To examine the outcomes of highly-active antiretroviral therapy (HAART) for individuals with free access to healthcare, we evaluated 2327 patients in a cohort study composed of military personnel and beneficiaries with HIV infection who initiated HAART from 1996 to the end of 2007.Outcomes analyzed were virologic suppression (VS) and failure (VF), CD4 count changes, AIDS and death. VF was defined as never suppressing or having at least one rebound event. Multivariate (MV) analyses stratified by the HAART initiation year (before or after 2000) were performed to identify risk factors associated with these outcomes.Among patients who started HAART after 2000, 81% had VS at 1 year , 85% at 5 years , and 82% at 8 years (N = 735). Five years post-HAART, the median CD4 increase was 247 cells/ml and 34% experienced VF. AIDS and mortality rates at 5 years were 2% and 0.3%, respectively. In a MV model adjusted for known risk factors associated with treatment response, being on active duty (versus retired) at HAART initiation was associated with a decreased risk of AIDS  and mortality , an increased probability of CD4 increase ≥ 50% , but was not significant for VF.In this observational cohort, VS rates approach those described in clinical trials. Initiating HAART on active duty was associated with even better outcomes. These findings support the notion that free access to healthcare likely improves the response to HAART thereby reducing HIV-related morbidity and mortality. Despite substantial progress since the introduction of highly-active antiretroviral therapy (HAART) -4, maintWe sought to examine a large, multicenter cohort composed of military personnel and beneficiaries with HIV infection followed since diagnosis in order to illustrate the HAART outcomes for patients within a free-access healthcare system in the United States. The U.S. military medical system provides comprehensive HIV education, care and treatment, including the provision of ARVs and regular visits with HIV clinicians at medical treatment facilities (MTF), at no cost to the patient. Mandatory periodic HIV screening according to Department of Defense (DoD) policy  allows tAside from the advantages afforded by the medical system, there are aspects of this cohort that allow for a unique perspective on HIV treatment response. The military population from which these patients are derived consists of highly motivated and disciplined individuals who possess either a minimum of a high school equivalent education (enlisted) or an undergraduate college degree (officers) and maintain rigorous physical standards -11. As aThe U.S. Military HIV Natural History Study (NHS) is a prospective multicenter observational study of HIV-infected active duty military personnel and other beneficiaries  from the Army, Navy/Marines and Air Force. All participants provided written informed consent. The cohort characteristics have been previously described . PatientSeroconverters (SC) were defined as patients having a documented HIV seronegative date prior to the first positive HIV date. The estimated date of seroconversion for SC was defined as the midpoint between the two dates. All CD4 count and VL measurements were done as part of routine clinical care. The clinically-approved methodology for this testing varied by site and over time. Sexually transmitted infections (STIs) were defined as having a documented clinical history of gonorrhea, chlamydia, syphilis or herpes simplex at any time prior to initiation of HAART. Chronic hepatitis B co-infection was defined as having at least two positive hepatitis B surface antigen tests at least 6 months apart. Hepatitis C virus (HCV) co-infection was defined as having at least one positive HCV antibody test. ARV use referred to any antiretroviral therapy not meeting the NHS definition of HAART . HAART iOutcomes were described for all patients and separately for those initiating HAART from 1996-1999  and for those starting HAART in 2000-2007 . Virologic outcomes and CD4 cell count response were described at 6-month intervals through 8 years after the initiation of HAART. Due to differing lengths of follow-up after HAART initiation, the sample size was 1063 (46%) at 5 years and 735 (32%) at 8 years. CD4 and viral load (VL) at HAART were the last recorded value up to 6-months before HAART. Six-month follow-up values where those recorded closest to the 6-month interval after HAART initiation (within a window of ± 3 months). Patients with missing laboratory values for a given time point were excluded from analyses at that time point. Virologic suppression (VS) was defined as an undetectable viral load (< 400 copies/mL). Virologic failure (VF) was defined as 2 consecutive VL detectable after VS (virologic rebound) or never achieving VS (never suppressed). Always suppressed was defined as having all measured VL undetectable for the entire period beginning 6 months after HAART initiation. CD4 count outcomes were expressed as the group mean and the mean increase after HAART initiation at a given time point. The percentage of patients who experienced at least a 30% or 50% CD4 count increase from HAART initiation was also determined. Switches and discontinuations of ARVs were not counted as failures.Kaplan Meier (KM) life-table methods were used to estimate the cumulative rate of VF, CD4 increase of 50%, AIDS-defining conditions, and all-cause mortality. Patients without the event of interest were censored at the last recorded visit. For time-to-VF, patients never suppressed were considered to have failed at time zero. Stratified Cox-regression  was used to determine the association of relevant covariates with these same outcomes. Baseline covariates used in the model were those found to be associated (p < 0.1) in univariate analyses as well as those shown to be risk factors in the literature.Characteristics for patients who initiated HAART overall and by HAART initiation era are shown in Table As expected, both prior ARV use and initial HAART regimen differed significantly p < 0.001) between eras . For this same era at 5 and 8 years, there were 34% and 50% of LHE patients (versus 61% and 68% EHE), respectively. Similarly, the degree of immune reconstitution was greater in the LHE, despite similar CD4 levels at HAART start. In the first year, 52% of patients from the LHE had achieved a 50% gain in CD4 count. This increased to 63% of patients at 5 years.The rate of AIDS events and deaths were lower in the LHE compared to the EHE. At 1 year, the AIDS event rate Figure  was 4.7%In a multivariate model Table  stratifiIn this study, we describe the clinical characteristics and response to HAART among HIV-infected military personnel and beneficiaries initiating treatment over the course of twelve years (1996-2007) with an average follow-up of over 6 years. The NHS is conducted within the military medical system allowing for an evaluation of HAART response in a U.S. clinical setting with free and open access to healthcare and medications.After stratifying patients into two HAART initiation eras, 1996-2000 (EHE) and 2000 onward (LHE), it was evident that these eras differed significantly for several reasons. First, the large majority of patients starting treatment in the EHE had prior exposure to suboptimal therapy which has been shown to compromise the response to HAART . SecondlFor the LHE patients in this cohort, the virologic and immunologic responses were similar to those reported by randomized clinical trials using a regimen containing either efavirenz or a boosted-PI. A meta-analysis of 20 clinical trials by Gupta et al. described a VS rate of 76% and CD4 change of 176 cells/mL at 48 weeks . The ratAlthough there are drug assistance programs in the U.S. for eligible individuals with HIV/AIDS, the delay before medical care becomes available can postpone HAART initiation, and even the minimal associated costs can be a significant barrier for some patients ,34. Co-pThis cohort provided an opportunity to examine the relationship between demographic and clinical factors with outcomes after HAART in a clinical setting that minimized confounding related to access to care and IDU. Previously, we and others have shown associations between both age at HAART initiation  and ethnActive duty status was associated with improved survival, immune reconstitution and a lower rate of AIDS-defining events. Although a distinctive factor in our cohort, important implications related to adherence and general health can be proposed as to why individuals on active duty had improved outcomes; some of which could be translated to other settings. Factors that might improve an active duty member's medication adherence include: (1) better access to ARVs, (2) closer clinical monitoring, and (3) a more disciplined and regimented environment. Although all participants in this cohort study do have free access to the DoD healthcare system, retirees can live further from network facilities and can choose private insurance resulting in copayments for ARVs. Furthermore, active duty personnel may be more closely monitored as they are required by their supervisors to seek medical care on a regular basis. As evidence, research study visit attendance has been shown to be significantly greater for active duty vs. others . GeneralOne limitation of this study is that medication adherence data were unavailable for most patients (adherence questionnaires were added to the data collection in 2006). The relative impact of HIV drug resistance was also not assessed in this study. Finally, a disadvantage of any cohort study is that these results cannot be readily extrapolated to other clinical settings where rates of IDU, demographic characteristics, and access to healthcare differ. However, this cohort does provide an opportunity to observe sustainable treatment success after early HAART initiation under these conditions.In summary, we find rates of VS and CD4 reconstitution to be high and clinical events to be low for DoD beneficiaries receiving treatment for HIV. These rates approach those reported in clinical trials. Active duty personnel have better immunologic and clinical outcomes but equivalent rates of VF to other beneficiaries. These findings support the notion that free and open access to healthcare provides a favorable environment for optimizing HIV treatment outcomes.The authors declare that they have no competing interests.The following authors were involved in study conception and design: VCM, GG, ACW, BKA; acquisition of data: VCM, ACW, HC, MLL, AG, JFO, NCC, RJO, GWW, BKA; analysis and interpretation of data: VCM, GG, ACW, BKA; manuscript drafting and critical revision: VCM, GG, ACW, MLL, AG, JFO, NCC, RJO, AL, GWW, BKA. All authors read and approved the final manuscript.


