Ritonavir-boosted saquinavir (SQVr) is nowadays regarded as an alternative antiretroviral drug probably due to several drawbacks, such as its high pill burden, twice daily dosing and the requirement of 200 mg ritonavir when given at the current standard 1000/100 mg bid dosing. Several once-daily SQVr dosing schemes have been studied with the 200 mg SQV old formulations, trying to overcome some of these disadvantages. SQV 500 mg strength tablets became available at the end of 2005, thus facilitating a once-daily regimen with fewer pills, although there is very limited experience with this formulation yet.Prospective, multicentre study in which efficacy, safety and pharmacokinetics of a regimen of once-daily SQVr 1500/100 mg plus 2 NRTIs were evaluated under routine clinical care conditions in either antiretroviral-naïve patients or in those with no previous history of antiretroviral treatments and/or genotypic resistance tests suggesting SQV resistance. Plasma SQV trough levels were measured by HPLV-UV.95: 63.6 - 71.7%) by intention-to-treat, and 92.2% (CI95: 89.8 - 94.6%) by on-treatment analysis. The reasons for failure were: dropout or loss to follow-up (18.4%), virological failure (7.8%), adverse events (3.1%), and other reasons (4.6%). The high rate of dropout may be explained by an enrollement and follow-up under routine clinical care condition, and a population with a significant number of drug users. The median SQV Cmin (n = 49) was 295 ng/ml . The only variable associated with virological failure in the multivariate analysis was adherence .Five hundred and fourteen caucasian patients were included . Efficacy at 52 weeks (plasma RNA-HIV <50 copies/ml) was 67.7% (CIOur results suggests that SQVr (1500/100 mg) once-daily plus 2 NRTIs is an effective regimen, without severe clinical adverse events or hepatotoxicity, scarce lipid changes, and no interactions with methadone. All these factors and its once-daily administration suggest this regimen as an appropriate option in patients with no SQV resistance-associated mutations. Saquinavir was the first protease inhibitor (PI) commercially available for the treatment of patients with HIV infection. Its oral bioavailability is markedly increased when concomitantly administered with low dose retainer, which allows for reduced dosing frequency and dosage. Ritonavir-boosted saquinavir (SQVr) at the standard dosing of 1000/100 mg twice daily has shown as effective as ritonavir-boosted-lopinavir, although requiring a higher pill burden when prescribed as the 200 mg hard or soft-gel capsules, which frequently leads to a bad compliance and high rates of therapy discontinuation ,4. On thSQV 500 mg strength tablets became available at the end of 2005. This formulation would facilitate a once-daily regimen (1500/100 mg) with fewer pills, although the experience with this dosage is still very scarce .The aim of the present study was to assess the efficacy, safety and pharmacokinetics of once-daily SQVr 1500/100 mg plus 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) in antiretroviral-naive patients or in those with no previous antiretroviral treatment history and/or genotypic resistance tests suggesting SQV resistance, under routine clinical care conditions.A total of 518 patients started a regimen of SQVr (1500/100 mg qd) plus 2 NRTIs at the mentioned centres during the mentioned period. One hundred and twenty patients  had a genotypic resistance test available just before starting SQVr. Four experienced patients had HIV protease mutations associated with SQV resistance (L90M) and were excluded from further analysis. Among the remaining cases, 33 (27.5%) had wild-type isolates, and 71 (59.1%) had resistance mutations in the reverse transcriptase (TAMs in 29 patients with a median (range) of 2 (1 -5); the K65R mutation was present in 5, the L74V in 6, and the M184I/V in 44; other mutations which confer resistance to non-nucleoside reverse transcriptase inhibitors was observed in 53 patients). Sixty eight patients had PI-related mutations, either minor mutations or polymorphism in most cases. One minor SQV-related mutation was present in 16 cases (L10I/V or I54V or I62V or A71T/V or V77I), and 3 minor resistance mutations  in 1 case. Genotypic resistance tests were not available in the rest of the patients, since amplification was not possible in cases with a VL <1000 copies/ml, or the test had not been requested in cases of treatment interruption for a long period, so that it was not expected to add relevant data.The baseline characteristics of the 514 patients included in the analysis  are summarized in table 95: 63.6 - 71.7%) by ITT, and 92.2% (CI95: 89.8 - 94.6%) by on-treatment analysis  and with no significant differences between the other groups. By ITT, 135 patients (26.2%) failed because of treatment dropout or loss to follow-up in 95 cases (18.4%), AEs in 16 cases (3.1%), and other reasons  in 24 patients (4.6%). Virological failure occurred in 40 patients (7.8%): group A, 6/50 (12%), group B, 10/80 (12.5%), group C, 10/81 (12.3%), and group D, 14/303 (4.6%). In 17 of them VL had not achieved <50 copies/ml after 24 weeks of treatment , and the other 23 showed a confirmed viral load >200 copies/ml after a previously undetectable viral load. The variables associated with virological failure in the univariate analysis were baseline VL >100000 copies/ml, baseline CD4 count <200/μl, and non-adherence. No relationship was found between virological outcome and either treatment group, baseline PI-related mutations, earlier PI failure, methadone treatment or active illegal drug use. The only variable associated with virological failure in the multivariate analysis was adherence .For the whole of patients, the treatment efficacy at 52 weeks was 67.7% . Rash appeared in 4 patients (0.7%), in 3 of them it was related with abacavir. Grade 1 serum creatinine elevations occurred in 5 patients, 4 of them concomitantly receiving tenofovir. Five of the 6 cases of lypodystrophy were patients from the "simplification" group and 1 from the "ART-restart" group ; in LDL-cholesterol, -1 mg/dl , in HDL-cholesterol, 0 mg/dl , and in triglyceride levels -9 mg/dl , respectively. Among the patients starting or restarting ART (groups A and B), the median change at week 52 in total cholesterol value from baseline was 12 mg/dl , in LDL-cholesterol 2 mg/dl , in HDL-cholesterol 8 mg/dl , and in triglyceride levels -6 mg/dl , respectively  weighing a median of 65.5 kg . Twenty four (49%) of them were affected by viral chronic hepatitis and 9 (18.3%) by cirrhosis. The median SQV trough level was 295 ng/ml ; four patients (8.1%) had values under 100 ng/ml, and 3 of them had a satisfactory virological response. No correlations were found between SQV levels and either weight, gender or the presence of chronic hepatitis and/or cirrhosis.95 value (25 ng/ml) for wild HIV-1 isolates and the estimated trough level required to obtain the half-maximal antiviral response (EC50: 50 ng/ml) [min below 100 ng/ml, and 3 of them had a satisfactory virological response. Although 100 ng/ml is suggested as the minimum target trough concentration for wild-type HIV-1 [in vivo, resulting in a median intracellular drug accumulation ratio of 2.75-3 as compared with that in plasma, suggesting that intracellular exposure to SQV may be a better predictor of the virological response to therapy [Although the approved dosage of SQVr is 1000/100 mg twice daily, several once-daily schemes  plus 2 NRTIs have demonstrated a good virological efficacy in patients with no SQV resistance mutations -11. From0 ng/ml) . In our 0 ng/ml) . Four oupe HIV-1 , this va therapy . Thus, oThe efficacy in the ITT analysis in our series (65.7%) is similar to that observed in the