruption, whereby nucleos(t)ide reverse transcriptase inhibitors (NRTIs) were continued for median nine days after NNRTI interruption, did not prevent NNRTI-RAMs, but increased detection of NRTI-RAMs . After restarting NNRTI-based ART (n = 90), virologic suppression rates <400 copies/ml were 8/13 (61.5%) with NNRTI-RAMs, 7/11 (63.6%) with NRTI-RAMs only, and 51/59 (86.4%) without RAMs. The ORs of re-suppression were 0.18  and 0.17  for patients with NNRTI-RAMs or NRTI-RAMs only respectively vs. those without RAMs (p = 0.04).Detection of resistant mutants in the rebound viremia after interruption of efavirenz- or nevirapine-based ART affects outcomes once these drugs are restarted. Further studies are needed to determine RAM persistence in untreated patients and impact on newer NNRTIs. All necessary permits were obtained for the described study, which complied with all relevant regulations. The samples used in this study have been described in previous publications (references 1 and 8).Efavirenz and nevirapine concentrations were measured by validated RT100–190UDS) was performed as previously described Plasma samples collected 4–12 weeks after NNRTI interruption were used for resistance testing. Selection was based upon sample availability and viral load levels >3000 copies/ml to allow reliable testing by the sensitive assays. Samples underwent Sanger sequencing and AS-PCR as previously described a priori and included in the multivariable models. In the first model, the variables analyzed were age, gender, ethnicity, HIV-1 transmission risk group, nadir CD4 count, duration of ART before interruption, viral load and CD4 count at the time of interruption, and interruption modality. In a second model exploring factors associated with the detection of NNRTI-RAMs, the analysis also included nevirapine and efavirenz plasma concentrations (n = 70). To overcome the limitation related to the small number of observations, the drug concentration data were pooled and analyzed as categorical variables (either above or below the median concentration measured in the study population). This approach was stipulated a priori as there was insufficient statistical power to analyze the two drugs separately or assess the interaction between nevirapine and efavirenz in this model. The proportion of patients who regained virologic suppression <400 copies/ml after re-starting NNRTI-based ART was investigated using logistic regression analysis as an intention-to-treat switch = failure analysis. Only patients restarting ART without a PI and with at least one viral load measurement in the following 4–12 months were included (n = 90). All factors of interest were stipulated a priori and included in the multivariable models. The variable included were age, gender, duration of ART before interruption, viral load and CD4 count at the time of interruption, time between interrupting and restarting ART, the NNRTI restarted, the interruption modality and the presence of RAMs. P-values were not corrected for multiple comparisons. All statistical analyses were performed using STATA software .Factors associated with the detection of RAMs by all testing modalities combined (n = 131) were investigated using standard univariable and multivariable logistic regression analysis. All factors of interest were stipulated The analysis included 132 patients that interrupted efavirenz , nevirapine , or delavirdine  in SMART. All patients were also receiving ≥1 NRTI, comprising lamivudine , tenofovir , zidovudine , emtricitabine , abacavir , didanosine , or stavudine . In addition, 15/132 (11.4%) patients were receiving a PI. The modality of interruption was simultaneous in 63/132 (47.7%) patients, staggered in 46/132 (34.8%) patients, and switched in 23/132 (17.4%) patients. The latter two groups continued the NRTIs or replaced the NNRTI with a PI/r respectively for median nine days  before stopping all therapy. The characteristics of the study population at the time of NNRTI interruption and according to the interruption modality are summarized in NNRTI concentrations were measured in 70 patients with sufficient plasma samples available at week 4, the first study visit after NNRTI interruption. Median 32 days  after interruption, nevirapine was detected in 19/31 (61.3%) patients at a median concentration of 2.2 ng/ml . Median 30 days  after interruption, efavirenz was detected in 34/39 (87.2%) patients at a median concentration of 21 ng/ml . Overall median concentrations  were 1.0 ng/ml for nevirapine (n = 31) and 16 ng/ml for efavirenz (n = 39).RT100–190UDS results (RT100–190UDS results (no RAMs detected). Nine samples did not have sufficient volume and were tested only by AS-PCR (no RAMs detected). One sample failed to give a result by both Sanger sequencing and AS-PCR (RT100–190UDS not done). Of the 21 samples tested by both AS-PCR and RT100–190UDS, 21 yielded a result by RT100–190UDS and 19 yielded a result by AS-PCR.Resistance testing was performed in samples collected median 8 weeks  after NNRTI discontinuation. At the time of testing, the median viral load was 27,618 copies/ml . Resistance results by were obtained in 131/132 (99.2%) samples, comprising 122 Sanger sequencing results, 124 AS-PCR results, and 21  results . OverallRT100–190UDS confirmed the AS-PCR results, with the exception of one sample that showed G190A by RT100–190UDS but not by AS-PCR; the frequency of the G190A mutant was 4% by RT100–190UDS. In addition RT100–190UDS detected NNRTI-RAMs not targeted by AS-PCR, including V179D, L100I, and K101E. Detection of NNRTI-RAMs according to the interruption modality was 13/62 (21.0%) for simultaneous interruption, 8/46 (17.4%) for staggered interruption, and 1/23 (4.3%) for switched interruption.The prevalence of ≥1 NNRTI-RAM by all testing modalities was 22/131 (16.8%) overall. Sensitive testing detected ≥1 NNRTI-RAM in 8/116 (6.9%) samples lacking NNRTI-RAMs by Sanger sequencing, and also increased the number of NNRTI-RAMs detected in each sample . With 19RT100–190UDS confirmed the AS-PCR results, with the exception of one sample that showed M184V by RT100–190UDS but not by AS-PCR; the frequency of the M184V mutant was 3% by RT100–190UDS. Detection of NRTI-RAMs according to the interruption modality was 10/62 (16.1%) for simultaneous interruption, 13/46 (28.3%) for staggered interruption, and 1/23 (4.3%) for switched interruption.The prevalence of ≥1 NRTI-RAM by all testing modalities was 24/131 (18.3%) overall. The NRTI-RAMs comprised TAMs in 20/24 patients, with mean 2.4 TAMs per patient; M184I/V in 15/24 patients; K65R in 3/25 patients; and L74V in 1/25 patient. Sensitive testing detected ≥1 NRTI-RAM in 2/116 (1.6%) patients lacking NRTI-RAMs by Sanger sequencing. These included K65R after simultaneous interruption of tenofovir, didanosine, and efavirenz , and K70R after staggered interruption of zidovudine/lamivudine and efavirenz  . SensitiIn total, 13/131 (9.9%), 9/131 (6.9%) and 15/131 (11.4%) patients had ≥1 NNRTI-RAM, both NRTI-RAMs and NNRTI-RAMs, and NRTI-RAMs only, respectively.3 higher ; and staggered interruption relative to simultaneous interruption with an adjusted OR of 4.25 .Detection of NNRTI-RAMs was less likely in patients with a viral load recorded as <50 copies/ml at the time of treatment interruption with an adjusted odds ratio (OR) of 0.28  . There wPatients restarted ART median 29 weeks  after interruption. The analysis of responses was restricted to 90 patients who restarted NNRTI-based ART (58 with efavirenz and 32 with nevirapine) without a PI and had at least one viral load measurement while on the NNRTI-based regimen in the 4–12 months after restarting. Overall, 73/90 (81.1%) patients regained virologic suppression <400 copies/ml; 50/90 (55.5%) had a viral load recorded as <50 copies/ml. The proportion of patients with viral load <400 copies/ml was 8/13 (61.5%) with NNRTI-RAMs, 7/11 (63.6%) with NRTI-RAMs only, and 51/59 (86.4%) without RAMs (p = 0.04); and 32/42 (76.2%), 23/29 (79.3%), and 18/19 (94.7%) following simultaneous, staggered, and switched interruption, respectively (p = 0.18). In multivariable analyses , the adjThis study demonstrated RAMs in a substantial number of patients experiencing rebound viremia after stopping suppressive NNRTI-based ART. Interpretation of the findings should take two limitations into consideration. Viral load assays with different lower limits of quantification were used in SMART and viral load suppression <50 copies/ml could not be demonstrated in all patients at study entry. Furthermore, due to sample availability, NNRTI concentrations were obtained only in a subset of patients. Nonetheless, the data provide sufficient evidence to indicate that different factors influenced the detection of RAMs after ART interruption. NNRTI-RAMs were less likely in patients who had a viral load recorded as <50 copies/ml at the time of interruption, indicating a risk of selecting NNRTI resistance even at low levels of viremia between 50 and 400 copies/ml. In addition, NNRTI-RAMs were more likely in patients showing higher plasma NNRTI concentrations at week 4 after interruption. These findings provide support to the notion that selection of NNRTI resistance can occur in patients experiencing slower NNRTI clearance after ART interruption. Two previous studies did not observe an association between NNRTI concentrations after interruption and