The objective of this work was to study the virological outcomes associated with two different types of treatment interruption strategies in patients with allergic reactions to nevirapine (NVP). We compared the virological outcomes of (1) HIV-1-infected patients who discontinued an initial NVP-based regimen because of cutaneous allergic reactions to NVP; different types of interruption strategies were used, and second-line regimen was based on efavirenz (EFV); and (2) HIV-1-infected patients who began an EFV-based regimen as a first-line therapy (controls).This retrospective cohort included patients who began an EFV-based regimen, between January 2002 and December 2008, as either an initial regimen or as a subsequent regimen after resolving a cutaneous allergic reaction against an initial NVP-based regimen. The study ended in March 2010. The primary outcome was virological failure, which was defined as either (a) two consecutive plasma HIV-1 RNA levels >400 copies/mL or (b) a plasma HIV-1 RNA level >1,000 copies/mL plus any genotypic resistance mutation.A total of 559 patients were stratified into three groups: (a) Simultaneous Interruption, in which the subjects simultaneously discontinued all the drugs in an NVP-based regimen following an allergic reaction (n=161); (b) Staggered Interruption, in which the subjects discontinued NVP treatment while continuing nucleoside reverse transcriptase inhibitor (NRTI) backbone therapy for a median of 7 days (n=82); and (c) Control, in which the subjects were naïve to antiretroviral therapy (n=316). The overall median follow-up time was 43 months. Incidence of virological failure in Simultaneous Interruption was 12.9 cases per 1,000 person-years, which trended toward being higher than the incidences in Staggered Interruption (5.4) and Control (6.6). However, differences were not statistically significant.Among the patients who had an acute allergic reaction to first-line NVP-based therapy and later began an EFV-based regimen, virological outcomes resulting from a staggered interruption of treatment (with a continuation of NRTI backbone therapy for 7 days after discontinuing NVP) did not differ from those of the patients who began an EFV-based regimen as their initial therapy (Control). However, the virological failure of Simultaneous Interruption was possibly higher than those of Control and Staggered Interruption. Non-nucleoside analog reverse transcriptase inhibitor (NNRTI)-based regimens are currently recommended as a first-line therapy for HIV-1-infected patients because of their virological potency, lower risk of drug interactions and lower cost, as well as the availability of fixed-dose combinations. Nevirapine (NVP) is a first-line NNRTI recommended for antiretroviral-naïve patients, according to the European AIDS Clinical Society (EACS) guidelines . NVP retThe method of antiretroviral treatment interruption following an allergic reaction to an NNRTI-based regimen is a major consideration. The simultaneous discontinuation of all the drugs in a NNRTI-based regimen would result in a period of functional NNRTI monotherapy; NNRTIs exhibit a longer plasma half-life and a longer duration of detectable levels than nucleoside analog reverse transcriptase inhibitors (NRTIs) . NNRTIs HIV-1-infected patients with a history of allergic reactions to NVP who plan to begin an EFV-based regimen may later experience different effects from those associated with the prevention of mother-to-child transmission or with treatment interruption strategies. Thus, we conducted a retrospective cohort study to examine the effectiveness of different types of treatment interruption strategies in HIV-1-infected patients who halted an NVP-based regimen because of cutaneous allergic reactions to NVP and later began an EFV-based regimen.This retrospective cohort study was performed at the Bamrasnaradura Infectious Diseases Institute, Ministry of Public Health, Nonthaburi, Thailand. The Simultaneous Interruption group consisted of HIV-1-infected patients who simultaneously discontinued the use of all the drugs in an NVP-based regimen after experiencing a cutaneous allergic reaction to the treatment. The Staggered Interruption group consisted of HIV-1-infected patients who first discontinued the use of NVP after experiencing a cutaneous allergic reaction to an NVP-based regimen but who continued with other NRTIs for several days. The Control group included patients who began an EFV-based regimen as first-line therapy and who were never exposed to NVP. The study end date was March 2010.A total of 12,803 medical records of HIV-1-infected patients were retrieved from a medical database and reviewed  demographic features, (b) any history of allergic reactions to NVP-based regimens or other drugs, (c) previous and current antiretroviral (ARV) regimens, (d) CD4 cell counts and plasma HIV-1 RNA levels, and (e) both laboratory and clinical adverse events.Plasma HIV-1 RNA was initially measured using reverse transcriptase polymerase chain reaction (RT-PCR) with a COBAS AMPLICOR HIV-1 MONITOR test, version 1.5 , which detects plasma HIV-1 RNA levels in the range of 50–100,000 copies/ml (ultrasensitive technique) or 400–1,000,000 copies/ml (standard technique). After June 2008, plasma HIV-1 RNA levels were evaluated using real-time PCR with a COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test , which can be used to measure plasma HIV-1 RNA levels in the range of 40–10,000,000 copies/ml.Patients who did not appear for clinical visits for more than 3 months after a missed appointment or who discontinued an EFV-based regimen for more than 3 months were considered lost to follow-up. Patients who halted an EFV-based regimen for fewer than 3 months were classified as having an EFV-based regimen interruption.We defined an adverse laboratory event as any laboratory result of at least a grade 3, according to the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (the DAIDS AE grading table). A clinical adverse event was defined as any clinical adverse event that resulted in the physician changing or modifying the antiretroviral regimen. A patient was allowed to continue participating in the study if the physician only changed or modified the NRTIs used. However, if the physician changed EFV to other NNRTIs or PIs, the patient was discontinued from the study cohort.Between January 2010 and March 2010, there was a final evaluation of the HIV-1-infected patients who continued an EFV-based regimen through the end of study. The evaluation consisted of the following: (a) a plasma HIV-1 RNA assay, (b) a CD4 cell count, (c) a complete blood count, and (d) blood chemistry tests. Patients were informed about the evaluation if they (a) came to the hospital during this period, (b) were continuing an EFV-based regimen, and (c) did not have the results of any plasma HIV-1 RNA measurements obtained between October 1, 2009 and December 31, 2009. At the end of the study, 381 patients were still participating  two consecutive plasma assay results indicating HIV-1 RNA levels >400 copies/mL after 6 months of an EFV-based regimen or (b) a single plasma HIV-1 RNA level >1,000 copies/mL in addition to any genotypic resistance mutation to an EFV-based regimen. These criteria included only virological results obtained after 6 months of an EFV-based regimen. Genotypic resistance mutations were included in the virological criteria to reduce the bias from viral blips and noncompliance.The secondary outcomes were as follows: (a) the percentage of patients who exhibited virological suppression; (b) the CD4 cell counts; (c) the clinical outcomes of death, major opportunistic infection, immune recovery syndrome, and non-AIDS-associated disease; and (d) adverse laboratory and clinical events.The relative risk and 95% confidence intervals were calculated to compare the differences in primary outcome among the groups. In addition, stratified analysis models were used to evaluate the effects of confounding factors on the primary outcome.Virological suppression was defined as either (a) a plasma HIV-1 RNA level <50 copies/ml  or (b) a plasma HIV-1 RNA level <40 copies/ml .t-test and a one-way ANOVA for continuous data with a normal distribution. All the reported p-values were two-sided and were considered statistically significant if p<0.05. The analyses were performed using SPSS version 14.The baseline characteristics and outcomes were analyzed with (a) a chi-squared test for dichotomous data, (b) the Mann–Whitney test or the Kruskal-Wallis test for continuous data with a non-normal distribution, and (c) an unpaired Figure Among the 139 patients who (a) were lost to follow-up (n = 53), (b) were referred to other hospitals (n = 50), (c) experienced a change in therapy from EFV to another ARV (n = 26), and (d) died (n = 10), 104 patients (74.8%) underwent plasma HIV-1 RNA assays within 6 months before they were removed from the cohort. Ninety-five of these patients (91.3%) demonstrated virological suppression.There were 26 patients in the study (4.7%) whose therapy was changed from EFV to another ARV (another NNRTI or PI) Figure . This grThe baseline demographic features of the patients are summarized in Table Table p<0.001).The overall median (IQR) duration of the follow-up period was 43  months. The Simultaneous Interruption group had a significantly longer follow-up period than the other two groups (p = 0.007). The median (IQR) duration of the EFV-based regimen interruptions was 42  days. Common causes of the regimen interruptions included (a) financial issues (n =7), (b) toxicity (n = 7), and (c) poor compliance with the medication regimen (n = 7). There were 13 patients who discontinued all ARVs during regimen interruptions, whereas the remaining patients switched to other therapies, such as PI- or NVP-based regimens.There were 27 patients who experienced EFV-based regimen interruptions during the study (Table p = 0.100. The relative risk between the Simultaneous Interruption and Staggered Interruption groups was 2.38 (0.32–105.75), p = 0.224. The Staggered Interruption group displayed virological outcomes comparable to those of the Control group.The incidence of virological failure was trended toward being higher in the Simultaneous Interruption group than in the other groups Table , but thiThere were imbalances in the three groups’ baseline characteristics, such as age, sex, previous history of major opportunistic infections, baseline CD4 cell count, frequency of ARV administration, and history of EFV-based regimen interruptions Table . A stratOverall, 12 of the 16 subjects (75%) who experienced virological failure underwent genotypic resistance assays prior to changing to other regimens . The mutations detected in these 12 subjects included TAMs (n = 4), M184V mutations (n = 6), and NNRTI-resistance mutations (n = 10). The incidence of NNRTI-resistance mutations in the Simultaneous Interruption group was 8.1 cases/1,000 person-years, which trended toward being higher than the incidences in the Staggered Interruption (5.4 cases/1000 person-years) and Control (3.74 cases/1000 person-years) groups. However, these differences were not statistically significant. The Y181C and G190A NNRTI-resistance mutations, which are frequently related to the selective effect of NVP ,18, werep = 0.411)  months after receiving EFV-based regimens  was not significantly different among the three groups  . These patients generally exhibited low compliance with ARV therapy, which is associated with virological failure ,24.This study had the following limitations: (a) a retrospective design; (b) a non-randomized design; (c) some incomplete data ; (d) a follow-up loss of 9.5%; and (e) the frequent use, in the beginning of the study, of NRTI drugs such as d4T/3TC that are not preferred NRTIs under the current guidelines . These limitations should be carefully considered when interpreting the data from this study.In our cohort of patients who experienced acute allergic reactions to first-line NVP-based therapy and later began an EFV-based regimen, the virological outcomes of the Staggered Interruption group (who continued 2 NRTIs for 7 days after discontinuing NVP) did not differ from those of the Control group . However, the virological failure in the Simultaneous Interruption group was possibly higher than that in the Staggered Interruption and Control groups.ABC: Abacavir; ARV: Antiretroviral; AZT: Zidovudine; DHHS: Department of Health and Human Services; D4T: Stavudine; EACS: European AIDS clinical society; EFV: Efavirenz; ETR: Etravirine; NNRTI: Non-nucleoside analogue reverse transcriptase inhibitor; NRTIs: Nucleoside analogue reverse transcriptase inhibitors; NVP: Nevirapine; PI: Protease inhibitor; SMART: Strategies for management of antiretroviral therapy; TDF: Tenofovir; 3TC: Lamivudine.Krittaecho Siripassorn received research and/or honoraria/travel grants from Merck, Sharp and Dohme and Bristol-Myers Squibb in the past five years. KiatRuxrungtham received consultancy fees and/or honoraria and travel and/or research grants from Tibotec, F Hoffmann-La Roche, Merck, Sharp and Dohme, Bristol-Myers Squibb, Gilead, Abbott, and Glaxo SmithKline.KR and WM prepared drafts of the report and reviewed and edited the final manuscript. AP and SN screened medical records and contributed important information to the methods section. AC, NH, KW, and AL reviewed medical records and provided information to validate the use of the chosen analytical methods. All authors read and approved the final manuscript.