MaxCmin2 trial and in the Gemini study with a 1000/100 bid SQVr dosing [Evaluation of the available genotypic tests from patients with virological failure revealed a low incidence of selection of protease inhibitors major/minor resistance mutations following treatment with SQV/r, which is consistent with previous observations made for boosted protease inhibitors, mainly in naive patients (3).The combination of once-daily SQVr was well tolerated during the 52-week follow-up, with no clinical grade 3 or 4 adverse events recorded. Only 12 patients (3.0%) changed their regimen because of AEs. Although lypodystrophy occurred in 6 patients after switching treatment to SQVr, this may reflect long-term antiretroviral drug exposure rather than an effect caused only by this regimen, since 5 out of 6 patients belonged to the "simplification"group, and the remaining one to the ART-restart group. Particularly meaningful are the results regarding liver toxicity in a population with 58.7% of the patients presenting chronic hepatitis , 10,7% of them being cirrhotic. Among patients with and without chronic hepatitis and/or cirrhosis, only 1 (0.6%) and 13 cases (5.4%), respectively, developed grade 3-4 transaminase increases. Moreover, none of these cases was symptomatic, and the alterations observed were transient and improved without treatment discontinuation in every case, which may indicate that much of these elevations could be due to the natural evolution of chronic hepatitis and/or cirrhosis rather than caused by the treatment. Also, it is remarkable that patients receiving this regimen showed no changes, or just negligible increases, in the levels of total cholesterol, LDL cholesterol, and triglycerides.The absence of relevant pharmacokinetic interactions between SQVr and methadone is an additional advantage in patients on methadone maintenance therapy ,24.We are aware that the open-label characteristics of the study, the heterogeneity of the analyzed population and the lack of available genotypic resistance tests in some of the patients at baseline and after virological failure are limitations of our study, although they reflect the real-life clinical setting.This open-label multicentre study suggests that SQVr (1500/100 mg) once-daily plus 2 NRTIs is an effective regimen, without severe clinical adverse events or hepatotoxicity, scarce lipid changes, and no interactions with methadone. All these factors and its once-daily administration make this regimen make this regimen worth to be considered as an alternative in patients with no SQV resistance-associated mutations. In addition, the 1500/100 mg qd dosage is one of the cheapest PI combinations and, given that the patent will soon expire, a more affordable generic formulation would then be available, making it possible a more extended use of this drug.From November 2005 to May 2007, HIV-1 infected patients older than 18 years attended at the HIV clinics in 17 hospitals from Andalusia, Ceuta and Extremadura (Spain), and scheduled to receive a regimen of SQVr 1500/100 mg once-daily plus 2 NRTIs, were consecutively enrolled in this observational, prospective, single-arm, open-label study. NRTIs prescribed as part of HAART were selected by the responsible physicians on the basis of previous antiretroviral treatments (ART) and/or genotypic resistance testing. Patients were enrolled and followed-up under routine clinical care conditions, and no entry restrictions were made except for pregnancy, history of previous ART and/or genotypic resistance tests suggesting resistance to SQV according to the 2005 International AIDS Society , and thePatients' assessment was performed at baseline, after the first month on treatment and every three months thereafter, including adverse effects (AEs), biochemical and hematologic profiles, flow cytometric count of CD4/μl and plasma HIV-1-RNA (VL) measured by polymerase chain reaction . Adherence was evaluated by personal interview at each following visit. Efficacy at 52 weeks, analyzed by intention-to-treat (ITT), was the primary clinical endpoint. Virological failure was defined as inability to suppress plasma VL to <50 copies/ml after 24 weeks on treatment, or a confirmed viral load >200 copies/ml in patients who had previously achieved a viral suppression or had an undetectable viral load at inclusion. If confirmed, the time of the first measurement meeting the failure criteria was selected as the time of failure. Secondary outcomes included virological efficacy according to on-treatment (OT) analysis, changes in CD4 cell counts, incidence of AEs and lipid profiles. The changes in serum ALT and AST from pre-treatment levels to the highest level during treatment were categorized via a standardized toxicity grade scale, modified from that used by the AIDS Clinical Trials Group. Patients with pre-treatment serum AST and ALT levels within normal range (AST <35 IU/L and ALT <31 IU/L) were classified according to the changes observed with respect to the upper limit of normal (ULN): grade 0 (<1.25 ULN); grade 1 (1.25-2.5 × ULN); grade 2 (2.6-5 × ULN); grade 3 (5.1-10 × ULN); and grade 4 (>10 × ULN). In patients with chronic viral hepatitis or cirrhosis, toxicity was classified according to changes relative to baseline values rather than ULN: grade 0 (<1.25 × baseline); grade 1 (1.25-2.5 × baseline); grade 2 (2.6-3.5 × baseline); grade 3 (3.6-5 × baseline); and grade 4 (>5 × baseline). Genotypic resistance tests were performed in patients with virological failure whenever viral load levels allowed. Patients missing two consecutive scheduled visits were considered lost to follow-up.Blood samples for SQV plasma levels were obtained 24 ± 0.3 hours post-dose, after at least one month on treatment, from random patients who usually took SQVr in the morning and who were included in the study at Hospitales Universitarios Virgen del Rocío. Plasma samples were stored frozen at -80°C for determination of SQV by high-performance liquid chromatographic assay according to a validated method .Descriptive statistic was used for demographic, epidemiological and clinical data, prior ARTs, CD4 cell count, viral load and SQV trough concentrations. Kaplan-Meier plots were produced for the 'time to event' analyses and comparisons among the 3 treatment groups were made using the log-rank test. The relationships between virological failure and different variables were assessed by the chi-square test for qualitative variables and by the Spearman's rank-correlation coefficients for quantitative variables. The variables tested by univariate analysis as predictors of virological failure with a p value <0.1 were included in a multivariate analysis to identify possible independent predictors of virological failure. Statistical calculations were performed with the Statistical Product and Service Solutions for Windows .LFLC, PV, FZ, and AR have received unrestricted funds for research and honoraria for speaking at symposia organized on behalf of Abbott laboratories (Spain), Bristol-Myers Squibb, GlaxoSmithkline, Gilead Sciences, Janssen-Cilag España, Merck Sharp & Dohme España, and Roche Pharma SA. Other authors: none to declare.Conception, design, analysis, interpretation of the data, drafting of the article and obtaining of funding: LFLC.Provision of study materials or patients: LFLC, PV, JP, JR, FL, DM, AV, AT, LG, AR, AMS,Determination of saquinavir plasma concentrations: RRV.Critical revision of the article for important intellectual content: PV, RRV, JP, JR, FLo, DM, AV, AT, LG, AR, and AMS.Final approval of the article: LFLC, PV, RRV, JP, JR, FL, DM, AV, AT, LG, AR, and AMSCollection and assembly of data: LFLC, PV, JP, JR, FL, DM, AV, AT, LG, AR, AMS.All authors have read and approved the final manuscript.