detection of NNRTI-RAMs CYP2B6), for instance, catalyzes the main oxidative metabolism reaction for efavirenz. Three polymorphisms within the CYP2B6 gene have been associated with efavirenz estimated Cmin, although together they explain only one-third of inter-individual variability Genetic predictors of NNRTI clearance are being identified which may help tailoring NNRTI discontinuation. The cytochrome P450 (CYP)-2B6 isoenzyme (de novo selection during viral load rebound. In support of this hypothesis, most NRTI-RAMs were detected by Sanger sequencing, and there was a high prevalence of patients showing two or more TAMs. As TAMs are known to emerge in stepwise fashion during prolonged therapy with zidovudine or stavudine Detection of NRTI-RAMs was surprisingly common in this study. NRTI-RAMs were more likely in patients with a long previous ART history, suggesting re-emergence of resistant mutants archived during previous virologic failures, rather than, or in addition to, We previously reported that patients who had undergone a simultaneous interruption showed reduced virologic responses after restarting ART compared with those with a staggered or a switched interruption In our previous study we used Sanger sequencing to detect RAMs after ART interruption The AS-PCR methodology employed in this study has undergone extensive validation In summary, this study provides substantive evidence in support of the widely cited hypothesis that stopping NNRTI-based ART carries a risk of drug resistance. We show that viral load levels at the time of interruption, plasma NNRTI concentrations at week 4 after interruption, overall treatment history, and interruption modality combine to influence the risk of resistance and ultimately predict virologic responses when NNRTI-based ART is resumed. Further studies are required to assess the persistence of NNRTI-RAMs in patients remaining off therapy, the potential for their onward transmission, and the implication of these findings for etravirine and rilpivirine use.


Non-adherence is one of the strongest predictors of therapeutic failure in HIV-positive patients. Virologic failure with subsequent emergence of resistance reduces future treatment options and long-term clinical success.Prospective observational cohort study including patients starting new class of antiretroviral therapy (ART) between 2003 and 2010. Participants were naïve to ART class and completed ≥1 adherence questionnaire prior to resistance testing. Outcomes were development of any IAS-USA, class-specific, or M184V mutations. Associations between adherence and resistance were estimated using logistic regression models stratified by ART class.Of 314 included individuals, 162 started NNRTI and 152 a PI/r regimen. Adherence was similar between groups with 85% reporting adherence ≥95%. Number of new mutations increased with increasing non-adherence. In NNRTI group, multivariable models indicated a significant linear association in odds of developing IAS-USA (odds ratio (OR) 1.66, 95% confidence interval (CI): 1.04-2.67) or class-specific  mutations. Levels of drug resistance were considerably lower in PI/r group and adherence was only significantly associated with M184V mutations . Adherence was significantly associated with HIV RNA in PI/r but not NNRTI regimens.Therapies containing PI/r appear more forgiving to incomplete adherence compared with NNRTI regimens, which allow higher levels of resistance, even with adherence above 95%. However, in failing PI/r regimens good adherence may prevent accumulation of further resistance mutations and therefore help to preserve future drug options. In contrast, adherence levels have little impact on NNRTI treatments once the first mutations have emerged. Combined antiretroviral therapy (ART) aims at continuous and lasting suppression of viral replication, which is one of the most important factors influencing long-term prognosis of HIV-infected individuals ,2. The iVirologic failure is associated with increased risk of emergence of drug resistance  and therThe adherence-resistance relationship in historic monotherapy regimens containing a single unboosted protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) is thought to be similar. Studies showed that most drug resistance mutations were occurring in individuals with adherence above 90% ,18–20. ARitonavir boosted PI regimens allow for more potent viral suppression than ritonavir unboosted PIs and this reduces the emergence of resistance mutations. Boosting increases the half-life of the PI and so PI concentrations remain in a suboptimal therapeutic range for a briefer time during periods of non-adherence . ResistaFor NNRTIs, resistance is associated with interruptions in therapy  and deveThe potency of the ART regimen, defined as the likelihood to suppress HIV-1 viremia below the limits of standard assay detection for prolonged periods of time, is the largest single determinant of the development of resistance for all ART classes. The fitness cost of resistance and the genetic barrier to resistance are important but they matter most during active viral replication . TherefoThe goal of the study is to quantify the impact of adherence to ART on the development of class-specific resistance mutations in patients starting a new class of ART. Local ethical committees of all seven participating study sites , Ethikkommission des Kantons St. Gallen, Kantonale Ethikkommission Zürich, Comitato etico cantonale del Ticino, Commission d'éthique de la recherche clinique de la faculté de biologie et de medicine de l'université de Lausanne, Comité d'éthique du départment de médicine des hôpitaux universitaires de Genève) have approved the study and written consent has been obtained from all participants.Patients for this study were selected from the SHCS, which is a nationwide observational study of HIV infected individuals in medical care in Switzerland. In semi-annual visits, laboratory measurements are performed and clinical questionnaires are administered . The genThe SHCS drug resistance database was screened for genotypic drug resistance tests (GRT) which were performed while patients were receiving ART and to which an adherence assessment could be linked. In particular, GRTs/patients were included if the following conditions were fulfilled : PatientOn twice-yearly study visits, levels of self-reported adherence are assessed via interview with the clinician. Patients are asked about the frequency of missed drug doses over the past four weeks  and whether consecutive doses of ART were missed. Using the first question plus the dosing frequency of the regimen, the percentage adherence was calculated and then categorized into 100%, 95-99%, and <95%. We considered the inclusion of both adherence measures (percentage adherence and consecutive missed doses) together as well as separate in all models. The predictive value of the adherence questions and non-adherence definitions with regard to viral load levels have been demonstrated previously .The primary study outcome was the new emergence of any International AIDS Society – USA drug resistance mutations while receiving antiretroviral therapy . The priIn addition to the analysis of any new IAS-USA mutations, further sub-analyses considered specific types of mutations, which were any major mutations to the group-specific third drugs (i.e. either PI/r or NNRTI) and the new emergence of M184V mutations. The latter mutation was selected due to its low genetic barrier and the widespread use of M184V selecting drugs in Switzerland. These sub-analyses were restricted to individuals with the appropriate drug exposures, which selected for the mutations of interest. As a secondary outcome, we compared levels of plasma HIV RNA around the time of the adherence assessment across the different strata of medication adherence. Because a number of included patients lacked information from prior genotypic drug resistance testing  we perfoAll analyses were done using SAS v9.2  and Stata v12.0 . All p-values are two-sided and the threshold for statistical significance was set at 0.05.Of over 6700 GRT from therapy-exposed individuals, we were able to select 314 test results belonging to 162 individuals receiving NNRTI and 152 receiving PI/r . Patient>95% in both groups. Due to the limited number of cases, all adherence strata <95% were collapsed into one group for further analyses. Among the most notable differences between the two groups were the somewhat higher proportion of individuals who have acquired HIV via injecting drug use in the PI/r group and the lower proportion of individuals in the PI/r group who had previously experienced virological failure on antiretroviral therapy. Levels of adherence were similar between the PI/r and NNRTI groups, and approximately 85% of individuals reported adherence levels As shown in Indeed, the Stanford cumulative genotypic sensitivity scores of the drug combination at the time of genotyping (i.e. the number of active drugs) were generally higher in the PI/r group  with lower adherence in the PI/r group . No such trend was observed for the NNRTI group. Potentially those results in the PI/r group could have been influenced by previous virological failures, which may have affected genotypic sensitivity scores for NRTI drugs . Nevertheless, those associations between GSS and adherence in the PI/r were still observed when