Rifampicin and protease inhibitors are difficult to use concomitantly in patients with HIV-associated tuberculosis because of drug-drug interactions. Rifabutin has been proposed as an alternative rifamycin, but there is concern that the current recommended dose is suboptimal. The principal aim of this study was to compare bioavailability of two doses of rifabutin (150 mg three times per week and 150 mg daily) in patients with HIV-associated tuberculosis who initiated lopinavir/ritonavir-based antiretroviral therapy in Vietnam. Concentrations of lopinavir/ritonavir were also measured.This was a randomized, open-label, multi-dose, two-arm, cross-over trial, conducted in Vietnamese adults with HIV-associated tuberculosis in Ho Chi Minh City . Rifabutin pharmacokinetics were evaluated before and after the introduction of lopinavir/ritonavir -based antiretroviral therapy using patient randomization lists. Serial rifabutin and 25-O-desacetyl rifabutin concentrations were measured during a dose interval after 2 weeks of rifabutin 300 mg daily, after 3 weeks of rifabutin 150 mg daily with lopinavir/ritonavir and after 3 weeks of rifabutin 150 mg three times per week with lopinavir/ritonavir.Sixteen and seventeen patients were respectively randomized to the two arms, and pharmacokinetic analysis carried out in 12 and 13 respectively. Rifabutin 150 mg daily with lopinavir/ritonavir was associated with a 32% mean increase in rifabutin average steady state concentration compared with rifabutin 300 mg alone. In contrast, the rifabutin average steady state concentration decreased by 44% when rifabutin was given at 150 mg three times per week with lopinavir/ritonavir. With both dosing regimens, 2 – 5 fold increases of the 25-O-desacetyl- rifabutin metabolite were observed when rifabutin was given with lopinavir/ritonavir compared with rifabutin alone. The different doses of rifabutin had no significant effect on lopinavir/ritonavir plasma concentrations.Based on these findings, rifabutin 150 mg daily may be preferred when co-administered with lopinavir/ritonavir in patients with HIV-associated tuberculosis.NCT00651066ClinicalTrials.gov  In 2011, there were an estimated 34 million adults and children living globally with HIV/AIDS and an estimated 8.7 million new cases of tuberculosis: 1.1 million persons had HIV-associated tuberculosis and 430,000 persons with HIV-associated tuberculosis died Since 2003, there has been a remarkable scale up of antiretroviral therapy with 8 million people estimated to be on therapy by the end of 2011 Observational studies have clearly shown that antiretroviral therapy improves the prognosis of patients with HIV-associated tuberculosis Mycobacterium avium complex and for the treatment of drug-susceptible tuberculosis. Plasma concentrations of rifabutin are increased in the presence of protease inhibitors Rifabutin is an attractive alternative to rifampicin as it is a less potent inducer of CYP3A4 The present study was therefore undertaken with the primary objective of comparing the pharmacokinetic parameters of two doses of rifabutin (150 mg TPW and 150 mg daily) in patients with HIV-associated tuberculosis in Vietnam who initiated antiretroviral therapy with LPV/r. Secondary objectives were to investigate (i) the pharmacokinetics of LPV/r in combination with RBT, and (ii) the safety and toxicity of rifabutin in combination with antiretroviral therapy during the initial phase of anti-TB treatment.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study was approved by the Institutional Review Board and Ethical Review committee at Pham Ngoc Thach Hospital, the Health Department of Ho Chi Minh City and the Ministry of Health, Vietnam, as well as the Union Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease, Paris, France.This study was a randomized, open-label, multi-dose, two-arm, cross-over trial, conducted in Vietnamese patients with HIV-associated tuberculosis - Clinical trial registry number: NCT00651066.The study was carried out in Pham Ngoc Thac Hospital, Ho Chi Minh, Vietnam, a tertiary care facility that has 800 beds and cares for TB patients, about 10% of whom have associated HIV-infection. In Vietnam, patients with suspected tuberculosis are investigated according to National Tuberculosis Guidelines Study patients were adults aged 18 – 65 years, HIV-positive, with a CD4 count less than or equal to 250 cells/µL and with newly diagnosed tuberculosis. Eligibility requirements included:- provision of written informed consent; having a firm home address that was readily accessible; if female, having a negative pregnancy test on day of enrolment; having a diagnosis of pulmonary tuberculosis confirmed by smear microscopy, culture or a chest radiograph compatible with active tuberculosis and associated with a typical clinical history and two negative sputum smears; no previous history of antiretroviral therapy; weight ≥40 kg; a Karnofsky score Q ≥80%; no grade 3 or 4 clinical or laboratory findings according to Division of AIDS tables Patients were randomized to receive one of two individual treatment arms on the day of enrolment. Randomization lists were produced prior to the start of the trial by the Medical Research Council in South Africa . The clinical research team in Vietnam used pre-prepared envelopes in chronological order, indicating to which treatment arm the patient should be assigned.The detailed planned trial timeline describing the intended allocation of treatments in the two arms of the trial in relation to the initial and continuation phases of anti-tuberculosis treatment and randomization is shown in Laboratoires SERB supplied rifabutin 150 mg capsules for oral administration  and the new film-coated tablet formulation of LPV/r, Aluvia® was purchased from Abbott Laboratories (USA).0-24) for rifabutin determined in previous studies 0-24 for the participants taking rifabutin without antiretroviral therapy and the AUC0-24 for the participants taking rifabutin when combined with antiretroviral therapy. To provide a target of 12 evaluable patients in each arm, because patients with low CD4 cell counts recruited into the study might experience high mortality and morbidity resulting in a high attrition rate, it was decided that 32 patients should be enrolled (16 in each arm).Based on the area under the curve  was drawn prior to administration of study drugs and a standard hospital breakfast was served exactly two hours (2 h) after drug ingestion. Subsequent bloods were drawn at 2, 3, 4, 5, 6, 8, 12, 24 and 48 h (in the case of intermittent RBT dosing) after drug ingestion. The samples were placed on ice immediately and centrifuged at 3000 rpm at 4°C for 10 minutes within 30 minutes of collection. Separated plasma was transferred to polypropylene tubes and stored immediately at -70°C until analysis. The drug assays for RBT and its metabolite (25-O-desacetylrifabutin) as well as lopinavir and ritonavir are described in the following section Rifabutin and 25-O-desacetylrifabutin were analyzed simultaneously with a validated HPLC assay. Rifabutin and 25-O-desacetylrifabutin standards were kindly provided by Pfizer. In brief, after addition of medazepam as internal standard both chemicals were extracted from 0.2 mL of plasma with a hexane/dichloromethane solution (6/4 v/v). After vortex and centrifugation, the organic phase was evaporated to dryness. Dry residue was reconstituted with 100 µL of mobile phase constituted of /acetonitrile: 600/400 (v/v). 50 µL is injected onto the Eclipse XDB RP-C18, 150×4, 6 mm, 5 µm – Agilent column. The spectrophotometer for UV detection was set at 272 nm. Lower limits of detection were 12.5 ng/mL and 6.25 ng/mL for rifabutin and desacetyl rifabutin respectively. Linearity of standard curves was demonstrated up to 500 ng/mL and 250 ng/mL for rifabutin and desacetyl rifabutin respectively. Variability of day to day quality controls inserted in each analytical run was lower than 9% for median and high concentrations and lower than 15% for low concentrations. The accuracies  for rifabutin and 25-O-desacetylrifabutin were between 97% and 106% at low, medium and high QC levels during inter-run validation.Plasma lopinavir and ritonavir concentrations were quantified by a validated reverse phase HPLC method as described elsewhere max), and time to Cmax (Tmax) were obtained directly from the concentration-time profiles. Drug concentrations at the end of a dosing interval were reported as Cmin and pre-dose concentrations on the day of pharmacokinetic evaluation reported as C0. The steady-state AUC (AUCτ) during a dosing interval τ 24 hours or 48 hours for rifabutin and 12 hours for lopinavir/ritonavir were calculated for each drug by the linear up/log down trapezoidal method. As an index of exposure during a dosing interval, the average concentration at steady state (Cave) was calculated for rifabutin and its metabolite as Cave = AUCτ/τ where τ is the dosing interval. The metabolite ratio was calculated as the ratio of metabolite to parent drug AUCs.The main pharmacokinetic measures for rifabutin, 25-O-desacetylrifabutin and lopinavir were derived by non-compartmental analysis using WinNonLin software . The peak concentration  as a covariate was applied. As no sequence or day effect was found, the drug groups were pooled and dose levels were compared. Rifabutin parameters for assessing the interaction when combined with LPV/r were Cmax, C0, and Cave. These parameters were logarithmically (log) transformed and a linear mixed model fit was used which included treatment, period and sequence as fixed effects and the patient as a random effect. Ninety percent confidence intervals (90% CIs) for the difference in mean log-transformed (log) PK parameters for a particular rifabutin combination therapy (150 mg OD or 150 mg TPW) compared to rifabutin monotherapy (300 mg OD) were calculated. These differences in mean log PK parameters and 90% CIs were back transformed and presented in their original units as geometric means and 90% CIs. The geometric mean ratio presented in Thirty nine patients were assessed for eligibility for the trial, with Mycobacterium tuberculosis was isolated from sputum, and in one of these patients resistance to rifampicin and isoniazid was diagnosed after completing the initial phase of treatment. Patients were on no other drugs except for anti-tuberculosis drugs and ARV drugs.Plots of mean concentrations of rifabutin and its desacetyl metabolite against time are shown in The median trough and peak concentrations (C0 and Cmax) of lopinavir and ritonavir with rifabutin 150 mg OD and 150 mg TPW are shown in The 33 enrolled patients had a total of 124 adverse events . Eighty percent of the adverse events were low grade (grades 1 and 2). Hepatic events with raised levels of liver enzymes were the commonest adverse events with 56 events occurring in 25 patients. Of these, seven were grade 3 or 4. Of these hepatic events, 33 occurred in the first 2 months and 23 after rifabutin was stopped; their average duration was more than 66 days. There was one case of IRIS (immune reconstitution inflammatory syndrome) grade 3 and no uveitis. There were 4 cases of neutropenia but only one that was grade 3 and none that was grade 4. Serious adverse events are shown in Mycobacterium tuberculosis and 2 had positive cultures (one patient was sputum smear negative but had drug-resistant TB with resistance to isoniazid and rifampicin and one patient was sputum-smear positive for acid-fast bacilli with the culture indicating non-tuberculous mycobacteria). For the 24 study patients, the median (IQR) increase in CD4 cells/mm3 was 127 (64–170) – there were two patients who had a decrease from 229 to 188 and 223 to 219 cells/mm3. Plasma HIV-RNA was undetectable (<250 copies/mL) for 19 (79%) of the 24 study completers. Five patients had a detectable HIV-RNA without any resistance mutations at HIV genotyping.Among the 24 patients who completed anti-tuberculosis treatment with all PK visits scheduled, 22 (92%) had negative cultures for max) and the area under the curve (AUCτ) of the drugs were in the same range, regardless of the dose used. There was a significant and almost one third higher average concentration at steady state of rifabutin when used with LPV/r at a dose of 150 mg daily compared with 300 mg alone. The intermittent dosing of rifabutin co-administered with LPV/r led to a lower average concentration compared with 300 mg alone, although pre-dose concentrations remained in the same range. The different doses of rifabutin had no significant effect on the concentrations of lopinavir or ritonavir. Although there were a large number of recorded adverse effects, these were largely low grade and mainly related to an increase in serum liver enzyme levels. Of the 24 patients who completed the pharmacokinetic studies and who completed six months of anti-tuberculosis treatment, over 90% had negative Mycobacterium tuberculosis cultures, all but two patients had a measurable increase in CD4 cell counts and over 70% of patients had undetectable viral loads.This is one of the first studies to investigate whether doses of rifabutin at 150 mg once daily or 150 mg three times per week are suitable in combination with the tablet formulation of LPV/r in an antiretroviral therapy regimen in the treatment of patients with HIV-associated tuberculosis. The main findings were that peak concentrations Click here for additional data file.Protocol S1Trial protocol.(PDF)Click here for additional data file.