An effective therapeutic vaccine that could augment immune control of HIV-1 replication may abrogate or delay the need for antiretroviral therapy. AIDS Clinical Trials Group (ACTG) A5187 was a phase I/II, randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of an HIV-1 DNA vaccine (VRC-HVDNA 009-00-VP) in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. Twenty healthy HIV-1 infected subjects who were treated with antiretroviral therapy during acute/early HIV-1 infection and had HIV-1 RNA<50 copies/mL were randomized to receive either vaccine or placebo. The objectives of this study were to evaluate the safety and immunogenicity of the vaccine. Following vaccination, subjects interrupted antiretroviral treatment, and set-point HIV-1 viral loads and CD4 T cell counts were determined 17–23 weeks after treatment discontinuation.10 HIV-1 RNA copies/mL, respectively.Twenty subjects received all scheduled vaccinations and discontinued antiretroviral therapy at week 30. No subject met a primary safety endpoint. No evidence of differences in immunogenicity were detected in subjects receiving vaccine versus placebo. There were also no significant differences in set-point HIV-1 viral loads or CD4 T cell counts following treatment discontinuation. Median set-point HIV-1 viral loads after treatment discontinuation in vaccine and placebo recipients were 3.5 and 3.7 logThe HIV-1 DNA vaccine (VRC-HIVDNA 009-00-VP) was safe but poorly immunogenic in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. Viral set-points were similar between vaccine and placebo recipients following treatment interruption. However, median viral load set-points in both groups were lower than in historical controls, suggesting a possible role for antiretroviral therapy in persons with acute or early HIV-1 infection and supporting the safety of discontinuing treatment in this group.NCT00125099Clinicaltrials.gov  Despite the striking decline in morbidity and mortality in persons receiving antiretroviral therapy One approach to inducing HIV-1-specific immunity is through the delivery of multiple viral antigens by DNA plasmids. The DNA vaccine VRC-HIVDNA009-00-VP is a 4 plasmid mixture encoding a subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from HIV-1 subtypes A, B and C. This multiclade DNA vaccine has previously been evaluated in a phase I dose escalation study in healthy, HIV-1-uninfected adults and was found to be safe and well toleratedHere we report the findings from ACTG A5187, a phase I/II, randomized, placebo-controlled, double-blinded trial of the DNA vaccine VRC-HIVDNA009-00-VP . The first phase of this study was to evaluate the safety of the vaccine, which was the primary aim. The second phase of the study was to determine if there was a difference between the two treatment arms in HIV-1 RNA levels and CD4 T cell counts at viral load set-point after antiretroviral therapy was discontinued. Exploratory analyses assessed the immunogenicity of the vaccine. The study enrolled 20 healthy, HIV-1 infected subjects who were treated with antiretroviral therapy during acute or early infection. The rationale for studying persons treated during acute or early HIV-1 infection was to test this vaccine in persons presumed to have relatively preserved immune functionThe protocol for this trial and supporting CONSORT checklist are available as supporting information; see 3 and HIV-1 RNA levels <50 copies/ml for at least 6 months. All subjects gave written informed consent, and the study protocol was approved by the AIDS Clinical Trials Group, the NIH Division of AIDS (DAIDS) and the human protection committees of each participating institution.Twenty healthy HIV-1 infected adults, aged 26–47, who were treated with antiretroviral therapy during acute or early HIV-1 infection participated in this study . Subjects with treated acute HIV-1 infection were defined as initiating antiretroviral therapy after being diagnosed by a positive HIV-1 viral load and either a negative or indeterminate Western blot. Early infection was defined as having a positive ELISA or a positive Western blot with a non-reactive detuned ELISA (OD<0.75), provided the interval between the presumed acute retroviral syndrome and initiating antiretroviral therapy was 6 months or less. Subjects were required to be on a stable antiretroviral regimen and have a CD4+ T cell count >350 cells/mmThe vaccine used in this study was developed by the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). This vaccine consisted of a 4 plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope constructs from HIV-1 subtypes A, B and C.This was a randomized, double-blind, placebo-controlled phase I/II clinical trial to assess the safety and immunogenicity of the HIV-1 DNA vaccine in HIV-1-infected subjects who were treated with at least 2 antiretroviral agents during acute or early infection and who maintained an HIV-1 RNA viral load of <50 copies/mL. Five ACTG sites enrolled subjects into this study: Massachusetts General Hospital, Brigham and Women's Hospital, Aaron Diamond AIDS Research Center, University of California at San Diego and University of Washington. The first part of the study (phase 1) was designed to assess the safety and immunogenicity of the vaccine. With 10 active vaccine recipients, there would 80% probability of observing at least one safety endpoint if the per-subject probability is 15%. The second part (phase 2) of the study involved a supervised treatment interruption in order to determine if vaccination with HIVDNA009-00VP resulted in improved immune control of viral replication as evidenced by a reduction in the set-point level of HIV-1 RNA in the absence of antiretroviral therapy.For the first part of the study, subjects were randomized in a 1∶1 fashion to vaccine (Arm A) versus placebo (Arm B) using permuted blocks. The vaccine or placebo was administered as an intramuscular 1 ml injection using a needle-free Biojector 2000™. Subjects received 4 vaccinations with 4 mg of DNA vaccine or placebo at weeks 0, 4, 8 and 24.3, or a confirmed HIV-1 RNA level of >100,000 copies/mL for at least 8 weeks. All subjects were followed until completion of the study at week 52 and those subjects who did not meet criteria to restart therapy or elected not to restart therapy were followed an additional 20 weeks until week 72.At week 30, all individuals safely completing the therapeutic vaccination phase of the trial were given the opportunity to discontinue therapy. During the supervised treatment interruption phase of this study, subjects were monitored closely and asked to restart antiretroviral therapy if they met the following criteria: a confirmed decline in CD4 count from baseline of >50% or an absolute CD4+ T lymphocyte count of <250 cells/mm3 while receiving antiviral therapy; or 2 consecutive CD4 counts more than 50% below the baseline CD4 count. Only events that occurred on or subsequent to the first vaccination and within 24 weeks of the last vaccine administration were considered.The primary objective of this study was to evaluate the safety of the vaccine. The primary safety endpoint was the development of a grade 3 or higher sign, symptom or laboratory abnormality that was at least possibly related to the vaccine; 2 consecutive viral loads ≥400 copies/mL while on antiretroviral therapy; or 2 consecutive absolute CD4 counts ≤250 cells/mmThe immunogenicity of VRC-HIVDNA009-00-VP was determined by the following assays: unfractionated interferon-γ ELISPOT, CD4 (CD8-depleted) ELISPOT, CD8 (CD4-depleted) ELISPOT and,lymphocyte proliferation. ELISPOT assays were performed using the following peptide pools : Gag, Pol-1, Pol-2, Nef, Env-A, Env-B, and Env-C. Baseline ELISPOT responses were determined by taking the geometric mean of pre-entry and entry. Positive responses were defined as a 2-fold increase from baseline that were also ≥100 spot forming cells (SFC) per million PBMC. Since 7 HIV-1 antigens were tested, a positive ELISPOT sum response was defined as a 2-fold increase from baseline that was also ≥700 spot forming cells per million PBMC. Lymphoproliferative responses to CMV, Nef, rt, Gp160, and p24 were assessed by the stimulation index. A stimulation index of ≥5 was considered a positive response.10 viral load measured at weeks 18, 20 and 22 after antiviral withdrawal . Similarly, CD4+ T cell counts were determined at the same time points as the viral load measurements. The CD4+ T cell count prior to interruption of therapy was determined by taking the average of the last 2 observations prior to treatment interruption.The main secondary endpoint of this study was the viral load set-point defined as the average of the logThe study team conducted a weekly review of adverse events including all reported signs and symptoms and laboratory abnormalities. The study team remained blinded to the randomization assignment of the study subjects. After reviewing all reported events, the team assessed the possible relationship of adverse events to the study vaccine. In addition, an independently appointed Study Monitoring Committee was convened to review the study data, broken down by vaccine and placebo arm.Individuals receiving vaccine versus placebo were summarized using medians and compared using exact