repeating the analysis on the set of individuals starting their first antiretroviral therapy .Descriptive analyses shown in Kaplan-Meier curves for the unadjusted association between adherence and development of mutations were estimated . These oIn the NNRTI group, only the development of any IAS-USA mutations showed a statistically significant linear univariable association with adherence (1.61 [1.06-2.46]). The association between NNRTI mutations (1.46 [0.94-2.23]) and M184V (1.01 [0.49-2.08]) and adherence did not reach statistical significance. In multivariable models provided in 10 copies/mL (standard deviation) were increasing with lower adherence and reached 2.8 (1.5) log copies/mL in the 100% adherence group, 3.2 (1.5) log copies/mL in the 95-99% group and 3.7 (0.9) log copies/mL in the <95% group. Consequently, there was a statistically significant linear trend of 0.5 [95% confidence interval 0.1-0.8] log10 copies/mL per lower adherence stratum . No such relationship was observed in the NNRTI group, where HIV RNA levels remained approximately constant across all adherence strata (in descending order) with 3.6 (1.3), 3.6 (0.9), and 4.0 (0.9) log10 copies/mL. Neither of these adherence group means differed significantly (F-test p=0.33). The sensitivity analysis yielded almost identical results. For the PI/r group a decrease in adherence was associated with a 0.46 [95% confidence interval 0.1-0.8] log10 copies/mL increase in HIV RNA, whereas no association was observed for the NNRTI group.We further investigated the impact of adherence on levels of HIV RNA measured at time of genotypic testing in our sample. In the PI/r group, average levels of HIV RNA logWe compared the durability of the regimens and found the viral failure rate to be significantly higher in those on NNRTI compared to PI . In those who experienced virological failure, the average durability of the regimen was 21 and 15 months in NNRTI and PI/r respectively (p=0.039) Within the NNRTI group, the average durability of those on regimens with EFV was slightly longer than those on NVP (22 versus 19 months) but the difference was not statistically significant. In this carefully selected data set of genotypic drug resistance tests and adherence measurements from individuals treated with potent combination antiretroviral therapies, we observed associations of NNRTI drug resistance emergence with levels of prior self- reported adherence, while associations for PI/r regimens varied according to type of resistance mutation. Therapies containing boosted PI seemed more forgiving to incomplete compliance compared with NNRTI-based therapies, which allowed much higher levels of resistance emergence, even at adherence levels of 95% and higher. In the NNRTI group, levels of resistance defined by the presence of at least one IAS-USA mutation increased markedly from the highest (29%) to the lowest adherence stratum (50%). Not surprisingly, many of those individuals from the NNRTI group with at least 1 new mutation also harboured NNRTI resistant viruses  with lower adherence in the PI/r group but not in the NNRTI group.Taken together, these observations suggest that good adherence may be beneficial even with virologically failing PI/r regimens, because this may prevent the accumulation of further resistance mutations and can therefore help to preserve future drug options. In contrast, adherence levels seem to have little impact on NNRTI treatments once the first mutations, and NNRTI mutations in particular, have emerged . Our data are largely consistent with previous studies showing that antiretroviral classes may have different adherence–resistance relationships. Gallego et al  found reSeveral limitations should be noted about this analysis. Despite drawing from large observational databases including almost 6800 genotypic drug resistance tests from drug exposed individuals, the final numbers of patients included in this analysis were small, but of the same order of magnitude of other studies ,15. OwinOur observations have important clinical implications. It is self-evident that good adherence promotes viral suppression and reduces the emergence of resistance mutations. In this analysis, we also observed evidence that adherence levels influenced resistance levels of virologically failing or failed PI/r regimens. Thus, even when partial resistance has occurred, good adherence levels may slow down or even stop progression to higher resistance levels, thereby possibly extending the durability of PI/r regimens . This efIn summary, our data suggest that promoting adherence may still be worthwhile for individuals receiving virologically failing PI/r regimens for damage control, but failing NNRTI regimens should be switched immediately.Table S1Development of new mutation by adherence level and drug class for individuals who either started their first antiretroviral therapy ever or who started a new class of ART and who had a prior on-treatment genotypic test result available .(DOCX)Click here for additional data file.


A paradoxical immunologic response (PIR) to Highly Active Antiretroviral Therapy (HAART), defined as viral suppression without CD4 cell-count improvement, has been reported in the literature as 8 to 42%, around 15% in most instances. The present study aims to determine, in a cohort of HIV infected patients in Brazil, what factors were independently associated with such a discordant response to HAART.3 (hazard ratio for AIDS or death of at least 8.5) and undetectable HIV viral load on HAART for at least one year. Controls: similar to cases, but with CD4 counts ≥ 350 cells/mm3. Eligibility criteria were applied. Data were collected from medical records using a standardized form. Variables were introduced in a hierarchical logistic regression model if a p-value < 0.1 was determined in a bivariate analysis.A case-control study (1:4) matched by gender was conducted among 934 HIV infected patients on HAART in Brazil. Cases: patients with PIR, defined as CD4 < 350 cells/mmAmong 934 patients, 39 cases and 160 controls were consecutively selected. Factors associated with PIR in the logistic regression model were: total time in use of HAART , nadir CD4-count , and time of undetectable HIV viral load .PIR seems to be related to a delay in the management of immunodeficient patients, as shown by its negative association with nadir CD4-count. Strategies should be implemented to avoid such a delay and improve the adherence to HAART as a way to implement concordant responses. Highly active antiretroviral therapy (HAART) has been introduced in the therapeutic arsenal for HIV infection since the 90's. The efficacy of HAART turned HIV infection into a chronic condition which made it possible to considerably improve its survival rate over the last 20 years -3. Such The frequency of a paradoxical immunologic response to HAART, defined as viral suppression without CD4 cell-count improvement, has been reported in the literature as 8 to 42%, around 15% in most instances -25.Several outcomes have been related to this poor immunologic response. Most of the studies had demonstrated worse outcomes when compared to those with complete response such as the increased risk of AIDS-defining illness or death ,16,23,25The efforts to determine the prevalence of this condition and its associated factors are hampered by the absence of standardization in its definitions. Different studies establish different boundaries in the CD4 cell counts or different time frames to cross such boundaries in the characterization of an adequate immune response, making it difficult to compare most of the results ,16,22,28Different associated factors have also been pointed out by several authors, like older age ,22,28-35The present study aims to determine, in a cohort of HIV infected patients in Brazil, what factors were independently associated with such a discordant response to HAART.Study individuals were selected from a cohort of 934 patients under treatment with HAART in a specialized facility in the city of Vitória, Espírito Santo State, Brazil. The study was designed as a matched case-control approach. Each case was matched to four controls on gender. Retrospective analysis was performed by a single investigator who collected the data from charts using a standardized form containing demographic, socioeconomic, and clinical and laboratory variables.3 in a patient with viral load suppressed who was on HAART over a year in the moment of the sampling. Controls were patients with the same characteristics as described for the cases, except for a CD4 count greater than or equal to 350 cells/mm3.Cases were all patients with paradoxical immunologic response (PIR) being defined as a CD4 cell count below 350 cells/mm3 with those with counts above 350 cells/mm3. Even comparison with individuals with counts between 200 and 350 cells/mm3 revealed a hazard ratio as high as 8.5 [This level of CD4 was used because hazard ratios as high as 10.7 were found for AIDS or death when comparing individuals with counts below 200 cells/mmh as 8.5 . RegardiThe controls were consecutively selected from the total number of patients who met the matching criteria from cases. As cases and controls were defined by the level of CD4 cell count, such count should be consistently determined by a constant value measured at least for twice during the previous six months (April-September 