Access to antiretroviral therapy (ART) is increasing in resource-limited settings (RLS) and can successfully reduce HIV-related morbidity and mortality. However, virologic failure and development of viral drug resistance can result in reduced treatment options and disease progression. Additionally, transmission of resistant virus, and particularly multi-drug resistance, could become a public health concern. This study evaluated treatment success and development of ART drug resistance after short-term treatment among patients attending the Comprehensive HIV Care Centre (CCC) of Coast Province General Hospital, Mombasa, Kenya. One hundred and fifty HIV-infected individuals receiving ART were consecutively recruited to participate in the study. After determination of plasma viral load, patients with detectable viral load levels were subjected to genotypic drug resistance testing. At the time of sampling, 132 of the 150 participants were on ART for more than 6 months . An efficient viral load reduction to below 50 copies/ml was observed in 113 (85.6%) of them. Of the 19 patients with a detectable viral load, sequencing of the protease (PR) and reverse transcriptase (RT) gene was successful in 16. Eleven (11) of these 16 patients were infected with a subtype A1 virus. Major PR mutations were absent, but mutations associated with drug resistance in RT were detected in 14 of the 16 patients (87.5%). High-level resistance against at least 2 drugs of the ART regimen was observed in 9/14 (64.3%). The 3TC mutation M184V and the NNRTI mutation K103N were most frequent but also the multi-drug resistance Q151M and the broad NRTI cross-resistance K65R were observed. The results of this study revealed a high rate of treatment success after short term ART in patients treated at a public provincial hospital in a RLS. Nevertheless, the observed high risk of accumulation of resistance mutations among patients failing treatment and the selection of multi-drug resistance mutations in some, remains of great concern for future treatment options and potential transmission to partners. Recent data show an HIV prevalence in Kenya of 7.4%, resulting in 1.4 million Kenyans living with HIV ,3. InforFrom the patients attending the CCC in Coast Province General Hospital, Mombasa, a total of 150 consecutive patients, over 18 years of age and receiving ART, were asked to participate in this surveillance study by a trained adherence counsellor. Fifty (50) patients were recruited in April 2006, 100 patients were recruited in May 2007. Participants gave written informed consent, and refusal to participate did not influence the standard of care. Ethical approval was obtained from the Kenyatta National Hospital Ethics and Research Committee.Using a structured questionnaire, basic socio-demographic and clinical data was obtained from each participant. ART adherence was measured by self-report and pill count over the last month and recorded as satisfactory (>95%) or unsatisfactory (<95%). Ten (10) ml of EDTA blood was collected for CD4 cell count . The remainder of the blood was centrifuged to collect plasma, which was stored at -80°C until processing for viral load measurement and genotyping.Plasma HIV RNA quantification was performed, using the Ultrasensitive Cobas Amplicor HIV-1 Monitor Test version 1.5  with a lower detection limit of 50 copies/ml.EU872121–EU872135 and 878548 for PR and EU872136–EU872150 and 878549 for RT].Extraction, amplification and genotyping of HIV RNA was performed by nested RT-PCR and an in-house sequencing assay as described elsewhere . The intAll statistical analyses were performed using SPSS 15.0 .3 (IQR = 63–184). A combination of d4T+3TC+NVP was the most commonly prescribed first-line regimen (n = 79), followed by d4T+3TC+EFV (n = 63), AZT+3TC+NVP (n = 5), AZT+3TC+EFV (n = 2), and d4T+ddI+EFV (n = 1). Patients receiving their first ART regimen had been treated for a median of 17 months (IQR = 10–24). In 16 patients treatment was changed after a median of 18 months (IQR = 13–26) by substituting one (n = 7), two (n = 5) or three (n = 4) drugs because of adverse events (n = 8), start of anti-tuberculosis treatment (n = 1), unavailability of drugs (n = 1), or immunological failure (n = 6). The 6 patients with immunological failure were switched to a LPV/r based regimen as recommended by the Kenyan national guidelines [The median age of the participants was 37 years IQR = 32–43) with 69% being women  or mean baseline CD4 (P = 0.18). A significantly higher mean CD4 count was seen in patients with an undetectable viral load  compared to those with a detectable viral load  (P = 0.03). However, the mean increase in CD4 from baseline level was not significantly different between the two groups (P = 0.33). Seventy-four patients (58.6%) reported to have satisfactory adherence, which was significantly associated with treatment success (P = 0.02).Eighteen of the 150 patients initiated ART less than 6 months  before blood collection and were excluded from further analyses. For the remaining 132 patients, an undetectable viral load was seen in 110 (87.3%) of the 126 patients without treatment changes or with treatment changes for other reasons than immunological failure and in 3 of the 6 patients in whom the treatment was changed because of immunological failure. The median viral load of the 19 patients with ongoing viral replication was 3,060 copies/ml . A detectable viral load was not significantly associated with the mean duration of ART  and for RT in 16 84.2%). Amplification failures were due to low viral loads . The subtype distribution for these 16 samples was as follows: subtype A1 (n = 11), CRF16_AD (n = 2), C (n = 1), D (n = 1) and a new recombinant of A1 and D (n = 1). No major PR resistance mutations were seen in any of the sequences, but a mean of 4 minor PR mutations were observed per sample (data not shown). Resistance mutations in RT were detected in 14 out of the 16 patients. Overall, the V184M was most commonly observed (n = 12), followed by K103N (n = 9). In one patient the multi-drug resistant Q151M mutation was seen and two patients carried virus with a K65R mutation, all three combined with V184M (Table S2; Additional File %. AmplifIntensive campaigns to improve availability of ART worldwide is paying off and most African countries are currently able to provide first-line ART regimens to a considerable number of HIV-1 infected individuals in need of treatment. Efforts to scale-up laboratory facilities for treatment monitoring in these patients however are running behind. A random sample survey is often the only way to assess treatment efficacy and the selection of drug resistance in treatment programs in Africa. The information obtained from these surveys is important for eventual future adaptation of treatment strategies. Moreover, resistance data obtained from these surveys will be crucial in evaluating the value of second-line regimens in Africa where only limited drugs are available.In this study, 85.6% of the patients receiving ART ≥ 6 months had a VL<50 copies/ml. These figures are comparable to what has been published for other African regions -10. The Despite the overall high efficacy of ART treatment regimens, a significant accumulation of resistance mutations was observed in patients with a detectable viral load at 6 months or more after treatment initiation. Though we cannot excluded that some of these mutations were already present at baseline, we assume that most of them were selected during treatment. As mutations can only be selected in the presence of the drug, their detection excludes the poor-intake of medication as the main reason for failure.The combinations of AZT/d4T+3TC+EFV/NVP are extensively used as a first-line regimen in RLS . DespiteK65R mutations have previously been described among populations with similar subtypes . HoweverAccumulation of mutations against drugs from different drug classes and/or the presence of broad cross-resistance mutations will jeopardize the effectiveness of the NRTI backbone of second-line regimens that often include ABC and TDF. Moreover, the limited availability and the high cost of boosted PIs force clinicians in RLS to recycle NNRTIs in the second-line regimen. Based on the resistance data from this study, we can assume that the effect of such a second-line regimen will be at the most temporary.The small number of patients on a second-line regimen that were included and the limited time period between initiation of this regimen and the date of sampling, did not allow us to make conclusions about the efficacy of second-line regimens.In conclusion, the results of this observational study show that effective first-line ART in clinical care centres with limited resources is feasible. However, the resistance data point out the danger of the absence of viral monitoring, with regard to the accumulation of resistance mutations.Besides high quality adherence counselling, efforts are needed to guarantee a robust supply of drugs from different classes, as well as the worldwide availability of affordable and simple viral load and genotyping assays to ensure long-term success of global ART programs.The authors declare that they have no competing interests.KS designed the study, carried out molecular work and sequence analysis and prepared the manuscript. SL and CV assisted in designing the study and drafting the manuscript. KD and JR performed viral loads and genotyping. KM supervised the study in Mombasa. JW, JP and MT provided substantial intellectual content to the manuscript. All authors critically reviewed and approved the final manuscript.Table S1. Characteristics of women and men at the time of study enrolment.Click here for fileTable S2. Overview of resistance mutations detected in the RT gene of patients with a detectable viral load after more than 6 months of ART.Click here for file


Although combination antiretroviral therapy (cART) initiated in the acute phase of HIV-1 infection may prevent expansion of the latent reservoir, its benefits remain controversial. In the current study, HIV-1 RNA transcription patterns in peripheral blood mononuclear cells (PBMC) were monitored during acute cART to assess the effect of early treatment on cellular viral reservoirs.Acutely HIV-1 infected patients (n = 24) were treated within 3–15 weeks after infection. Patients elected to cease treatment after ≥1 year of therapy. HIV-1 DNA (vDNA), HIV-1 RNA species expressed both in latently and productively infected cells, unspliced (UsRNA), multiply spliced , and PBMC-associated extracellular virion RNA (vRex), expressed specifically by productively infected cells, were quantified in PBMC by patient matched real-time PCR prior, during and post cART. In a matched control-group of patients on successful cART started during chronic infection (n = 15), UsRNA in PBMC and vDNA were measured cross-sectionally. In contrast to previous reports, PBMC-associated HIV-1 RNAs declined to predominantly undetectable levels on cART. After cART cessation, UsRNA, vRex, and MsRNA-tatrev rebounded to levels not significantly different to those at baseline (p>0.1). In contrast, MsRNA-nef remained significantly lower as compared to pretreatment (p = 0.015). UsRNA expressed at the highest levels of all viral RNAs, was detectable on cART in 42% of patients with cART initiated during acute infection as opposed to 87% of patients on cART initiated during chronic infection . Accordingly, UsRNA levels were 105–fold lower in the acute as compared to the chronic group.Early intervention resulted in profound depletion of PBMC expressing HIV-1 RNA. This is contrary to chronically infected patients who predominantly showed continuous UsRNA expression on cART. Thus, antiretroviral treatment initiated during the acute phase of infection prevented establishment or expansion of long-lived transcriptionally active viral cellular reservoirs in peripheral blood. These