Many HIV treatment programs in resource-limited settings are plagued by high rates of loss to follow-up (LTFU). Most studies have not distinguished between those who briefly interrupt, but return to care, and those more chronically lost to follow-up.We conducted a retrospective cohort study of 11,397 adults initiating antiretroviral therapy (ART) in 71 Southern African Catholic Bishops Conference/Catholic Relief Services HIV treatment clinics between January 2004 and December 2008. We distinguished among patients with early death, within the first 7 months on ART; patients with interruptions in laboratory monitoring (ILM), defined as missing visits in the first 7 months on ART, but returning to care by 12 months; and those LTFU, defined as missing all follow-up visits in the first 12 months on ART. We used multilevel logistic regression models to determine patient and clinic-level characteristics associated with these outcomes.In the first year on ART, 60% of patients remained in care, 30% missed laboratory visits, and 10% suffered early death. Of the 3,194 patients who missed laboratory visits, 40% had ILM, resuming care by 12 months. After 12 months on ART, patients with ILM had a 30% increase in detectable viremia compared to those who remained in care. Risk of LTFU decreased with increasing enrollment year, and was lowest for patients who enrolled in 2008 compared to 2004 .In a large community-based cohort in South Africa, nearly 30% of patients miss follow-up visits for CD4 monitoring in the first year after starting ART. Of those, 40% have ILM but return to clinic with worse virologic outcomes than those who remain in care. The risk of chronic LTFU decreased with enrollment year. As ART availability increases, interruptions in care may become more common, and should be accounted for in addressing program LTFU. In 2009, South Africa was home to the greatest number of people living with HIV in the world – 5.7 million Despite tremendous advances in the scope and reach of HIV care and treatment in South Africa and other resource-limited countries, high rates of loss to follow-up (LTFU) have been reported in systematic reviews of the literature, approaching 70% before ART initiation and approximately 25% one to three years after ART initiation Routine patient monitoring is accepted as a cornerstone of HIV disease management and program evaluation Our objective was to study patients newly initiated on ART with suboptimal adherence to HIV care – either with brief interruptions in care or chronic LTFU – in addition to patients with early mortality. We investigated patient and clinic level factors associated with these patient groups, and virologic response among those in care and those with interrupted care.IRB approval was obtained from the Massachusetts General Hospital and from the University of Cape Town.The Catholic Relief Services and Southern African Catholic Bishops Conference (CRS/SACBC) are faith-based, non-governmental organizations that have been providing care to HIV-infected individuals in South Africa since 2003. The PEPFAR-funded HIV treatment program commenced largely in concert with South Africa's 2004 national ART roll-out. It was initially launched in 20 facilities, most of which were pre-existing home-based care programs run by the Catholic Church. The program is now comprised of 71 sites , which have provided care to over 70,000 patients since 2004 The study population is comprised of adults (≥15 years) who enrolled in the CRS/SACBC treatment programs between January 2004 and December 2008. Patients included in the analysis were those who were eligible for ART at clinic enrollment (baseline CD4 count <200/uL or WHO stage III or IV disease), subsequently initiated ART, and had at least 400 days of potential follow-up time on ART before the end of the study. We included a minimum follow-up time in order to have an observation period sufficient to capture brief interruptions in care.After clinic enrollment, patients received baseline clinical assessment and CD4 count testing. Those with CD4 counts ≤200/uL or WHO stage III or IV disease, were started on a standard regimen of stavudine, lamivudine, and efavirenz (unless contraindicated) after completing ART literacy training. Upon ART initiation, patients received monthly adherence assessments, 3 to 6 monthly clinical follow-up, and 6 monthly laboratory follow-up with CD4 testing, and, in some cases, HIV RNA monitoring. Only those clinic visits associated with routine laboratory monitoring were entered into the database and available for analysis. An adherence counselor typically contacted patients who defaulted from care either by phone, home-visit, or both. The details of this practice varied by clinic. A minimum set of demographic and clinical variables was collected at each clinic site and entered into a standardized program database.in care were guideline-concordant. They were alive and had at least one visit between 30 and 210 days after ART initiation. Patients with interrupted laboratory monitoring (ILM) had no follow-up visits between 30 and 210 days after ART initiation, but returned to clinic for laboratory monitoring by 400 days. LTFU was defined as having no follow-up visits between 30 and 400 days after ART initiation. Patients with early death had a documented death within the first 210 days after starting ART. Patient deaths were ascertained passively. Clinics were notified of patient deaths by home-based caregivers, family members, or by residential facilities associated with the clinics. Patients with documented transfers to another facility were assumed to be in care for this analysis.A patient visit was defined as a follow-up appointment associated with CD4 monitoring. Since first published in 2004, South African national HIV treatment guidelines have recommended 3-monthly clinical follow-up and 6-monthly CD4 monitoring Bivariate and multivariate, multilevel multinomial logistic regression modeling was used to assess the relationship of patient-level factors  and clinic-level factors  on the odds of ILM, LTFU, and dying within 7 months of ART initiation, compared to remaining in care. Multilevel modeling was required to account for patient clustering within satellite clinics, which were clustered within primary clinics Between 2004 and 2008, there were 22,888 ART-naive adults enrolled in the CRS/SACBC HIV treatment programs with advanced HIV disease (CD4 count ≤200/uL or WHO stage III or IV), for whom data were available. The analysis focused on 11,397 persons who initiated ART and had at least 400 days of potential observation time after ART initiation and before the end of the study. Follow-up data were restricted to the first 400 days after ART initiation for all patients. Thirty-eight percent of patients were enrolled in urban programs, 44% in peri-urban programs, and 18% in rural programs.The number of ART-naïve, ART-eligible patients enrolled in central clinics over the study period ranged from 372 at the smallest site to 5153 at the largest. On average, 65% of ART-eligible patients were initiated on ART.The majority of patients were female (67%). Median age was 35 years, and median baseline CD4 count at enrollment was 101/uL  . CD4 couAt the end of the study period, 63% of patients remained consistently in care , 28%  missed visits, and 9% (n = 988) had early death. Of the patients in care, 26%  had documented transfers to another facility after a median of 399 days on ART. Of the 3,194 patients who missed visits 1,236 (11% of cohort) had ILM, and 1,958 (17% of cohort) were LTFU . Across The number of patients that initiated ART in the CRS/SACBC clinics increased with each calendar year, from 753 in 2004 to 3,042 in 2008 . Over thIn multivariate analysis, later year of enrollment was associated with decreased risk of LTFU. Compared to patients enrolled in 2004, patients enrolled in 2008 had a 51% reduction in the risk of LTFU . We did not find similar trends related to ILM or early death.Of all patients who remained in care, 71% had a viral load obtained a median of 376 days after ART initiation; 13% had detectable viremia (HIV RNA >400 copies/uL). Among patients with ILM, 64% had a viral load obtained a median of 393 days after ART initiation; 17% had detectable viral loads. There was a 30% increase in detectable viremia at 12 months among patients with ILM compared to patients in care (p = 0.0112).In multivariate analysis, female sex was associated with a decreased odds of ILM , LTFU , and early death  compared to male sex. Aside from female sex, we did not find any other patient-level factors associated with ILM .Baseline CD4 count was a predictor of early death, but not LTFU. In univariate analysis, patients with baseline CD4 <50/uL had an increased risk of LTFU  compared to those with baseline CD4 >200/uL, but this relationship did not persist in the multivariate model . In multivariate analysis, the risk of early death increased with decreasing baseline CD4 count , for patients with baseline CD4 <50/uL compared to those with baseline CD4 >200/uL.Among all factors assessed, we did not find any clinic characteristics associated with higher rates of LTFU. Longer time to ART initiation was associated with a decreased risk of early death , and there was a trend suggesting decreased risk of ILM among patients attending larger programs (>100 patients on ART), .late deaths (after 7 months on ART) were analyzed in a separate multinomial, multivariate model comparing ILM, LTFU, early death, and late death with the reference group (in care), we saw no change in the factors associated with ILM, LTFU, and early death. Enrollment year after 2005 was associated with a decreased odds of late death; patients enrolled in 2006, 2007, and 2008 had a 49% [95%CI 28–64], 70% [95%CI 57–79], and 76% [95%CI 65–83] reduction in the odds of late death compared to those in care. Additionally, patients with a baseline CD4 count ≤100/uL had an increased risk of late death with OR 1.66 [95%CI 1.09–2.54] for patients with baseline CD4 50–100/uL, and OR 1.75 [95%CI 1.14–2.62] for patients with baseline CD4 count <50/uL.When patients with In an analysis of over 11,000 patients newly-initiated on ART between 2004 and 2008 across South Africa, 63% of patients remained stably in care in the first year after ART initiation. Nine percent of patients suffered early death within the first seven months after ART initiation. These patients presented with very advanced disease, with a median CD4 count of 48/uL. Missed visits were seen in 28% of patients. Eleven percent of patients had ILM but returned to care by 400 days, and there was a trend towards increase over time. Importantly, patients with ILM would often have been categorized as LTFU if the analysis did not account for those who resumed laboratory monitoring after an initial absence from care, and would have increased the “LTFU” rate in this analysis from 17% to 28%. In addition, the majority of patients with interruptions had an increase in CD4 count and had suppressed HIV viral load upon return to care, suggesting that they had continued ART during the period of interrupted monitoring. Despite this, patients with ILM had a lower rate of virologic suppression at 12 months (83%) compared to patients who remained in care (87%). Seventeen percent of patients were lost to follow up at one year, but the odds of LTFU decreased significantly with more recent enrollment year. As in several other studies, women had a decreased risk of LTFU, ILM and death With the critical goal of retaining patients in HIV care over time, LTFU has been recognized as an important programmatic metric. Patient tracing studies confirm that many patients identified as lost to follow up have died (7%–40%), transferred care (30%), or voluntarily withdrawn from care Despite a growing body of literature on LTFU in HIV treatment programs, patients that interrupt, but subsequently return to care, have received relatively little attention. While failure to attend clinic appointments is common in the management of chronic illness, there are few data about patients with ILM in resource-limited settings It is possible that patients with ILM sought care or obtained ART from other facilities. This phenomenon may be especially relevant for highly mobile populations and in communities where expansion in HIV treatment facilities provides patients with the option to choose clinics that best meet their needs. It is also possible that some patients came to clinic visits only, but did not complete laboratory follow-up, or that incomplete record-keeping led some patients to be misclassified, despite complying with routine laboratory monitoring. One study from South Africa found that among patients classified as lost to follow-up in an HIV treatment program, 21% were still in care – half were incorrectly identified as lost, and the other half had interrupted care but returned While many studies have investigated the association between patient characteristics and LTFU, fewer have measured the association between program characteristics and this risk. One large meta-analysis of 33 patient cohorts in 13 Sub-Saharan African countries found no association between cohort size or year of program initiation and LTFU. But the ART-LINC study of 23 treatment programs in low and middle-income countries found that larger programs had higher rates of LTFU and were less likely to trace patients who were lost Definitions of LTFU may also affect the assessment of temporal trends of important patient outcomes. Our analysis showed a decrease in LTFU and patient deaths, but an increase in ILM, during the period of study. This contrasts with a study from the IeDEA-SA cohort, which similarly showed a decline in patient mortality, but an increase in LTFU, from 1% to 13%, between 2002 and 2007 This study has several limitations. First, we did not have systematic ascertainment of patient outcomes, including transfers, LTFU, and death. While all programs in the cohort had some mechanism for tracing patients lost from care, including notification by home-based caregivers , family members, and residential facilities associated with clinics, it was not standardized across all sites. This may have introduced ascertainment bias, and furthermore, the deaths in the LTFU group are a minimum estimate. Second, detailed information on ART retrieval, and clinic visits that occurred outside of laboratory follow-up were not recorded in the clinical database. Consequently, determinations of LTFU and ILM were based solely upon clinic visits that were tied to laboratory visits, which may have introduced misclassification bias. We were reassured that the predictors of early death  and LTFU  were consistent with those reported in other studies of patient retention and attrition. Finally, use of logistic regression analysis rather than survival analysis did not allow us to calculate incidence of LTFU. Despite these limitations, this study provides a novel assessment of patients lost from HIV care in a large, multi-site, NGO-based clinical program in South Africa, with a focus on the importance of interrupted monitoring and care.In summary, in the first year of ART initiation in a large multi-site cohort in South Africa, we found that 60% of patients remain in care, 30% miss visits, and 10% die early from advanced immune suppression. Of the patients who miss visits, nearly 40% have interruptions at their primary clinical sites. While most returned to care with virologic suppression in the short term, our study suggests that patients who interrupt care generally, and laboratory monitoring specifically, may have worse virologic outcomes than patients who remain in care. ILM may become more common as ART programs become more widely available, and studies that do not account for such interruptions will overestimate LTFU rates. In the current study, LTFU rates decreased from 2004–2008, while ILM increased. For program evaluation and quality improvement, it is critical to distinguish between these two important outcomes, and specifically to account for patients who are successfully receiving ART in new locations.