Wilcoxon Rank-Sum tests. A Hodges-Lehmann confidence interval was used for the difference in viral load setpoint between arms (vaccine minus placebo). Time-to-event endpoints were compared with exact log-rank tests. All tests were two-sided, 5% level and exploratory.The viral load set-point analysis was based on subjects who entered the treatment interruption phase of the study and who had an observed viral load set-point. Subjects who restarted antiviral therapy before week 12 of treatment interruption were not included in the viral set-point analysis. A supplemental intent-to-treat sensitivity analysis, which included all subjects, was also performed. In this analysis, the last observed HIV-1 RNA value during treatment interruption was carried forward for any subjects who did not have an observed viral load set-point. Analysis of CD4+ T cell count was also carried out in the same manner.3 . All subjects had HIV-1 RNA levels <50 copies/mL at study entry and all received the 4 scheduled vaccinations. At week 30, all 20 subjects elected to discontinue antiretroviral therapy. Visit compliance and data availability were ≥90%.Twenty subjects were enrolled in this study . Ten subThe vaccine was safe and well tolerated. No subjects experienced grade 3 or 4 signs, symptoms, or laboratory abnormalities that were at least possibly related to the vaccine. Moreover, no subjects exhibited detectable viral loads during the vaccination phase of the study while on antiretroviral therapy. No subject met any primary safety endpoint.All 20 subjects discontinued antiretroviral therapy at week 30. No subject restarted therapy due to safety endpoints, which included sustained high viral loads or declines of CD4 counts see . One sub10 RNA copies/ml in Arms A and B, p = 0.50, 95% confidence interval: −0.9 to 0.6 log10 copies/ml; sensitivity analysis, n = 20, p = 0.63). There were also no evidence for differences between the two groups in exploratory analyses of peak viral loads during the treatment interruption , time to peak viral load , or time to detectable viral load . Moreover, no differences in CD4 counts were observed at set-point following treatment discontinuation . There were also no detectable differences in viral loads or CD4 counts following treatment interruption between the two arms when separately analyzed by initial treatment during acute vs. early infection (data not shown).HIV-1 viral loads and CD4 counts are shown in 10 copies/ml for Arms A and B, respectively (p = 0.48). Similarly, CD4+ T cell counts were also determined and were 499 and 698 cells/mm3, Arms A and B respectively (p = 0.28)All subjects remaining off antiretroviral therapy at week 52 were asked to extend the period of follow-up for an additional 20 weeks . Seventeen subjects agreed to be followed for this extended time period. HIV-1 RNA and CD4+ T cell measurements were obtained at week 42 of the treatment interruption phase. The median HIV-1 RNA viral load at week 42 of treatment interruption was 4.4 and 4.2 logCD4 and CD8 ELISPOT responses are depicted in ACTG A5187 assessed the safety, immunogenicity and viral load set-point following discontinuation of antiretroviral therapy in HIV-1-infected subjects who received either HIV-1 DNA vaccine or placebo. All subjects enrolled in the study were healthy HIV-1-infected subjects who were diagnosed and treated with antiretroviral therapy during acute or early HIV-1 infection. Twenty subjects were followed through phase 1 (the immunization phase) of the study and entered phase 2 (the treatment interruption) of the study. Of the 20 subjects who discontinued antiretroviral therapy, 2 subjects restarted therapy and 18 remained off therapy at the end of the study. This study was designed using a relatively small sample size based on limited availability of vaccine and anticipated difficulty in recruitment of subjects with treated acute or early HIV-1 infection.The primary objective of this study was to determine the safety of the HIV-1 DNA vaccine when used therapeutically in healthy HIV-1-infected subjects. In this study, no subject experienced any serious (grade 3 or 4) adverse events during the administration of the HIV-1 DNA vaccine, and the vaccine was safe and well tolerated. These safety data are consistent with a prior study utilizing this vaccine in HIV-1-uninfected individualsAll subjects safely discontinued antiretroviral therapy at week 30, and no safety endpoints were reached during this phase of the trial. No subjects met predetermined criteria to restart therapy. These findings contrast with those reported in the SMART study that assessed the efficacy of continuous versus episodic use of antiretroviral therapy in persons with chronic HIV-1 infectionAlthough the HIV-1 DNA vaccine was safe and well tolerated, the vaccine exhibited minimal immunogenicity that appeared lower than what has been reported in prior studies in HIV-1-uninfected volunteers in which significant humoral and cellular immune responses were observed10 respectively. When these viral load set-points are compared to those described in the natural history Multicenter AIDS Cohort (MACS) study10 10  in subjects completing the treatment interruption phase of the present study. Since there was not a significant difference between the vaccine group and placebo group, it is unlikely that administration of the HIV-1 DNA vaccine contributed to the low viral load set-point in this study. It is therefore possible that early treatment during acute or early HIV-1 infection may have resulted in better control of viral replication once antiretroviral therapy was discontinued, although this hypothesis needs to be evaluated in larger prospective studies. Such a finding would be consistent with previously described observationsOne notable finding of this study is the low viral load set-point in subjects who completed the treatment interruption phase of the study. Although there were no evidence for vaccine efficacy in terms of a difference in viral set-points between subjects receiving the vaccine compared to the placebo (p = 0.50), both the vaccine group and the placebo group had low median viral set-points of 3.5 and 3.7 log Protocol S1Trial Protocol.(0.60 MB DOC)Click here for additional data file.Checklist S1Consort Checklist.(0.19 MB DOC)Click here for additional data file.


The optimal stage for initiating antiretroviral therapies in HIV-1 bearing patients is still a matter of debate.pro et contra of immediate as compared to deferred Highly Active Antiretroviral Therapy (HAART).We present computer simulations of HIV-1 infection aimed at identifying the + cytotoxic T lymphocytes response is detected when therapy is delayed. Compared to very early initiation of HAART, in deferred treated patients CD8+ T cells manage to mediate the decline of viremia in a shorter time and, at interruption of therapy, the virus experiences a stronger immune pressure. We also observe, however, that the immunological effects of the therapy fade with time in both therapeutic regimens. Thus, within one year from discontinuation, viral burden recovers to the value at which it would level off in the absence of therapy.Our simulations highlight that a prompt specific CD8In summary, simulations show that immediate therapy does not prolong the disease-free period and does not confer a survival benefit when compared to treatment started during the chronic infection phase.Our conclusion is that, since there is no therapy to date that guarantees life-long protection, deferral of therapy should be preferred in order to minimize the risk of adverse effects, the occurrence of drug resistances and the costs of treatment. Positive effects of Higly Active Antiretroviral Therapy (HAART) include suppression of plasma viremia below detection level, rising circulating CD4of death -3. It issmission ,4-6. Howimmunity . Possiblimmunity . The demin silico patients treated with HAART either in the acute phase of primary infection or at a later date during the chronic disease. The computational model employed closely reproduces the hallmarks of HIV-1 infection for both drug-free  and treOur results suggest that deferral of HAART favors HIV specific cytotoxic T lymphocytes while minimizing toxic side-effects, when compared to very early therapeutic intervention.i.e., including both the inflammatory phase and after seroconversion). Table et al [We started from the analysis of the cohort data of naïve patients that received HAART  within six months from primary infection , all subjects were enrolled in Structured Therapy Interruption (STI). For the majority of the subjects, plasma viremia reverts to detectable levels in a median time of ten days following the initial HAART interruption. Despite this rapid viral rebound, five subjects managed spontaneously to control viremia maintaining their plasma virus load below the detection limit for a longer time . Then, we fixed immunological parameters at therapy start time on the basis of the average values measured in in vivo patients: (5.8 ± 0.2) RNA copies/ml , (870 ± 50) CD4 cells/μl and (430 ± 50) CD8 cells/μl. Finally, the drug control effect of HAART is to suppress viremia, during the first ten weeks, below the detection level. This occurs in three phases with a specific time