results strongly indicate that patients initiating cART in the acute phase of infection may attain a substantial virological benefit from early intervention.To elucidate these issues in the current study, intra- and extracellular viral dynamics were assessed before, during, and after treatment of acute HIV-1 infection. For this purpose HIV-1 RNA transcription patterns in PBMC and levels of plasma viremia were monitored. Comparison with a group of patients, who had initiated cART during chronic infection, was performed cross-sectionally. Unexpectedly, cell-associated viral RNA burden in PBMC became mostly undetectable in the acutely infected patient group. The levels of UsRNA, the most abundant cellular HIV-1 RNA Acutely HIV-1 infected patients (n = 24) were treated with cART within 3–15 weeks after infection. Plasma viremia was monitored using standard diagnostic quantitative PCR-assays (qPCR) and PBMC-associated HIV-1 nucleic acids  were quaTo investigate the activity of these HIV-1 infected cells, distinct PBMC-associated viral RNA species (vRNAs) were quantified: unspliced RNA (UsRNA) and 2 types of multiply spliced encoding tat and rev (MsRNA-tatrev) or nef (MsRNA-nef). These RNAs were found to be expressed both in latently Initiation of cART resulted in a decay of viral RNA production , but wasTo corroborate these observations and to extend them to UsRNA and MsRNAs, empirical distributions of the proportions of patients with detectable viral RNAs after initiation of cART were plotted . SimilarAfter cessation of cART, plasma viremia reached levels above the threshold of 50 copies/ml in a median time of 4.2 weeks . Rebound of UsRNA and MsRNA proceeded with some delay without reaching statistical difference : Median time to rebound was 8.0 weeks  and 9.8 weeks  for UsRNA and MsRNA, respectively. Lastly, within a median time of 17.9 weeks  vRex rose to detectable levels. Its rebound was statistically different from that of plasma viremia and of UsRNA  but not from MsRNA . It has to be noted that vRex was measured less frequently than plasma or PBMC viremia. Therefore the median time to rebound may have been overestimated.To investigate whether cART initiated in acute infection had an impact beyond its cessation, the levels of HIV-1 nucleic acids at baseline and after treatment cessation were compared. Maxima were used to represent post-cessation HIV-1 nucleic acid levels, to account for the variability among the different parameters regarding the time to reach stable set-points.et al., unpublished data), but also to a naturally occurring “first peak” of viremia observed in acute HIV-1 infection Plasma viremia  was signAs opposed to baseline and post-cessation viral nucleic acid levels, which were represented by single measurements, on-cART levels were estimated by calculating means. Taking into account the rapid decay of vRex, the mean of samples at time-points ≥2 weeks after initiation of treatment was used. For all other parameters measurements taken at time points ≥24 weeks were used for calculation of mean values. The rationale for choosing this time-window was that the parameter with the slowest decay kinetics, plasma viremia, reached undetectable levels within a median time of around 24 weeks as shown in 6 PBMC (mean±sem) during cART initiated in acute infection. However, proviruses in these infected cells were transcriptionally almost completely silent. Expression of MsRNAs and vRex was mostly undetectable and attenuated to mean levels <1 copy/106 PBMC . Only expression of UsRNA was occasionally detected at a mean level of 2.6±1.2 copies/106 PBMC. The absence of vRex during cART observed in the current study was similar to findings of previous studies HIV-1 DNA persisted at a level of 823±253 copies/106 cells, range 0–1486) was 105-fold higher than in the acute group  in the chronic group as compared to the patients treated during acute infection. To adjust for this difference in the total number of infected cells, the average viral transcriptional rate i.e., the ratio of viral RNA to vDNA, was calculated + T-cell loss, preservation of immune functions, and inhibition of initial viral spread throughout the possible sites of replication. Despite considerable efforts from various research groups, to date evidence from clinical studies in humans to empirically substantiate these concepts remains limited. Here we performed a longitudinal in-depth analysis using patient matched highly sensitive qPCR assays to detect different HIV-1 gene trancripts to assess effects of early treatment on cellular reservoirs of HIV-1 in peripheral blood.The main aims and rationales for antiretroviral therapy in acute HIV-1 infection are confinement of genetic diversity of viral quasispecies to prevent escape from adaptive immunity, restriction of CD4HIV-1 transcription was monitored longitudinally before, during, and after cessation of treatment of acute HIV-1 infection in well characterized patients The major finding of this study was that early cART initiated during acute HIV-1 infection significantly depleted the number of transciptionally active proviruses by an order of magnitude when compared to levels detected in patients treated during chronic infection.+ PBMC, expressing solely intracellular viral RNA, can therefore be viewed as latently infected Specifically, productively infected PBMC, hallmarked by expression of cell attached virions (vRex) were depleted within a median time of 4.5 weeks after cART initiation. The remaining HIV-1+ cells in response to treatment occurred in a significantly staggered mode according to their pattern of viral RNA expression and life-span as previously described Decay of HIV-1+ T-lymphocytes, a cohort of patients on early cART showed lower levels of latently infected cells than a control group with deferred cART initiation. In addition, two small studies in humans and in the SIV model also suggest that HIV-1 suppression as measured by unspliced HIV-1, respectively SIV RNA can be reduced by treatment during primary HIV infection in the gut associated lymphoid tissue These findings imply that early cART led to the clearance of long-lived cells harboring transcriptionally active latent proviruses. In an early study, Markowitz and colleagues showed persistence of viral transcription in PBMC when antiretroviral therapy was initiated within 90 days after onset of primary symptoms de novo” infection of PBMC by plasma virions rather than by reactivation of latently infected PBMC. The rebounding virus most likely originates from distinct compartments such as the secondary lymphoid organs , lymph nodes, spleen), the cerebral nervous system, the kidneys, or the genital tract. Very recent data by Yukl et al.After cessation of cART, virological parameters reappeared inverted to their decay. UsRNA rose to significant levels after 8 weeks, followed two weeks later by MsRNA and finally vRex reappeared with a significant delay. The observations that viral rebound in plasma preceded that of cellular viral RNAs and plasma viremia was the last parameter to decay during cART, implies that PBMC can be viewed as independent compartment to some degree separate from the source of virus appearing in plasma. These dynamics potentially suggest that repopulation of the PBMC compartment of rebounding transcriptionally active PBMC was most likely due to “The fact that post-cessation control of viral RNA expression was achieved in several patients during the post treatment observation period supports this hypothesis: Plasma viremia was undetectable in patient #72, control of UsRNA expression was experienced in patient #25, control of MsRNA expression was achieved by patients #25, #56, and #92, and control of vRex was experienced by patients #72 and #99.The observation that MsRNA-nef levels, a parameter reported to be associated with viral latency Taken together, our data demonstrate a profound virologic effect of treatment initiation during acute HIV-1 infection. The unprecedented finding that residual viral transcription was almost completely depleted, suggests that the latent reservoir of HIV-1 during cART can be reduced by early intervention and that proof of concept studies aiming at HIV-1 eradication or remission of HIV-1 replication should be initiated in patients during acute infection.In accordance with the guidelines of the Ethics Committee of the University Hospital of Zurich, written informed consent was obtained from all participants of the studies analyzed: the Zurich Primary HIV Study (ZPHI), the Swiss Spanish Intermittent Therapy Trial (SSITT), and the Zurich HIV-1 Transcription Study.http://clinicaltrials.gov, NCT00537966) pol, tat, and env.Patients n = 24;  includedBlood samples were collected at the day of the very first cART application ( = baseline sample), longitudinally during treatment, and after treatment cessation. EDTA-blood samples were collected and separated into cells and plasma according to standard procedures pol-sequences were available from the SHCS genotypic drug resistance database PBMC samples for a cross-sectional control group of 15 patients treated in the chronic phase after seroconversion were obtained from the Swiss Spanish Intermittent Therapy Trial (SSITT) Total PBMC RNA was extracted with the RNeasy extraction kit on the ‘QIAcube’ extraction device  using an initial volume of 0.35 ml for cell lysis, DNAse I digestion on the extraction column and a final elution volume of 0.1 ml.Selective isolation of PBMC-associated extracellular, virion-encapsidated genomic HIV-RNA, vRex, free of intracellular nucleic acid, was performed as described pol-sequences) To ensure accurate quantification a patient matched approach reaching single copy sensitivity was used for qPCR measurements of RNA and vDNA To obtain sequence information to match qPCR primers and probes for MsRNAs of predominant quasispecies, two regions were amplified from each patients total DNA by a nested PCR scanning approach as described As previously described et al.2 (1.5 µM) by incubation for 15 min at 95°C and 60 cycles of 10 s at 95°C, 5 s at 55°C and 40 s at 60°C.Assays for RT-qPCR were performed as described Patient-nucleic acids were directly used to generate HIV-1 standard-curves. To obtain standard curves with maximal dynamic range, baseline specimens were used as standards for vDNA-measurements. Post-cessation time points with high plasma viremia and high vDNA were chosen. Standards were prepared as serial 3-fold dilutions in quadruplicates. Specimens other than the ones chosen as standards were measured in duplicate.I is the Y-axis intercept of the regression line, depicting the point where Ct equals one copy ( = 100) and S is its slope. I and S were determined as described in Linear regression used in standard real-time PCR analysis was applied for calculation of HIV-1 copy numbers:6 cell equivalents were empirically determined in PBMC specimens with known cell numbers  resulting in a conversion factor of 2.85×107 copies GAPDH-RNA/106 PBMC.To control for input and/or differences in qPCR efficiencies in RNA PCR the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) against a standard dilution series of in vitro transcribed GAPDH RNA was measured in duplicates as described previously 6 PBMC.Cellular input for DNA was quantified by beta-actin PCR using primers and probe described in GraphPad Prism 5.0 software  was used for statistical analyses. P<0.05 was considered as the level of significance. Mann-Whitney testing was applied unless otherwise indicated for group comparisons. Fisher's exact test was used in analysis of contingency tables. No adjustment for multiple testing was applied.Table S1Characteristics of patients.(0.01 MB PDF)Click here for additional data file.Table S2Oligoucleotides used for patient-specific qPCR.(0.10 MB PDF)Click here for additional data file.Methods S1Supplementary methods.(0.05 MB PDF)Click here for additional data file.