2009). For the purpose of this study, previous year was that between Oct. 1, 2008 and Sept. 30, 2009.Sample size was calculated assuming an 80% power for the test, a 95% confidence interval in the estimation of the effect, a minimum frequency among controls of 25% for any of the assessed risk factors, a ratio of one case to four controls and a frequency of the risk factors three times higher in the group of cases. With these parameters, 30 cases and 120 controls were necessary to discriminate the difference estimated a priori. This calculation was conducted using Epi Info™ Version 3.5.1.This study was approved by local Institutional Committee of Ethics on research on Sept. 9, 2009 under the registration number 087/09. Data collection was performed from Oct. 1, 2009 to Jan. 31, 2010.3; use of HAART for more than one year at inclusion; at least two CD4 counts in the previous six months; HIV viral load < 400 copies/ml (most common limit of laboratory detection over time) during six months before inclusion, and to be registered at the local outpatient clinic.Inclusion criteria were: age greater or equal to 18 years old; having started HAART according to Brazilian guidelines, with CD4 < 350 cell/mmIndividuals were excluded if they: had participated in the programmed interruption of HAART at any time during their follow-up; underwent treatment with interferon or chemotherapy in the last year; were considered as immune failures in the previous year (defined as a decline of more than 25% of the CD4 count); were considered as clinical failures in the previous year (defined as the occurrence or recurrence of AIDS defining illness after 3 months of HAART); had irregular use of HAART in the previous year (defined as discontinuation of the regimen for more than 30 consecutive days); failed to attend the medical appointments in the previous 6 months, and were pregnant at the inclusion moment of the sampling process.Chi-square test or Fisher's exact test were used for comparison of categorical variables between the two groups. For continuous variables, "Student's" T test was used for comparison of normal distributions between the two groups. For non-normal distributions, it was used the Mann-Whitney test.Multivariate analysis was performed by the binary logistic regression model using Enter method for all variables with a p-value < 0.1 in the bivariate analysis model. It is important to note that the inclusion of variables in this model was performed according to a hierarchical causal theoretical tree previously developed for this study. Such a hierarchy had previous application in epidemiological studies to improve the accuracy of statistical analysis .The model was fitted with the selected variables that fulfilled the requirements for logistic regression: being independent, having p-value < 0.1 in bivariate analysis, having values in the cells of the cross tabs greater than or equal to one, and having not more than 20% of cells values below five. Variables were not included in the model if missing information exceeded 10% of the sample.The independency among variables was tested to see if there was a correlation or association between them. Pearson correlation test or Chi-square test was performed, as more appropriate. When variables were associated or correlated with each other, the one chosen to enter the model was the most significant in the bivariate analysis or the most relevant in clinical practice.The Hosmer-Lemeshow test was used to verify if the variables were well adapted to the theoretical model.SPSS Statistics package version 15.0).All statistical analyses were performed through the SPSS software  563 had undetectable HIV viral load (HIVVL) (limit of detection: 50 copies/ml). Out of 563 with viral suppression, 39 cases and 160 controls were consecutively selected, with individual matching by gender. Controls were derived from 272 medical records sequentially verified. In the group of cases, there were eight females and 31 males, whereas in the control group 36 females and 124 males.Sampling of cases and controls according to the eligibility criteria of the study is represented in Figure Demographic and socioeconomic characteristics of the 199 patients (39 cases and 160 controls) included in the study are shown in Table The median age at diagnosis for AIDS and the onset of the first HAART regimen was 38 years old. From these subjects (cases plus controls) 37.3% were single, 44% married, 11% separated, and 7.7% widowed. Regarding race/color, there were 47.1% of white people, 38.1% mixed, and 14.8% black. The educational level of the patients was as follows: 19.7% had four years of education at most; 40.4% had up to eight years; 29.5% had completed high school, and 8.7% with University education (completed or not).3. Regarding the HIV transmission category, 97% of them had a suspected sexual exposure, 11.2% were injecting drug users, and only 0.5% was related to a blood transfusion  adapted criteria for AIDS definition; comorbidities in the previous year; the year of HIV diagnosis; time from HIV diagnosis until the beginning of the first HAART Table . AIDS diCases and controls differed in the presence or not of an AIDS defining illness before their first HAART regimen ; in the presence or not of an AIDS defining illness before any HAART (p = 0.04); if they had been using concomitant medication in the previous year for over thirty consecutive days p = 0.002); in the total time in use of HAART in months (p = 0.001), and in time of use of current HAART in months (p < 0.001) , and had a marginal difference in their frequency resulting from the association of nucleoside analogues reverse transcriptase inhibitors with non-nucleoside analogues (p = 0.099). There were no significant differences for the following variables: therapy containing tenofovir plus didanosine; regimen containing protease inhibitors; prior use of mono or dual antiretroviral therapy; occurrence of substitutions in the components of HAART since the first regimen in use; number of substitutions since the first HAART regimen in use; percentage of visits attended in the last year, and the percentage of absences in the pharmaceuticals dispensations in the last year (data not shown).Differences were observed in the presence of comorbidities and in the frequency of concomitant medications as follows: presence of dyslipidemia in the previous year , use of fibrates , and prophylaxis with trimethoprim-sulfamethoxazole  (data not shown).There were also differences in the reasons for HAART substitutions: prior abandonment or poor adherence , and Zidovudine myelotoxicity  (data not shown).Regarding laboratorial data, the following are the different characteristics between cases and controls: lymphopenia before first HAART (p = 0.026) and before the current HAART (p = 0.007), CD4 count prior to the first HAART (p = 0.001) and before the current HAART (p < 0.001), nadir CD4 count (the lowest CD4-count ever presented by patient during outpatient follow-up) (p < 0.001), time of undetectable HIVVL (p < 0.001) and time of HIVVL below 1 000 copies/ml (p < 0.001). There were no statistically significant differences between the two groups regarding the HIV viral loads before the first HAART and before the current HAART, either in absolute numbers or in logarithm values Table .Figure Variables that remained significantly different after logistic regression were: total time in use of HAART , nadir CD4-count  and time of undetectable HIVVL (adjusted OR 0.969: CI 95%: 0.94-0.99) . For each additional month of undetectable HIVVL there is a reduction of 3.1% in the risk of PIR Table .3 in nadir CD4-count corresponds to a risk reduction of PIR of 77.7%.Regarding nadir CD4-count, every increase of one cell corresponded to a reduction of 1.5% in the risk of PIR  has been surrounded by uncertainties, both regarding its determinant factors and its consequences for the outcome of HIV infection. Despite its retrospective design and use of preexisting data, this study was able to uncover a negative association between PIR and total time in use of HAART as well as time for undetectable HIV viral load. Such an association was not adequately revealed by other studies because of their rigid eligibility criteria in terms of time frames, thus preventing any analysis of the influence of time frames on the outcomes ,19-21,23Time of HIVVL suppression can be a surrogate marker of adherence to HAART. Thus, patients who have good adherence and, therefore, a longer time of undetectable HIVVL are less likely to come forward with paradoxical immune response. Such an association has also been reported by others ,41. LongAnother variable independently associated with PIR in this study was the nadir CD4-count. In other studies, both baseline CD4-count (measured before starting HAART) ,34-40,46Studies have been undertaken to elucidate the mechanisms involved in the occurrence of this poor immune response. Some of these studies have found direct association between the frequency of paradoxical immune response and the increase in T-cell activation (or persistence) and, consequently, excessive apoptosis of these cells ,48,49. BOther studies have reported a relationship between PIR and cytokines profile. As an example, Sachdeva et al (2008)  demonstrA relationship between age and PIR has been emphasized by studies highlighting a correlation with thymus