Investigate the cost and effects of a single-pill versus two- or three pill first-line antiretroviral combinations in reducing viral load, increasing CD4 counts, and first-line failure rate associated with respective regimens at 6 and 12 months.Patients on first-line TDF+3TC+EFV, TDF+FTC+EFV, Truvada®+EFV or Atripla® between 1996–2008 were identified and viral load and CD4 counts measured at baseline, six and twelve months respectively. Factors that independently predicted treatment failure at six and twelve months were derived using multivariate Cox's proportional hazard regression analyses. Use and cost of hospital services were calculated at six and twelve months respectively.All regimens reduced viral load to below the limit of detection and CD4 counts increased to similar levels at six and twelve months for all treatment regimens. No statistically significant differences were observed for rate of treatment failure at six and twelve months. People on Atripla® generated lower healthcare costs for non-AIDS patients at £5,340  per patient-semester and £9,821  per patient-year that was £1,344  less per patient-semester and £1,954  less per patient-year compared with Truvada®+EFV; healthcare costs for AIDS patients were similar across all regimens.The single pill regimen is as effective as the two- and three-pill regimens of the same drugs, but if started as first-line induction therapy there would be a 20% savings on healthcare costs at six and 17% of costs at twelve months compared with Truvada®+EFV, that generated the next lowest costs. One of the early problems that people living with HIV (PLHIV) had was the large pill burden associated with triple-therapy when first introduced into routine treatment and care in 1996. The association of lowered adherence with increased pill burden and poorer outcomes has been recognized for some time, in terms of number of pills to be taken To reduce pill burden, various strategies have been developed over time to produce once-daily dose regimens, combining a number of different drugs into fewer tablets: ‘fixed-dose combinations’ (FDCs). In some FDCs different antiretroviral drugs (ARVs) are combined into one tablet that can be taken once-a-day and improves adherence WHO Expert Committee on the Use of Essential Drugs provided the following criteria for FDCs: “Most essential medicines should be formulated as single compounds. Fixed dose combination products are selected only when the combination has a proven advantage over single compounds administered separately in therapeutic effect, safety, adherence or in delaying the development of drug resistance in malaria, TB and HIV/AIDS.”The production of FDCs precedes their development and use for HIV infection. One of the first FDCs was an oral contraceptive produced in the 1960s followed by the development of maloprim (pyrimethamine+dapsone) and cotrimoxazole (trimethoprim+sulfamethoxazole) in the late 1960's Atripla® is a FDC currently on the market that combines Tenofovir (TDF) with Emtracitabine (FTC) and Efavirenz (EFV). Prior to the introduction of this FDC, TDF was initially prescribed separately with Lamivudine (3TC) and EFV, while after the release of FTC on the market, the three pill regimens also included TDF, FTC and EFV. The next development was a combination of TDF and FTC into one pill – Truvada®; subsequently EFV was added to Truvada® to create Atripla®, a single-pill ARV regimen.Truvada®+EFV were licensed for first-line induction therapy in 2005 in the UK, while Atripla® was licensed for first-line therapy in the US in 2006 and in 2007 in the UK. Atripla® is currently not licensed for first-line induction therapy in the UK but clinicians can switch from Truvada®+EFV to Atripla® as part of first-line simplification. This particular first-line regimen can therefore be prescribed in four combinations and the aim of this study was to compare the effectiveness and costs of these four combinations in terms of reducing viral load, increasing CD4 counts and treatment failure at 6 and 12 months respectively.effectiveness, efficiency, equity and acceptability of treatment and care in participating HIV units since 1996. Using an agreed minimum dataset, standardized data are routinely collected in clinics and transferred to the NPMS-HHC Coordinating and Analytic Centre (CAC) The National Prospective Monitoring System on the use, cost and outcome of HIV service provision in UK hospitals - HIV Health-economics Collaboration (NPMS-HHC) has monitored prospectively the 2 test as well as test for trend and where appropriate these were adjusted using Yates' correction.Parametric quantitative data are presented as means with 95% confidence intervals (CIs) or standard deviation (SD) while non-parametric data are presented as medians with inter-quartile range (IQR). Between group comparisons of parametric data with more than two independent groups were tested using one-way-ANOVA while two independent groups were compared using unpaired t-test. Between group comparisons of non-parametric data with more than two independent groups were tested using the Kruskal-Wallis test while two independent groups were compared using the Mann-Whitney U test. Qualitative data by CD4 count strata were tested using the χBaseline CD4 counts and baseline viral loads were obtained within 4 months before or up to two weeks since starting the first-line regimens under consideration with the closest value to starting the regimen taken as baseline. For 7% of cases baseline CD4 counts and for 10% of cases baseline viral loads could not be obtained and for these patients their baseline CD4 counts or baseline viral loads were imputed using the Multiple Imputations (MI) procedure in SAS. This procedure assumes that the missing baseline CD4 count and baseline viral load data were missing at random (MAR). The missing baseline CD4 counts and baseline viral loads were substituted with an estimated value using a multiple imputation procedure which replaced each missing value with a set of plausible values that represented the uncertainty about the right value to impute Linear mixed models were used to calculate the difference in averages (DAVG) which represent the time weighted difference in CD4 counts from baseline to clinic visits at 6 and 12 months respectively, and where necessary data were transformed to stabilize the variance.MIXED procedure in SAS was used to fit values of all available CD4 count results since starting first line regimen as a dependent variable. Independent variables included the fixed effects of the treatment groups TDF+3TC+EFV, TDF+FTC+EFV, Truvada®+EFV or Atripla®, clinic visit time points at 6 and 12 months, and treatment groups by study time point interaction. A covariance matrix was used to model the within patient errors. Estimates of change in CD4 count from baseline were obtained from intervention by clinic visit time point interaction. Trends over time are presented as point estimates with 95% confidence intervals (CIs). Multivariable analyses presented were adjusted for other time varying co-variables assumed to have potential confounding or residual effect on the trend of CD4 count changes, including baseline age, sex, ethnic group, clinical status, log10 viral load (VL) and year of starting ART.Time to first-line treatment failure was estimated from the date of starting the first-line regimen. First-line treatment failure was defined as any change to treatment, this included intensification of regimen by further adding anti-retroviral drug to the regimen or swapping to another anti-retroviral drug class. Simplification of ARVs with no other changes made to the regimen did not constitute treatment failure. Causes of treatment failure included clinical, immunological or virological reasons and others, where adverse effects were the most likely cause p<0.2 in univariable Cox's proportional hazards model were used to build a multivariable model to determine independent predictors of treatment failure while controlling for the other variables in the model. Quantitative data were categorised using median and inter-quartile ranges (IQR), including a separate category for any variables with missing data. This ensured no degrees of freedom were lost when building the multivariable models. The final multivariable models presented were tested for their distributional assumptions using Cox Snell residual plots and adjusted for sex, age, ethnic group, baseline CD4 count and viral load, stage of HIV infection at start of treatment and year of starting ART.Cox's proportional hazards regression models with single variables were initially used to estimate likelihood of treatment failure. All variables found to have a probability of st January 2004 and 31st December 2008 were obtained from computerized information systems from 9 UK hospitals participating in this analysis. All patients who started on first line TDF+3TC+EFV, TDF+FTC+EFV, Truvada®+EFV or Atripla® during that period were identified and patients who were known to have transferred from other HIV units were excluded as it was not possible to establish with certainty whether these regimens were indeed their first-line regimen.Data on the use of hospital inpatient, outpatient and dayward services between 1The mean number of inpatient days, outpatient visits and dayward visits were calculated for the first six and twelve months on ART respectively. The denominator for those on first-line TDF+3TC+EFV, TDF+FTC+EFV, Truvada®+EFV or Atripla® consisted of the total duration of follow up from when these first-line regimens were started until the end of 6 or 12 months respectively and data were censored as described above. Analyses were stratified by treatment regimens and whether patients had had an AIDS diagnosis or not.Numerators were calculated by summing the use of inpatient, outpatient or dayward services while on these first-line regimens. Mean use of services and 95% confidence intervals (95%CI) were calculated per patient-semester (6 months) and patient-year (12 months) and summarized by the formula:n = total number of individuals;k = day of censoring;ij = use of service of individual i at jth day;Sij = number of days on first-line TDF+3TC+EFV, TDF+FTC+EFV, Truvada®+EFV or Atripla® for individual i;tM = mean of services at i) 6 months