Retention in long-term antiretroviral therapy (ART) program remains a major challenge for effective management of HIV infected people in sub-Saharan Africa. Highly Active Antiretroviral Therapy (ART) discontinuation raises concerns about drug resistance and could negate much of the benefit sought by ART programs.Based on existing patient records, we assessed determinants of retention in HIV care among HIV patients enrolled in an urban ART at two urban hospitals in Cameroon. Extended Cox regression procedures were used to identify significant predictors of retention in HIV care.Of 455 patients, 314 (69%) were women, median (IQR) age and baseline CD4 cell count were respectively 36 years (30 – 43) and 110 cells/μL (39 – 177). Forty patients (9%) had active tuberculosis (TB) at enrollment. After a median (IQR) follow-up of 18 months (10–18), 346 (75%) were still in care, 8 (2%) were known dead, and 101 (22%) were lost to follow-up (LFU). Severe immunosuppression (CD4 cell count ≤ 50 cells/μL) at baseline  and active tuberculosis upon enrollment  were independent predictors of cohort losses to follow-up within the first 6 months after HAART initiation.These data suggest that three-quarter of HIV patients initiated on HAART remained in care and on HAART by 18 months; however, those with compromised immunologic status at treatment initiation, and those co-infected with TB were at increased risk for being lost to follow-up within the first 6 months on treatment. With more than 60% of the world’s HIV-infected people, sub-Saharan Africa is severely hit by the HIV epidemic; in 2007 alone, 76% of the estimated 2.1 million AIDS related death occurred in the region. CountrieProportions of non-death losses to follow-up (LFU) varying from 2 to 59% have been reported across ART programs in sub-Saharan Africa.–10 HowevIn this study, we describe retention in care in an urban pilot ART program in Cameroon, and identify temporal distribution and determinants of losses to follow-up.From March 1, 2003 to June 30, 2004, HAART eligible patients aged 15 years and older, with limited financial resources as defined by a standardized indigence score scale, were initiated on HAART and followed during 18 months in a pilot Antiretroviral Therapy (PART) initiative. This initiative of the Ministry of Public Health was launched in March 2003 with support from the World Bank and aimed at improving access to highly subsidized HAART and laboratory tests to indigent patients. Clinicians assessed each patient’s clinical and immunological eligibility for HAART while social workers assessed patient’s ability to pay using an indigence score scale which defines the patient’s co-payment . Labora®, Cipla, Mumbai, India). Second-line protease inhibitor based regimen “PI-based” combined indinavir (IND)/ritonavir (RIT) with 1 nucleoside reverse transcriptase inhibitor (NRTI) backbone (3TC plus either AZT or d4T). The monthly cost of HAART regimens ranged from 27$ to $51.A physician prescribed first- or second-line HAART regimens according to the 2002 Cameroonian guidelines. First-liCotrimoxazole was prescribed for all the patients enrolled. Patients with pulmonary TB and a CD4 cell count > 200 cells/μL were treated for TB and HAART initiation was deferred. For TB patients with a CD4 cell count between 50 and 200 cells/μL, HAART was initiated after the completion of two months of TB therapy. TB patients with a CD4 cell count < 50 cells/μL were started simultaneously on TB therapy and HAART. Regimen for TB consisted of two months of isoniazid, rifampin, ethambutol, and pyrazinamide daily, followed by four months of isoniazid and rifampin daily. TB patients received their TB regimen at a TB clinic affiliated to the ART center.Adherence nurses counseled patients on the importance of adherence prior to HAART initiation and encouraged them to designate a close relative who was also counseled on treatment adherence. Each patient received monthly appointments for drug dispensation and every three months for clinical evaluation. During the period under review, home-visits were not performed for patient who missed a scheduled visit.The study outcome, loss to follow-up, was defined as any patient who missed all follow-up visits and could not be contacted by telephone for 2 consecutive months and who did not re-enter the study at a later date. Based on clinical records, we determined the length of follow-up for each patient as the elapsed time between HAART initiation and the last visit. Patients were regarded as being in care in a given month if they attended to follow-up visit within that month. For patients known to have died, the date and reported causes of death, when available, were recorded.th month after enrollment or on the date of data collection . We computed rates and 95% confidence intervals using exact binomial procedure. To detect differences between groups, we used the Pearson Chi-square test statistic for categorical variables, and the 2-tailed t test statistic for continuous variables. In case of any violation of the assumption of equality of variance, we reported the unpaired t-test statistics; otherwise, the paired t-test was reported. We used the Kaplan-Meier method to estimate the cumulative probability of remaining in care. We plotted smoothed hazard-function estimators of losses to follow-up with the SAS macro %Smooth using a kernel smoothing method.[Patients were included in the analysis regardless of any previous HAART at enrollment, and we excluded those with incomplete files . All patient information was censored at the 18g method. We definWe first built a multivariate model to assess the probability of retention in care, considering the event of interest as any loss to follow-up and defining a censored observation for patient remaining in care, or known dead. In a second model, we assessed the ‘net probability’ of retention in care, considering death as a competing outcome. In this later worst-case scenario model, the event of interest was defined as any loss to follow-up or notified death. We used We retained baseline CD4 cell count, active tuberculosis upon enrollment as significant predictors  for the Cox’s model building and controlled for treatment site and previous history of HAART. We assessed the proportional hazard assumption using the graphical method. Since the variables baseline CD4 cell count and active tuberculosis violated the proportional hazard assumption, we defined time-dependent variable based on the pattern of crude hazard function. Based on the pattern of the hazard function, we defined two time-intervals . For each predictor, we defined two time-dependent variables as product-terms with each time-interval and we used an extended Cox’ proportional hazard model to derived adjusted hazard ratio. CollineaOf the 465 individuals who started HAART between March 1, 2003 and June 30, 2004, 455 individuals (98%) were included in final analysis . BaseliOf the 455 patients enrolled and followed-up during a median (IQR) period of 18 (10–18) months, 346 (76%) remained in care, 8 (1.8%) were reported dead and 101 (22.2%) were lost to follow-up . PatienEarly losses were associated with significantly lower mean CD4 cell count/μL at baseline compared to patients remaining in care . Conversely patients lost to follow-up after 6 months on HAART (late losses to follow-up) had comparable mean CD4 cell count/μL than those remaining in care, respectively at baseline  and 6 months , however, late losses to follow-up had significantly higher mean CD4 cell count/μl at 12 month as compared to those remaining in care .Kaplan-Meier curves  show thFor the 455 patients followed during 537.5 person-year, the mortality rate was 1.5 per 100 person-years (95% CI 0.5 – 2.7). In a worst-case scenario, the probability of remaining in care and alive (net retention) was 0.83 (95% CI 0.79–0.86) at 6 month, 0.77 (95% CI 0.73–0.81) at 1 year, and 0.75 (95% CI 0.71–0.78) at 18 months. Patients with severe immuno suppression at baseline (CD4 cell count < 50 cells/μL) were 2.3 times more likely to be lost to follow-up by 6 months after HAART initiation  .One-third of the patients in this program were enrolled at an advanced stage of HIV disease. After 18 months, 75% of patients remained alive and in care despite the lack of outreach activities and defaulters tracking system in the community. Unlike other reports from ART programs in low-income  and highNine percent of patients were diagnosed with TB upon enrollment. None of the reported deaths was attributed to TB, although, patients with active TB at enrollment were more likely to drop out from the program in the first 6 months after initiation of HAART as compared to those without TB. Patients diagnosed with TB upon enrollment in the PART initiative received TB treatment in a TB clinic located either within the same hospital where the ART clinic is found or away from the ART clinic. For instance, in the Douala Laquintinie Hospital, TB clinic and ART services coexisted in the same hospital, contrary to Yaoundé Central Hospital which had to refer TB patients to an external facility  located at a distance of 6 km away. However, these patients were still considered enrolled in the ART cohort at Yaoundé Central Hospital where they came for ARV drugs refill. This difference in location might have contributed to the high proportion of loss to follow-up observed in Yaoundé Central hospital compared to Douala Laquintinie hospital .Community support for ART programs, including tracing and referral of treatment defaulters has been reported to significantly improve ART programs outcomes in sub-Saharan Africa , 18. LowOur results suggest however, the need for more pro-active strategies to prevent early losses to follow-up, including identifying HIV patients early through routine provider initiated HIV counseling and testing (PITC), and strengthening integration of TB/HIV co-infection management.This program evaluation was conducted on selected sites enrolled in an initiative to increase access to HIV care and treatment for indigent Patients, less likely to have accessed HAART otherwise at that time. This ART program was initiated at an early stage of ART scale-up in Cameroon, and additional strategies were subsequently integrated in 2006 to strengthen communities outreach activities .Despite the affordability of ART services, HIV patients enrolled with severe immunosuppression, and those co-infected with TB were more likely to be lost to follow-up early after HAART initiation. HIV programs should prioritize strategies for early identification and enrollment of HIV patients prior to advanced stage of disease, as well as strengthening the management of TB/HIV co-infected patients. Strategies for long term adherence counseling should be designed and implemented to ensure retention in care of patients with substantial immuno recovery. Further studies are needed to understand the determinants of late losses to follow-up.