size and function ,29,31-34Recent guidelines for the treatment of HIV infection do not establish a rigid CD4 cell count threshold at which therapy should be initiated. The International AIDS Society-USA Panel, for example, states that deciding when to initiate HAART is mainly a matter of the readiness of the patient . Such reThe limitations of the present study are those inherent to the case-control design, particularly regarding the vulnerability to selection bias and misclassification. Such possibilities are even more evident when one considers the preexistence of the data and the selection of prevalent cases. However, the consistence of the associations observed between the candidate variables of exposure and the outcome reinforces the validity of the conclusions, even if some potential risk factors could have persisted undetected.The main contribution of this study lies in the demonstration of an independent association of PIR with markers of intense immunossupression such as low nadir CD4 counts and short periods of time for undetectable viral loads. It seems that immune reconstitution is a process that depends on a residual immune function to be effective. In other words, if the immunossupression had gone too far, it could be too late to obtain a consistent reconstitution.Therefore, there is a need for more studies, mainly with a prospective design to fully establish the consequences of PIR. If there is an important impact over morbidity and quality of life, as anticipated, the conclusions from studies like this one should strengthen the need for early intervention with HIV infected patients regarding HAART. The more we learn about it, the more we understand the complex nature of HIV infection, and better strategies are made available to allow for safe and effective intervention for the immune reconstitution. In the future, inadequate responses like PIR should be eliminated at the best scenario.The authors declare that they have no competing interests.JASC contributed to the study design, collection and analysis of data and writing of the manuscript. LNP participated in the study design and in the revision of the manuscript. FJPO made the analysis of data, organized the database and formatted the tables. CCJ participated in the study design, in the data analysis and in the revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/306/prepub


The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru.5 RNA/ml (range: 2.1·103 – 1.2·106), and a median CD4-count of 232 cells/μL (range: 1–1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots.Between 2002–5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·10During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure.Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/− NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure. Antiretroviral therapy (ART) has, for the past years, increased the hope for survival of millions of people living with the human immunodeficiency virus (HIV) worldwide, adults as well as children. A clear survival advantage was achieved for HIV-infected patients with a dramatic decrease in new AIDS cases . Immune en bloc and large groups of patients are started on ART simultaneously, cross-sectional drug resistance testing may be particularly useful.Once a first-line regimen has failed however, the reasons for such failure may be complex, including malnutrition and co-morbidities leading to poor absorption of medications. Lack of economic resources and education may further complicate the already difficult adherence to complex medication schedules -11. SomeThis study aims to test the value and feasibility of cross-sectional resistance testing as well as innovative tools to display disease progression or clinical/immunological improvement in the first cohort of children starting ART in Peru. With Global Fund support, structured ART first became available in August 2002 to a select group of HIV-infected children at the Instituto Nacional de Salud del Niño (INSN) in Lima, based on the criteria established by the Guideline for the Management of the HIV-Infected Child by the Peruvian Ministry of Health (MINSA) -17.2 every 12 hours) with lamivudine  and nelfinavir  [In contrast to a neonatal cohort starting ART several years later, the majority of patients in this first cohort at the INSN were school-age, had already progressed to AIDS when starting ART and were born before the broad introduction of prevention of mother-to-child transmission (pMTCT) programs in Peru . Therefo8 hours) .At the time of introduction of ART in Peru, access to drug resistance testing was still limited. To save cost, alternative testing methodologies and transportation modalities were sought, such as the Oligonucleotide Ligation Assay (OLA) -21 and fThe aims of the study were:1. To determine the prevalence of antiretroviral drug resistance in children with virologic failure versus no virologic failure.2. To evaluate the sensitivity of the DNA-OLA from frozen peripheral blood mononuclear cells (PBMC) as compared to the OLA from virion RNA (plasma) and RNA consensus sequencing from dried blood spots.3. To determine factors associated with virologic failure and drug resistance development.4. To design a simple and integrative display of clinical/immunological progression of HIV disease after ART initiation.a and age < 18 months, or age >18 months and CDC immune category 2 or 3. Exceptions were planned for asymptomatic patients with a rapid decline in CD4+ or viral load >100,000cp/ml  [From 2002–2005, study participants had undergone standard medical procedures and routine HIV medical care at the Infectious Diseases Service at the INSN. According to the MINSA Guideline for the Management of the HIV-Infected Children, CD4+counts had been determined every 3 months, and viral load every 6 months at the Peruvian National Institutes of Health  . Antiret months) . Ethics PACTG P1042S) was used at the time of cross-sectional analysis to systematically measure adherence based on information provided by parents and caregivers [For the cross-sectional analysis in 2005, all eligible subjects undergoing ART according to the MINSA program who agreed to participate and whose parents/guardians had signed the informed consent, were included. Basic clinical and virologic parameters from the start of ART in the individual patient until the date of testing were extracted from routine medical records and laboratory reports . Additional parameters were obtained, such as CDC stage , opporturegivers ,29.At the time of the first regular follow-up visit after entry into the study, routine blood sampling was again performed at the INS. In addition, 5 ml of citrated blood were collected from study participants for resistance testing. In addition, two Guthrie filter cards were collected with 4 capillary blood spots (finger prick) of 50 uL each.Ficoll-Hypaque centrifugation and separation of the citrated blood was performed at the PRISMA laboratory in Lima. Plasma and PBMC were immediately stored separately at -20C and shipped on dry ice to the Tulane and LSU PACTU laboratory for RNA and DNA extraction. Viral loads in plasma were quantified by real-time RT-PCR as described .The OLA was conducted according to the NIH protocol for mutations at HIV-1B protease positions D30N, I50V, V82A, V82S, V82T, I84V, N88D, and L90M as well as reverse transcriptase positions K103N, Y181C, K65R, T215F, T215Y, M184V, and Q151M . Dried BFor the purposes of the study, virologic failure was defined by two or more consecutive HIV RNA measurements above the detection limit (400cp/ml), 4 – 6 months after the initiation of ART therapy in patients where ≥ 2 viral load measurements were available. In patients P016T, P021T, P041T, P053T and P057T only two viral load measurements were available in total. These patients all showed signs of virologic failure indicated by HIV RNA measurements > 400cp/ml > 10 months after treatment initiation.N needed for assessing differences in resistance development between patients failing ART and those successfully treated.We assessed the population size P (failure) = 0.5 and that those failing ART would with 90% probability develop drug resistance P (res. |failure) = 0.9.We assumed that 50% of patients would eventually fail ART P (res.