Current WHO guidelines recommend antiretroviral therapy (ART) initiation at CD4 counts ≤350 cells/µL. Increasing this threshold has been proposed, with a primary goal of reducing HIV-1 infectiousness. Because the quantity of HIV-1 in plasma is the primary predictor of HIV-1 transmission, consideration of plasma viral load in ART initiation guidelines is warranted.Using per-sex-act infectivity estimates and cross-sectional sexual behavior data from 2,484 HIV-1 infected persons with CD4 counts >350 enrolled in a study of African heterosexual HIV-1 serodiscordant couples, we calculated the number of transmissions expected and the number potentially averted under selected scenarios for ART initiation: i) CD4 count <500 cells/µL, ii) viral load ≥10,000 or ≥50,000 copies/mL and iii) universal treatment. For each scenario, we estimated the proportion of expected infections that could be averted, the proportion of infected persons initiating treatment, and the ratio of these proportions.Initiating treatment at viral load ≥50,000 copies/mL would require treating 19.8% of infected persons with CD4 counts >350 while averting 40.5% of expected transmissions (ratio 2.0); treating at viral load ≥10,0000 copies/mL had a ratio of 1.5. In contrast, initiation at CD4 count <500 would require treating 41.8%, while averting 48.4% (ratio 1.1).Inclusion of viral load in ART initiation guidelines could permit targeting ART resources to HIV-1 infected persons who have a higher risk of transmitting HIV-1. Further work is needed to estimate costs and feasibility. Antiretroviral therapy (ART) for treatment of HIV-1 infection has significant prevention benefits, reducing the risk of secondary HIV-1 transmission by over 90% Plasma viral load is the primary predictor of HIV-1 transmission Data for this analysis were from the Partners in Prevention HSV/HIV Transmission Study, which was a randomized placebo-controlled trial of herpes simplex virus type-2 (HSV-2) suppression with acyclovir among 3381 heterosexual HSV-2/HIV-1 co-infected individuals and their HIV-1 seronegative partners from 7 countries in southern and east Africa . At enrollment, all HIV-1 infected participants had CD4 counts ≥250 cells/µL and were not yet on ART. The median CD4 count was 462 cells/µL (interquartile range 348–631) and 68% were women. The primary outcome of the trial was virologically-linked HIV-1 transmission within the partnerships; acyclovir did not decrease the risk of HIV-1 transmission. The design, methods, and primary outcomes have been described previously For the present analysis, we used cross-sectional data on sexual behavior, CD4 count, and plasma viral load collected at the time of enrollment into the Partners in Prevention HSV/HIV Transmission Study for HIV-1 infected partners with CD4 counts >350 cells/µL. Sexual behavior data were from a standardized questionnaire that asked the number of sex acts with the HIV-1 uninfected partner with and without condoms in the prior month. We used enrollment data only, because our goal was to describe a population presenting to clinical care for consideration of early ART  and thus to minimize the potential impact on sexual behavior reporting as a result of participation in the clinical trial, which included monthly risk-reduction counseling.1 = 1– exp{−exp[−6.974+.923*]}, and for acts protected with a condom: λ0 = 1–exp{−exp[−8.389+.923*]}. The estimated annual numbers of sex acts with and without condoms were calculated from reported behaviors in the prior month and multiplied by 12. Next, each participant's probability of transmitting to his or her partner within the next year was estimated with the following equation: 1– [(1–λ0)annual #acts with condom(1–λ1) annual #acts without condom]. Because reported condom use was higher in this clinical trial cohort than in previous observational studies of HIV-1 serodiscordant couples, potentially reflecting risk-reduction counseling during the pre-enrollment study screening period, we also calculated each participant's probability of transmitting with reduced condom use . Each participant was assigned a weight of 100,000 divided by the sample size.We used these cross-sectional data to estimate the number of transmissions that would occur in one year and the number that could be averted with ART in a hypothetical population of 100,000 HIV-1 infected persons in serodiscordant partnerships with CD4 counts and viral load similar to the study participants. To estimate expected transmissions, we first assigned each HIV-1 infected participant per-act infectivity estimates for sex acts with and without condoms, based on our prior analysis of infectivity in the same study population Participants were stratified according to plasma viral load  and CD4 count . Several strategies for early ART initiation were considered: CD4 count <500 cells/µL; plasma viral load ≥50,000 or ≥10,000 copies/mL; and universal coverage. The CD4 threshold was based on current international guidelines; viral load thresholds were based on the low risk of transmission with plasma viral loads <10,000 copies/mL The clinical trial protocol and informed consent documents were reviewed and approved by human subjects research committees at the University of Washington and all local study site and affiliated institutions. All clinical trial participants provided written informed consent for participating in this research.A total of 2484 HIV-1 infected partners had CD4 counts >350 cells/µL, of whom 69% were female. The mean age was 33 years, 42% had CD4 counts <500 cells/µL and 20% had viral loads ≥50,000 copies/mL. Among those with CD4 counts 351–500, the proportion with viral loads ≥50,000 copies/mL was 26%; among those with CD4 counts >500 cells/µL this proportion was 16%. The mean number of reported sex acts per month differed little across CD4 and viral load strata, ranging from 5.2 to 6.6 . On averInitiating treatment at viral load ≥50,000 copies/mL would require treating 19.8% of infected persons with CD4 counts >350 while averting 40.5% of expected transmissions, resulting in a ratio of proportion averted to proportion treated of 2.0 . In contOur analysis of different scenarios for ART initiation at earlier than current WHO guidelines indicates that incorporation of plasma viral load, the primary predictor of HIV-1 transmission Others have evaluated the potential impact of initiating treatment based on high viral load in the general population and found strong evidence for an important prevention benefit, consistent with our findings Because we enrolled stable HIV-1-serodiscordant couples who had not transmitted, the cohort could be biased toward HIV-1 infected partners with a lower risk of transmission due to behavioral or biologic factors. However, our population reflects those couples who will present jointly for testing and care, and thus would likely be a population targeted for early ART. In addition, in a community-wide survey in Bostwana, the distribution of viral load among HIV-1 infected persons not on ART with CD4 counts above 350 was similar to that seen in our population A limitation of our analysis is potential inaccuracies in self-reported sexual behavior. The sexual frequency reported in our study is similar to that found in other studies of African heterosexual populations Another limitation is that in using cross-sectional data, our estimates do not incorporate transitions in CD4 count and viral load over time. In reality, individuals would move through CD4 strata gradually, be evaluated for ART periodically , and become eligible for ART at some point based on CD4 alone. If CD4 counts were to decline relatively faster than plasma viral load increased between re-evaluation visits, our results could be attenuated. While a more complex model could capture these subtleties, because high viral load is such a strong predictor of transmission, guidelines for early ART initiation that include viral load will always avert a higher concentration of new infections than those without viral load.In conclusion, inclusion of viral load in ART initiation guidelines for those with CD4 >350 cells/µL could be a useful way to increase the prevention benefits of ART by identifying HIV-1 infected persons with the highest risk of HIV-1 transmission. Until resources are available for universal access to ART, it is critical to determine the public health and clinical benefits of ART for persons with higher CD4 counts who are not currently eligible for ART in most resource-constrained settings. A revision of ART guidelines, expanding access to HIV-1 infected persons with CD4 counts >350 cells/µL who have high viral load, could optimize prevention and clinical benefits. Further work is needed to estimate costs, feasibility, and the impact of such guidelines on a population level.