In this article we summarize several recent major developments in human immunodeficiency virus treatment, prevention, outcome, and social policy change. Updated international guidelines endorse more aggressive treatment strategies and safer antiretroviral drugs. New antiretroviral options are being tested. Important lessons were learned in the areas of human immunodeficiency virus vaccines and microbicide gels from clinical studies, and additional trials in prevention, especially pre-exposure prophylaxis, are nearing completion. Insight into the role of the virus in the pathogenesis of diseases traditionally thought to be unrelated to acquired immunodeficiency syndrome has become a driving force for earlier and universal therapy. Lastly, we review important achievements of and future challenges facing China as she steps into her eighth year of the National Free Antiretroviral Treatment Program. Antiretroviral therapy (ART) has evolved from monotherapy with zidovudine (AZT) to the use of combination nucleoside reverse transcriptase inhibitors (NRTIs), to triple therapy with highly active antiretroviral therapy (HAART), to today's numerous combinations drawn from 6 classes and 32 drugs, including fixed dose formulations, approved by the United States (US) Food and Drug Administration (FDA) . TreatmeThe US Department of Health and Human Services (DHHS)  and the 3. In addition, ART is now recommended for all patients with CD4 between 350 and 500 cells/mm3. As for patients with CD4 > 500 cells/mm3, the panel of experts is evenly split between favoring ART initiation and considering it optional. These changes stem from mounting evidence that earlier ART initiation, even before any significant CD4 drop and immune deficiency symptoms, translates to better immune recovery, better tolerance for side effects, smaller risk for TB reactivation, and reduced HIV and TB transmission on a public health level [3 or when CD4 declines to 250 cells/mm3 [3 to deferral of ART until CD4 declines below 350 cells/mm3 in terms of morbidity and mortality [The new US DHHS guidelines for ART initiation have expanded to include all patients with pregnancy, HIV-associated nephropathy, and hepatitis B virus (HBV) coinfection requiring treatment for HBV, regardless of CD4 count, and in all patients with CD4 <350 cells/mmth level -10. It sth level ,10 and tells/mm3 . The Strortality . These tPreferred regimens for ART-naïve patients have also been revised. They now include the integrase inhibitor raltegravir (Isentress), recently approved by the US Food and Drug Administration (FDA) for ART-naïve patients, in combination with the NRTIs tenofovir (TDF) and emtricitabine (FTC) . Three o3, regardless of symptoms. However, these guidelines have not been widely adapted in part because of limited drug supplies. To reduce rates of mother to child transmission and improve child survival, treatment to prevent mother to child transmission starting at 14 instead of 28 weeks and continuing through breastfeeding was added as an option. Although ART can be delivered safely without routine monitoring of hematology and biochemistry in resource-limited settings based on the Development of AntiRetroviral Therapy in Africa (DART) study [In contrast to the US DHHS guidelines, the WHO recommendations target resource-limited countries and are more conservative. In the most recent WHO draft guidelines , immunolT) study , expandeThe WHO now urges replacing the NRTI stavudine (d4T) with AZT or TDF. d4T is inexpensive and widely available but causes mitochondrial toxicity that can lead to sometimes permanent peripheral neuropathy and lipodystrophy. Its phase-out will be difficult given that a large proportion of patients on ART in developing countries are reliant on d4T-containing first line regimens, but the new WHO recommendation may prove to be more cost-effective in the long run .In addition to updated guidelines, several new antiretroviral (ARV) drugs were approved by the US FDA this past year for treatment-naive HIV patients. Raltegravir, as previously mentioned, was approved based on results from the STARTMRK trial, a double blind controlled study comparing raltegravir to EFV in combination with TDF/FTC ,22. ViraThe use of the CCR5 antagonist maraviroc (Selzentry) was also expanded by the FDA to include ART-naïve patients with CCR5-tropic HIV-1 virus . MaravirThe development of 2 new pharmaco-enhancing agents, GS9350  by GileaResults from the phase-3 Thai vaccine trial using a combination of ALVAC, a recombinant canarypox vector vaccine, and AIDSVAX, a recombinant glycoprotein-120 subunit vaccine, were released late 2009. Although the two protocol specified analyses (intention-to-treat and per-protocol) only showed a trend toward significance, the modified intention-to-treat analysis excluding 7 subjects who were determined to be HIV-infected at study entry showed a 31%  reduction in the risk of HIV infection -35, makiDisappointing news came from the vaginal microbicide gel PRO 2000 trial ,37 conduAs newer drugs with less toxicity have made earlier ART administration feasible, they are also being considered for pre-exposure prophylaxis (PrEP) to prevent HIV infection. TDF was effective in preventing Simian Immunodeficiency Virus (SIV) infection in the macaque model , and to The Vaginal and Oral Interventions to Control the Epidemic (VOICE) study, a large double blind placebo controlled trial supported by the NIH , is undeHIV transmission is strongly correlated to the concentration of virus in blood (VL), and this is usually reflected in genital secretions . ART sigUniversal HIV testing and treatment has also been proposed as a method to control the spread of HIV . Using dCurrent evidence is insufficient to support a radical policy change , as diff3 [With optimal ART, the life expectancy of persons living with HIV in developed countries approaches that of HIV-negative individuals . As ment3 . The cau3 . Cumulat3 ,18,65-693 -73 obser3 . Equally3 ,75-77. AScientific advances sometimes cannot move society forward without corresponding changes in policy. Worldwide, injection drug use is a major route of transmission for both HIV and viral hepatitis due to needle sharing. Needle exchange programs that provide clean needles to injection drug users (IDUs) are controversial because they are perceived as condoning the illegal and harmful behavior. However, studies have shown that needle exchange decreases transmission of blood borne pathogens and does not increase drug abuse . The US HIV-positive individuals are a vulnerable population not only concerning their health status but also because of social discrimination and stigma. Currently, more than 60 countries have laws that restrict the entry, stay or residence of people living with HIV. In the early 1990's, at the height of the epidemic when effective treatment was still lacking, an entry and immigration policy was passed in the US prohibiting HIV-positive individuals from entering the country. That ban was lifted effective January 2010, sending a clear message combating fear, stigma, and discrimination against people living with HIV and AIDS. As a result, the XIX International AIDS conference will be held in Washington, DC in July 2012.3. The rapid scale-up of HIV treatment and care has led to a dramatic decrease in HIV mortality from roughly 27-30 deaths per 100 person-years before ART to about 4-5 deaths per 100 person-years after ART [Since the scale-up of China's National Free Antiretroviral Treatment Program (NFATP) in 2003, over 80,000 patients have been treated. Nine ARV drugs are currently available, including 6 NRTIs , 2 NNRTIs (EFV and NVP), and one boosted PI (LVP/r). In addition, three drugs - raltegravir (integrase inhibitor), darunavir (PI), and etravirine (NNRTI) - are in the process of entering the Chinese market. China's current national guidelines updated in 2008 recommend ART initiation in all patients with CD4 count under 350 cells/mmfter ART ,80.However, major challenges remain in diversifying and optimizing treatment options provided through the free ART program. d4T is currently used as part of the first-line regimen in close to half of all patients on ART in China . Its tot3 [As lower baseline CD4 count correlates to worse prognosis, decreasing the treatment threshold and expanding treatment coverage may be an urgent next step in China to control both AIDS and non-AIDS related mortality and morbidity. Most patients still present with late disease, and fewer than 1 in 3 HIV-positive individuals are aware of their status . At the 3 . Resourc3 .Substantial progress has been made in the area of harm reduction, especially among IDUs. In 2006, the Ministry of Health, Ministry of Public Security and the State Food and Drug Administration (SFDA) issued the revised "Opium Abusers Community-Based Drug Maintenance Treatment Protocol," which expanded the MMT program from pilot phase to general application. By the end of 2008, 558 MMT clinics in 23 provinces have served more than 170,000 clients . NSPs haHowever, major hurdles remain among other risk groups. Sexual transmission has become the fastest growing means of HIV transmission in China due to changing sexual behavior and attitude since China opened up to the outside world in the late 1970s . Among nWe have summarized several recent major themes in HIV/AIDS. We share in the disappointment from setbacks, excitement of new successes, and hope in coming advancements. Future directions in China as well as the rest of the world will continue to focus on methods of HIV prevention such as vaccine and microbicide development, expanding HIV testing and treatment, discovering and developing new ARV drugs, optimizing the ART delivery system, and improving therapy for non-AIDS related conditions and coinfections.The authors declare that they have no competing interests.All authors fulfill the criteria of authorship. KS performed the literature search and was the lead author for the paper. SZ contributed to reviewing the literature as well as planning and drafting the manuscript. RYC, MSC, and FZ carried out critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.