|sucess) = 0.1. We can therefore compute the expected number of patients with failure and resistance a = P(res. |failure)●P(failure)●N, with failure and no resistance b = (1-P(res. |failure))●P(failure)●N, with no failure and resistance c = P(res. |success)●(1-P (failure))●N and with no failure and no resistance d = (1-P(res. |success))●1-P(failure))●N. According to Fisher’s exact statistics p ≤ 0.05) for a sample size of N = 12. For values P(res. |failure) = 0.8, P(res. |failure) =0.7 and P(res. |failure) = (1-P(res. |sucess)) population sizes of N = 12 and N = 22 would be required.Conversely, successfully treated patients may with 10% probability develop resistance For the purpose of this analysis, CDC categories were applied in a novel way, assigning new CDC categories at each assessment time point ignoring previous CDC scores.rC and rI respectively (average change of CDC score per year throughout the study population) were computed with the following formulaThe rates of clinical and immunological progression mI and mC denote the magnitude (number of scores) of change observed and Fm,I and Fm,C the fractions that have changed by that magnitude within a certain time interval. For our evaluation, we computed the rates of immunological and clinical progressionfrom enrolment throughout years 1, 2 and and beyond (>=3).x represents the child’s weight and μ and σ denote the average weight within the child’s age category based on the WHO reference and standard deviation, respectively [x = μ-2σ).Malnutrition in the study population was assessed in terms of standard scores (z-scores) of child weight at enrolment in relation to the WHO reference weight . The staectively . A stand5 RNA/ml (range: 2.1∙103 – 1.2∙106) and the median CD4-count was 232 cells/μL (range: 1–1591). Notably, five children had CD4 counts below 10 cells/μL. The median weight at enrolment was 18 kg (range: 5.5-45). Notably, 43/46 (93%) had negative z-scores for child weight compared to the WHO reference corresponding age group [A total number of 46 children were enrolled between September 2002 and March 2005. Median age at enrolment was 5.6 years (range: 0.7-14y). The median viral load at enrolment was 1.7∙10ge group , indicatge group ) were atge group . Four chVertical HIV transmission was the mode of infection for all but two children, who were infected by blood transfusion. Seven mothers had received antiretroviral prophylaxis with AZT +/− NVP for the prevention of mother-to-child transmission (pMTCT). Three children had been exposed to postnatal AZT for pMTCT . Four children had been exposed to unsupervised ART prior to enrolment: two children  received 3TC+AZT prior to enrolment. One child (P067T) continued NFV+3TC+AZT without any gap, while P053T and P016T had received NFV+3TC+AZT prior to initiation of the program. One child P016T continued with only a few weeks interruption, whereas for P053T there was a gap of one year between his prior ART medication and ART medication provided through this program. Throughout the study period, standard treatment was modified in five children . In these children, one component of their ART regimen was substituted respectively: AZT was replaced by stavudine (d4T) in P011T and P031T, 3TC was replaced by didanosine (DDI) in P057T, and NFV was replaced by nevirapine (NVP) in P007T and P019T.The central tendency of viral dynamics is shown in Figure As can be seen, 44% of children experienced virologic failure during the first year of ART, half of the children failed before the end of the second year of ART. By the end of the study, 60 ± 16% had experienced virologic failure.Both patients who had been infected by blood transfusion (2/2) and all children with previous ART exposure (4/4) eventually experienced viral failure. None of the 7 children whose mothers had received pMTCT prophylaxis with AZT +/− NVP (0/7) and none of the children who had received post-natal AZT prophylaxis for pMTCT (0/3) experienced virologic failure.2 test), with severely immunosuppressed patients being most likely to fail ART.Children who were younger at entry were slightly more likely to fail ART (p = 0.06 by Wilcoxon rank sum test). Virologic failure was significantly associated with the immunologic CDC-score at baseline .In contrast, the CDC clinical category at baseline was not predictive of virologic failure during subsequent ART. Children who had reported missing > 50% of doses (according to the adherence questionnaire administered) were also more likely to experience virologic failure .Study participants were classified in terms of CDC clinical and immune categories at enrolment, during year 1, during year 2, and after year 2, as shown in Figures It can be seen in Figure The overall rate of clinical/immunologic disease progression per treatment year is shown in Figures Immunologic improvement was minimal during year three (−0.1 stages), whereas the clinical status of the study participants worsened slightly by 0.16 stages on average , with patients failing therapy showing higher scores , while the final clinical CDC-scores were not linked.The immunologic CDC-scores at the time of final assessment were significantly correlated with virologic failure  seemed to lag behind, possibly due to the fairly advanced disease stages at baseline.The median weight after 1, 2 and 3 years of treatment was 20 kg, 22.3 kg and 23 kg, respectively. The median z-score was −1. During the first year of ART, 72% of the children showed negative z-scores, 75% in year 2 and 67% in/after year 3, which is a considerable improvement over child weight at enrolment, 93% showed negative z-scores.On average, drug resistance testing was performed at 2.4 years after the initiation of structured ART. Prior to the cross-sectional analysis of this treatment cohort, drug resistance information had not been available to direct the choice of treatment regimens. In ART-failing patients, the vast majority of drug resistance tests (96%) were performed at time points after virologic failure.Samples for RNA consensus sequencing were transported as dried blood spots on Guthrie cards. RNA amplification for consensus genotyping was possible in 14/46 samples , in 4 instances only the protease gene (PR) could be sequenced. All RNA consensus sequencing data is provided in Additional file Samples for DNA and RNA OLA testing were transported as frozen plasma and PBMC samples after Ficoll-Hypaque centrifugation and separation. Of these, RNA-OLA testing was performed successfully in 20/46 43%), in one case only the protease mutations could be tested by RNA-OLA. All OLA data is provided in Additional File %, in oneThe M184V reverse transcriptase mutation was detected in 80% of the sequenced RNA samples and tested positive in 74% and 47% by RNA-OLA and DNA-OLA, whereas thymidine associated mutations  were deThe protease mutation D30N was detected in 43% of RNA genotyping samples and in 0% and 2% of available RNA- and DNA-OLA samples. The N88D and L90M protease mutations were detected in 36% and 21% of genotyping samples, in 25% and 20% of RNA-OLA samples, and in 42% and 44% of DNA-OLAs, respectively.Children who were previously exposed to short-term antivirals showed significantly higher numbers of resistance mutations detected by DNA-OLA (p = 0.01 by Wilcoxon rank sum (WRS) test), but not by RNA-OLA  or genotyping  at the time of cross-sectional analysis. Virologic failure was strongly associated with the number of resistance mutations detected by DNA-OLA .b, p < 0.05c and p < 0.001c). Also, the mutations N88D and L90M (NFV resistance) were more frequently detected by DNA-OLA in patients with virologic failure . The protease mutation D30N was not detected more commonly in cases of virologic failure (by any of the assays used), neither were TAMs selected differentially in failing vs. non-failing patients.The detection of the M184V reverse transcriptase mutation (indicating 3TC resistance) by any of the three methods  was significantly more frequent in patients with virologic failure . The ability to obtain positive results with the RNA OLA, along with the detection of mutations M184V, N88D and L90M, may thus suggest virologic failure in this cohort of patients.The detection of the same mutations  by DNA-OLA yielded a slightly lower sensitivity of 0.86, 0.9 and 0.75 for virologic failure, but the assay could be performed in almost all patient samples (regardless of virologic success or failure) indicating that virologic failure may indeed be attributed to resistance development at these three residues  all patients received the same first-line antiretroviral regimen and b) patients, on average, were in advanced stages of HIV/AIDS when starting their first antiretroviral regimen . When anThe effect of delayed access to ART in this first cohort becomes evident in comparison to a recent study observing the transmission of resistant virus in a much younger cohort of neonates and children with timely access to pMTCT and ART in Peru, revealing a predominance of NNRTI mutations, whereas mutations conferring high-level resistance to ARV were still found to be rare . This obChances of virologic failure were high in this first pediatric cohort gaining access to ART in Peru in 2002/3, with ~44% showing virologic failure after the first year of ART, ~53% after two years. The majority of children were in poor health, as evidenced by malnutrition 93% of children below the reference weight for the respective age group ) and a hImmunologic classification at baseline was very predictive for virologic failure. In agreement with studies in industrialized countries ,37, thesDespite relatively high rates of virologic failure in this cohort, both immunological and clinical conditions improved during ART, in particular throughout the first and second years of treatment. Thereafter little additional improvement was achieved. Overall, from the time of initiation of ART up until the time of the cohort assessment, 57% had showed marked improvement with respect to their clinical status (as measured by CDC category/visit), whereas 35% were unchanged clinically, and only 8% showed disease progression. With respect to the immunologic CDC-scores, 76% had improved, 22% had experienced no change, and 2% showed a decline in CD4 counts.For improved visualization of the overall development of treatment cohorts during ART, we summarized the clinical and immunological response to therapy in an innovative fashion using a Clinical Course Integrated Display (CCID) with 