The success of HIV treatment is limited by tolerability/toxicity of ARVs and patient adherence remains paramount to achieve viral suppression. Several small studies have investigated the combination Raltegravir and Atazanavir. This is particularly attractive to exclude NRTIs and ritonavir in metabolic toxicity. Optimum dosing has yet to be decided however recent data from SPARTAN suggests once daily regimens are associated with raltegravir resistance.To assess HIV virologic control in patients switched to RAL/ATZ. To compare metabolic parameters before and after switch.We retrospectively identified patients RAL/ATZ from pharmacy database. Using medical notes and lab results system, we recorded the HIV-1 VL and lipid profiles pre- and post- switch. TDM, hepatitis status, drug resistance and treatment experience were also recorded.We identified 11 patients on RAL/ATZ, 9 male. At switch, 1 patient had a detectable VL (582 copies/mL), all other patients were undetectable. At the most recent appointment all patients had an undetectable viral load. The mean number of previous regimens was 5; no patient commenced the regimen with known PI resistance. In 5 patients other ARVs were included, and in 6 RAL/ATZ were used alone.Therapeutic drug levels of raltegravir and atazanavir were measured in 9/11 patients and the predicted trough levels for both raltegravir and atazanavir were greater than the minimum recommended concentration. There was a reduction in total cholesterol and triglycerides post switch and a trend towards reduction in LDL-cholesterol and total cholesterol:HDL ratio. Two patients were lost to follow up and three patients discontinued the combination. The reasons for discontinuation: to avoid drug interactions pre-renal transplant, to aid adherence (once daily regimen) and presumed raltegravir intolerance (anxiety and sleep disturbance).A switch was made when 10/11 patients were suppressed and required therapy change because of another reason. All patients maintained a viral load of less than 50 copies/ml (7-102 weeks).The combination was well tolerated and there was a trend towards improved lipid parameters observed. In the absence of resistance, this combination shows promise in terms of prolonged virologic efficacy, tolerability and an improved metabolic profile.


Introduction. Hepatotoxicity is a concern in HIV/hepatitis C virus (HCV) coinfected patients due to their underlying liver disease. This study assessed the incidence of hepatotoxicity in HIV/HCV co-infected patients in two outpatient infectious diseases clinics. Methods. HIV/HCV co-infected adults were included in this retrospective study if they were PI or NNRTI naïve at their first clinic visit and were initiated on an NNRTI- and/or PI-based antiretroviral regimen. Patients were excluded if they had active or chronic hepatitis B virus (HBV). The primary objective was to determine the overall incidence of severe hepatotoxicity. Results. Fifty-six of the 544 patients identified met inclusion criteria. The incidence of severe hepatotoxicity was 10.7% (6/56 patients). Severe hepatotoxicity occurred with efavirenz (N = 2), nevirapine (N = 1), indinavir (N = 1), nelfinavir (N = 1), and saquinavir/ritonavir (N = 1). Conclusion. The incidence of severe hepatotoxicity appears to be low in this retrospective analysis of HIV/HCV co-infected patients receiving a PI-and/or NNRTI-based regimen. The advent of highly active antiretroviral therapy (HAART) in the treatment of human immunodeficiency virus (HIV) infection has significantly decreased the incidence of opportunistic infections as well as improved morbidity and mortality among HIV patients. However, along with these positive outcomes, HAART is associated with a host of adverse reactions such as hepatotoxicity, hyperlipidemia, hyperglycemia, and lactic acidosis. Hepatotoxicity can interrupt HIV therapy and cause an increase in morbidity and mortality . ProteasSeveral studies have evaluated hepatotoxicity associated with HAART in patients coinfected with HIV and either HCV or hepatitis B virus (HBV) –9. HIV/HThis was a retrospective study conducted at the DUMC and DVAMC Infectious Diseases Clinics and was approved by the Institutional Review Boards of both facilities with a waiver for patient consent. Medical and pharmacy records of patients were reviewed to identify eligible patients. Patients were included in the study if they met all of the following criteria: ≥18 years of age, HIV-infected with HCV coinfection, followed at either the DUMC or DVAMC Infectious Diseases Clinics from their initial visit between August 1, 1994 and December 31, 2006, PI or NNRTI naïve at their first clinic visit, received a PI- and/or NNRTI-based regimen during the study period, and had baseline liver function tests (LFTs) both prior to starting HAART and during treatment. Patients were excluded if they had active or chronic HBV. The following data was collected: demographic information, baseline liver enzymes  prior to first antiretroviral therapy (ART) regimen and for subsequent regimens that the patient was changed to, subsequent liver enzymes during ART therapy for the study period, HCV genotype, baseline and subsequent PI/NNRTI regimens through December 31, 2006 and the duration of each regimen in months, data regarding rationale for changing PI or NNRTI regimens, plasma HIV-RNA level at baseline defined as viral load closest in time to the first HAART regimen, CD4 lymphocyte count at baseline defined as CD4 count closest in time to the first HAART regimen, HCV treatment at any time, alcohol consumption at baseline defined as no intake, moderate intake (not >1 drink/day), heavy intake (>1 drink/day), or unable to determine, and subsequent alcohol consumption was evaluated if LFT elevations were noted.The primary endpoint of the study was to determine the incidence of severe hepatotoxicity  in coinfected HIV/HCV patients receiving a PI- and/or NNRTI-based antiretroviral regimen . In patiData was entered into a Microsoft Access database  and analyzed using SAS . Descriptive statistics were used to analyze the demographics, HCV genotype, CD4 count, viral load, and incidence of severe hepatotoxicity. 3 (range: 5–960 cells/mm3) and 42,078 copies/mL , respectively. There was insufficient information available to quantify baseline and subsequent alcohol consumption as well as receipt of HCV therapy.A total of 544 HIV/HCV coinfected patients were identified, however, only 56 patients met all of the inclusion criteria. The primary reason patients were excluded from the study was that they were not treatment naïve at their first clinic visit (67.4%). Other reasons patients were excluded from the study included: active or chronic hepatitis B virus (21.5%), not receiving a PI- or NNRTI-based regimen (4.7%), insufficient data available, or not receiving antiretroviral therapy during the study period. Baseline demographics of the study population are described in N = 6). Severe hepatotoxicity occurred with the following antiretrovirals: efavirenz (N = 2), nevirapine (N = 1), indinavir (N = 1), nelfinavir (N = 1), and saquinavir/ritonavir (N = 1)  and lopinavir/ritonavir (269 months), respectively. Atazanavir/ritonavir was the PI with the second highest period of exposure of 213 months. For the primary endpoint, the incidence of severe hepatotoxicity (grades 3 or 4) associated with any PI- or NNRTI-based regimen was 10.7% ( (N = 1) . All of  (N = 1) . NotablyRationale for changing or discontinuing an NNRTI- or PI-based regimen was identified in 33 cases. The most common reason for changing a regimen was an adverse effect related to the medication (40%), resistance/treatment failure (21%), nonadherence (18%), regimen simplification (15%), and hepatotoxicity (6%).N = 298), hepatotoxicity was assessed in patients who initiated either a PI-based (n = 211) or dual nucleoside analog (n = 87) antiretroviral regimen [P = .0001). On multivariate analysis, coinfection with HBV or HCV was associated with severe hepatotoxicity (P = .001), but there was no association with individual NRTI or PI medications possibly due to small sample size [HIV patients coinfected with HCV represent about a third of the HIV population in the US and are at a greater risk for liver toxicity with the use of potentially hepatotoxic antiretrovirals . Questio regimen . Forty-e regimen . Lai et  regimen . Co-infeple size . A largeOverall, our finding of an incidence of severe hepatotoxicity in coinfected patients of 10.7% is similar to the rate of severe hepatotoxicity related to HAART in noncoinfected patients in these studies. In our study we found cases of hepatotoxicity occurred with older, less commonly used PIs . Interestingly, when reviewing newer agents such as lopinavir/ritonavir and atazanavir/ritonavir which had the largest months of exposure for the PIs, no severe hepatotoxic events were identified. This may suggest that these newer PIs may be relatively safe to use in this patient population. The commonly used NNRTI, efavirenz, was associated with two cases of severe hepatotoxicity; however, this drug had the most months of usage which led to a low normalized incidence.A major limitation of our study is the small sample size since the patients were used as their own controls. Future studies could include a control group of noncoinfected patients so patients that are treatment experienced could be included. Additionally, LFT changes documented in the study could have been attributed to other causes such as HCV or the use of other concomitant hepatotoxic drugs or substances, such as alcohol. In a retrospective study assessing severe hepatic injury after beginning HAART, coinfected patients had a 3.99 greater chance of developing severely elevated transaminase levels compared with patients not coinfected with HCV. This risk was especially greater in coinfected patients with identified alcohol abuse . AlthougIn conclusion, additional studies are needed to gain a better appreciation for the incidence of severe hepatotoxicity in HIV patients coinfected with HCV. However, based on this retrospective analysis, the incidence of severe hepatotoxicity appears to be relatively low in HIV/HCV-coinfected patients receiving either a PI- or NNRTI-based regimen.