In this bioinformatics study we treat response to antiretroviral therapy as a two-body problem: response to therapy is considered to be a function of both the host and pathogen proteomes. We set out to identify potential responders based on the presence or absence of host protein and DNA motifs on the HIV proteome.The HIV viral genome mutates at a high rate and poses a significant long term health risk even in the presence of combination antiretroviral therapy. Current methods for predicting a patient's response to therapy rely on site-directed mutagenesis experiments and An alignment of thousands of HIV-1 sequences attested to extensive variation in nucleotide sequence but also showed conservation of eukaryotic short linear motifs on the protein coding regions. The reduction in viral load of patients in the Stanford HIV Drug Resistance Database exhibited a bimodal distribution after 24 weeks of antiretroviral therapy, with 2,000 copies/ml cutoff. Similarly, patients allocated into responder/non-responder categories based on consistent viral load reduction during a 24 week period showed clear separation. In both cases of phenotype identification, a set of features composed of short linear motifs in the reverse transcriptase region of HIV sequence accurately predicted a patient's response to therapy. Motifs that overlap resistance sites were highly predictive of responder identification in single drug regimens but these features lost importance in defining responders in multi-drug therapies.in vitro mutation experiments.HIV sequence mutates in a way that preferentially preserves peptide sequence motifs that are also found in the human proteome. The presence and absence of such motifs at specific regions of the HIV sequence is highly predictive of response to therapy. Some of these predictive motifs overlap with known HIV-1 resistance sites. These motifs are well established in bioinformatics databases and hence do not require identification via  HIV hIV virus . POL alsIV virus . The thiIV virus . MutatioIV virus .in vitro cultures of HIV infected immune cells have diminishing growth rates in the presence of antiretroviral therapy but eventually a resistant viral phenotype emerges [Most of the drugs that are currently used for controlling HIV infection target the three viral enzymes coded by the HIV POL gene. Antiretroviral drugs such as zidovudine (AZT), lamivudine (3TC), emtricitabine (FTC), zalcitabine (ddC), stavudine (D4T), didanosine (DDI) and nevirapine (NVP) target RT  whereas  emerges . The res emerges -16. Comb emerges -19. As a emerges .HAART therapies often reduce viral replication to undetectable levels. They decrease morbidity and mortality rates but nonetheless can be ineffective in some individuals ,22. SearIn this study, we first cluster patients into responder and non-responder categories based on viral load response to antiretroviral therapy. We then used stepwise logistic regression to differentiate responders and non-responders using linear sequence motifs common to host and viral genomes as features. We focused on viral load in the responder/non-responder classification because recent studies indicate that CD4 cell count monitoring does not accurately identify individuals with virologic failure among patients taking antiviral therapy . A novela priori information obtained via virus-specific in vitro cell assays. This type of a feature set is attractive, as it can be used to explore the drug response of viruses to antiviral therapy in the absence of extensive data on resistance mutations. Previous research on quantitative prediction of patient response to antiretroviral drugs in HIV infection [Another novel feature of our study is the treatment of drug response as a two body problem, namely that response to drugs is assumed to be affected by both the viral and host genotypes. We sought to identify linear motifs on the HIV sequence that are also found in the host and are functionally annotated: host transcription factor binding sequence motifs , miRNA bnfection -43 has eClinical annotation of more than 2,000 RT sequence samples in the Stanford HIV Drug Resistance database contained measurements of VL at six time points during the course of twenty-four week therapy. The drugs used in various single and combination therapies as well as the numbers of HIV-1 individuals taking the therapy are shown in Table The overlap between these three methods is shown in the Venn diagram in Figure Our results show that the HIV sequence, although highly variant in nucleotide sequence, expresses eukaryotic linear motifs (ELMs) that are largely conserved over hundreds of subtype B and subtype C sequences, as shown in Figure features. SWLR employs an iterative algorithm to determine which features should be included in the final logistic regression model [We used Step-Wise Logistic Regression (SWLR) to classify patients into responder or non-responder categories based on the presence or absence of ELMs, miRNA binding sites, TF binding sites, and resistance sites, collectively referred to as on model . In brieWe used SWLR in 500 iterations of training and testing at equal proportions for all responder/non-responder samples shown in Table The AUC values for all three phenotype classification methods are shown in Table The average number of regression coefficients (features) found significant over 500 training/testing iterations ranged from five to ten, depending on the drug regimens presented in Table One of the most consistent predictors of positive outcome across therapy regimens is the presence of ELM-Lig-SH3-3 Figure . This isThe two resistance sites and the ELMs that overlap them continue to be predictors of negative outcome in terms of response to subsets of antiretroviral therapies in phenotype classification based on incremental reduction of the VL  in stepwise logistic regression for 16 different combinations of antiretroviral drug regimens containing at least one drug against HIV reverse transcriptase. Responder phenotype was defined multiple ways to gain insights into drug response at 8 weeks (SD phenotype classification) and 24 weeks (BM phenotype classification) after the beginning of the therapy and somewhere in between (IR Phenotype classification). Host motifs that appear to be highly relevant to viral replication such as the transcription site binding motifs ,57 and ma priori information about how the HIV sequence would mutate in the presence of antiretroviral drugs. We were able to make accurate predictions without the resistance site information available in the literature. The input to our machine learning algorithm is simply the HIV sequence. We use publicly available bioinformatics tools to annotate these sequences with host motifs relevant to outcome. We then identify the motifs on the sequence that differentiate between responders and non-responders. These motifs can then be linked to specific viral host protein interactions and the pathways of these interactions. The promise of our approach will be fully explored with the availability of clinically annotated HIV whole genome sequences obtained at different time points during HAART therapy.Our approach to relate HIV sequence motifs to the course of infection does not require Linear binding motifs found in both the host and viral proteomes constitute a set of features highly predictive of response to therapy involving different combinations of antiretroviral drugs. Stepwise logistic regression as used here utilizes only the HIV-1 sequence and does not require annotations of resistance sites specific to various antiretroviral drugs. This study emphasizes finding sequence motifs which facilitate binding between viral and host proteins. This binding may allow the hijacking of host protein binding sites from their usual binding partners and thus alter the signalling pathways of the host cell. Our study points to competitive binding of HIV proteins to host proteins using motifs found in the host as the mechanism of interplay between the host and pathogen genotypes in dictating response to therapy. Our method is applicable to other viral infections where the viral sequence is known but resistance sites to antiviral therapies have not yet been documented. in order to get a motif expression map of the whole genome. As of 9/1/2006, this dataset consisted of 1,112 subtype B and 922 subtype C whole genome sequences, along with a smaller number of samples from other subtypes. This dataset also contained five reference sequences each for alignment of subtypes B and C.This study utilizes sequence and clinical data from two distinct sources. All whole genome HIV-1 sequences were downloaded from the Los Alamos HIV Sequence Database We used data from the Stanford HIV Drug Resistance Database  in orderWe focused on VL in the responder/non-responder classification  and exama priori-based resistance mutations. In order to evaluate the relative positions of nucleotide motifs and protein motifs on the same platform, we annotated the protein motifs back to their corresponding nucleotide positions. This could create some ambiguity since HIV has multiple overlapping reading frames. However, our clinical dataset only contained sequences from the RT region. We used a local BLASTx query [Our classification method uses the presence and absence of short linear motifs on the HIV genome. These motifs can be grouped into three basic types: eukaryotic linear motifs (ELMs), nucleotide-based motifs and Tx query  on a dat® database as of 11/14/08 [The first feature group consisted of ELM ligation sites and subcellular targeting sequences. These were identified on HIV-1 protein amino acid sequences using the ELM webserver tool . The web11/14/08 . We requ11/14/08 . For the11/14/08 . As of 111/14/08  and the 11/14/08 . Any bin11/14/08 . In orde11/14/08 .We used stepwise logistic regression (SWLR) to assess the potential of the extracted short linear sequence features along the RT sequence in differentiating between responders and non responders . SWLR waSWLR algorithm was applied to differentiate responders from non-responders in three different assignments of the phenotype for 500 iterations of 2-fold cross validation. Since the efficiency of the SWLR algorithm is sensitive to the class composition of the training data  we ensurThe authors declare that they have no competing interests.WD wrote the manuscript with AT and performed the analysis with PE. LU provided technical insight. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:Source Code. All python and MATLAB code required to produce the figures and tables shown in the manuscript. Documentation and unit-tests are provided to facilitate their usage.Click here for file


This retrospective study assessed the efficacy, safety, and the predictors of sustained viral response (SVR) to a 48-week-course of peginterferon α-2a (Pegasys) and ribavirin combination therapy in 335 consecutive Saudi patients with chronic hepatitis C virus (HCV) infection.Clinical, biochemical, and virological parameters were collected at time 0 (pretreatment) and at 12, 24, 48, and 72 weeks posttreatment. The mean ± SD age was 49.1 ± 13.0 years; 229 (68.4%) were males, mean ± SD body mass index was 27.8 ± 7.4, 85 (25.4%) were diabetic, 25 (7.5%) had renal impairment, 136 (40.6%) had previously received interferon ± ribavirin therapy, and 247 (73.7%) underwent pretreatment liver biopsy. Patients with genotypes 1, 2 or 3, 4 and mixed genotype were 60 (22.15%), 30 (11.0%), 148 (54.4%), and 34 (12.5%), respectively.P = 0.000), nondiabetics (P = 0.015), had higher serum albumin (P = 0.007), had less pretreatment inflammatory grade (P = 0.011), infected with genotypes 2 or 3 (P = 0.014), and treatment-naïve patients (P = 0.001). However, in stepwise multivariate logistic regression analysis, only treatment naiveté and low pretreatment inflammatory score were the independent predictors of SVR .Early viral response (≥2-log10 HCV-RNA decline 12 weeks posttreatment) was achieved in 253 (75.3%). Patients who completed 48 weeks of treatment were 292 (87.1%); of these, 121 (75.6%) achieved ETVR, 161 (55.1%) continued to have SVR and 60 (20.5%) had a viral relapse following end-of-treatment response, that is 48.1 and 17.9% of all patients (n = 335), respectively. Nonresponders (NR) were 71 (24.3%) patients and 43 (12.8%) were unable to complete treatment (due to side effects or loss to follow up). Compared to the relapsers, patients with SVR were significantly younger (Combination therapy, if tolerated and completed, is effective in treating chronic HCV patients, especially those with no previous interferon therapy and lower pretreatment inflammatory grade. Chronic hepatitis C virus (HCV) infection affects approximately 300 million people worldwide and currently is the most frequent cause for liver transplantation in the United States and Europe. Natural The recommended treatment for patients with HCV genotypes 1 and 4 is pegylated interferon plus ribavirin for 48 weeks. Such tre6There are limited reports on HCV genotype 4, the most predominant genotype in the Middle East. Most of Therefore, the primary objectives of this retrospective study conducted among 335 consecutive Saudi patients with chronic HCV infection were to (1) assess the overall efficacy of a 48-week course of peginterferon α-2a (Pegasys) and ribavirin combination therapy, (2) compare between patients who achieved SVR and those who relapsed after end-of-treatment viral response, and (3) define the independent predictors of SVR (persistently undetectable HCV RNA 6 months after cessation of treatment) in these patients.A total of 335 consecutive patients with chronic HCV infection referred to King Faisal Specialist Hospital and Research Centre (KFSH and RC), Riyadh, Saudi Arabia, between February 2003 and November 2005 were included in this study. Baseline subject characteristics are shown in The accepted indications for starting treatment were the presence of detectable HCV RNA of any genotype, elevated serum alanine aminotransferase (ALT) >1.5-fold and histologically consistent with chronic hepatitis C using METAVIR scoring system. PatientsBaseline assessment included clinical history, physical examination, body mass index (BMI), as well as routine hematological, biochemical, serological, and virological tests including that of HCV genotype, HCV polymerase chain reaction (PCR).