table [silico patients has been compared with those obtained from in vivo patients whose viral load was known in the same time window  within six months from primary infection . Finally, the virus rebounds in one year off therapy, to reach the value at which it would level off in the absence of treatment (green rectangle in panel c). These findings are confirmed by in vivo data in which the median viremia one year after stopping HAART is about 10,000 RNA copies/ml  = 4, blue rectangles in panel c). Despite considerable advances in the understanding of the interplay between HIV-1 and its host, the effect of antiretroviral treatment on the developing immune response remains elusive. During the acute stage of HIV-1 infection in untreated subjects, viral load expands exponentially and antiviral immune defenses are still developing [in silico when HAART is delayed. In particular, in order to determine the optimal time to initiate HAART, we performed simulations in which we started a long course of HAART (2.5 years) either during seroconversion  and the CD4+ T cell count was (400 ± 50) cells/μl. Moreover, we compared CD4+ T cell count and viremia between treated and untreated in silico patients .Due to the inability to eliminate virus infected cells, antiretroviral treatment fails to eradicate the infection and cessation of suppressive therapy will, most likely, be accompanied by viral rebound. We have analyzed the pace of viral rebound and the ability of early-treated veloping . Once thveloping . We have HAART 2. years eiin silico patients still manage to spontaneously control viremia, whereas the virus rebounds completely one year after therapy discontinuation in early treated infected persons .Although the impact of a therapeutic intervention on plasma viremia is well documented -15, much+ and CD8+ T cells in patients treated with HAART, are subject of heated debate [in silico patients following such an antiviral therapy regimen show a modest loss of CD8+ T cell numbers coincident with a decline in viremia (green line in panel c and a). When therapy ends, the viral load rises again within a year, but in in silico patients undergoing early treatment the natural immune activity remains impaired. It thus takes several years to restore the natural equilibrium between the host and its uninvited guest. On the contrary, prior to administration of HAART in chronically infected patients, HIV-1 infection is associated with a failure in T cell homeostasis [+ T cell numbers (red line panel b), whereas the cytotoxic activity is well developed (red line in panel c). Once therapy starts, CD4+ T lymphocyte counts quickly increase, bringing the host almost to immune restoration.The evolution of the immune response and, in particular, the kinetics of CD4d debate ,16. Our eostasis , resultiSimulation of delayed therapy produces enhancements of both functional immune response and immune control of infection. Our interpretation is that, during the period prior to initiation of therapy, the virus elicits a cytotoxic response. This, by contrast, is almost absent in early treatment, as the magnitude of the infection is considerably reduced by the action of the antiretroviral drugs.+ count and prevents potentially irreversible damage to the immune system; it reduces the risk of HIV-associated complications [In vivo studies have often been constrained by a lack of adequate human and economic resources. Clinical trials are demanding and require long follow-up. There is also the difficulty of recruiting patients during the acute phase of the infection. In this respect, a computer model may provide a great deal of insight into the dynamics and control of HIV-infection. In particular, the use of a computer model for the simulation of HIV-infection as a predictive tool acquires a clinical and an epidemiological role, as it offers the possibility to reduce the time and the costs of antiretroviral research, potentially leading to a reduction in public health expenditure. The objective of the present analysis is to gauge by means of an immune system simulator the differences in the impact of long-term HAART when initiated either in the primary or in the chronic phase of HIV-1 infection.The timing of when antiretroviral therapy has to be initiated remains a challenging issue. HAART is costly, it is demanding for both patient and health care provider, and it leads, quite frequently, to adverse events. The clinical benefit of treatment must therefore be weighed against the burden imposed by therapy on the patient and its the side-effects. Immediate treatment has been always advocated as it preserves a higher CD4ications ; it may ications ,5. Howevications . To date+ T cells have been thought to mediate the decline of viremia and recent studies show that the interplay between viral replication and CD8+ activity influences the equilibrium viral-set-point [There is increasing evidence that HIV-specific cytotoxic response plays a crucial role in controlling viremia ,18-21. Cet-point .in silico patients regain specific CD8+ response. The time it takes to mount an effective natural immune response depends on the disease's progression before therapy was initiated. Computer simulations indicate that, while the immune functions are preserved during immediate therapy, the initiation of antiretroviral treatment before full emergence of a specific immune response prevents cytotoxicity from developing, whereas, in patients receiving deferred therapy, the immune system has already mounted a specific cytotoxic response in the phase prior to HAART, and is able to dump viral load and to reduce virus rebound at therapy interruption. In addition, in the presence of prompt natural immunity, the virus cannot evolve under the selective pressure of the drug (no drug escapes are detected). This difference fades within one year following discontinuation. We therefore found no valid arguments to promote very early therapy. Although other factors may explain the different immunological effects of early as compared to late initiation of HAART, our findings suggest a rationale to support deferred therapy in clinical practice. This is in line with an in vivo study that underlines the importance of an effective natural host-immune response [+ T cells since these cells constitute both a reservoir for HIV and a stimulus for CD8 cell proliferation. We suggest that a higher level of CD8 in lately treated patients with respect to those who receive early treatments is a consequence of the fact that the immune system has spent considerable time to control viremia before HAART initiates and therefore there is a higher level of cytotoxicity. However, in the simulations, the difference in viremia between immediate and deferred treated patients is very small, likewise the difference in CTLs (~10%) and therefore it is difficult to draw final conclusions. In [We found that cytotoxic responsiveness declines during long term HAART exposure in both the acute and the chronic infection stages. However, when therapy terminates, and viremia restores, response . It's reions. In  the authHowever, there are two major differences between the cohorts used in  and in tTreatment interruptions are not currently recommended as routine clinical practice, although several strategies are being investigated in clinical trials. The occurrence of adverse events may, however, require cessation of therapy. At present, a large multicentric study for the treatment of primary HIV-1 infection is under investigation. This is SPARTAC , an international, randomized, controlled trial comparing three different strategies of intervention in patients recently infected with HIV-1.The computer model used in the present analysis, the effectiveness of which has been tested in previous studies ,22, may i.e., beyond stage VI of [i.e., more than about 25 days from HIV exposure). Patients treated with HAART in very early stages of the infection (stage I-III) are likely to better control viremia after treatment interruption [In conclusion, we employed a simulator of the immune system tested against clinical data aimed at identifying the golden moment to start the therapy. We observe that if the therapy starts in the acute phase (stage IV-VI of ) then thge VI of ), then arruption . As a fupro tempore guidelines. Specifically, patients in arm B resumed treatment only when necessary  and stopped HAART when HIV-RNA became again undetectable. This on/off treatment regime was undertaken for the first 12 months. At this time point, all patients suspended HAART and were monitored for viral and immunological parameters up to month 24. Eligibility criteria to participate in the study were as follows: CD4+ T cell count ≥ 500 cells/μl and HIV viremia in the last two years below the detection limit, to have initiated HAART during HIV primary infection. In addition, eight patients followed unplanned treatment interruption mandating re-initiation of therapy according to medical approval  with primary HIV-1 infection were selected at the Clinical Department of the National Institute for Infectious Disease "L. Spallanzani" in Rome. Criteria for diagnosis were: documented seronegative HIV-1 antibody test within the previous 6 months; acute symptomatic seroconversion illness; evolving