To describe patient antiretroviral therapy (cART) outcomes associated with intensive decentralization of services in a rural HIV program in Malawi.Longitudinal analysis of data from HIV-infected patients starting cART between August 2001 and December 2008 and of a cross-sectional immunovirological assessment conducted 12 (±2) months after therapy start. One-year mortality, lost to follow-up, and attrition (deaths and lost to follow-up) rates were estimated with exact Poisson 95% confidence intervals (CI) by type of care delivery and year of initiation. Association of virological suppression (<50 copies/mL) and immunological success (CD4 gain ≥100 cells/µL), with type of care was investigated using multiple logistic regression.P<0.0001) than other patients. Two years after cART start, program attrition was lower in decentralized than centralized facilities . One year after treatment start, differences in immunological success , and viral suppression  between patients followed at centralized and decentralized facilities were not statistically significant.During the study period, 4322 cART patients received centralized care and 11,090 decentralized care. At therapy start, patients treated in decentralized health facilities had higher median CD4 count levels (167 vs. 130 cell/µL, In rural Malawi, 1- and 2-year program attrition was lower in decentralized than in centralized health facilities and no statistically significant differences in one-year immunovirological outcomes were observed between the two health care levels. Longer follow-up is needed to confirm these results. In sub-Saharan Africa, approximately 5,200 people became newly infected with HIV each day in 2008, and 3835 died daily from AIDS-related conditions The effectiveness of cART in terms of mortality reduction at both population In the Chiradzulu district, located in Southern Malawi, 1 in 4 adults are estimated to be living with HIV infection. In 2003 decentralized HIV care started being provided by mobile health teams every 2 weeks in 10 peripheral health facilities Here we describe temporal trends in patient characteristics at cART start and compare outcomes in HIV-infected patients receiving decentralized versus centralized care in Chiradzulu, Malawi.The Chiradzulu HIV program was established in 1996 as a collaboration between the Ministry of Health of Malawi and Médecins Sans Frontières (MSF). Free provision of cART at the district hospital started in August 2001 Eligibility criteria for cART initiation followed World Health Organization (WHO) treatment guidelines: CD4 cell count <200 cells/µL (<250 since January 2007), clinical stage 4 irrespective of CD4 cell count, and since January 2007, clinical stage 3 irrespective of CD4 cell count First, a retrospective cohort analysis of monitoring data from patients starting cART between August 2001 and December 2008 at the district hospital and at 10 rural health centers was conducted. Sociodemographic, clinical, and treatment information were routinely recorded on the patient files. Carbon-copied duplicate forms were then sent to the hospital for data entry in an electronic database  and record filing. Standard data consistency checks  and verifications (e.g. regular cross-verification of paper forms and laboratory information against the information entered in the database) were routinely done on and off site.Second, a cross-sectional survey was carried out in February–July 2009 to determine immunovirological outcomes among patients receiving cART for 12±2 months. A random selection of 702 eligible patients was invited to participate in the evaluation . The sample size was estimated to detect a 10% difference in virological failure between patients receiving centralized versus decentralized care, assuming a significance level of 5% and 80% power. Fifty-two (7.4%) women were excluded due to current pregnancy or breastfeeding, 9 (1.3%) had died, 3 (0.4%) had been transferred outside the program, 13 (1.8%) were lost to follow-up and 7 (1.0%) declined participation. An independent clinical team scheduled appointments for the study patients, obtained informed consent, and conducted the cross-sectional evaluation. Physical examination by clinicians, including assessment of cART adherence during the previous month using a 10-point visual analogue scale, and blood draws for laboratory testing, were performed at the facilities. Blood specimens were collected and labeled. CD4 cell counts were quantified using semi-automated machines  at the laboratory of the Chiradzulu district hospital. Plasma samples were stored at -20°C and transported to the DREAM Laboratory  weekly for viral load quantification with the VERSANT HIV-1 RNA bDNA assay .We used individual patient monitoring data to compare patient characteristics at cART start by calendar period of cART initiation  and type of care delivery . Type of care was defined as patient attendance to the district hospital or to health centers, respectively, for at least 90% of all consultations since cART initiation. Characteristics between the two care groups were compared using chi-squared tests or Fisher exact tests for categorical and Wilcoxon rank sum tests for continuous variables.Time of follow-up for each patient started at the time of cART initiation and was censored at the earliest of the following dates: death, transfer outside the program, last clinic visit or 2 years. One-year mortality, lost to follow-up, and attrition (deaths and lost to follow-up) rates were estimated with exact Poisson 95% confidence intervals (CI). Lost to follow-up was defined as missing a scheduled appointment by 2 months or more.2 for patients aged >18 year old, and equivalent age and sex specific BMI cut-off points for the 2–18 year old group The proportions of patients included in the cross-sectional evaluation with undetectable viral load, virological failure (>5000 copies/mL), and CD4 cell counts <200 cells/µL, and gains in CD4 counts since the start of therapy were stratified according to the type of care received. In sensitivity analyses, the rate of virological failure was estimated assuming both, that patients who were alive and in care 12 months after cART start but who did not participate in the cross-sectional study had experienced the same rate of virological failure as patients who participated in the evaluation; and that all patients who died or were lost to follow-up between 6 and 12 months after cART start had experienced virological failure. Patients who died or were lost to follow-up within 6 months of therapy initiation were excluded from this estimation because in WHO guidelines only patients who receive cART for more than 6 months are considered in the definition of treatment failure. Multiple logistic regression was used to compare virological suppression (<50 copies/mL) and immunological success (CD4 gain since treatment start ≥100 cells/µL) at 12 months of cART among patients aged 2 years or more, and 5 years or more, respectively. Factors considered in multivariable models were residence in the Chiradzulu district (yes/no), sex, age in quartiles , body mass index (<18.5 and ≥18.5 kg/mP<0.20) were included in the corresponding multivariable models. Likelihood ratio tests for associations across categories of variables were calculated. Sensitivity analyses excluding observations with missing CD4 cell count and clinical stage data (n = 60) were also performed. All data analyses were carried out using STATA statistical software, version 11.2 . Statistical significance was defined by two-sided P values of <0.05.All factors associated with a CD4 gain ≥100 cells/µL  in univariable analyses , and median age was similar in patients who received centralized or decentralized care (P<0.001 after 2002).In each time period, higher proportions of men were initiated on cART in centralized than in decentralized facilities  and 19.5 per 100 person-years (95% CI: 18.5–20.2) at 1 year of cART start and 6.9 per 100 person-years (95% CI: 6.6–7.3) and 12.6 per 100 person-years (95% CI: 12.2–13.1) at 2 years, respectively. As the program scaled up, patient attrition and mortality decreased in both centralized and decentralized health facilities .During the first year of cART use, mortality and attrition rates were higher in patients treated at central level than in those treated in decentralized health centers . At 6 moSix-month rates of attrition were 47.2 per 100 person-years (95% CI: 44.1–50.4) at central level, and 23.8 per 100 person-years (95% CI: 22.5–25.1) at decentralized level; and 6–12 month attrition rates were 13.8 per 100 person-years (95% CI: 12.1–15.7) at central level, and 6.2 per 100 person-years (95% CI: 5.5–6.9) at decentralized level.Six hundred and eighteen patients had immunovirological data at 1 year of cART. Of them, 279 (45.2%) received centralized care and 339 (54.8%) decentralized care.P = 0.02). Median CD4 cell gain in the same age group was higher in individuals followed at decentralized health facilities than in those receiving centralized care . After 1 year of therapy, 66.9% of patients had CD4 gains of 100 cells/µL or greater, 70.2% of decentralized and 62.7% of centralized patients. In contrast, 57.8% of patients had undetectable viral load one year after ART initiation, 56.6% of decentralized and 59.1% of centralized patients. Only 35 (5.7%) patients had viral load >5000 copies/mL. In sensitivity analysis we estimated an overall failure rate in the program of 9.4% (95% CI: 8.9–9.9%).Of the 584 patients older than 5 years old, 10 (3.8%) patients who received centralized care and 3 (0.9%) treated in health centers had CD4 counts <100 cells/µL . Women , and patients with no history of ART use at therapy initiation  were more likely to achieve CD4 gains ≥100 cells/µL after 1 year of cART  and more likely to achieve CD4 gains ≥100 cells/µL , these associations were not statistically significant  and patient median CD4 cell counts at central and decentralized levels. Compared to patients who received decentralized care, those treated at central level were more likely to be men and to be severely immunosuppressed, but less likely to have low BMI at therapy initiation. The higher median CD4 cell count observed in patients who received centralized care could be explained by a combination of the existence of in-patient care and a later presentation of male patients compared to women Overall program attrition  decreased over time and, despite the increasing number of patients started and followed on therapy in decentralized health facilities, this decrease was more important at decentralized care level. After 1 year of cART, 83% of patients continued receiving HIV care (79% after 2 years). These findings are comparable or better than those reported in other African HIV programs, including other MSF programs, where patients were primarily treated in hospitals or urban locations Virological suppression 1 year after therapy start was observed in 58% of patients. Although this figure is lower than the estimated median proportion of patients virologically suppressed reported in a recent systematic review including data from 29 HIV programs in sub-Saharan Africa, it falls within the range of published estimates  In our analysis women and patients who had no history of cART use before enrolment in the HIV program of Chiradzulu were more likely to have gains of CD4 counts ≥100 cells/µL and virological suppression more likely to be observed in patients aged 35 years or more. Higher immune recovery was also reported in previous studies The burden of HIV infection on health systems and service providers in Malawi is overwhelming. The growing volume of patients receiving cART and the effects on non-HIV related services causes concern, raising questions about the most cost-effective strategies to ensure access to cART while maintaining acceptable treatment outcomes in HIV patients, and the optimal achievable level of task delegation to less-qualified personnel. Scarcity of qualified health care staff, limited access to laboratory testing, unreliable water and electricity supply, and poor road infrastructure are some of the challenges faced when HIV care is provided in rural areas, where the majority of patients in this population live. The decentralization of HIV care and cART provision from central hospitals to rural decentralized health facilities provides an opportunity for improved access. Wide community uptake of cART services and delegation of clinical tasks traditionally performed by physicians to well trained and supervised non-physician health staff have contributed to the achievements of the program.The findings of this study need to be interpreted in light of some limitations. First, the retrospective evaluation was based on analysis of routine data, and, despite the mechanisms in place to document mortality among patients (e.g. reporting by family and community tracing workers), ascertainment of death was incomplete. Indeed, in a previous study in the same setting, we found that 54% of adults started on cART and lost to follow-up were dead In conclusion, one-year and two-year HIV program attrition was lower in decentralized than in centralized health facilities of Malawi and no statistically significant differences in one-year immunovirological outcomes were observed between the two health care levels. Longer follow-up is needed to confirm these favorable results. The value of using health care providers with less formal training in rural settings remains an important area for further investigation in rural African settings of high HIV prevalence.Table S1Characteristics of patients included and excluded from the cross-sectional study.(DOC)Click here for additional data file.