or ii) 12 monthsThe method used to calculate mean use of services has been employed in a large number of previously published studies The unit cost for an average inpatient day was £511, £101 for an outpatient visit and £413 per dayward visit, unit costs that were obtained from the 2010 NPMS-HHC report The total number of patients who started on the four regimens was 1,448, of whom 25% had been diagnosed with AIDS . Among bNo significant differences were observed in terms of age when starting first-line ART, sex and past or current history of injecting drugs for patients starting on the different regimens. Some minor differences were noted among the ethnic background of AIDS patients .Baseline viral loads for non-AIDS patients on Atripla® were significantly lower than for those patients on the other regimens . HoweverBaseline viral loads for AIDS patients on Atripla® were significantly lower than for those patients on other regimens . HoweverNo significant statistical differences were observed between regimens in terms of likelihood of treatment failure at 6 and 12 months  and 4.For non-AIDS patients, the cost of use of services at 6 months ranged from £5,340  for those on Atripla® to £7,554  for those on TDF+3TC+EFV. Patients on Atripla® generated the lowest cost, as their use of inpatient services was significantly lower than that generated by patients on the other regimens. For Truvada®+EFV the cost of services at 6 months was £6,684  which was £1,344  per patient-semester more expensive compared with Atripla® .By twelve months, annual cost per patient-year for non-AIDS patients ranged from £9,821  for patients on Atripla® to £13,467  per patient-year for patients on TDF+3TC+EFV. For Truvada®+EFV the cost of services at 12 months was £11,775  which was £1,954  per patient-year more expensive compared with Atripla® . Again tFor AIDS patients at 6 months, cost of services ranged from £9,123  for TDF+FTC+EFV to £15,061  for those on TDF+3TC+EFV. The cost at 6 months for Atripla® was £10,836  and the difference with Truvada®+EFV was only £489 (95% £321 to £655) per semester .At 12 months, the cost per AIDS patient-year ranged from £17,710  for those on TDF+FTC+EFV to £25,811  per patient-year for patients on TDF+3TC+EFV. The annual cost on Atripla® was £19,933  and differed by £328 (95% CI 146 to 512) per patient-year with the Truvada®+EFV regimen .All regimens displayed similar effectiveness in terms of reducing viral load to undetectable serum levels by 6 months and maintained that at 12 months; all regimens increased CD4 count to similar levels by 6 and 12 months and no statistically significant differences were observed in terms of treatment failure rates at 6 months and 12. These outcome were achieved, despite that median VL was lower and mean baseline CD4 counts for non- AIDS patients on Atripla were higher than for those on other regimens. While the number of patients failing their regimen was similar across all regimens, those on Atripla used fewer inpatient or dayward services. The observed percentage of patients failing Atripla within one year was 16% and this was similar to the 20% recently reported from a single London centre In term of use and cost of services for non-AIDS patients, Atripla® generated the lowest healthcare costs at six and twelve months whereas six monthly or annual costs for AIDS patients were similar across all regimens. Starting with higher CD4 counts, not using inpatient services, even for those who failed, and using fewer dayward services, all contributed to lower healthcare costs. The lowest costs among AIDS patients were generated by patient on TDF+FTC+EFV. However, as there were only 18 patients on this regimen - none of whom used inpatient services – questions can be raised concerning the representative nature of this and the other results observed among the AIDS patients. Furthermore waiting until a person living with HIV develops an AIDS defining condition before starting them on ART would be inappropriate; one wants to diagnose people living with HIV early and review them regularly in a controlled clinical situation and start ART well before they develop AIDS The analyses as presented have their limitations. Firstly, some of the comparator groups had small number of patients many of whom were seen in London clinics. Secondly first viral load or CD4 count when starting these regimens could not be retrieved for a small number of subjects and these had to have their viral load and CD4 count imputed. Thirdly, the data available for operational research are by definition observational data Fixed-Dose Combinations for HIV/AIDS, Tuberculosis, and Malaria not only provided evidence of the effectiveness of generic FDCs against HIV but also tried to place the role of FDCs within the broader context of diverse pharmacological interventions The report of a 2003 WHO meeting on “reducing pill burden by developing ‘one pill a day’ (or less often) fixed-dose combinations (FDCs)” and its 2020 Goal is the availability of “effective, affordable, one pill, once-daily potent ARV regimens with minimal toxicities or drug interactions and high barriers to resistance are available in lower and middle-income countries”Optimizing drug regimens is one of the five pillars of WHO and UNAIDS' Treatment 2.0 policy, and includes cost of the regimen as well as their effectiveness. If it is considered to be appropriate to start a person living with HIV on a regimen of TDF, FTC and EFV, strong clinical and financial arguments can be made for starting this person on single-pill Atripla® as first-line therapy. While Atripla® is currently not licensed for induction therapy in the UK, a substantial number of UK clinicians have started using Atripla® as first-line induction therapy. If Atripla® is started as induction therapy, this avoids the extra costs associated with first starting patients with Truvada®+EFV as induction therapy and only switching to Atripla® after 6 months as part of treatment simplification.During times when Governments in high-income countries are also cutting health and welfare budgets to ameliorate rising healthcare costs


Hepatitis B coinfection is common in HIV-positive individuals and as antiretroviral therapy has made death due to AIDS less common, hepatitis has become increasingly important. Several drugs are available to treat hepatitis B. The most potent and the one with the lowest risk of resistance appears to be tenofovir (TDF). However there are several questions that remain unanswered regarding the use of TDF, including the proportion of patients that achieves suppression of HBV viral load and over what time, whether suppression is durable and whether prior treatment with other HBV-active drugs such as lamivudine, compromises the efficacy of TDF due to possible selection of resistant HBV strains.A systematic review and meta-analysis following PRISMA guidelines and using multilevel mixed effects logistic regression, stratified by prior and/or concomitant use of lamivudine and/or emtricitabine.Data was available from 23 studies including 550 HBV/HIV coinfected patients treated with TDF. Follow up was for up to seven years but to ensure sufficient power the data analyses were limited to three years. The overall proportion achieving suppression of HBV replication was 57.4%, 79.0% and 85.6% at one, two and three years, respectively. No effect of prior or concomitant 3TC/FTC was shown. Virological rebound on TDF treatment was rare.TDF suppresses HBV to undetectable levels in the majority of HBV/HIV coinfected patients with the proportion fully suppressed continuing to increase during continuous treatment. Prior treatment with 3TC/FTC does not compromise efficacy of TDF treatment. The use of combination treatment with 3TC/FTC offers no significant benefit over TDF alone. Approximately 10% of people infected with HIV are coinfected with hepatitis B virus (HBV). Among populations with access to antiretroviral therapy (ART), in whom serious opportunistic infections have become a rare event, liver diseases including HBV infection represent a major cause of morbidity and mortality. It is uncertain what proportion of patients achieves suppression of HBV DNA  and whether those in whom suppression is not seen after one year may achieve HBV suppression later. It is also unclear to what extent, if at all, those with complete suppression may relapse despite continued treatment, e.g. in case of development of resistance mutations. Finally, it remains uncertain whether sequential treatment, for example with 3TC initially and TDF later, compromises the chance of successful treatment with TDF.A recent meta-analysis examined all randomised controlled trials of treatment for HBV but excluded patients with HIV coinfection and only compared responses at 12 months. We carried out this meta-analysis of data from patients coinfected with HIV to amalgamate all available evidence and to answer the following questions:what proportion of patients achieve HBV viral load suppression on TDF?does the rate of suppression differ in those with prior 3TC experience?does the rate of suppression differ in those treated with combination therapy compared with TDF monotherapy?how common is HBV rebound on TDF?The systematic review was carried out following the guidance laid out in the PRISMA statement Studies included were those that described HBV/HIV coinfected individuals treated with TDF with or without 3TC and/or FTC for a period of at least one year and that gave results of quantification of plasma HBV viral load at yearly intervals (at a minimum) while on TDF treatment. Studies included could be randomised controlled trials or prospective or retrospective cohort studies. Patients with undetectable plasma HBV viral load at baseline were excluded since their inclusion gives a falsely high estimate of the effect of treatment. Baseline HBV viral load data was not given for 20 patients in three studies see . The anaTDF received approval for the treatment of HIV infection from the United States Food and Drug Administration (FDA) in October 2001 and from the European Medicines Agency in February 2002.  The first reports of the use of TDF in treating HBV infection were presented in 2002. Web of Science, Embase and Medline were searched, including all years. Conference abstracts from The Liver Meeting (American Association for the Study of Liver Diseases), The International Liver Congress (European Association for the Study of the Liver) and the Conference on Retroviruses and Opportunistic Infections were searched for the years 2002–2010.To search databases, a combination of key terms was used including “hepatitis”, “HIV”, and “tenofovir”, limited to articles with human subjects and written in English . ConfereStudies were screened initially by title and then data was collected by HP from the full article of all published studies and from conference posters, or conference abstracts if posters were not available. Some studies met the eligibility criteria except that the published report did not include data on the number with undetectable HBV viral load at one year, or information on prior or concomitant drug exposure. The authors of these studies were contacted by email and asked to provide additional data if available. Additional, unpublished data was obtained from the authors of 11 of the 23 sources included . The autData collected consisted of type of study, source of study funding, number of HBV/HIV coinfected participants, number HBeAg positive at study entry, prior 3TC/FTC exposure, drug regimens used during study period, length of follow-up, type of HBV viral load test used and lower limit of detection, numbers tested for HBV viral load at yearly intervals, and numbers with undetectable HBV viral load at yearly intervals. To maximise power and in the absence of any evidence suggesting a difference in effect on HBV between 3TC and FTC, exposure to these two were grouped together.Results were stratified by treatment into four groups. Group A consisted of patients who had no prior exposure to 3TC/FTC and who were treated with TDF without concomitant 3TC/FTC, Group B those without prior exposure to 3TC/FTC treated with TDF in combination with 3TC/FTC, Group C those with prior exposure to 3TC/FTC but treated with TDF without 3TC/FTC, and Group D those with prior exposure to 3TC/FTC treated with TDF in combination with 3TC/FTC.Statistical analysis was carried out using STATA version 10.1. The main outcome measure used was the proportion of patients tested who had a HBV viral load below the limit of detection at each of any available yearly time intervals. 