HIV-specific antibody response by ELISA; positive HIV-DNA PCR in PBMC, or positive plasma HIV-RNA quantification in the absence of an antibody response. Fourteen subjects were enrolled into a pilot study of Structured Therapy Interruption (STI). Two different therapy interruption protocols were used: arm A (seven patients), 4 weeks off/8 weeks on HAART; arm B (seven patients), intermittent therapy guided by plasma HIV-RNA levels, according with The ethical committee of the institute  approved this study and the patients gave a written informed consent to the blood sampling for the study.ml; Bayer Diagnostics, Milan, Italy) from 2001 until 2008.Plasma HIV-1 RNA levels were determined by a second-generation assay based on nucleic acid sequence based amplification (NASBA), for samples collected until 2001 and by the branched-chain DNA assay  and some of the myeloid lineage . All these entities behave as finite state machines following a set of "rules" that describe the different phases of the recognition and response of the immune system against a pathogen.The model of immune system response we employ has been quite extensively described in ,22-25. Ietc. . The affi) HIV replication inside infected lymphocytes; ii) T production impairment; iii) specific response against HIV strains; iv) HIV mutation and evolution. The evolution of HIV in untreated patients has been studied and described by means of this model in [The model represents a single lymph node of a vertebrate animal that is mapped onto a three-dimensional ellipsoid lattice with periodic boundary conditions Figure . The primodel in .l bits long); the first one corresponds to the epitope  and the second one to the peptide . The simulator allows the definition of an arbitrary number of epitopes and peptides. In the simulations the bit string length l is equal to 16 corresponding to a potential repertoire of 65536 distinct receptors and molecules. Actually, since the virus is represented by one epitope and a single peptide, each strain is identified by 2 l = 32 bits. So, the potential number of different virus strains becomes equal to 232. Each time step of the simulation corresponds to eight hours of "real life".The virus is represented by two bit strings , molecules with small molecular weight like interleukins or chemokines are represented in terms of concentrations and their dynamics described by the following parabolic partial differential equations plus a degradation term accounting for the finite half-life of molecules:While the virus and the antibodies are uniquely represented (c = c(x) is the concentration of chemokines, D is the diffusion coefficient and λ is the half-life. We assume D = 3000 μm2/min and λ = 3 hrs [μm/min, followed by B cells with 6 μm/min and DC with a velocity of 3 μm/min [where  = 3 hrs ,29. Diffmix of three or more powerful antiretroviral drugs, commonly reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). Although most studies agree that HAART is not able to eradicate the virus, it is very successful in allowing the stabilization of patients' symptoms and viremia. The simulated life cycle of the virus is represented by the following stages: 1) the virus infects CD4+ T cells, macrophages, dendritic cells; 2) reverse transcriptase copies the viral single stranded RNA genome into a double-stranded viral DNA. The viral DNA is then integrated into the host chromosomal DNA; 3) the virus remains at rest until an event activates the transcription; 4) the replicating virus buds from the cell membrane. Fully assembled virions are then able to infect other cells to restart the life cycle. HAART composed of transcriptase and protease inhibitors influences the life cycle of the virus as follows: RTIs block reverse transcriptase enzymatic function and avoid completion of synthesis of the double-stranded viral DNA thus preventing HIV-1 from replicating ; PIs prevent viral replication by inhibiting the activity of HIV-1 protease, an enzyme used by the virus to cleave nascent proteins for final assembly of new virions . While RTI leads to cells carrying the equivalent of the HIV-1 RNA, PI leads to "inactive" virus which is produced in infected cells and that accounts, among other things, for the HIV-1 DNA observed in real life in patients during HAART. Notice that since the clinical data available only included HIV-1 RNA counts, as an indicator of the viral rebound we use just HIV-1 RNA.HAART is a + T cell replenishment due to the HIV by decreasing the physiological "normal" cell count, whereas immune reconstitution due to the HAART is modeled by increasing it. The parameters of this process are set so as to reproduce, in absence of therapy, a decrease of CD4 cell count in line with clinical expectations of time to AIDS see [+ T cell increase of about 200 cells/μl in one year [We model the damage of the CD4AIDS see  whereas one year ,32.i.e., an acute phase of primary infection lasting from three weeks to one year [etc.); (2) parameters that correspond to the initial conditions of the system and that determine the problem under investigation (for the setting of these parameters see the Section Results); (3) parameters whose value is well known and available from immunology literature. An accurate description of the all parameter setting is given as supplementary material  unknown values (free parameters) which are set after a tuning procedure that starts with an initial guess and iteratively improves by looking at the outcomes that have to reproduce the well know dynamics of HIV-1 infection ;  unknown An educational version of the immune system simulator is available on our website:- Name: C-ImmSimhttp://www.iac.rm.cnr.it/~filippo/C-ImmSim.html- Home page: - Operating system(s): Linux, Unix Mac OS X, Windows- Programming language: Chttp://www.iac.rm.cnr.it/~filippo/how-to-get-cimmsim_files/LicenseAgreement-1.pdf- Licence: C-ImmSim is available under a LICENSE AGREEMENT that needs to be signed: HIV: human immunodeficiency virus infection; AIDS: acquired immune deficiency syndrome; HAART: higly active antiretroviral therapy; STI: structured therapy interruption; PIs: protease inhibitors; RTIs: reverse transcriptase inhibitors; NRTIs: nucleoside reverse transcriptase inhibitors; NNRTIs: non-nucleoside reverse transcriptase inhibitors; SPARTAC: short pulse antiretroviral therapy at hiv seroconversion.The authors declare that they have no competing interests.FC, MB and GD contributed to the design of this study and critically revised the manuscript. PP and FC performed research, analyzed data and drafted the manuscript; RC has made sbstantial contributions to the interpretation of the results and critically revised the manuscript. All authors read and approved the final version of the manuscript including the data as presented.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/172/prepubMathematical model details. This file lists all the interactions between cells and molecules considered in the model and an accurate description of the parameter setting.Click here for file


Mycobacterium avium complex (MAC) immune reconstitution inflammatory syndrome (IRIS).To describe long term outcomes of Cases of MAC IRIS were retrospectively identified at four HIV clinics  from 1996–2004. Patients were included if they were initially diagnosed with AIDS and found to have evidence of focal MAC infection documented by tissue culture or PCR after initiating HAART, and at least 6 months of follow up.3 at pretreatment baseline and 100/mm3 at time of MAC IRIS diagnosis. Sites of disease included lymph nodes , gastrointestinal tract (7) and liver (3). Sixteen patients (80%) responded to treatment and were disease free after a mean of 17.4 months of therapy for MAC IRIS; IRIS therapy was withdrawn in 6 without relapse. Four patients (non-responder group) had persistent or relapsing disease despite 27 months of ongoing MAC IRIS treatment. At the time of resolution or last follow-up, the mean CD4 cell count and viral load was 143/mm3 and 7,000 c/mL for responders, and 65/mm3 and 17,000 c/mL for non-responders, respectively. Most patients with peripheral adenopathy were responders ; many with abdominal adenopathy  were nonresponders.Among the 20 patients included, the mean age was 40 years, mean CD4 cell count was 24/mmThe majority of patients with MAC IRIS eventually responded to treatment. Our sample size was not adequate to perform statistical analysis, but there was a tendency towards adequate CD4 response to HAART and peripheral rather than intraabdominal adenopathy among responders. Mycobacterium avium complex (MAC) may occur in AIDS patients with severe immune suppression after starting HAART [Focal manifestations of ng HAART ,2. This ng HAART ,4.Studies have addressed the clinical features of IRIS at onset, as well as the course of six patients after one year -10 and 2Patients who received treatment at four university HIV/AIDS programs in the United States  were retrospectively identified from 1998 to December 2004. One patient (#18) has been previously reported elsewhere . We used3 which increased to 100 ± 85/mm3 at the time of MAC IRIS diagnosis. In 6 patients (30%), HAART was initiated after disseminated MAC was diagnosed (mean time from MAC diagnosis to HAART initiation was 36 days ± 