The study was conducted in 2007, in the Northern Province of Zambia, to assess the impact of cultural values contributing to ARV non-compliance among elderly patients afflicted by AIDS. Providing care for sick child, loss of income by the family, caring for the orphans, seeking treatment were studied. Parental experiences and impact of caregiving during the time of child illness that led to poor compliance are examined.A community-based survey was conducted in three randomly selected catchment areas of Nakonde, where 250 out of 682 patients receiving ARVs within the twelve months period, were recruited through the District's Health Management Board Zonal Health Centre. All patients were interviewed using a pre-tested structured questionnaire, consisting of: Socio-demographic characteristics, Socio-economic factors, Knowledge about HIV transmission and prevention. Cultural beliefs regarding ARVs in Zambia and the social taboos surrounding treatment and actual drug adherence were observed.Most male AIDS patient respondents tended to be older and more educated than the female patient respondents. Overall, 32.6% of the patients stopped taking their medication. There were 49.1% of the females and 23% of the males, who reported that AIDS patients stopped taking their medication within the first 4 months of commencing treatment. Age, marital status and educational levels were not significantly associated with compliance. The major factors leading to non-compliance included patients beginning to feel better (55% and 48.6%), lack of knowledge on the benefits of completing a course (25.7%), running out of drugs at home (25.4%) and ARVs drugs too strong (20.1% and 20.2%). There was a significant difference  in HIV knowledge, with more males than females reporting shaking hands as a means for HIV transmission, after adjusting for age, marital status and educational levels.This study established that 32% of AIDS patients failed to comply with AIDS drug taking regimen once they started feeling better. Providing means and knowledge for primary caregiver to administer effective drugs and ARVs or to prevent and treat opportunistic infections, TB and pneumonia, would help reduce caregivers distress.


Initial virologic suppressors had significantly less CD4+ cell decline by ATI week 16 as compared to non-suppressors . However, of the ten initial virologic suppressors with a pVL at ATI week 49, only three maintained pVL <3.0 loggag vaccine trial, initial viral suppression was found in a subset of patients, but this response was not sustained. The association between CTLA-4 and PD-1 expression on CD4+ T cells and virologic outcome warrants further study in trials of other therapeutic vaccines in development.Among individuals participating in a rAd5 therapeutic HIV-1 NCT00080106ClinicalTrials.gov  The initial optimism that antiretroviral therapy (ART) could lead to the eradication of HIV infection has been tempered by the realization that virologic control is lost with the discontinuation of ART even after an extended period on treatment gag therapeutic vaccine on plasma viral load (pVL) in subjects undergoing an analytic treatment interruption (ATI) AIDS Clinical Trials Group (ACTG) protocol A5197 was a randomized, placebo-controlled trial to test the effect of a recombinant adenovirus serotype 5 (rAd5) HIV-1 The goals of this analysis were to characterize study participants with initial virologic suppression, evaluate viral and immunologic correlates of such a response, and determine the durability of virologic control 49 weeks after treatment interruption.3, plasma HIV-1 RNA levels of ≤50 copies/mL at screening with a history of pVL ≤500 copies/mL for 24 months prior to enrollment. Participants received a rAd5 vaccine containing an HIV-1 gag insert or placebo at weeks 0, 4, and 26 (Step I). Starting at week 39, 110 participants  with CD4+ cell counts ≥500/mm3 and no confirmed viral rebound in Step I (two consecutive pVL>500 copies/mL) underwent a 16 week ATI (Step II). The pVL set point acted as one of the primary endpoints and was defined as the mean of the ATI weeks 12 and 16 pVL . After ATI week 16, study participants had the option of resuming ART or continuing the treatment interruption. Resumption of ART was recommended under any of the following conditions: confirmed CD4+ cell count <300/mm3, three consecutive HIV-1 RNA levels ≥300,000 copies/mL, AIDS-defining illness, or development of retroviral rebound syndrome, clinically significant Immunosuppression, based on subject's clinician preference, or pregnancy. Participants with an HIV-1 set point of <3.0 log10 copies/ml soon after ATI were categorized as early virologic suppressors. This level of plasma viremia has been used to define virologic suppression on ART Study design and patient inclusion criteria for ACTG A5197 have been described in detail th International Histocompatibility Workshop (http://www.ihwg.org). Protective HLA alleles were defined a priori as HLA B*13, B*27, B*51, B*57, and B*5801. Unfavorable HLA alleles were defined as the HLA-B*35-Px variants  HLA class I typing was performed following the sequence-specific oligonucleotide probing and sequence-based typing protocols recommended by the 13Cell-mediated immune responses at study weeks 0, 8, and 38 were evaluated by an intracellular cytokine staining assay for interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) as previously described Most participants had plasma viral sequences analyzed at three time points: at the time of initial detectable viremia , ATI week 16 (study week 54), and ATI week 49 (study week 87). The Gag and reverse transcriptase (RT) coding sequences were amplified from plasma HIV-1 RNA by nested RT-PCR using gene-specific primers. Population (“bulk”) sequencing was performed on an ABI 3730 automated DNA sequencer. Chromatograms were analyzed using Sequencher (Genecodes). We calculated the number of amino acid mismatches between the vaccine or consensus HIV-1 subtype B sequence and the patient-derived sequence. HLA-associated polymorphisms in patient HIV-1 sequences were determined based on a published list A comparison of host genetic, immunologic, and viral sequence characteristics of those with and without initial viral suppression was performed using the 2-sample Wilcoxon rank sum test. Fisher's exact tests were used to compare dichotomous outcomes between initial viral suppressors and the non-suppressors. All of the p-values are exact 2-sided p-values. No adjustments were performed for multiple comparisons. Note that the comparisons presented here were not originally planned in the protocol.10 copies/ml and were categorized as an initial virologic suppressors (P = 0.5). Initial virologic suppressors had a median pVL set point of 2.6 log10 RNA copies/ml versus a pVL set point of 4.2 log10 RNA copies/ml for virologic non-suppressors. The time on ART and distribution of HLA allele groups  was not significantly different between the initial virologic suppressor and non-suppressor groups. When compared to CD4+ cell counts at study entry, participants with initial virologic suppression had a median gain of 7 CD4+ cells/mm3 at ATI week 16. This was in contrast to a median loss of 247 CD4+ cells/mm3 among non-suppressors (P = 0.04).Of the 104 participants with evaluable pVL set point, 11 were found to have a pVL set point <3.0 logpressors . Eighty-10 copies/ml (3 (N = 9) as compared to a decline of 99 cells/mm3 for initial non-suppressors .Of the 10 participants with initial virologic suppression and a measured pVL at ATI week 49 off of ART, only 3 subjects continued to have a pVL <3.0 logopies/ml . Two of 10 HIV-1 copies/ml, respectively, P = 0.07). In addition, those with initial virologic suppression had a significantly greater decrease between pre-ART pVL and the set point pVL compared to those without initial virologic suppression . Almost all individuals were found to have HLA-associated polymorphisms in HIV-1 Gag early in the treatment interruption with a median of four polymorphisms per person. In the eight individuals with plasma available at ATI week 49, four were found to have accumulated additional Gag escape mutations (median 1.5 mutations). No significant differences were seen in the proportion of Gag polymorphisms between initial virologic suppressors and non-suppressors at either the first available ATI time point  or at ATI week 49 . This finding suggests that the lack of sustained pVL suppression in the latter group could not be explained by the accumulation of HLA-associated escape mutations in Gag.Of those with available pre-ART pVL data, individuals with initial virologic suppression had lower pre-ART pVL, but this difference was of marginal statistical significance (median 3.9 [N = 8] versus 4.8 [N = 62] loggag sequence and the earliest available patient-derived HIV-1 sequence after ATI (median 4 weeks). These early sequences represent viral populations least likely to have been shaped by significant vaccine-driven immune responses. There was no significant difference in the number of Gag amino acid mismatches between the vaccine and patient HIV-1 sequences among initial virologic suppressors and non-suppressors who received the vaccine . The change in the number of vaccine-to-patient Gag amino acid mismatches between the first ATI time point and ATI week 49 was used as a potential reflection of vaccine-induced viral evolution. However, we detected little overall change in the number of mismatched amino acids between the two time points and no significant differences between initial virologic suppressors and non-suppressors .The number of predicted amino acid mismatches was calculated between the vaccine 3, P = 0.58). In the initial A5197 analysis, an inverse association was seen between the ATI set point viral load and the number of HIV-1 Gag-specific CD4+ IFN-γ-producing CD4+ T cells at study weeks 8 and 38 10 cells/106 lymphocytes, P = 0.21; week 38 median 2.5 versus 2.2 log10 cells/106 lymphocytes, P = 0.20), or Gag-specific IFN-γ-producing CD8+ T-cells . Similarly, no significant differences were detected between initial virologic suppressors and non-suppressors in the number of subjects who had an increase in HIV-1 Gag-specific IFN-γ-producing CD4+ and CD8+ T-cells from study entry to week 38. Thirty percent (3/10) of initial virologic suppressors had a significant increase in the number of CD4+ IFN-γ-producing cells between baseline and week 38 as compared with 35% (31/89) non-suppressors (P = 1.0). Fifty percent (5/10) of initial virologic suppressors had a significant increase in the number of CD8+ IFN-γ-producing cells as compared with 38% (34/89) of non-suppressors (P = 0.51).There was no significant difference between initial virologic suppressors and non-suppressors in the CD4+ T-cell count at study entry . However, participants with initial virologic suppression had a significantly higher percentage of Gag-specific CD4+ TNF-α+ cells expressing either CTLA-4 or PD-1  and differing characteristics  of the latent HIV-1 reservoir from which the rebounding virus originated. Although we detected no significant differences between initial virologic suppressors and non-suppressors in the number of accumulated HLA-associated HIV-1 polymorphisms, we cannot rule-out the possibility that qualitative differences exist between escape mutations with differential impact on viral fitness.Three participants were able to maintain virologic suppression 49 weeks after treatment interruption. All three individuals had received study vaccine and two of the three had CD4+ cell counts at ATI week 49 above that found at study entry. In evaluating which virologic or immunologic characteristics may be predictive of initial virologic suppression, we found a trend toward lower pre-ART viral load for participants with initial virologic suppression. Pre-ART viral load has also been seen in other studies to be associated with the extent of viral rebound The magnitude of HIV-1 Gag-specific IFN-γ-producing CD4+ T cells has been previously associated with virologic control CTLA-4 and PD-1 are inhibitory immunoregulatory molecules that regulate T cell activation and peripheral immune tolerance This post-hoc analysis has several limitations. Only a subset of participants were initial virologic suppressors, which limited our ability to identify significant viral and immunologic predictors of virologic control. The analysis of viral load and CD4+ cell counts between ATI weeks 16 and 49 may be confounded by selection bias, especially in the non-suppressor group. After the ATI week 16 time point, participants had the option of restarting ART and those with particularly high viral loads or low CD4+ cell counts were encouraged to do so. Therefore, the viral load increases and CD4+ cell declines in the non-suppressor group are likely to be underestimated, which may have obscured continued viral load and CD4+ T cell benefits in the initial suppressor group. Immunologic studies on the magnitude of CTLA-4 and PD-1 expression of CD4+ cells were performed on a subset of participants, which may have limited our ability to detect a significant difference between groups.An effective HIV-1 therapeutic vaccine would be a significant advance in our efforts at HIV eradication and could provide insight into the optimal preventative vaccine strategy. In ACTG A5197, we found that participants with initial virologic suppression had a lower proportion of CD4+ T cells expressing CTLA-4 and a higher percentage of Gag-specific CD4+ TNF-α+ cells expressing either CTLA-4 or PD-1. Further studies are needed to determine whether optimizing CTLA-4 and PD-1 expression on CD4+ T cells will improve virologic control in recipients of other therapeutic vaccines in development.