95% confidence intervals for these proportions were calculated for each time point in each study and for the aggregate results.To detect potential sources of bias, assay cut-off was plotted against proportion suppressed at one year. Publication bias was examined using funnel plots, in which asymmetry with a lack of poorly performing studies (to the left) would suggest such studies were not published.Multilevel mixed effects logistic regression (XTMELOGIT command) was used to assess the effect of prior exposure to, and combination treatment with 3TC/FTC on the probability of viral suppression, with individual studies fitted as a random effect to account for clustering . This imModels were re-run with an interaction term to examine whether the effect of concomitant 3TC/FTC was the same in both those naïve and those exposed to prior 3TC/FTC. As sensitivity analyses, the model was re-run (i) including only larger studies (reporting at least ten patients), (ii) including all studies apart from one that appeared as an outlier on the funnel plot. All authors had access to the data in the study and reviewed and approved the manuscript.The initial searches produced 2,110 references which, after duplicates were removed, referred to 1,607 publications. Publications were then screened by title and if necessary by abstract to remove those clearly not meeting the eligibility criteria. This left 379 published articles. The full text of articles and posters was then checked for eligibility (or abstracts if the full article or poster was not available). 356 were removed as ineligible , 79.0% (95% CI: 73.6–83.8%), and 85.6% (95% CI: 79.2–90.7%) after one, two, and three years of treatment with TDF .It was possible to assess rates of virological suppression by HBeAg status for patients from ten of the included studies. The proportion suppressed increased over time and reached 100% overall . The numVirological rebound on TDF was rare, with no cases seen in 16 of 23 studies. Three studies reported a single patient with an increase in HBV viral load on TDF treatment, The funnel plot  shows thThis review of HBV/HIV coinfected patients treated with TDF results demonstrates lasting virological suppression of HBV replication to below the level of detection, with the proportion suppressed increasing to 100% over time, though with small numbers at later time points. Few patients experience virological failure on treatment.However several reservations should be noted. Firstly most of the studies included were observational in design and patients dropping out were not well characterised. Secondly, in this meta-analysis we compare different treatment groups though allocation to these was randomised in only two studies. The proportion with undetectable HBV at one year (59%) was lower than the proportion found in HIV negative patients receiving TDF for treatment of HBV infection. For example, a multicentre cohort study found that, of 54 HIV-negative patients treated with TDF and FTC, 60% of whom were HBeAg positive, the probability of attaining an undetectable HBV viral load was 76% at one year and 94% at two years. In the latter study, ten patients (2.3%) had virological breakthrough . The high rate of virological suppression and low rate of breakthrough may be related to the low chance of developing TDF-resistance mutations. In HBV/HIV coinfected patients treated with lamivudine as the only drug active against HBV, resistance develops in about 90% after four years No statistically significant effect of prior 3TC/FTC exposure or of concomitant 3TC/FTC use was found and thus no evidence to support the hypotheses that prior exposure may make subsequent treatment less effective or that concomitant use of 3TC/FTC may give a higher rate of suppression. However given the modest number of patients available for inclusion in the meta-analysis, the confidence intervals were wide and we could not exclude the possibility of moderately strong effects in either direction. In HIV-negative patients TDF monotherapy is as effective for HBV as combination therapy with TDF and 3TC/FTC with suppression rates (<400 copies/mL) of 81% at one year in both arms of an RCT using TDF alone or TDF/FTC combination therapy, and 88% and 85% respectively at three years The main concern with sequential treatments that fail to fully suppress the viral load is that resistance may develop and that cross-resistance could reduce the efficacy of subsequent drugs. TDF resistance is yet to be clearly demonstrated but it may be that the risk of cross-resistance is higher with drugs that are more similar to TDF in structure than 3TC/FTC. However HBV mono-infected patients failing to achieve virologic suppression with adefovir have also been shown to respond well to TDF A second mechanism by which prior treatment exposure could reduce the apparent effectiveness of subsequent TDF is through introducing bias, in that patients failing one regimen for reasons other than lack of potency (such as poor adherence to therapy) may go on to fail other regimens but again, no such reduction in the effect of TDF in those with prior exposure to 3TC/FTC was found and so the effect of any such bias must be small.As stated above, TDF received FDA approval in late 2001 and thus clinical experience to date is limited to just over one decade. Although this review includes data to a maximum of seven years, a lack of data limited the main regression analyses to three years. Patients with HIV require lifelong treatment and patients with HBV coinfection are likely to require the same. The possibility of safe discontinuation of HBV treatment may be limited to patients who clear serum hepatitis B surface antigen (HBsAg). However the probability of HBsAg loss is low with a rate of approximately 2.5% per year A limitation of this study is that it does not include analysis of the adverse effects of treatment. Future studies with longer follow-up duration will be required to determine the risk of treatment associated adverse effects, such as renal and bone toxicity, in patients exposed to TDF for many decades.In conclusion, this meta-analysis shows that tenofovir suppresses HBV to undetectable levels in the majority of HBV/HIV coinfected patients, with the proportion fully suppressed increasing with time on treatment and with little if any virological rebound on treatment. Prior treatment with 3TC/FTC does not alter the efficacy of TDF treatment. Combination treatment with 3TC/FTC offers no significant benefit over tenofovir alone.Appendix S1Literature search strings.(DOC)Click here for additional data file.Appendix S2Stata code.(DOC)Click here for additional data file.


Before initiating an antiretroviral combination therapy which includes Maraviroc as one of its components, a coreceptor tropism test has to be performed. As Maraviroc is only effective against HIV strains that are using CCR5 as coreceptor a thoroughly validated and reliable assay should be performed. In case of treatment change due to toxicity, testing should also be possible using proviral DNA.[coreceptor] was used. Data acquisition for week 12 and week 24 is already finished. It is planned to collect data for 96 weeks for all the patients. Data analysed are besides viral load and the other components of the HAART, genotypic resistance assay at baseline, immunologic parameters and treatment side effects.Included in the study cohort of this non-interventional observational study are 157 patients. Only inclusion criteria was a treatment with Maraviroc and a previous tropism test. Phenotypic tropism tests were performed by Monogram biosciences (TROFILE®). For Genotypic tropism determination from V3 loop sequence data geno2phenoGenotypic tropism results were available for 88 patients from viral RNA sequences and for 53 patients from proviral DNA sequences. A TROFILE® result was available for 70 of the patients as 71 of the patients had a viral load below 1000 cop./ml and a testing with TROFILE® was not possible. In the intent to treat (ITT) analysis 70% of the patients were successfully treated at week 24, defined as a viral load decline of at least 3 logs or a viral load below detection limit of 50 copies/ml. The positive predictive value (PPV) of geno2pheno using a FPR cut-off of 10% was 77% (PPV TROFILE®: 66%). In the subgroup of patients where only a genotypic tropism test from proviral DNA was available 86% of the patients had a viral load below detection limit of 50 copies/ml (70% at baseline).On this clinical dataset performance in terms of predictive power is slightly better when using geno2pheno compared to TROFILE®. Therefore genotypic tropism testing proves to be an easy and inexpensive alternative to phenotypic tropism testing.Especially in the group of patients, where only a genotypic tropism test could be performed from proviral DNA, a high rate of successful treatments was observed, showing that this method makes it possible to perform tropism testing guided treatment change despite undetectable viral load.[coreceptor]: http://www.geno2pheno.orggeno2pheno


The ANRS PRIMO is a prospective multicenter French Cohort which had enrolled 1433 patients during primary-HIV-1 infection (PHI) since 1996, with the objectives to improve pathophysiological knowledge on PHI, to assess the impact of an early antiretroviral treatment and to contribute to document the epidemiology of new infections in France. Clinical and laboratory data are collected at inclusion, month (M)1, M3, M6, and then every 6 months. All patients are antiretroviral-naïve at inclusion.3 and 5.1 log10 copies/mL at PHI, with a wide range of values in the whole population , depending only partially to the time from infection. Median cellular DNA at inclusion was 3.4 log cp/106 PBMCs . Early immunological (CD4 counts) and virological parameters (HIV RNA and DNA) could predict the risk of progression to a CD4 count < 350 cells/mm3 in untreated patients and the chance of spontaneous persistent control of viral replication after infection. 52% of patients initiated an antiretroviral treatment at inclusion. Although a transient treatment during PHI did not translate into a benefit in terms of viral set-point compared to untreated patients, it could lead to long term preservation of CD4 cell counts. Furthermore, we showed that rare treated patients were capable of controlling viral replication after treatment interruption, those patients had lower viral reservoir at inclusion before treatment and at treatment interruption compared to non controllers. Cellular activation was high during PHI and declined substantially thereafter, with lower levels in patients treated early after infection compared to patients treated during the chronic phase.Patients were included early after infection ; 84% were men. The % of patients infected with non-B subtypes increased in the last decade, while the frequency of resistant viral strains remained stable. At inclusion, median CD4 cell counts and viral HIV load were 518/mmAntiretroviral treatment is now given in less advanced infection and even discussed as a universal approach to prevent HIV transmission. The question of treatment initiation in all patients diagnosed during PHI is relevant.


Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART.  Once infected with human immunodeficiency virus (HIV), the large majority of individuals are unable to control the virus. Exceptional patients, so-called elite controllers (EC), continue to have an “undetectable” plasma viral load (VL<50 copies/ml) without treatment Despite all described associations, it remains controversial which functional characteristics of T cell responses are important for control of viral replication and protection against disease progression. The following features have been suggested: strong proliferative T cell responses, preferential targeting of particular viral proteins (e.g. Gag better than Env) Most HIV-infected subjects ultimately become dependent on highly active antiretroviral therapy (HAART) for their survival. HAART has improved life expectancy and quality of life of all HIV-infected patients with progressive disease In contrast to this general rule, we recently identified four exceptional subjects, who were first treated for progressive disease and then stopped HAART, but nevertheless kept their plasma virus undetectable for a long time. We have called these patients “secondary controllers” (SC). Similar phenomena have been described by others ex vivo and to formally study the replication kinetics of those viruses in vitro. In addition, we investigated several aspects of HIV-specific CD4+ and CD8+ T cell functions. The latter included ex vivo breadth, amplitude, avidity and polyfunctionality as well as proliferative responses upon triggering with HIV peptides.To address this question, we compared five groups of HIV infected individuals, including SC, rebounders or “secondary non controllers” (SNC), patients under stable HAART, therapy-naïve progressors (TN), as well as EC. As possible “correlates of secondary protection” we quantified levels of intracellular viral markers, tried to cultivate the virus from the patients CD4+ T cells Blood samples were obtained from HIV-1 infected individuals enrolled at the Institute of Tropical Medicine (ITM), Antwerp, Belgium. The study protocol was approved by the Institutional Review Board of ITM and by the Ethical Committee of the Antwerp University Hospital. All subjects gave written informed consent. Five groups of HIV-1 infected individuals were selected. (1) Secondary controllers kept their viral load (VL) below 1,000 copies/ml after cessation of HAART and showed a VL of <50 copies at month 6. (2) Secondary non controllers (SNC) had a VL >10,000 copies/ml 3 months after cessation of HAART. (3) HAART patients were on stable antiretroviral treatment with VL <50 copies/ml. (4) Non-controller therapy naive (TN) patients had a VL >10,000 copies/ml. (5) Elite controllers (EC) had a VL <50 copies/ml without any treatment for more than 3 years. All patients in the study were chronically infected with HIV-1 i.e. for at least 3 years.Each patient enrolled in the study donated 3 times 100 ml blood with an interval of 3 months. Plasma samples obtained from these patients were frozen at −80°C in aliquots. PBMC were isolated on a Lymphoprep® density gradient . From freshly prepared PBMC, CD4+ T cells were purified using positive magnetic selection  and these were used to cultivate virus. At each time point 10 million PBMC were freeze-dried for extraction of DNA or RNA to perform HLA typing and measurement of intracellular viral markers. The rest of the PBMC were frozen as described elsewhere Intermediate to high resolution HLA class I-typing for the B locus was performed by the Red Cross Blood Transfusion Center at the Free University of Brussels in Jette, Belgium. The presence of CCR5Δ32 was determined for all SC, EC and SNC as described elsewhere gag, pol and env were amplified as described elsewhere DNA was extracted from PBMC samples using commercial kits . Afterwards 6 PBMC for prDNA or as number of copies per µg total RNA for usRNA and msRNA. The sensitivity of all three assays was four copies per reaction, which translated into approximately 40 copies/106 PBMC for the prDNA assay and 200 copies/µg total RNA for RNA assays . The linear range was at least five orders of magnitude. The sensitivity, reproducibility, and accuracy of these assays have been documented earlier For quantitation of cellular HIV-1 RNA and DNA load, total cellular nucleic acids were extracted from ten million dried-frozen PBMC using the Boom procedure in vitro cultivated viruses from three secondary controllers and four secondary non controllers was evaluated. To this end, PHA and IL-2 stimulated PBMC from 3 healthy donors were infected at multiplicity of infection (MOI) 10−3 with the viruses from the patients and BaL as a reference strain . Viral production was assessed by measuring HIV p24 concentrations by ELISA Purified CD4+ T-cells from all patients were cultivated with phytohemagglutinin (PHA 10 µg/ml) and interleukin (IL)-2  stimulated PBMC to produce autologous virus. In some cases (SC and EC) purified CD4+ T-cells were electroporated with siRNA against RcKp54  and cultivated with CD8 depleted PHA and IL-2 stimulated PBMC. The relative replication capacity (fitness) of the γ enzyme-linked immunospot (ELISPOT) assays  were performed with thawed PBMC as described elsewhere 6 PBMCs and was at least 3 times higher than the mean SFC in the 3 negative control wells (containing PBMC but no peptides).Interferon (IFN)-Mapping of T cell responses was performed by IFN-γ ELISPOT using a strategy based on a matrix of peptide pools as previously described T cell avidity refers to activation threshold in response to defined concentrations of exogenous peptide. It was measured by pulsing PBMC in IFN-γ ELISPOT assay with fivefold serial peptide pool dilutions. Only those peptide pools were used that were previously positive in the screening assay. T cells were determined as high avidity when the two lowest concentrations (3.2 and 0.64 ng/ml) were positive in ELISPOT assay.6 per experimental condition) were washed and stimulated in the presence of Brefeldin A  with medium, the standardized mixture of Cytomegalovirus-, Epstein Bar Virus and Influenza (Flu) peptides  as well as HIV Gag or Pol peptide pools always at a final concentration of 2 µg/ml. Only peptide pools positive in the screening IFN-γ ELISPOT assay were used. After stimulation for 6 hours, cells were stained for membrane markers using the following conjugated monoclonal antibodies: anti-CD3-PerCP-Cy5.5, anti-CD4-Alexa 700, anti-CD8-Pacific blue, and anti-CD69-APC-Cy7 (BD Biosciences). Next, cells were fixed and permeabilized  and intracellular cytokines were stained with the following directly conjugated antibodies: anti-CD107a-phycoerythrin (PE)-Cy5, anti-IFN-γ-FITC, anti-TNF-α-PE-Cy7, and anti-IL-2-PE. Polychromatic flowcytometric analysis was done using a Cyflow ML flow cytometer . Between 200,000 and 500,000 events were collected per sample. The events were subjected to a lymphocyte gate by FSC versus SSC plot. Following identification of CD8+ T cells (CD3+ CD8+) and CD4+ T cells (CD3+ CD8−), a gate was made for each respective function within the CD3+CD8+ T-cells and within the CD3+CD8− T-cells, by representing SSC in the X-axis versus cytokine function in the Y-axis. After the gate for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating 16 response patterns when testing 4 functions . For more detailed explanation on this methodology see: http://www.flowjo.com/v765/en/boolean.html and http://www.flowjo.com/v8/html/boolean.htmlThawed PBMC (1*103H) Thymidine incorporation assay, as previously described 5 cells/well in 96 flat bottom well plates  in 0,2 ml complete medium supplemented with 2,5% heat-inactivated pooled human serum . Cells were cultured at 37°C in a humidified 5% CO2 incubator in absence or in presence of each peptide pool separately. Only peptide pools that were positive in IFN-γ ELISPOT screening were tested in the proliferation assay. Cell cultures were pulsed for 8 hours on day 6 with 1 µCi/well (3H) Thymidine . Cells were harvested onto glass filters and 3H incorporation was measured using a TopCount scintillation counter (Perkin-Elmer). T cell proliferation was reported as stimulation index (SI), determined by dividing the mean counts per minute for the peptide-stimulated wells by the mean for non stimulated control wells.Lymphoproliferative responses were determined using the . To investigate the neutralization capacity against heterologous variants following pseudoviruses were obtained from the NeutNet program: SF162 (subtype B), DU174 (subtype C), 92BR025 (subtype C) and 92UG024 (subtype D) Because also patients on HAART are included in the study, neutralization assays can only be performed with immunoglobulin (Ig)G isolated from plasma to avoid interference by drugs. To this end a commercial kit  was used and manufacturer's instructions were followed.Neutralizing capacity of each patients IgG against autologous and heterologous viruses was measured by a reduction in luciferase gene expression after a single round of infection of TZMbl cells with pseudotyped viruses. The 50% inhibitory dose (ID50) was calculated as the reciprocal IgG dilution resulting in a 50% reduction of RLU compared to the virus control. For cellular HIV-1 load, statistical analyses were performed on log10-transformed values. Undetectable values of cellular HIV-1 DNA and RNA were left-censored at the detection limit of corresponding assays. Statistical analysis and graphical presentations were performed using Prism . For comparison of multiple groups, significance levels were determined by Kruskal-Wallis tests; when two groups were compared, Mann-Whitney tests were used. All statistical tests were two-sided. P values <0.05 were considered statistically significant.Within the HIV cohort of the Institute of Tropical Medicine of Antwerp, including over 1700 patients, we identified 160 subjects, excluding pregnant women, who had been successfully treated for progressive HIV infection with full HAART, but at some point decided to stop treatment against medical advice. Four patients, further referred to as secondary controllers (SC), either showed a limited and short-lived rebound (P1 and P3) or no viral rebound at all (P2 and P4) and