39). Seven of the 20 total patients had been receiving MAC prophylaxis prior to the development of MAC IRIS.Table The median time from initiation of HAART to diagnosis of MAC IRIS was 2.6 months (range 10 days to 4.7 years). Seven (35%) patients developed IRIS less than 60 days after initiation of HAART, 10 (50%) from 60 days to one year, and 3 (15%) after more than a year. Presenting symptoms of IRIS included fever (15 patients), abdominal pain (9), symptomatic lymphadenopathy (6), night sweats (4), loose stools (4), nausea and vomiting (4), fatigue, weight loss, and rash (1 each). The most common focal site of IRIS was lymph nodes . Other sites included: gastrointestinal tract (7 patients) and liver (3). All patients in whom MAC was isolated from cultures of bone marrow also had focal inflammation of another site , reflecting the case definition of IRIS. Other sites of MAC isolation included liver biopsy (3 patients), and aspirates or biopsies of other tissues. Isolator blood cultures were positive in 2 patients who also had MAC isolated from a focal site of inflammation (Table All patients received a macrolide (azithromycin or clarithromycin) and ethambutol for the treatment of MAC IRIS. Ten patients received rifabutin as an additional agent. Four patients received ciprofloxacin and two received amikacin as part of their regimen of treatment. Eight patients received therapy with adjunctive agents: 8 received oral corticosteroids for an average of 17 ± 14.6 months, and 2 received granulocyte-colony stimulating factor (G-CSF). Table 3 and was 88/mm3 at the time of IRIS diagnosis  experienced clinical and/or radiographic resolution of signs of active infection after 19.5 ± 21.7 months (range 22 days to 5.2 years) of MAC IRIS treatment : A 40-year-old man initially diagnosed with Pneumocystis pneumonia and HIV infection  initially responded well to trimethoprim/sulfamethoxazole and corticosteroids. He then developed recurrent fevers up to 102°F after beginning zidovudine, lamivudine, and efavirenz. An abdominal CT scan showed multiple enlarged mesenteric, retroperitoneal and periaortic lymph nodes. A lymph node core needle biopsy of an enlarged lymph node showed necrotizing granulomas. Mycobacterium avium complex was isolated from culture of the biopsy specimen. He received azithromycin and ethambutol and continued HAART. Clinical improvement was documented within one month and radiologic resolution at one year. The CD4 cell count at that time was 229/mm3, and the viral load was undetectable. Antimycobacterial therapy was then discontinued.Patient #3 : A 44-year-old man was initially diagnosed with Pneumocystis pneumonia and HIV infection with a CD4 count of 69/mm3 and a viral load of 215,201 c/mL. HAART was initiated and antimycobacterial therapy was begun 2 months after an AFB isolator blood culture drawn at the time of his initial diagnosis because of ongoing fever yielded MAC. One month later, fevers persisted, and MAC was isolated from cultures of bone marrow. The antimycobacterial regimen was intensified with rifabutin and ciprofloxacin in addition to azithromycin and ethambutol, and his fever resolved. Two months later he developed recurrent fever and abdominal pain. A repeat blood culture was positive for MAC. An abdominal CT scan demonstrated multiple enlarged intra-abdominal lymph nodes. His CD4 count had increased to 199/mm3 with a viral load of 684 c/mL. Because of significant abdominal pain associated with MAC IRIS involving the gastrointestinal tract, oral corticosteroids were initiated. Seven days later, his symptoms of abdominal pain completely resolved. Corticosteroids were unable to be withdrawn because of persistent abdominal pain and were therefore continued for 27 months. Antimycobacterial therapy was discontinued after 3 years of treatment.3 in this non-responder group prior to treatment, 152/mm3 at IRIS diagnosis, and 65/mm3 at the time of last follow up : A 46-year-old man was diagnosed with HIV infection in 1989 with a CD4 cell count of 4/mm3. Because of alcoholism, he took antiretroviral therapy only intermittently. In 8/02 his CD4 cell count was 11/mm3 and his viral load was 292,571 c/mL. A screening blood culture yielded MAC, and he was started on ethambutol in addition to azithromycin. In 9/02 he discontinued alcohol use and began HAART with a regimen of didanosine, efavirenz, and lopinavir/ritonavir. His CD4 cell count increased to 99/mm3 with a reduction in viral load to 3,636 c/mL. In 9/03, with a CD4 cell count of 72/mm3 and viral load of 1,991 c/mL, he was evaluated for severe right sided upper abdominal pain. An abdominal CT scan revealed multiple enlarged necrotic intra-abdominal lymph nodes  had a complete response to continuation of HAART and antimycobacterial agents over a 36 month follow up period. Of note, our study size was not large enough to permit a statistical analysis of features that may predict a favorable outcome. Furthermore, there has been a decline in incidence of MAC IRIS associated with the recent availability of potent HIV agents effective in those with late stage or highly resistant HIV infection. Because larger studies may therefore not be forthcoming, there may be a utility in making observations from our study about features that may accompany favorable responses to MAC IRIS. For example, we, as others, have noted a tendency of responders to maintain a more substantial HAART-associated CD4 cell count increase than non-responders . In addi3 with an increase to 213/mm3 at the diagnosis of IRIS [3, while the other had a CD4 count of 382/mm3; both presented with lymphadenitis. No repeat CD4 cell count was reported for one patient with chronic MAC involving breast tissue. These patients developed MAC IRIS on average 5.6 months after starting HAART. In addition, there is insufficient data regarding whether a particular site of involvement, such as a peripheral lymph node, may have a more favorable outcome because of a lower burden of mycobacterial organisms in tissue, as well as being more amenable to surgical resection and medical management.There has been insufficient information published in the literature regarding the characteristics of patients who do not respond to initial antimycobacterial therapy and who develop persistent MAC IRIS disease. As is the case in our study, the previous studies were unable to perform statistical analyses of prognostic factors because of small sample sizes. Of the three papers that have reported three such cases (without duplication of cases from the present study), the average CD4 cell count prior to the initiation of HAART was 96/mm of IRIS ,7,9. FewThe lack of a standardized duration of antimycobacterial therapy required to manage IRIS reflects the variation in time in which a clinical and radiologic response may be observed. In some patients with vigorous CD4 responses to HAART, antimycobacterial agents may be withdrawn after resolution of IRIS without the development of reactivation. On the other hand, patients in our study with MAC IRIS who did not experience a significant CD4 response to HAART, who did not have improvement in symptoms, and/or who failed to eradicate MAC infection, were managed with intensification of antimycobacterial therapy and sometimes the addition of adjunctive therapy. Of note, however, the strategy of antimycobacterial therapy intensification, as well as the contribution made by adjunctive agents in the management of MAC IRIS has not been verified in controlled, prospective trials.3 or less) and a CD4 cell count increase in response to HAART at the time of IRIS diagnosis [Our findings regarding the clinical features of MAC at the time of diagnosis are consistent with prior reports. We and others have found that IRIS is a focal inflammatory condition most often involving lymph nodes, liver or gastrointestinal tract. In addition, blood cultures rarely grow MAC at the time of IRIS; rather, a biopsy of an involved focal organ, such as lymph node, GI tract, or liver, is usually required to establish a diagnosis . Furtheriagnosis -13. FinaIn conclusion, the majority of patients with MAC IRIS experience favorable responses to long-term antimycobacterial therapy. Patients who have an adequate CD4 response to HAART may have antimycobacterial treatment safely withdrawn following resolution of focal involvement. However, neither the present study nor previous reports have had ample numbers of patients to permit statistical analyses of factors predicting a favorable outcome. Furthermore, the incidence of MAC IRIS may be declining with the development of newer agents to manage HIV. Nonetheless, the observations of the current study – that adequate immunologic responses to HAART and peripheral lymph node location of MAC IRIS may be associated with favorable outcomes, may be useful for physicians managing patients with AIDS who develop MAC IRIS.The author(s) declare that they have no competing interests.JR coordinated study, performed analysis of data, drafted manuscriptDRK contributed subject dataPCK contributed subject data, participated in study designJEG contributed subject data, edited manuscriptJM contributed subject dataPHK conceived of study and participated in design, edited manuscriptAll authors read and approved the final manuscript.