The development of tipranavir and darunavir, second generation non-peptidic HIV protease inhibitors, with marked improved resistance profiles, has opened a new perspective on the treatment of antiretroviral therapy (ART) experienced HIV patients with poor viral load control. The aim of this study was to determine the virologic response in ART experienced patients to tipranavir-ritonavir and darunavir-ritonavir based regimens.A computer based literature search was conducted in the databases of HINARI , Medline and Cochrane library. Meta-analysis was performed by including randomized controlled studies that were conducted in ART experienced patients with plasma viral load above 1,000 copies HIV RNA/ml. The odds ratios and 95% confidence intervals (CI) for viral loads of <50 copies and <400 copies HIV RNA/ml at the end of the intervention were determined by the random effects model. Meta-regression, sensitivity analysis and funnel plots were done. The number of HIV-1 patients who were on either a tipranavir-ritonavir or darunavir-ritonavir based regimen and achieved viral load less than 50 copies HIV RNA/ml was significantly higher  than the number of HIV-1 patients who were on investigator selected boosted comparator HIV-1 protease inhibitors (CPIs-ritonavir). Similarly, the number of patients with viral load less than 400 copies HIV RNA/ml was significantly higher in either the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group . Meta-regression showed that the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimen.Tipranavir and darunavir based regimens were more effective in patients who were ART experienced and had poor viral load control. Further studies are required to determine their consistent viral load suppression effect as the duration of treatment is more prolonged. The advent of ART in 1995 has added 14 million life-years for HIV infected people in low- and middle-income countries As compared to other classes of antiretroviral drugs, PIs have the highest genetic barrier to resistance On the other hand, the development of tipranavir and darunavir, second generation non-peptidic HIV-1 PIs with markedly improved resistance profiles, has opened a new perspective in the treatment of ART experienced patients with poor viral control or developed resistant HIV strains. Patients who received tipranavir-ritonavir (TPV/r) with an optimized background regimen have achieved and maintained more treatment response than patients who received investigator selected comparator PIs (CPIs) with optimized background regimens Even though there are several studies on both tipranavir and darunavir, to the best of the authors’ knowledge, no published meta-analysis has assessed their consistent efficacy in ART experienced HIV patients with poor viral control. Thus, the primary aim of this meta-analysis was to determine the virologic response to tipranavir-ritonavir (500 mg/200 mg) twice daily and darunavir-ritonavir (600 mg/100 mg) twice daily based regimens, as compared to CPIs based regimens in ART experienced HIV-1 patients.A computer based literature search was conducted in the databases of HINARI, Medline and the Cochrane library. The search was further strengthened by searching literature using Google scholar search engine and literature from the reference lists of retrieved articles. Our search terms included: “tipranavir”, “PNU-140690”, “darunavir”, “TMC114”, “second generation PI” and “virologic response”. During searching, the search terms were combined alternatively with Boolean logic (and/or).In this meta-analysis, the pre-determined inclusion criteria were: 1) randomized controlled studies that assessed the effectiveness of tipranavir-ritonavir (500 mg/200 mg) twice daily or darunavir-ritonavir (600 mg/100 mg) twice daily based regimens relative to investigator selected boosted CPIs in ART experienced HIV-1 patients; 2) studies that recruited HIV type 1 patients with plasma viral load above 1,000 copies HIV RNA/ml; and 3) studies conducted in English. These criteria were set to address the primary aim of this meta-analysis, which was to determine the effectiveness of tipranavir and darunavir in ART experienced HIV-1 patients (probably with resistant HIV strains) as compared to CPIs and the authors’ inability to translate articles written in other languages.Study selection was performed in two stages. Firstly, the titles and abstracts of all the retrieved articles were reviewed and then grouped as “eligible for inclusion” and “ineligible for inclusion”. Secondly, articles which were grouped under “eligible for inclusion” were reviewed in detail for decision.10 copies HIV RNA/ml from the baseline in each group, number of patients with viral load <400 copies HIV RNA/ml in each group at the end of interventions, and number of patients with viral load <50 copies HIV RNA/ml in each group at the end of interventions.For data extraction, a standard Excel spreadsheet was used; and data were abstracted individually. From the included studies, the following data were extracted: the first author’s name, year of publication, names of the PIs that were given in each group in the study period, duration of therapy, frequency of drug administration, strength of the drugs (dose), sample size in each group, baseline viral load, baseline CD4 count, number of patients who achieved viral load reduction by ≥1 logIn this meta-analysis the term “ART experienced” means a patient who was on anti-HIV treatment  with poor viral load control . “Investigator selected boosted comparator protease inhibitors” (CPIs) – means any of the currently available first generation HIV PIs with ritonavir as a booster (CPI-r).10 copies HIV RNA/ml from the baseline, (b) viral load <400 copies HIV RNA/ml at the end of the interventions, and (c) viral load <50 copies HIV RNA/ml load at the end interventions were computed with the DerSimonian-Laird method (random effects model).Meta-analysis was carried out to assess the efficacy of tipranavir-ritonavir and darunavir-ritonavir plus an optimized background regimen relative to ritonavir boosted CPIs in ART experienced HIV patients. The odds ratios (OR) and the 95% CIs for: (a) achieved a viral load reduction by ≥1 log2 and p-values. I2 ≥50% was considered as statistically significant. When there was significant heterogeneity, subgroup analysis and meta-regression analysis were conducted to determine the possible contributing factors for the heterogeneity. Meta-regressions were conducted based on the duration of therapy, baseline viral load and baseline CD4 count. Sensitivity analyses (leaving one study out at a time) were also conducted to estimate the stability of the overall odds ratios in the withdrawal of any of the studies from the analysis. Publication/disclosure biases were assessed with funnel plots. All the meta-analyses and plots were generated using Review Manager (RevMan) Version 5.1 software. Comprehensive Meta-Analysis Software was used to do meta-regression.The heterogeneity among the studies was assessed by computing values for chi-square (Q), IAs presented in 10 copies HIV RNA/ml from the baseline showed a statistically significant reduction in patients who were on either tipranavir-ritonavir or darunavir-ritonavir based regimens  (2 = 91%) but no significant heterogeneity among the studies that compared tipranavir-ritonavir based regimens with boosted CPIs based regimens (I2 = 0%). In the sensitivity analysis, the overall OR swings between 3.9 and 4.6; however, when Madruga JV et al The meta-analysis  of viral load reduction by ≥1 log – 5.79) . As compThe meta-analysis of HIV-1 patients who achieved a viral load below 400 copies of HIV RNA/ml at the end of the interventions was conducted by including 2,189 HIV-1 patients who were on either tipranavir-ritonavir or darunavir-ritonavir based regimens and 2,156 HIV-1 patients who were on CPIs-ritonavir based regimens. In the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group, in all studies except for that of Willing JH et al 2 = 79%, Q = 38.65, P<0.00001) and the uniformity of studies on tipranavir.Similarly, the meta-analysis including 2,861 HIV-1 patients who were on either tipranavir-ritonavir or darunavir-ritonavir based regimens and 2,821 HIV-1 patients who were on CPIs-ritonavir based regimens, the number of HIV-1 patients who achieved a viral load less than 50 copies HIV RNA/ml at the end of the interventions was significantly higher in patients who were on either tipranavir-ritonavir or darunavir-ritonavir based regimens  . All the included studies on tipranavir  and seven of the nine studies on darunavir  showed a statistically significant viral load reduction. However, heterogeneity testing yet again affirmed the significant variability of studies on darunavir .However, the significant heterogeneity among studies on darunavir was largely due to Madruga JV et al 10 copies HIV RNA/ml from the baseline, the number of patients who achieved viral load less than 400 copies HIV RNA/ml and number of patients who achieved viral load less than 50 copies at the end of the interventions. But, the computed values revealed the lack of relation between the efficacy of tipranavir-ritonavir or darunavir-ritonavir based regimens and the duration of therapy, the baseline viral load and baseline CD4 count (Table 1). In other words, the viral suppression effect of boosted tipranavir or darunavir based regimens was not dependent on the duration of therapy, the initial viral load and the CD4 count at the start of interventions.Meta-regressions were conducted to determine how the duration of tipranavir-ritonavir based therapy, baseline viral load and baseline CD4 count affects the reduction of viral load by ≥1 logThis meta-analysis demonstrated that treatment with tipranavir-ritonavir or darunavir-ritonavir based regimen in ART experienced HIV-1 patients showed a statistically significant viral load reduction. In other words, though all patients included in the selected studies had a plasma viral load above 1,000 copies HIV RNA/ml at the time of recruitment, the number of patients who achieved a viral load of <50 copies HIV RNA/ml was significantly higher in the group of tipranavir-ritonavir or darunavir-ritonavir based regimens treated than in the CPIs-ritonavir based regimens treated group. Such an extent of viral load suppression with tipranavir-ritonavir or darunavir-ritonavir based regimens in ART experienced is an advantage to meet the ART guideline recommendation of making the viral load below 400 copies HIV RNA/ml within six months Specifically, relative to CPIs-ritonavir based regimens, the odds of viral load suppression of darunavir-ritonavir based regimens were greater than the odds of tipranavir-ritonavir based regimes. However, studies that directly compared tipranavir-ritonavir based regimens with darunavir-ritonavir based regimens did not show significant difference in terms of virologic suppression Other primary studies that compared the effectiveness of darunavir-ritonavir based regimens with lopinavir-ritonavir based regimens on ART naïve HIV patients also showed a significantly greater viral load reduction effect of darunavir-ritonavir based regimens Heterogeneity testing has shown the existence of significant variability among the included studies on darunavir. However, as the meta-regression showed, the variation in duration of therapy, baseline viral load and CD4 count among the included studies were not adequate to explain the sources or causes of heterogeneity. However, the lack of relation between the duration of tipranavir-ritonavir or darunavir-ritonavir based therapy and viral load reduction should be interpreted cautiously, because a Taiwanese study with a longer duration of therapy than any of the studies included in this meta-analysis showed a significant increase in the levels of genotypic resistance to tipranavir and darunavir in patients with virological failure to first generation PIs As limitations of this analysis, firstly, since most of the included studies did not report the types of drugs and the duration of therapies in the previous PI based regimens, this analysis was not able to assess their impacts on the therapeutic outcomes of tipranavir-ritonavir and darunavir-ritonavir based regimens. Secondly, the available data on CD4 counts were incomplete, making it difficult to determine the effects of tipranavir and darunavir based regimens on changes in CD4 count. However, based on previous studies that found an inverse relation of CD4 count and plasma viral load In conclusion, this meta-analysis has shown a significant viral load reduction in ART experienced HIV-1 patients who were treated with tipranavir-ritonavir or darunavir-ritonavir based regimens as compared to CPIs-ritonavir based regimens. Furthermore, the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimens. However, further studies are required to determine their consistent plasma viral load suppression effect as the duration of treatment is more prolonged.