all 4 kept their VL at “undetectable” level (<50 copies) during at least 6 months after effective therapy interruption. Importantly, antiretroviral drug levels were undetectable in those SC. Some clinical observations of the first two (P1 and P2) have been described in a separate case report To that end, we compared SC with “secondary non-controllers” (SNC), who show a strong and sustained viral rebound after stopping HAART. However, the low-to-absent viral replication in the SC could itself influence immune function. Therefore two other groups with low VL were included: elite controllers (EC), controlling virus replication below 50 copies without HAART, and patients under successful HAART. In addition, we also considered a group of regular chronically infected therapy-naïve (TN) patients with high VL. In each of the four comparator groups, patients were included over a range of peripheral blood CD4+ T counts, as can be seen in First, we investigated whether some host genetic factors could be associated with the secondary controller status. HLA-B5701, known as over-represented in EC In order to understand the molecular details of intracellular HIV-1 dynamics, levels of proviral (pr)DNA, unspliced (us)RNA and multiplespliced (ms)RNA, were quantified on PBMC extracts . Due to CD4+T cells from all patients were purified and used as such (first attempt) or after transfection with with siRNA against RcKp54 (subsequent attempts) to isolate the virus with activated donor PBMC. Virus could easily be cultivated from TN and SNC patients  and from HAART patients (first attempt and within 2 weeks). By contrast, despite two attempts, virus cultures from only 1 EC (P5) turned positive. Interestingly, this particular EC also had a detectable level of usRNA and was the one who subsequently lost his EC status. With regard to the SC, no virus could be cultured from P2, despite 3 attempts, while the others turned positive with delayed kinetics as compared to SNC : it tookAt first view these data suggest that of the three cultivable SC viruses, P3 is the least replication competent and P4 the fittest. However, the “cultivability” also depends on other factors . Therefore, we formally compared replication kinetics of SC and SNC after adjusting their infectious titre to the same MOI and performed three independent “fitness” experiments in PBMC with the reference strain BaL as a positive control. As can be seen in in vivo the proviral load of SC was very low, but sequencing failed to show gross abnormalities in their virus genes. Clearly, we have positive evidence in all SC patients that replication competent virus was present. For SC: P1, P3 and P4, the virus could be obtained in primary ex vivo culture patient's CD4+ T cells with delayed kinetics, as compared to SNC. Although the virus from P2 could not be cultivated, she showed a viral blip later in the evolution. In the secondary in vitro cultures, the virus from P4 was fully replication competent, whereas the viruses P1 and P3 showed a very low replication capacity. Taken together, these virological data apparently cannot fully explain the SC status and therefore a thorough immunological evaluation was performed as well.In summary, ex vivo IFN-γ ELISPOT responses, using thawed PBMC, stimulated with peptide pools representing the whole HIV genome. Clearly, HIV-specific IFN-γ producing T cells were detected in all subjects. Comparing mean number of peptide pools recognized or breadth  peptide concentrations ex vivoPolyfunctionality, i.e. the capacity of CD4+ and/or CD8+ T cells to exhibit multiple effector functions simultaneously upon antigen encounter, is currently considered as one of the best correlate of T cell efficacy measurable directly Next, the proliferative capacity of T cells towards Gag and Pol peptide pools was evaluated. Mean proliferative responses in the various groups are shown in We next quantified heterologous and autologous neutralizing antibodies in the different patients. To avoid the effect of anti-retroviral drugs (in HAART patients) and possible other plasma factors, IgG from the plasma of all patients was purified. IgG-mediated neutralizing capacity against 5 reference strains was low to absent in all patients and therefore no broad cross neutralizing antibodies were observed (data not shown). Autologous neutralization could be tested in two SC , two SNCSamples for this study were taken in 2008. During the following 3 years, P1 and P3 kept an undetectable VL and their already low CD4 T counts tended to decrease further. Remarkably, P2 showed 1 viral blip of about 1000 copies and P4 showed multiple blips between 100 and 1000 copies/ml, but both kept their CD4 T count over 1000 cells/µL. Only P3 was put on HAART again, because of infectious complications.In this study we describe a special group of patients, which we labeled “secondary controllers”. These HIV-1 infected individuals first showed clear-cut disease progression, were successfully treated for several years, stopped treatment and, unexpectedly, controlled their virus for many months or even years. This type of post-treatment interruption or “secondary” viral control might provide new clues to correlates or mechanisms of protection, which could be particularly useful to monitor immunotherapeutic strategies. We performed a comprehensive analysis of various host-viral interaction parameters. Remarkable, in common with equally aviremic but untreated elite controllers, the secondary controllers displayed very low level of HIV-1 usRNA and their virus was difficult to cultivate and had a lower fitness (except for SC P4). Relatively high T cell proliferation towards Gag and Pol peptides were also observed. In this respect, both controller groups behaved clearly different from viremic therapy-naïve progressors or secondary non-controllers as well as from aviremic patients under HAART. Interestingly, the two SC, from whom we could clone functional Env clones, showed the highest antibody-mediated autologous virus neutralization amongst all patients in various groups tested.Surprisingly, we were unable to confirm correlations of either EC or SC status with other T cell functions, previously associated with viral control, including the breadth, the amplitude or the avidity of responses in ELISPOT. Nor was there any evidence of more polyfunctional T cell responses in either EC or SC group. Admittedly, there were some limitations to this study, including the small patient sample size and the use of frozen PBMC to assess polyfunctionality, which might explain why we failed to confirm previously reported correlations for EC.Viral control and low setpoint have been attributed to specific HLA types , with genome-wide associations studies ascribing 12% of viral setpoint variability to HLA related variation Previously it was demonstrated that the level of HIV-1 usRNA in PBMC, a marker of “active viral reservoir” , when measured in HAART-treated patients with undetectable plasma viremia, represents a strong predictive marker for the outcome of therapy In our preliminary study, we provided evidence that the replication capacity of the endogenous virus in two of our SC was very low (35) and we were not able to culture the virus of the other two patients. In the mean time we were able to cultivate virus from 3 out of 4 SC and we performed a fitness analysis on the cultivated virus. The virus from P4 was easy to cultivate and showed a high replication capacity, the viruses from P1 and P3 were more difficult to cultivate and had a low replication capacity. The virus from P2 was not cultivable in our hands. Clearly, evidence of lower fitness was present in 3 out of 4 SC. Nevertheless, all four patients showed either an early (P1 and P3) or late viral blip(s) (P2 and P4) in vivo and therefore, again, a contribution of immune control seemed likely.Many studies have focused on identifying the functional characteristics of CD8+ and CD4+ T cell mediated immune control Avidity of T cells is another factor that could discriminate between virus control or lack thereof. Almeida et al. have shown that highly sensitive CD8+ T cells display potent HIV-suppressive activity in vitroT cell polyfunctionality is often described as the ability of a cell to produce at least 3 cytokines and/or other effector molecules  and it has been associated with CD8+ T cell mediated virus control The role of neutralizing antibodies in the control of viral replication has been controversial. Some early studies have shown high titers of heterologous neutralizing antibodies in long-term-non progressors compared to progressors In conclusion, despite the low number of patients studied, we were able to show that both EC and SC have a few viral and immune characteristics in common, which distinguish both types of controllers from non-controller groups, including aviremic HAART-treated patients. These include low levels of intracellular viral RNA, lower fitness and higher proliferative T cell responses towards Gag and Pol. Since these particular viral and T cell characteristics were absent, not only in viremic TN and SNC, but also in long-term aviremic HAART patients, it is tempting to speculate that they could be part of the mechanism by which SC and EC control the virus.Table S1Supplementary data avidity of Gag (A) and Pol (B) specific T-cells in all patients.A: number of peptides of Gag and Pol that have reacted in the epitope mapping ELISPOT. B: Number of deduced epitopes. This number can be equal or lower than in the “peptides reacting” column, due to the overlapping nature of the peptides format used i.e. a single epitope can be present in 2 or 3 overlapping peptides. C: Number of peptides tested: if not enough cells were available to test all epitopes, only those were used that were predicted to be restricted by the patients HLA-type (Los Alamos database). D: Number of epitope-containing peptides that showed no reaction, although they reacted in the first ELISPOT. As remark for this experiment blood from a later date was used, clearly illustrating that epitope recognition varies over time. E: Number of epitope-containing peptides that provided a positive ELISPOT at each concentration.(DOC)Click here for additional data file.