®, F. Hoffmann-La Roche, Basel, Switzerland) at a dose of 180 μg per week plus ribavirin  at a dose of 1000–1200 mg daily as per body weight-1000 mg if ≤75 kg and 1200 mg if ≥75 kg-for 48 weeks. Clinical, biochemical, and viral parameters were collected pretreatment and at weeks 12, 24, 48, and 72 of follow up.After an initial assessment, patients were treated with pegylated interferon  patients. The average period between the time of biopsy and start of treatment was 2.53 months. The hepatic necroinflammatory activity and stage of fibrosis in the biopsies were evaluated according to the METAVIR scoring system.Serum HCV RNA was extracted using an automated extraction system. HCV detection and quantification were performed using Abbott Real-Time M2000 RT-PCR assay, utilizing two sets of primers and probes, which target a conserved region of the 5' untranslated region of the genome and an internal control. This assay detects and quantifies HCV genotypes (1–6) with a detection limit that ranges from 30 to 100,000,000 IU/mL, where 1 IU/mL = 4 copies/mL. Prior to treatment, HCV genotype was assayed in 272 (81.2%) patients using INNO-LiPA HCV II  as previously described.[10 in serum HCV viral load is indicative of a positive response.The National Institute of Health guidelines state that a drop of ≥2-Log10 drop in serum HCV viral load at 12 weeks after start of treatment.Early viral response (EVR) was defined as ≥2-logEnd-of-treatment viral response (ETVR) was defined as undetectable serum HCV RNA at 48 weeks.SVR was defined as persistently undetectable HCV RNA at 72 weeks (6 months after the end of course of treatment).Nonresponse (NR) was defined as persistent positive HCV (PCR) after 48 weeks of treatment.t-test, nonparametric tests (Wilcoxon's and Mann-Whitney) or one-way analysis of variance (ANOVA) with Post-Hoc test (Turkey's) as appropriate. The Chi-Square or Fisher's exact tests were used to compare frequencies and proportions. Multivariate stepwise logistic regression analysis was performed to determine the independent predictors of sustained response. A P value of <0.05 was considered statistically significant. An intention-to-treat analysis was used. Patients who discontinued treatment either due to adverse effects or lost to follow up were not included in the analysis for NR, ETVR, and SVR.Data were collected initially using a specialized data collection form, then introduced into a Microsoft Excel worksheet and finally transferred to the statistical package for social sciences version 15.0  for analysis. Means of continuous variables were compared using Student's As shown in P < 0.001).Alanine aminotransferase level at baseline and at weeks 24, 48, and 72 in the sustained responders and those who developed virological relapse are shown in n = 335), EVR was achieved in 253 (75.3%). Nonresponse and incomplete treatment (due to side effects or loss to follow up) occurred in 71 (24.3%) and 43 (12.8%) patients, respectively. Patients who completed treatment were 292 (87.1%), of these, 161 (55.1%) achieved SVR and 60 (20.5%) developed virological relapse after end-of-treatment response, that is 48.1% and 17.9% of all patients, respectively [In the entire cohort (ectively .P = 0.036), higher SVR (P = 0.001), and lower relapse rate after ETVR (P < 0.001).Viral response rates in the entire cohort, in those who completed treatment and based on whether the patients were previously treated with interferon are shown in Tables P < 0.05 for genotype 2 or 3 vs. either genotype 1 or 4 or the mixed genotype groups) [P > 0.05) [To address this issue, we selected the subgroup of patients in whom genotyping was done, having completed 48 weeks of treatment and achieving ETVR. These comprised 234 patients: 51 had genotype 1, 29 had genotype 2 or 3, 130 had genotype 4 and 24 had mixed genotype. Of these, 26 (51.0%), 25 (86.2%), 66 (50.8%), and 11 (45.8%) achieved SVR ( groups) . However > 0.05) .Only 12 cases of those who had pretreatment liver biopsy had fibrosis stage 4 (cirrhosis). Of these, eight (66.67%) completed 48 weeks of therapy. Three patients were NS and five patients had ETVR. Out of the five patients, only three had SVR and the remaining two relapsed after ETVR.P = 0.000), mainly nondiabetics (P = 0.015), had higher levels of serum albumin (P = 0.007), were infected with genotype 2 or 3 (P = 0.014), had less pretreatment necro-inflammatory grade (P = 0.011), and were treatment-naïve (P = 0.001). Both groups were similar regarding pretreatment fibrosis stage and viral load [P = 0.005 and P = 0.018, respectively).Clinical, laboratory, and pathological characteristics of patients who achieved SVR and those who developed virological relapse after ETVR are shown in Tables ral load . Howevern = 63) and leucopenia (n = 73), respectively.A total of 327 side effects were encountered in 170 patients (50.7%) during follow up . The effThis is the biggest cohort of chronic HCV-infected patients treated with the combination of pegylated interferon α-2a and ribavirin to be reported from the Middle East, where infection with genotype 4 is predominant. Our resu13The current impression that the patients infected with HCV genotype 4 are difficult to treat and that they respond poorly to interferon therapy was made based on earlier studies where conventional interferon-α was used alone.1216202224–26202213n = 30), this rate of nonresponse is acceptable.A total of 71 (24.3%) patients were classified as NR after 48 weeks of therapy and their treatment was discontinued. However, due to the tertiary nature of our hospital and the inclusion of many cases in whom interferon therapy had failed before, cases with organ transplantation and on immunosuppressive therapy, cases co-infected with HIV and/or HBV, cases infected with mixed HCV genotypes and the inclusion of a small number of cases with genotype 2 or 3  in our patients who previously received interferon therapy is better than that concluded in many studies for both genotypes 1 and 4.2134 The 21.6% in ouet al. reportedet al. Interestet al.38 Elevatet al.40 A receet al. Thus, asThe potential limitations of the current study can be summarized in the following points: (1) Posttreatment biopsy was not performed as the main objective of the study was to assess the rate of SVR, which was assayed using serum HCV PCR, relying on the well-established information in the literature that proved stabilization and/or regression in hepatic fibrosis stage in response to treatment, especially if it is associated with viral clearance.–39 In adIn conclusion, combination therapy, if tolerated and completed, is effective in treating chronic HCV patients, especially in those with no previous interferon therapy and with low pretreatment inflammatory grade. Persistent efforts and support measures need to be implemented, possibly through a specialized dedicated clinic, to improve patient compliance with follow-up, adherence to therapy, tolerance to treatment, early detection, and subsequent treatment of complications, if any. Further studies addressing the possible underlying mechanism(s) responsible for SVR, including the pretreatment assessment of IP10 levels, are warranted.


Mortality of HIV-infected patients initiating antiretroviral therapy in the developing world is very high immediately after the start of ART therapy and drops sharply thereafter. It is necessary to use models of survival time that reflect this change.In this endeavor, parametric models with changepoints such as Weibull models can be useful in order to explicitly model the underlying failure process, even in the case where abrupt changes in the mortality rate are present. Estimation of the temporal location of possible mortality changepoints has important implications on the effective management of these patients. We briefly describe these models and apply them to the case of estimating survival among HIV-infected patients who are initiating antiretroviral therapy in a care and treatment programme in sub-Saharan Africa.As a first reported data-driven estimate of the existence and location of early mortality changepoints after antiretroviral therapy initiation, we show that there is an early change in risk of death at three months, followed by an intermediate risk period lasting up to 10 months after therapy.By explicitly modelling the underlying abrupt changes in mortality risk after initiation of antiretroviral therapy we are able to estimate their number and location in a rigorous, data-driven manner. The existence of a high early risk of death after initiation of antiretroviral therapy and the determination of its duration has direct implications for the optimal management of patients initiating therapy in this setting. In a clinical trial, 1120 HIV-infected individuals initiated antiretroviral therapy (ART) in rural Uganda. One hundred and five subjects died during the study. Table T. The baseline hazard of this model for the ith subject isA frequently used mathematical model of patient survival is based on the Weibull probability distribution function for survival time h = h0(ti)eβ'z, i.e.,Covariates are incorporated straightforwardly so that β and z are the vectors of coefficients and covariate values respectively. The cumulative hazard of the Weibull model from the time origin up to time ti iswhere so thatti, given the covariates z, is h = ho(t)ϕ where ϕ = eβ'z is a proportionality constant that is not dependent on time. The log cumulative hazard is also linear in both time and the covariates, i.e., log H = ρlog ti - ρlog λ + β'z or, in the notation of Royston and Parmar s iThe hazard of mortality is very high after ART initiation for up to three months, and may persist up to a year after start of treatment. This has strong implications for patient management and may be helpful in refining patient care protocols in this setting by intensifying follow-up of newly treated patients during this period and possibly extending the duration of intensified follow-up for up to one year after start of therapy. Further investigation and re-analysis of data from a number of ongoing studies will be important to authoritatively address this question. The flexibility afforded by these Weibull models will be useful in this endeavor.The author conceptualized and performed all analyses, interpreted the results and wrote the paper.CTY is a biostatistician who has been extensively involved with clinical and epidemiological HIV research for 15 years. This has been through the Harvard School of Public Health Statistics and Data Analysis Center of the AIDS Clinical Trials Group, and, more recently, as Director of the East Africa Regional International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortium at the Indiana University School of Medicine. His research interests in East Africa focus in the use of clinical and research databases to provide a locally relevant evidence basis for medical decision making, health care policy and clinical management of HIV-infected patients cared for and treated in programmes in the region.