3-by-4 tables based on the revised CDC clinical and immunological categories . Here, wPrevious exposure to (often incomplete) ART was significantly associated with virologic failure, indicating that short courses of unsupervised ART prior to the initiation of coordinated long-term treatment programs may be counterproductive as they may lead to the rapid development of drug resistance. Archived drug resistance mutations, acquired during previous exposures to antiretrovirals and still present in the PBMC compartment may be detected reliably by DNA OLA.Exposure of the newborn to post-natal pMTCT with AZT did not increase the likelihood of subsequent virologic failure, neither did maternal exposure to pMTCT with AZT +/− NVP. There are three possible explanations why pMTCT did not affect subsequent treatment success:a) The pMTCT did not lead to a transmission/selection and “archivation” of drug resistance,b) Although drug resistance against the pMTCT regimen (i.e. AZT +/− NVP) developed and was archived, it did not impede the success of subsequent triple-drug ART consisting of AZT + 3TC + NFV.c) Drug resistance did not persist until the initiation of ART.In fact, in only one child (P028T) we detected archived drug resistance by DNA OLA  at the time of cross-sectional resistance testing. This child (P028T) did not encounter virologic failure (hinting towards scenario b).Drug resistance in the context of pMTCT may emerge- or be transmitted - by two possible mechanisms:(i) Drug resistant virus is selected in the mother and passed on to the child (e.g. during birth or breastfeeding).(ii) The newborn is infected with susceptible virus and subsequently selects drug resistant virus, e.g. during ARV exposure.Ad (i): When a single dose of antivital medication for maternal pMTCT is administered at the onset of labor, it is rather unlikely that drug resistant virus is passed on to the child. Although the pMTCT regimen may induce a selective pressure on the maternal virus, there is hardly enough time for this virus to be selected to sufficient numbers to be transmitted during birth, see also . HoweverNewborns P019T, P020T and P028T were not breastfed and their mothers received a single dose of AZT +/− NVP shortly before birth. However, these newborns received 6 mg/day  or 28 mg/day (P028T) of post-natal AZT. As explained above, postnatal AZT administered to P028T may explain the archiving of AZT resistance in the child’s PBMC DNA (case ii). However, this did not lead to subsequent therapeutic failure (case b).The mothers of newborns P002T, P003T, P027T and P046T were breastfeeding. They received extended AZT for periods shorter than the actual duration of breastfeeding. None of these children  showed evidence of archived AZT resistance based on DNA-OLA at the time of cross-sectional assessment. These children could have either been infected with susceptible virus during labour, or during breastfeeding , or else resistance may not have persisted until treatment initiation or until the DNA OLA was performed.A potential weakness of a cross-sectional study design is that clinical and laboratory data from the beginning of ART up until the date of cross-sectional analysis had to be extracted from medical records and parent/patient interviews. Adherence data using the PACTG questionnaire are always self-reported. This study design does not allow for detailed cause-effect analyses, prospective surveillance and follow-up visits, or the assessment of mortality data. The cross-sectional analysis however does reflect the real-world effectiveness of a medical intervention in a low-resource setting, which often includes patients who would not typically be able to participate in controlled clinical trials. The focus of this study was the assessment of the usefulness of cross-sectional resistance testing using the DNA versus RNA OLA.The DNA OLA may be particularly useful for the purposes of population-based surveillance in low resource settings where genotyping tests may not be readily available. The DNA-OLA was very indicative for the presence of resistance , but less indicative for the absence of resistance  in comparison to the RNA OLA. To the contrary, the RNA-OLA was more useful to determine the absence rather than the presence of drug resistance. Therefore, DNA-OLA can be used to rule-in resistance, whereas RNA-OLA may be used to rule-out resistance.The detection of the resistance mutations M184V, N88D and L90M by DNA-OLA was highly sensitive for virologic failure in this cohort treated with lamivudine-azidothymidine-nelfinavir as first-line therapy. The analysis of archived HIV-DNA resistance in PBMC provided useful results in most patients, even if virologic failure was not (yet) evident. The DNA-OLA may detect resistance mutations that have been acquired during previous exposure to erratic short-term ART, still present in the lymphocyte compartment. This may occur in low-resource settings before antivirals become universally available, when patients and their families are restricted to temporary access to limited, often insufficient amounts of antiviral medications. Turnover rates within the lymphocyte compartment may however be too low for the early detection of antiretroviral drug resistance during therapy .A possible strategy for the improvement of ART in resource-poor settings (where genotyping is often not available) could be to use the DNA-OLA as a baseline screening tool before starting therapy. This could be combined with the use of RNA-OLA in those patients experiencing virologic failure. Notably, a positive RNA OLA at positions M184V, N88D or L80M was highly sensitive for virologic failure . Therefore, drug resistance monitoring at key residues using RNA OLA in patients experiencing virologic failure may be particularly useful as an economical indicator of drug resistance and could suggest a treatment change.Success rates could likely be improved even further if treatment was initiated at higher CD4 counts, in line with recent revisions of the treatment guidelines in industrialized countries (initiation of treatment at an adult CD4 count of 350 cell/μL) ,37. ThisWhile consensus RNA genotyping (if available) will likely remain the mainstay of individualized resistance testing during ongoing antiretroviral therapy, the applicability of the OLA in population-based surveillance remains to be fully assessed in larger cohorts, including cost-effectiveness analyses and assessments of the personnel and training required for either method. At the time of the study, genotyping was not available. In recent years, capacities for monitoring drug resistance have been expanded at the Peruvian INS including sequencing facilities and an e-health driven, web-based laboratory information system ,41. The Our data emphasize the need for timely antiretroviral treatment initiation and early HIV testing to contribute to this aim ,12,35,441. HIV drug resistance was the major factor contributing to virologic failure of antiretroviral therapy in this cohort of children with delayed access to structured ART in Lima, Peru.2. In most instances, virologic failure occurred early in the course of treatment and commonly after previous exposure to unsupervised ART, but not in relation to pMTCT.3. The DNA OLA method detected antiretroviral resistance at key positions independently of virologic failure in the form of integrated DNA (in PBMC), whereas the RNA OLA detected antiviral resistance in viral RNA (in plasma) only after virologic failure. Antiviral resistance was more readily detected by OLA than by RNA consensus genotyping (from dried blood spots).4. The DNA-OLA could be used prior to treatment initiation to rule-out archived drug resistance to standard regimens, in particular when previous exposure to antiretrovirals is anticipated. The RNA-OLA could be used to guide the choice of second-line antiretrovirals in patients switching ART regimens after experiencing virologic failure.a confirmed by DNA-PCR/viral load at 6 months, or by ELISA at/after 18 months or AIDS-defining diagnosisb Fisher’s exact testc χ2 testINS: Instituto Nacional del Salud ; IESN: Instituto Especializado de Salud del Niño; PRISMA: Asociación Benéfica Proyectos en Informática, Salud, Medicina y Agricultura; MINSA: Ministerio de Salud del Peru; PACTG: Pediatric AIDS Clinical Trials Group; ART: Antiretroviral Therapy; MTCT: Mother-to-child transmission; pMTCT: Prevention of mother-to-child transmission; AZT: Azidotymidine; 3TC: Lamivudine (LMV); NFV: Nelfinavir; NRTI: Nucleoside-analogue Reverse Transcriptase Inhibitors; NNRTI: Non-nucleoside-analogue Reverse Transcriptase Inhibitors; PI: Protease Inhibitor; OLA: Oligonucleotide Ligation Assay; PCR: Polymerase Chain Reaction; RNA: Ribonucleic Acid; DNA: Desoxyribonucleic Acid; WHO: World Health Organization; HIV: Human immunodeficiency virus; AIDS: Acquired Immunodeficiency Syndrome.All authors declare no competing interests.Study concept and design: BAR, RAO, RVD, DKK. Acquisition of data: BAR, GSC, MEC, LK. Laboratory Analyses: BAR, PC; AMA, JER, DKK. Analysis and interpretation of data: MVK, BAR. Drafting of the manuscript: BAR, MVK. Critical revision of the manuscript for intellectual content: DKK, RAO, RVD, AMA, GSC, PC. Statistical analysis: MVK. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/1/prepubIndividual viral load dynamics in children after treatment initiation, stratified by responders (black solid dots) and children who experienced virologic failure (red squares).Click here for fileSequencing Data. Table with the raw viral sequencing data from dried blood spots.Click here for fileOLA Data. Table with the raw OLA data from plasma (RNA-OLA) and PBMCs (DNA-OLA).Click here for file