P<0.0001). The half-life of efavirenz was higher (50.5 hours) in the 8 patients in whom K103N emerged (>0.1%) than in the 11 patients in whom it did not (32 hours) (P = 0.04). Thus ultrasensitive methods could prove more useful than direct sequencing for predicting treatment failure in some patients. However the presence of minor NNRTI-resistant viruses need not always result in virological escape.The impact of minor drug-resistant variants of the type 1 immunodeficiency virus (HIV-1) on the failure of antiretroviral therapy remains unclear. We have evaluated the importance of detecting minor populations of viruses resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTI) during intermittent antiretroviral therapy, a high-risk context for the emergence of drug-resistant HIV-1. We carried out a longitudinal study on plasma samples taken from 21 patients given efavirenz and enrolled in the intermittent arm of the ANRS 106 trial. Allele-specific real-time PCR was used to detect and quantify minor K103N mutants during off-therapy periods. The concordance with ultra-deep pyrosequencing was assessed for 11 patients. The pharmacokinetics of efavirenz was assayed to determine whether its variability could influence the emergence of K103N mutants. Allele-specific real-time PCR detected K103N mutants in 15 of the 19 analyzable patients at the end of an off-therapy period while direct sequencing detected mutants in only 6 patients. The frequency of K103N mutants was <0.1% in 7 patients by allele-specific real-time PCR without further selection, and >0.1% in 8. It was 0.1%–10% in 6 of these 8 patients. The mutated virus populations of 4 of these 6 patients underwent further selection and treatment failed for 2 of them. The K103N mutant frequency was >10% in the remaining 2, treatment failed for one. The copy numbers of K103N variants quantified by allele-specific real-time PCR and ultra-deep pyrosequencing agreed closely (ρ = 0.89 NCT00122551ClinicalTrials.gov  The lysine (K) to asparagine (N) mutation at codon 103 (K103N) in particular confers great resistance to efavirenz The remarkable benefits of combinations of antiretroviral drugs in controlling human immunodeficiency virus type 1 (HIV-1) infections can be compromised by the development of drug resistance. Non-nucleoside reverse transcriptase inhibitors (NNRTIs), notably efavirenz, are highly effective and are commonly used to treat HIV-1 infections pol gene at baseline Direct sequencing is currently used for routine HIV-1 genotyping Various strategies of intermittent antiretroviral therapy have been investigated during the past decade as ways of reducing drug-induced toxicity and costs and improving long-term adherence The randomized multicenter open-label ANRS 106 Window trial compared a fixed intermittent strategy of six cycles of alternating 8 weeks off-therapy and 8 weeks on-therapy with continuous therapy over a 96-week period The protocol was approved by the ethics committee of Toulouse University Purpan Hospital and by the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales (ANRS). All patients gave written informed consent. The study is registered at clinicaltrials.gov, no. NCT 00122551.+ T-cell count of ≥100 cells/µL, and a CD4+ T-cell count of ≥450 cells/µL at screening, and their HIV-1 RNA had been <200 copies/mL for at least the previous 6 months. They underwent a total of 6 cycles starting with 8 weeks off-therapy, followed by 8 weeks on-therapy. Efavirenz was stopped 7 days before the other drugs at the start of each off-therapy period th on-therapy period.The 21 HIV-1 infected patients had prospectively been included in a substudy of the ANRS 106 trial, on the basis of (i) being in the intermittent arm ; (ii) receiving efavirenz ; (iii) providing informed consent for additionnal blood samples required for the efavirenz pharmacokinetics study. They all had a nadir pre-treatment CD4Virus particles were pelleted from 1 ml of plasma by ultracentrifugation at 23,500 g for 1 h at 4°C before RNA extraction using the QIAamp viral RNA extraction kit (Qiagen). All the RNA extracted was then used in RT-PCR amplifications.5′-ATTTTCCCATTAGTCCTATT-3′ and reverse 5′-ATGTCATTGACAGTCCAGCT-3′. A nested PCR was then performed with the Expand High Fidelity Plus PCR System (Roche Diagnostics) and the following primers: forward 5′- CCAAAAGTTAAACAATGGCCATTGACAGA-3′ and reverse 5′- AGTTCATAACCCATCCAAAG -3′. Bulk PCR products were sequenced in both directions by the dideoxy chain termination method  on an ABI 3130 DNA sequencer as previously described Genotypic resistance tests were performed on plasma at the end of each off-therapy period. A first RT-PCR amplification step was performed with the QIAgen one step RT-PCR kit (Qiangen) and the following primers: forward 5′-GGAAGTTCAATTAGGAATACCACA-3′ and reverse 5′- TATAGGCTGTACTGTCCATTTGTC-3′). The amplified PCR products were purified using Agencourt Ampure PCR Purification beads (Beckman Coulter) and quantified with the Quant-iT Picogreen dsDNA Assay Kit (Invitrogen) on a LightCycler 480 (Roche). Pooled PCR products were clonally amplified on capture beads in water-in-oil emulsion micro-reactors. A total of 500,000 enriched-DNA beads were deposited in the wells of a full GS Junior Titanium PicoTiterPlate device and pyrosequenced in both forward and reverse directions. The 200 nucleotide flow cycles allow to sequence a 500 bp fragment in a 10-hour sequencing run as about 2.5 nucleotides would be incorporated per nucleotide flow cycle. The sequence reads were quantified with GS Amplicon Variant Analyzer (AVA) software Version 2.5p1 (Roche). The AVA software assigns each read to the proper amplicon and sample using multiplex identifiers. The sequence reads were aligned with the HXB2 consensus sequence. We assessed the frequency of errors resulting from PCR amplification and GS Junior pyrosequencing at codon 103 by analyzing the pyrosequencing data from a panel of 9 plasmid clones previously sequenced by the Sanger method . The mean error rate of pyrosequencing at codon 103 was 0.0047 . The upper confidence limit of the error rate was used to calculate the sensitivity of pyrosequencing for a given number of reads. At least 2500 reads reliably detects minor K103N mutants when they accounted for over 1.5% of the quasispecies. The first RT-PCR amplification step was common for direct sequencing and ultra-deep pyrosequencing. The mean virus load of the samples assesed was 67,792 copies/mL . A 469-nucleotide long fragment encompassing the reverse-transcriptase gene between codons 88 and 243 was then generated by nested PCR from the RT-PCR products. The nested PCR was performed with the Expand High Fidelity Plus PCR System (Roche Diagnostics). The deep sequencing primers are composed of fusion primers (5′ part) to fuse to the emulsion PCR beads, a four-base “key” sequence to define the DNA library, multiplex identifiers to enable for the identification of samples after pooling and sequencing, and a target-specific sequence (forward 5′-ACATCCCGCAGGGTTAAAAAAGAA-3′) located immediately upstream of the third nucleotide of the 103th codon of the reverse transcriptase gene, and a reverse primer (5′-AGTTCATAACCCATCCAAAG-3′) using the QIAgen one step RT-PCR kit (Qiagen) with the following conditions : 30 min of reverse transcription at 50°C; 15 min of inactivation at 94°C; 30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C for 40 cycles. The RNA input was adjusted for 50,000 HIV-1 copies per reaction. With this input, the first amplification was linear for 40 cycles (data not shown). This first round of amplification corrected for potential polymorphims of the targeted sequence to ensure that only the third nucleotide of the 103th codon was discriminant in the nested allele-specific amplification. Then 4 allele-specific real-time PCR real-time amplifications were performed in parallel to quantify the AAA and AAG wild-type codons (encoding «K»), and the AAC and AAT mutated codons (encoding «N») using the 4 specific forward primers 5′-CCGCAGGGTTAAAAAAGAIA/G/C/T-3′ and the reverse primer 5′-GGTTCTTTCTGATGTTTTTTGTCTGG-3′ and a Taqman probe famATGTGGGTGATGCATATTTTTCAGTTCtamra-3′5′- on a LightCycler (Roche) with the following conditions :10 min at 95°C; 15 sec at 95°C, 1 min at 62°C for 50 cycles. Each quantification was performed using a standard curve obtained by serial dilutions of a plasmid containing one of the targeted AAA, AAG, AAC, and AAT sequences cloned into a pCR4-TOPO vector (Invitrogen). The specificity of each amplification was assessed by amplifying equal quantities of the targeted and non-targeted sequences. The amplification specific for the AAC mutated sequence resulted in 220 and 216 more efficient amplifications of the targeted AAC sequence than the non-targeted AAA and AAG wild-type sequences. The amplification specific for the AAT mutated sequence resulted in 229 and 221 more efficient amplifications of the targeted AAT sequence than the non-targeted AAA and AAG wild-type sequences. The input for quantifying the mutated sequences was adjusted for 5,000 copies per reaction based on the quantification of wild-type sequences. The AAC and AAT amplifications were specific enough to extinguish the wild-type sequences in the amplification. The sensitivity of our allele-specific PCR assay to detect K103N mutants was assessed on mixtures of wild-type and K103N mutants. Our assay detects K103N mutants down to a frequency of 0.01%. This 0.01% threshold of detection was thus used in the study. A plot of the measured values versus the input was linear across a wide range of K103N mutants frequencies (A first round of amplification was performed with a forward primer (quencies . Reciproquencies .We used high-performance liquid chromatography (HPLC) with UV detection to determine efavirenz concentrations in the plasma 1, 3, 7, and 10 days after interrupting efavirenz therapy to assess interindividual variations. Half-lives were calculated from the slope of concentrations decline versus time.P value of <0.001 according to the Poisson model were considered authentic. Statistical analyses were performed with Stata SE 9.2.Quantitative variables were compared using the Wilcoxon rank sum test. Correlations between quantitative variables were estimated by calculating