In Lagos, Nigeria, Médecins Sans Frontières (MSF) and the Ministry of Health (MoH) commenced free antiretroviral treatment (ART) in a hospital-based clinic. We performed a cross-sectional study to compare factors associated with raised viral load between patients with (“experienced”) and without (“naïve”) prior antiretroviral (ARV) exposure at commencement of ART at the clinic. We also examined factors influencing ARV adherence in experienced patients prior to clinic entry.We included adult patients receiving ART from MSF who answered a questionnaire about previous antiretroviral use. Multivariate logistic regression was used to estimate odds ratios (OR) for raised viral load (≥1000 copies/mL).1246 (96%) patients answered: 1075 (86%) reported no, and 171 (14%) some, prior ARV exposure. ARV-naïve patients were more immunosuppressed at baseline: 65% vs 37% (p<0.001) had CD4<200; 17% vs 9% (p = 0.013) were WHO stage 4. Proportionately more experienced than naïve patients had raised viral loads  on ART in the MSF/MoH clinic. Raised viral load was associated with prior ARV experience  and complete interruption of current ART . Higher CD4 at time of VL and a higher self-rated score of recent adherence were associated with lower OR of a raised viral load. Among experienced patients who missed pills before joining MSF/MoH, most common reasons were because ARVS were not affordable (58%) or available (33%), with raised viral load associated with being unsure how to take them .Patients previously exposed to ARVs had increased OR of raised viral load. The cost and availability of ARVs were common reasons for missing ARVs before joining the MSF/MoH clinic, and inadequate patient knowledge was associated with raised viral load. Access to non-nucleoside reverse transcriptase inhibitor (NNRTI) based antiretroviral therapy (ART) in resource-limited settings has lowered HIV-related morbidity and mortality Data are limited on the outcomes of antiretroviral (ARV) experienced patients commenced on NNRTI-based ART in resource-limited settings. A recent study from Africa showed a two-fold increased risk of virological failure in those previously exposed to ART In 2003 in Lagos, Nigeria, free, accessible ART was scarce, resulting in many patients self-funding ART in the private sector, or co-paying in government subsidised care. In this setting of limited availability of ARVs with stock-outs, self-funded treatment, and ARVs sometimes obtained through non-medical sources, it was not uncommon for patients to take inadequate treatment. Médecins Sans Frontières (MSF) and the Lagos State Ministry of Health (MoH) commenced provision of ART in an urban hospital-based clinic in 2003. ART, drugs for opportunistic infections (OIs), and diagnostic tests were provided free of charge, and there were no interruptions to ARV drug supplies. In the absence of genetic resistance testing, and with the greater costs of second-line treatment, most patients were commenced on WHO-recommended first-line ART with an NNRTI-based ART regimen irrespective of prior ARV exposure and whether those with prior exposure had unplanned or unstructured treatment interruptions In this cross-sectional study we assessed the OR of raised viral load comparing patients with (“experienced”) and without (“naïve”) prior ARV exposure at commencement of ART at the MSF/MoH HIV clinic in Lagos, Nigeria. We also examined factors influencing ART adherence in experienced patients prior to entry into the clinic and examined their association with raised viral load.We analysed data from adult patients receiving ART from MSF who consented to use of their HIV-related medical data for non-clinical purposes and who answered all relevant questions in a standardised pilot-tested questionnaire about ARV use before enrolling for treatment . The quePatients were categorised into two groups according to their prior ARV exposure at commencement of ART at the MSF/MoH HIV clinic: experienced (with prior exposure) or naïve (without prior exposure). Because experience ranged widely from less than 1 month to 72 months, experienced patients were further categorised by duration of reported prior exposure: <6 months, ≥6 months, or unknown. We used “MSF/MoH-ART” to specify the time on treatment at our clinic, as distinct from ARVs taken prior to joining our clinic. We used the term ARV rather than ART to denote prior experience because during prior treatment patients did not all take triple ARV combination therapy.Routine clinical data were collected by clinical staff at each clinical consultation and entered into a standardised electronic database . HIV viral load (VL) testing was performed to facilitate programme review over the year encompassing the questionnaire period as was feasible, but was not available routinely due to programmatic restrictions. The first HIV viral load (VL) measurement for each patient after at least 6 months of MSF/MoH-ART was included. VL was measured at an independent laboratory using Bayer Versant HIV-1 RNA 3.0 bDNA assay with Bayer System 340 bDNA Analyzer  using samples prepared according to Versant guidelines and with quality control monitored by MSF. CD4 count was routinely measured at 6 monthly intervals by FACScount, with the result at start of ART and within 90 days of the time of the VL included in this analysis.Data were analysed using STATA 10.1 . Baseline characteristics were described using medians and interquartile range (IQR) for continuous variables, and counts and percentages for categorical variables. Characteristics were compared between ARV history using Kruskal-Wallis rank sum test for medians of continuous variables, and odds ratios (ORs) with 95% confidence intervals (CIs) for binomial variables. All p values are exact and two-tailed; p<0.05 was considered significant.Multivariate regression was used to estimate ORs for raised viral load (VL≥1000 copies/mL) at any time ≥6 months of MSF/MoH-ART for ARV-experienced versus naïve patients, adjusting for potential confounders of biological or programmatic importance: sex; baseline age, body mass index and WHO stage (4 or 1–3); CD4 count  at baseline and closest measurement to (and within 90 days of) the VL measurement; months on MSF/MoH-ART when VL was measured; whether MSF/MoH-ART had ever been completely interrupted or partially interrupted; and self-assessment of current adherence. A second more limited multivariate regression model examined only experienced patients and some additional factors related only to previous ARV history before starting MSF/MoH-ART, retaining only key characteristics and variables with p≤0.10 on unadjusted analysis. Only patients with all data available were included, but a sensitivity analysis was done to include patients with missing WHO and CD4 stage as the key contributors to missing data.Patients gave written informed consent for use of their clinical data, and in addition gave verbal informed consent before answering the questionnaire, documented by the interviewer. The study including questionnaire and consent documents were approved by the MSF Ethics Review Board. Coded identification numbers were used to match questionnaire data with routine clinical data, and personal identifiers removed.1246 (96%) of 1297 adult patients on ART during Oct 2007-April 2008 answered the questionnaire: 1075 (86%) reported no and 171 (14%) some prior ARV exposure . Of thesAlthough initial CD4 count when commencing ART with MSF/MoH was higher in experienced patients , increase in CD4 after 12 months of MSF/MoH-ART was higher for naïve patients . Nonetheless, at the time VL was measured, CD4 remained higher in experienced patients . After ≥6 months of ART, proportionately more experienced patients had raised viral load , although they were assessed after a slightly longer time on MSF/MoH-ART  . A higheRaised viral load after ≥6 months of MSF/MoH-ART was associated with prior ARV experience  and complete interruption of MSF/MoH-ART . Higher CD4 when VL was measured was associated with a lower OR for raised viral load , as was a higher self-rated score of recent adherence  . Increas2, p<0.001), haemoglobin  and CD4 at baseline , but a similar proportion were in WHO clinical stage 4 . After ≥6 months of MSF/MoH-ART the prior exposure groups did not differ in immunological or virological status (data not shown). Those with ≥6 months prior experience were more likely to have taken ≥1 NNRTI(s), and a combination of ≥3 ARVs (Women were more likely to have had longer prior ARV exposure (68% of patients with ≥6 months prior ARV exposure were women vs 46% with <6 months [p = 0.012]). Three women had PMTCT as their only previous ARV exposure, and a further 21 also had used ARVs at other times. Patients with ≥6 months of prior exposure compared with those with <6 months had higher body mass index  reported they had missed pills sometimes, including 32 patients who missed pills but not enough to meet our definition of complete or partial ARV interruption. The most common reasons for experienced patients missing pills before joining our clinic were that they could not afford to buy ARVs (58%), could not get them due to a stock rupture (33%), or forgot (31%) . Raised Multivariate analysis for raised viral load among only patients with prior experience and sufficient data (n = 154) indicated that CD4 at the time of VL measurement was associated with raised viral load in a similar way to the entire cohort: a higher CD4 at the time of VL was associated with lower OR for raised viral load. Additionally, complete interruption of MSF/MoH-ART remained associated with a high OR for raised viral load , and although the point estimate of the association of recent adherence self-score was nearly the same as for the entire cohort (OR = 0.76), it was less strongly associated (p = 0.19) . CD4 at Our study comparing raised viral load between ARV naïve and experienced patients in a resource-limited setting showed that patients with any previous exposure to ARVs had a nearly 4-fold greater OR of raised viral load in the MSF/MoH-ART programme, despite reporting better adherence to current ART regimens and being healthier when starting MSF/MoH-ART. It is possible that baseline resistance to first-line ARVs contributed to this finding, although we did not do baseline genotypic resistance testing to enable us to confirm this suspicion. In our cohort, many of the ARV-experienced patients had interrupted their ARVs prior to treatment in the MSF/MoH programme and treatment interruptions are associated with the development of ARV resistance Although the effect of prior ART exposure on virological outcomes for first-line ART in Africa Most experienced patients in our cohort were not found to have raised viral load. Nevertheless, the higher rate of raised viral load in these patients supports the need to develop adapted ART initiation algorithms for those previously exposed to ART, particularly as NNRTI-based regimens are used which have a low genetic barrier to resistance Experienced and naïve patients did not differ significantly in non-health demographics, but when starting ART with MSF, experienced patients had higher body mass index, haemoglobin, CD4 counts and a lower proportion in WHO clinical stage 4, probably as a result of the beneficial effect of prior ARVs. This occurred despite 60% of experienced patients reporting missing some pills before joining the MSF/MoH clinic, and 23% reporting partial treatment interruption and 39% complete treatment interruption before commencing with MSF .Factors associated with raised viral load after ≥6 months of MSF/MoH-ART were previous ARV experience, low CD4 at the time of VL, lower self-reported adherence, and interruptions to ART while treated by MSF . Self-reThe primary limitation of this study is the cross-sectional design, resulting in a potential survival bias as patients who died or were lost to follow-up before the questionnaire was administered were not included, however in most baseline factors those not included and included were similar. A secondary limitation is the possibility of recall bias in reporting prior exposure to ARVs. This risk was minimised by using picture charts of all potentially available ARVs, and cross-checking questionnaire results with patient admission notes. Additionally we did not determine reasons for missing pills while at the MSF/MoH clinic, and therefore we were unable to compare recent challenges with those reported by ARV experienced patients who missed pills prior to programme entry, nor assess any association with raised viral loads in our study, however common reasons in experienced patients such as cost and availability were no longer relevant as the MSF/MoH clinic provided free reliable care. A third limitation is that while the patients who did not have complete data for inclusion in the regression model were demographically similar, they differed to those included on some clinical characteristics, notably baseline WHO stage and CD4 counts, and for WHO stage proportion missing differed between experienced and naïve patients but was still relatively low. As these factors can both independently affect the risk of virological failure Patients who had been exposed to ARVs for any period and of any type before coming to MSF for free comprehensive HIV care had increased OR of raised viral load. The cost and availability of ARVs were common reasons for missing ARVs before joining the MSF/MoH clinic, and inadequate patient knowledge was associated with raised viral load. Other factors associated with raised viral load were self-reported adherence, ever completely or partially interrupting current (MSF/MoH) ART, and low CD4 count at the time of VL measurement.