A critical indicator of the future success of highly active antiretroviral therapy (HAART) is the incidence of HIV drug resistance, which has not been studied in China on the national scale.HIV drug resistance baseline survey was conducted in the eight provinces with the largest numbers of patients on HAART in 2009, and a prospective cohort study with 12-month follow-up was completed in 2010. Patients completed an interviewer-administrated questionnaire and provided blood for CD4+ T-lymphocyte count (CD4 count), HIV viral load (VL), and HIV drug resistance genotyping. Factors associated with incidence of HIVDR were identified by Cox regression analysis.The overall prevalence of HIV RNA ≥1000 copies/ml and HIVDR at baseline was 12.4% and 5.6%, respectively. Incidence of HIVDR in the one year follow-up was 3.5 per 100 person years. Independently associated factors were started treatment with a didanosine-based regimen, received care at township hospital or village clinic, low baseline CD4 counts, and high baseline VL.The incidence of HIVDR in China was higher than that of some developed countries. China urgently needs to provide comprehensive education and training to doctors at village clinics and township hospitals to improve quality community-based care and treatment. Since the introduction of combination drug regimens to treat human immunodeficiency virus (HIV) infection, known as highly active antiretroviral therapy (HAART), the rates of HIV-related morbidity and mortality have been markedly reduced. In 2009, the baseline survey on HIV drug resistance was conducted in the eight provinces in China with the largest numbers of patients on ART under the NFATP. The county in each province with the most patients was selected to receive the survey, and up to 250 patients were recruited for each province. If not enough eligible patients were found in the county, the county with the next largest number of patients was selected. All patients who received treatment from 2005 onwards were chronologically contacted for recruiting to the study; detailed subject recruitment procedure has been previously described. Following the baseline survey, all patients were followed up one year later in 2010 to evaluate the incidence of HIV drug resistance (HIVDR). The survey sites were : Yunnan (Long chuan), Anhui (Jieshou and Linquan), Shandong, Hunan (Hengyang), Guangdong (Shenzheng), Zhejiang , Xinjiang (Yining), and Guangxi (Hezhou). Shandong province has fewer patients who are dispersed throughout the province, hence patients were recruited from the whole province as opposed to in one county only.+ T-lymphocyte count (CD4 count), HIV viral load, and HIVDR mutations.In the survey, an interviewer-administrated questionnaire interview was conducted to collect demographic data and data on ART treatment. Demographic variables include height, weight, ethnicity, education, residency, occupation, average monthly family income, and residency status (permanent vs. migrant). Treatment and behavior variables include initial treatment date, spouse ART status, taking traditional Chinese herbal medicines, receiving counseling and instructions on ART use (currently and before starting ART), clinical symptoms in the recent month, recent sexual behavior, recent alcohol use, recent drug, source of ART drug distribution, interval of refilling drug in the past month, treatment termination date, and reasons for terminating treatment. The variables on self-reported ART adherence include missed doses in the past month, and the proportion of medicines taken on time in the past month. Venous blood specimen samples were also collected for testing CD4http://hivdb.stanford.edu) and was interpreted using the HIV db program. We included mutation results that conferred low-, intermediate-, and high- level resistance. CD4 count was measured within 24 hours by flow cytometry in the local CDCs and was quality assured by the National HIV Reference Laboratory. Plasma was separated by centrifugation and stored at −80°C, then they were transported to NCAIDS through cold chain. HIV viral load and drug resistance mutation tests were performed at the National Center for AIDS/STD Control and Prevention (NCAIDS), China CDC. Plasma HIV-1 RNA copy was quantified with real-time Nucleic Acid Sequence Based Amplification (NASBA)  or COBAS  according to the manufacturers’ protocols. In samples with viral load ≥1,000 copies/ml, HIV drug resistance genotyping was carried out by an in-house polymerase chain reaction (PCR) protocol as previously described. P≤0.05) associated with death in the univariate analysis were considered for inclusion in multivariate Cox regression models. All tests of significance were two-sided, with a P-value ≤0.05.Primary outcome variables: we defined a case of drug resistance as the combined condition of having a HIV viral load ≥1,000 copies/ml and displaying genotypic HIVDR mutation(s). We used Cox proportional hazard models to evaluate hazard ratios of HIVDR incidence. Time zero was defined as the enrollment date at the surveys, and incidence of HIVDR was defined as those who developed resistant mutations during the one year interval before follow-up. Variables that were significantly  used were AZT/D4T+DDI+NVP (5.2%), AZT/D4T +3TC+NVP (72.2%), AZT/D4T +3TC+EFV (19.7%), and others (2.9%). However, in 2009, only 1.4% of patients still received DDI based regimens and 6.1% had been switched to second-line regimens. At the time of the baseline survey, the median duration of treatment was 17.6 months . The mean CD4 count was 341.6 cells/µl, and the proportions of patients with CD4 count of 0–199, 200–349, and ≥350 cells/µl were 40.3%, 34.9% and 24.8%, respectively. The great majority of patients  had plasma HIV viral load <1,000 copies/ml. Among the patients with virologic failure, 123 (45.2%) had resistance mutations identified, including 90 (33.1%) with dual-class resistance.Among the 2005 patients followed up in 2010, the mean CD4 count was 384.6 cells/µl, and the proportions of patients with CD4 count of 0–199, 200–349, ≥350 cells/µl were 23.2%, 31.4% and 45.4%, respectively. Approximately the same proportion of patients, 89.2% (1785/2002), had plasma HIV viral load <1,000 copies/ml. The incidence rate of death was 2.0 per 100 person years, with 46 patients having died during the follow-up period. Among patients retained at the 2010 follow-up and who had no HIVDR mutations in 2009, the incidence of resistance to any type of HIV drugs, as well as to NNRTIs, NRTIs, and PIs alone, were 3.5 per 100 person years (64/1837.3), 3.4 (63/1837.3), 2.6 (47/1837.3), 0.1 (1/1837.3), respectively, and 2.4% (46/1930) were resistant to both NRTIs and NNRTIs . The mosThe risk factors for incidence of HIVDR were assessed through a Cox regression model . The fouIn this one-year prospective follow-up survey of HIVDR across eight provinces in China from January 2009 to December 2010, we found that the overall prevalence of HIVDR at baseline was 5.6%, virtually all with NNRTI mutations and three-fourths with NRTI mutations, which is comparable to proportions observed in other countries. An important concern for widespread ART use is the potential for emerging HIVDR mutations given improperly administered regimens and lack of drug adherence support in resource-limited settings. Our results here revealed that the HIVDR incidence rates in China are higher than those of a number of countries. An ecological study among ART treated patients in British Columbia, Canada reported that the incidence of HIVDR decreased dramatically from 1.73 per 100 person-months of therapy in 1997 to 0.13 per 100 person-months of therapy in 2008. The first risk factor we identified is the use of DDI-based regimens, which resulted in higher HIVDR incidence compared with lamivudine (3TC)-based regimens. Previous studies have reported that DDI-based regimens are associated with higher rates of emergence of liver disease, Secondly, our findings show that patients who received care in rural village clinics or township hospitals were significantly more likely to experience incident HIVDR than those treated at county-level CDCs or hospitals staffed by trained physicians. Patients with low income are also more likely to develop drug resistance. As our previous study reported, patients in rural or lower-income regions in China have significantly lower levels education and socio-economic status, and health care providers have less advanced technical infrastructure and capacity. Finally, we found that lower baseline CD4 cell count and higher baseline viral load are significantly associated with incidence of drug resistance. Our results are consistent with previous studies reporting that initiating HAART at higher CD4 cell counts may decrease the risk of developing drug resistance In conclusion, Chinese policymakers and care providers need to consider the relatively high incidence of HIVDR in China and address the related factors, and provide comprehensive education and training to community health workers or nurses to improve health care quality.