Spearman's rank correlation coefficient. Poisson distribution was used to distinguish authentic variants from artefactual sequences resulting from errors arising during PCR amplification and ultra-deep pyrosequencing. Only those variants whose frequency of occurrence yielded a + T-lymphocyte count of 648 cells/µL , and all had plasma virus load <200 copies/mL. They were all receiving a backbone of nucleoside reverse transcriptase inhibitors associated with efavirenz, plus a protease inhibitor for 3 of them.The 21 patients  had a median age of 38.6 years-old , a median CD4The allele-specific real-time PCR to detect K103N mutations could not be performed in 2 patients (harboring wild-type viruses by direct sequencing) because of amplification failure. K103N mutants were detected at the end of an off-therapy period in 15 of the 19 analyzable patients by allele-specific real-time PCR, but in only 6 of these 19 patients by direct sequencing. The frequency of K103N mutants in the quasispecies remained below 0.1% throughout follow-up in 7 of these 15 patients, without further selection. The initial frequency of K103N mutants was 0.1–1% in 2 of the 15 patients, and further increased to become predominant in one of them. The initial frequency of K103N mutants was 1–10% in 4 of the 15 patients, with further selection in 3 of them. Lastly, the initial frequency of K103N mutants was above 10% in 2 of the 15 patients .Allele-specific real-time PCR detected minor K103N mutants in 9 more patients than did direct sequencing; the frequency of K103N mutants was <0.1% in 7 of them, and 1% and 2.8% in the 2 others. The K103N mutants detected by allele-specific real-time PCR in 6 patients were also detected by direct sequencing . Allele-10 , 3.65 log10 , 3.97 log10 , and 3.04 log10  per mL plasma when first detected by allele-specific real-time PCR   .P = 0.04)  on day 1, 416 ng/ml  on day 3, 112 ng/ml  at day 7, and 50 ng/ml  on day 10. Estimations of the half-life of efavirenz revealed great inter-individual variations, from 27 to 136 hours. Thus, even stopping efavirenz 7 days before the other drugs of the regimen could result in efavirenz monotherapy in some patients. The median efavirenz half-life was 32 hours (CI95 [28.7–75.3]) in patients harboring no or few (<0.1%) K103N mutants (n = 11), and 50.5 hours (CI95 [45.1–99.6]) in patients in whom allele-specific real-time PCR detected a significant frequency (>0.1%) of K103N mutants (n = 8) ( = 0.04) .We analyzed the impact of emergent K103N mutants on the subsequent virological response to cART in the 15 patients in whom K103N mutants had been detected by allele-specific real-time PCR. It had no influence on the virological response to cART in the 7 patients whose frequency of K103N mutants remained <0.1% without further selection. By contrast, the emergence of K103N mutations in the other 8 patients with >0.1% K103N mutants resulted in treatment failure in 3 of them; the remaining 5 maintained a virological response to cART during on-therapy periods (plasma virus load ≤400 copies/mL). K103N mutants were abundant in 2 patients (patients 17 and 19) who were on stavudine, didanosine, and efavirenz, one from the end of the first off-therapy period (99.6%) and the other from the end of the second (85.5%); they experienced treatment failure thereafter. The third patient (patient 15) who experienced treatment failure was on stavudine, lamivudine, and efavirenz. The gradual emergence of K103N mutants was first detected by allele-specific real-time PCR during the third off-therapy period (1.9% at week 40), and the mutated population was then selected at each cycle until the virus population was almost totally mutated (91.9%) at week 88. Treatment failed in this patient after the fifth on-therapy period. By contrast, treatment failure did not occur in 5 patients despite the presence of K103N mutants: the frequency of K103N mutants remained quite low (maximum 1% and 2.8%) in patients 12 and 14, it was high (48.4%) in another (patient 16) who was also given indinavir in addition to efavirenz and lamivudine, and the frequencies of K103N mutants were high  in the remaining 2 patients (patients 13 and 18) who were given only two nucleoside reverse transcriptase inhibitors plus efavirenz. The follow-up data available for these 2 patients showed that they still had an undetectable plasma virus load (<50 copies/mL) 40 weeks after the end of the trial when they were on continuous therapy with the same regimen.We have assessed the performances of allele-specific real-time PCR and ultra-deep pyrosequencing for detecting minor K103N mutants in patients given efavirenz while on intermittent antiretroviral therapy. An earlier analysis by direct sequencing revealed that the proportion of patients in the intermittent arm given NNRTIs in whose plasma NNRTI-associated resistance mutations were detected increased over time from 3% at the end of the first off-therapy period to 27% at the end of the sixth off-therapy period P value of <0.001 according to the Poisson model were considered authentic. Our assay on the GS Junior can detect K103N minor variants that account for 1.5% of total virus if the number of reads is above 2500. But ultra-deep pyrosequencing was not as sensitive as allele-specific real-time PCR for detecting K103N mutants at very low frequency. However, unlike allele-specific real-time PCR, ultra-deep pyrosequencing can determine whether HIV-1 drug-resistance mutations are linked on a given sequence or are borne separately by different virus clones. Globally, the results obtained by allele-specific PCR and ultra-deep pyrosequencing were well correlated, providing reciprocal validation of each method.Allele-specific real-time PCR detected K103N mutants in 9 more patients harboring a low frequency of mutated viruses than did direct sequencing. Allele-specific real-time PCR also provided earlier detection than direct sequencing during the selection of drug-resistant viruses. Plasma samples from 11 patients were assayed by both allele-specific real-time PCR and ultra-deep pyrosequencing. Ultra-deep pyrosequencing is prone to artefactual errors. We have measured the sequence error rate at codon 103 resulting from PCR amplification and ultra-deep pyrosequencing by comparing the GS junior reads to the Sanger sequences of 9 plasmid clones. We used the upper 99% confidence limit of the error rate to calculate the frequency of artefactual sequences for a given number of reads and the Poisson distribution to estimate the number of reads above which K103N mutants found at low frequencies were authentic rather than artefactual. Only those variants whose frequency of occurrence yielded a We assayed the pharmacokinetics of efavirenz to determine whether its variability could influence the emergence of the K103N mutation. We found that its half-life varied greatly between individuals, from 27 to 136 hours. Thus, stopping efavirenz even 7 days before the other drugs of the regimen could result in efavirenz monotherapy in some patients, a situation in which the risk of NNRTIs resistance emerging is high. This may have occurred as the half-life of efavirenz was significantly higher in the 8 patients in whom K103N emerged than in the 11 patients in whom it did not. However, the small sample size precludes higher levels of statistical significance required for a definite conclusion.+ T cells during the period of intermittent therapy than did patients with wild-type viruses The impact of emergent K103N mutations on the subsequent virological response to cART in the 8 patients harboring >0.1% of the K103N mutated viruses varied. It resulted in treatment failure in 3 patients while 5 paradoxically maintained a virological response to antiretroviral therapy during each on-therapy period. However, 2 of the patients who responded to cART had only a few K103N mutants. A third patient with a high frequency of K103N mutants was given a protease inhibitor in addition to efavirenz, and this could have contributed to the virological response. The last 2 patients had high frequencies of K103N mutants but paradoxically maintained a virological response on efavirenz-based regimen during the intermittent therapy scheme, and on continuous therapy with the same regimen upon completion of the study. Genotypic methods for inferring HIV-1 resistance rely on the presence of known mutations at specific positions of the HIV-1 genome, but cannot assess the impact of changes in the replicative fitness or infectiousness of some viruses 10) copies/mL plasma of drug-resistant viruses experienced further selection of this population 10 copies/mL K103N mutants when first detected by allele-specific real-time PCR. The fourth had under 3 log10 copies/mL of K103N mutants but nevertheless underwent further selection of the mutated virus population. A recent pooled analysis found that low-frequency NNRTI resistance mutations confer a greater than 2-fold risk of virologic failure in treatment-naive individuals initiating a first-line NNRTI-containing cART regimen. A dose-dependent association of drug-resistant minority variants with increased risk of virologic failure was observed. Absolute numbers of drug-resistant minority variants over 1 log10 copies/mL plasma appear to be associated with a statistically significant higher risk of virological failure The clinical relevancy of detecting minor populations of mutated viruses remains open to debate. There is growing evidence that the presence of minor mutated viruses at baseline has a deleterious impact on the subsequent response to cART In conclusion, we have found that allele-specific real-time PCR and ultra-deep pyrosequencing are more sensitive than direct sequencing for detecting K103N mutants in the particular setting of intermittent antiretroviral therapy, with an excellent correlation between both mehods for quantifying the mutated viruses. These ultrasensitive methods could be more useful than direct sequencing for predicting treatment failure in some patients. But the presence of minor resistant viruses need not always lead to subsequent virological escape. Large prospective studies are now needed to demonstrate the clinical relevance of detecting minor resistant virus populations for adapting antiretroviral therapy.