Although considered an essential tool for monitoring the effect of combination antiretroviral treatment (CART), HIV-1 RNA  testing is greatly influenced by cost and availability of resources.To examine whether HIV infected patients who were initially successfully treated with CART have less frequent monitoring of VL over time and whether CART failure and other HIV-disease and sociodemographic characteristics are associated with less frequent VL testing.The study included patients who started CART in the period 1999–2004, were older than 18 years, CART naive, had two consecutive viral load measurements of <400 copies/ml after 5 months of treatment and had continuous CART during the first 15 months. The time between two consecutive visits (days) was the outcome and associated factors were assessed using linear mixed models.We analyzed a total of 128 patients with 1683 visits through December 2009. CART failure was observed in 31 (24%) patients. When adjusted for the follow-up time, the mean interval between two consecutive VL tests taken in patients before CART failure (155.2 days) was almost identical to the interval taken in patients who did not fail CART (155.3 days). On multivariable analysis, we found that the adjusted estimated time between visits was 150.9 days before 2003 and 177.6 in 2008/2009. A longer time between visits was observed in seafarers compared to non-seafarers; the mean difference was 30.7 days ; and in individuals who lived more than 160 kilometers from the HIV treatment center .Less frequent monitoring of VL became common in recent years and was not associated with failure. We identified seafarers as a population with special needs for CART monitoring and delivery. Globally, monitoring of plasma HIV-1 RNA  and determination of CD4 cell counts is related to the strategy of delivery of combination antiretroviral therapy (CART) and greatly influenced by the cost and availability of resources. In the public health approach of CART in resource-limited settings there is currently no consensus on the type, frequency and cost effectiveness of different types of monitoring . The open-label randomized DART trial conducted in Uganda and Zimbabwe found 3% less mortality after 5-years of follow-up in patients monitored with CD4 cell counts every 3 months compared to patients with clinical monitoring only In developed countries, determination of plasma VL is considered an essential component for monitoring effectiveness of CART. The virological goal of CART is to achieve <50 copies of HIV-1 RNA per mililiter of plasma measured by polymerase chain reaction (PCR) by week 24. Recommendations on the frequency of VL testing after a patient achieves an undetectable VL are mainly based on expert opinion and on the analysis of the Eurosida cohort suggesting that patients who had a stable and fully suppressive CART for 1 year had a low chance of experiencing treatment failure in the ensuing months United States Department of Health and Human Services (DHHS) It is also not clear when a VL test should be repeated after a change in the CART regiment in a patient with fully suppressive HIV-1 RNA. To assess the efficacy of the new regiment, the DHHS guidelines recommend repeating a VL test 2 to 8 weeks after a change in CART We examined whether there was a change in the frequency of VL testing over time and calendar year in all HIV infected patients in Croatia who started CART in the period 1999 to 2004. Furthermore, we assessed different sociodemographic and HIV disease factors related to the frequency of viral load testing.The study was approved by the Ethics Committee of the University Hospital for Infectious Diseases (UHID), Zagreb, Croatia. Written informed consent was obtained from all participants.Croatia has a centralized system of treatment and care for patients with HIV infection and all patients are treated in Zagreb at UHID. Also, antiretroviral drugs are only given from the hospital pharmacy at UHID. Health insurance is universal and all health care expenses including the cost of antiretrovirals and monitoring are free of charge for the individual. Highly active antiretroviral therapy became available through the national health insurance scheme in April 1998. At the end of 1997 determination of HIV-1 RNA by PCR became available. Croatia has a low-level epidemic; the epidemic started in 1985. The first cases were identified among labor migrants who returned from western European countries, and seafarers who acquired HIV in Africa and Eastern Asia Since 1997, there is a comprehensive electronic database on HIV infected patients available at UHID. We conducted a retrospective cohort study on the frequency of VL monitoring in population of patients on CART in Croatia from 1999 to 2009. The total number of patients in care per calendar year ranged from 120 in 1999 to 533 in 2009.Inclusion criteria were the following: age 18 years old or older, documented HIV-1 infection, initiation of CART at UHID from 1999 to 2004, and no active opportunistic disease at start of follow-up. Participants had to have continuous CART during the first 15 months of treatment as judged by the pharmacy refill and an undetectable VL (<400 copies/mL) on at least two consecutive tests after 5 months of CART. We excluded patients who had been treated at the time of acute HIV infection and had subsequently discontinued CART.We defined treatment failure as: a) two consecutive VL measurements >400 copies/mL, with the earlier date defined as the date of failure, b) a single VL measurement >10 000 copies/mL, c) an AIDS-defining opportunistic infection or malignancy, or d) death. Measurements after failure were not included in further analysis. If the patient decided to stop therapy the visit before this occurred was considered to be the last follow-up visit. The baseline value for our analysis was the last measurement taken before the 15 months of treatment and the last follow up visit for patients who did not fail was the last measurement taken before 31.12.2009.We used three outcome measures to assess the frequency of VL testing. First, subjects were divided into those who had at least 50% of their measurements more than 5 months apart and those who had not. Second, rates of VL measurements were computed as the number of VL tests (numerator) divided by the person-time contributed and rates are reported per one person-years of follow-up. Third, we examined the time interval, defined as the number of days between two consecutive VL tests for a subject. We than examined how the frequency of VL testing is affected by age, gender, type of initial CART, change in CART, duration of treatment, follow-up time, calendar year, being employed as a seafarer, distance from UHID, risk group and baseline and current CD4 cell count. We also analyzed whether patients who failed had less frequent VL testing prior to this event.Baseline socio-demographic and clinical characteristics between patients with at least half (50%) of their measurements more than 5 months apart and those with less than half (50%) of their measurements more than 5 months apart were compared with chi square tests for categorical variables and Wilcoxon rank sum tests for continuous variables. We used Poisson analysis to compute the rate of testing and rate ratios and associated 95% confidence intervals (CI) for different baseline characteristics.versus urban), baseline CD4 cell count, clinical AIDS before CART, calendar year of CART initiation, CART failure, having at least one CART change during follow-up, type of initial CART and positive for hepatitis C antibody. Time-varying covariates were type of current CART regimen, current CD4 cell counts and the calendar year of VL tests. Covariates with a p<0.25 in crude analysis were considered as candidates for inclusion in the multivariable model. We modeled follow-up time linearly and quadratically as fixed effects in both crude and multivariable analysis. The linear value of follow-up time was also specified as a random effect.Longitudinal data analysis was initially explored graphically. We then used a random coefficient model to take into account repeated measures of the outcome (days between tests). Crude analysis was performed including a random intercept and slope, and one fixed or time-varying explanatory variable. Fixed explanatory variables were gender, baseline age, HIV transmission group, distance from HIV center, migrant work, level of education, place of living  was not significant in crude (p = 0.071) nor in multivariable analysis (p = 0.376), whereas the interpretation of other coefficients did not change. We present our models without removing outliers. We used SAS software system release 9.1.3  for all analyses.A total of 175 patients 18 years of age or older started CART in the period 1999 to 2004. One hundred twenty-eight (73%) met our cohort inclusion criteria. We excluded 47 patients; 1 had HIV-2 infection, 1 had an active illness during CART, 37 had discontinuous CART in first 15 months of treatment, and 8 did not have two consecutive VL <400 copies/ml. Sociodemographic characteristics and HIV disease-related factors are shown in CART failure was observed in 31 (24%) patients. Of the 31 patients who failed, 3 died, 3 failed because of an AIDS event, 17 decided to stop CART and 8 were virological failures mainly because of missing doses. All patients who stopped CART or had other causes of virological failure achieved an undetectable VL with a subsequent CART regimen. The median time to failure was 1.7 years . Of 175 patients starting CART, 97 (55.4%) did not fail treatment.At baseline, patients were equally likely to be treated with a non-nucleoside reverse transcriptase inhibitor-  containing regimen and a protease inhibitor-  containing regimen. Twenty percent used zidovudine (ZDV)+lamivudine (3TC)+efavirenz (EFV) at baseline, 20% ZDV+3TC+ ritonavir-boosted lopinivir (LPV/r) and 15% stavudine (d4T)+3TC+EFV. Overall 65 (51%) patients changed their ART regimen at least once; there were a total of 83 instances of CART change. At last follow up, 23% were on ZDV+3TC+EFV, 17% on abacavir (ABC)+3TC+EFV, 15% on ZDV+3TC+LPV/r; the most frequent changes were d4T to ZDV (34%) and ZDV to ABC (25%). The overall median time between a change in CART and the following VL test was 90 days .One hundred twenty-eight patients had 1555 follow-up visits that contributed to a total of 632.7 person years of follow-up through December 2009. The overall rate of viral load testing was 2.5 per patient per year . On bivariable analysis patients who lived 160 km or more from UHID, were seafarers, were heterosexuals, did not fail CART and had longer follow-up time, had fewer VL tests done .Longitudinal data analysis included a total of 1683 intervals between successive tests. The median number of viral load tests per individual was 12 , the median length of follow-up was 5.0 years  and the median length of CART was 6.0 years . The median of the median interval between VL tests of each individual patient was 139.5 days . Inspection of smoothed graphs of days between VL tests and time, as well as the significant interaction of time*time, suggested that time could be modeled both linear and quadratic. When this was entered into the model without other predictors the estimated mean interval between VL tests was 165 days at three years of follow-up (corresponding to four years of CART).In crude analysis patients who lived 160 km or more from the HIV centre had a significantly greater estimated mean interval between tests (164.2 days) than those who lived less than 160 km away  as did patients who were seafarers (187.8 days) compared to non-seafarers  . Other vWe found a substantial individual variation in the frequency of VL testing in patients in Croatia. Less frequent testing among patients on long-term CART became common in recent years. Importantly, we did not observe that less frequent VL testing was associated with an increase in CART failure. We found no association of failure and time between VL testing on linear mixed model analysis, which takes into account correlated measurements on the same subject. The analysis of patients with less VL testing compared to those with more frequent VL testing  and the Risk factors for less frequent testing were living farther from the treatment center and being employed as a seafarer. Both of these factors likely represent patients' difficulties in keeping scheduled appointments due to distance, travel and working conditions. Patients who had a change in their CART regimen were, on average, more frequently monitored, however, this testing was done after a substantially longer period  than recommendations from developed countries suggest. As expected, the frequency of monitoring was influenced by the current CD4 cell count; patients with lower counts were monitored somewhat more frequently. This effect was modest; our multivariable model suggested an increase of time between tests of only 2.5 days per 100 cells.There are only a few studies from developed countries examining the frequency of VL testing To our knowledge, factors associated with the frequency of VL in middle income countries have as yet not been reported. Middle income countries in southeastern Europe  are not part of the European Union, have a low-level HIV epidemic and have had difficulties in providing HIV/AIDS care and CART. For example in Croatia in 2009, out of 26 antiretroviral formulations registered in the EU, 14 were available to Croatian patients. There are also occasionally irregular supplies of antiretrovirals and the level of stigma towards MSM and patients with HIV/AIDS is high The aim of routine frequent VL testing in patients with undetectable VL is to detect virological failure early, leading to adherence interventions or early changes in therapy that will limit ongoing viral replications and reduce the risk of accumulation of resistance mutations. However, since failure was not preceded by less frequent VL tests, our findings suggest that considerable savings can be achieved by less frequent monitoring of stable patients without compromising the efficacy of CART. On the other hand one may argue that less frequent monitoring was observed in our study mainly because patients who lived farther from the HIV center and worked as seafarers had difficulties in accessing facilities that perform VL testing.Our findings are subject to some limitations. First, this was not a trial of frequency of VL testing, so we are unable to establish that more or less frequent VL testing is causally associated with a greater or lesser risk of CART failure. Although our data indicates that less frequent testing is not associated with virological failure or adverse HIV-related clinical outcomes this issue needs to be further explored. Secondly, we included into the study only ART naïve patients who were initially well suppressed and considered fully adherent during the first 15 months of CART; the result would have been different using the whole population receiving CART. Thirdly, observational studies may be biased by not including unmeasured confounders and we might have missed an important predictor of less frequent VL testing. For example we were not able to study employment status and income. However, the magnitude of the differences we observed for our main predictors (working as a seafarer and the distance from the HIV center) makes it unlikely that an unmeasured confounder could have altered these findings. Our patient registry is also highly complete with little missing data, and we were able to include all patients on CART in Croatia who met our inclusion criteria.In conclusion, monitoring viral load became less frequent in recent years in a population of patients who were initially virologically well suppressed and considered adherent to CART. Working as a seafarer was the most important sociodemographic factor related to less frequent VL monitoring. Compared to intervals in patients who did not fail, patients who failed CART did not have longer times between VL monitoring visits before this event occurred. Our findings support recent recommendations that VL monitoring in adherent patients with a stable undetectable VL can be extended to every 6 months. In our experience, approximately 55% patients who start CART could be monitored with less frequent visits.