With African health-care systems facing exploding demand for HIV care, reliable methods for assessing adherence and its influencing factors are needed to guide effective public-health measures. This study evaluated individual patient characteristics determining antiretroviral treatment (ART) adherence and the predictive values of different measures of adherence on virological treatment failure in a cohort of patients in a routine-care setting in Cameroon.Longitudinal study over 6-months following ART introduction, using patients questionnaires and hospital and pharmacy records.3 had a greater risk of dying during the follow-up period . Compared with asymptomatic CDC stage A patients, CDC stage B (odds ratio 5.72) and CDC stage C patients (odds ratio 16.9) had higher risk of becoming lost to follow-up (P < 0.001). In the multivariate analyses, pharmacy non-adherence was less frequent in women  but more frequent in patients with high monthly income .At the end of the 6 months study period, 219 of 312 patients (70%) returned to the pharmacy to refill their medication, 17% (51) were lost to follow-up, 9% (28) were dead and 4% (14) were transferred to other care centres. Virological treatment failure at 6 months was experienced by 26 patients, representing 13% of patients with available viral load value. Pharmacy refill irregularity was the most powerful predictor  of virological treatment failure, compared with CD4 cell count increase at 6 months  or self-reported adherence at one month . Low intensity of ART side-effects after one month was strongly associated with survival . Patients starting ART with CD4 cell count <100 cells/mmPharmacy-refill adherence might be considered as an alternative to CD4 count monitoring for identification of patients at risk of virological failure, especially in resources-scarce countries. The study confirmed the difficulty in demonstrating clear associations of individual patient factors and treatment outcomes. The substantial loss to follow-up and deaths occurring within 6 months after initiating ART emphasise the need to understand the best timing of ART initiation and further elucidate and educate on the underlying reasons for delaying initiation of ART in resource-limited countries During the last decade, access to HIV care in Sub-Saharan Africa has been improved by reduction in the cost of ART and by the implementation of WHO guidelines promoting scaling-up by task shifting for clinical decision-making to less specialised health-care workers . HoweverAs in other African countries, the average prevalence of HIV in Cameroon has risen dramatically during the last two decades, from 0.5% in the early 1990s to 5.5% in 2004 . In viewAdherence to antiretroviral therapy (ART) is crucial to ensure viral suppression, decrease the risk of disease progression and drug resistance. However, it is difficult to measure accurately, which is reflected in the number of conflicting reports available on the response to ART in people living with HIV/AIDS (PLHIV). Optimistic reports -11 may hClearly, there is a need for simple and reliable methods of assessing adherence as well as its influencing factors in order to guide effective public-health measures.The objective of this study was to compare the diagnostic accuracy of CD4 cell count changes, self-reported adherence and pharmacy refill history to identify patients with virological treatment failure amongst a cohort of PLHIV in a routine care setting in Cameroon during the first 6-month follow-up period. We then examined the relationship between patient individual factors and adherence, including in our analysis the substantial number of patients loss to follow-up.The Day Hospital of Yaoundé Central Hospital (YCH) opened in 1998 with a capacity of offering care to 4500 PLHIV. By 2006 this centre had registered more than 10,000 PLHIV, with approximately 2000 patients on ART, resulting in long waiting periods, overburdened staff and a severe shortage of clinic space.At the time of the study, patients had to pay for care, including drugs (USD 7 to 17 monthly), laboratory tests (USD 50 for pre-treatment check-up) and clinical visits . Before receiving ART, pre-counselling and post-counselling visits were necessary, followed by three consecutive medical visits and a final socioeconomic inquiry to assess ART readiness. Therapeutic committee determined eligibility for ART based on CDC clinical staging  and CD4 cell counts.We recruited all ART-naïve patients at the Day Hospital of YCH between June and September 2006 at their first antiretroviral prescription visit. Exclusion criteria were age under 18 years, imminent transfer to another treatment centre, prior antiretroviral therapy and poor health status, with the patient unable to provide consent and respond to the initial inclusion questionnaire. In most cases the latter patients were hospitalised in a common room that rendered confidentiality difficult.Routine clinical visits where scheduled on day 15, at months 1 and 3, and every 3 months thereafter. Antiretroviral therapy was based on triple-drugs regimens consisting of two NRTIs and one non-NRTI and was dispensed only at the central hospital's pharmacy. Patients were provided with more medication than required, i.e. tablets were usually dispensed for 30 days, whereas pharmacy visits were scheduled in multiples of 4 weeks. At each pharmacy visit were registered: date of present prescription refill, prescribed drug regimen and date of the next pharmacy appointment.This study was approved by the Cameroon National Ethics Committee and informed written consent was obtained from each participant prior to inclusion into the study.Biomedical and clinical data were extracted from the clinic file of each enrolled patient and included biochemistry, haematological tests, CD4 cell counts determinations , date of death or transfer to another health centre. Determination of CD4 cell levels were performed at the pre-treatment examination and routinely requested every 6 months thereafter. Plasma viral load assays  were offered to all study participants after ≥ 5 months of ART.At the day of introducing ART, three trained study interviewers administered an initial questionnaire exploring socioeconomic status, knowledge and beliefs toward HIV and ART, social support and disclosure of HIV status ,22.A second culturally adapted questionnaire gathered information on self-reported ART adherence and early ART side-effects . It was Pharmacy records were reviewed 6 months after ART initiation to define pharmacy-refill adherence. In order to further ascertain true adherence status, patients without renewed prescriptions in the last two months were actively traced by two phone calls and if still alive, encouraged to come back to the clinic to restart therapy.3 [Virological treatment failure was defined as a viral load >400 copies/ml (lower detection limit of 40 copies/ml), and was used as the gold standard to compare different methods of adherence measurements: CD4 count change, self reported adherence and pharmacy-refill history. Immunological treatment failure was defined as a reduction in CD4 count after 6-months of treatment to, or below, pre-therapy baseline, or persistent levels below 100 cells/mmSelf-reported adherence was classified as "adherent" when not a single dose was missed or non-adherent if the patient admitted having missed at least one dose during the last month.Pharmacy non-adherence was defined as renewal of prescriptions of later than 2 weeks after the scheduled pharmacy appointment, or patients confirming abandoned ART on phone call tracing. Patients not traced by phone calls were considered to be lost to follow-up. The follow-up time was censored at the last scheduled pharmacy visit.Statistical analyses were conducted using STATA version 9 .The three different methods to measure adherence were compared in their ability to predict virological failure by computing their sensitivity and specificity, positive and negative predictive values and positive and negative likelihood ratios. Finally, the association between each method and virological failure was also reported as OR and 95% confident interval. We then used the method of adherence assessment showing the strongest association with virological failure as a surrogate of true treatment adherence. Chi-square tests were used to determine the associations between each individual patient baseline characteristic and adherence, at a P < 0.2 level of significance. Logistic regression models were fitted including characteristics shown to be associated with adherence in univariate analysis (P < 0.2). Variables measured at one month were excluded from this analysis as the substantial number of patients with early loss to follow-up reduced the number of available data and thus statistical power.As the substantial rate of patients loss to follow-up may introduce bias into estimates of risk factors for treatment non-adherence, we conducted a sensitivity analysis where associations were tested in two scenarios: a best-case scenario, where all patients lost to follow-up and not successfully traced by phone call were considered as adherent, and a worst-case scenario where all such patients were defined as non-adherent Fig. .Patient's disposition is shown in Fig . Of the Among the 312 enrolled patients at the start of ART, the mean age (± SD) was 37 ± 9 years, with a majority of women (63%) [Table 3 (interquartile range 50 - 177 cells/mm3). All patients received two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) with the exception of one patient who received two NRTIs and one protease inhibitor.Most patients (n = 195 or 63%) had been followed at the clinic for less than one month before initiating ART. However, only 36 patients (12%) reported the use of alternative therapies for HIV in the past. At the time of starting ART the majority of the cohort were classified as symptomatic (64% at CDC stage B and 14% at stage C), the median baseline CD4 count was 104 cells/mmWhen asked about social support and stigmatisation, 275 participants (88%) reported having disclosed their HIV status to at least one person and 260 (83%) declared satisfied by family or friends support. Most patients (n = 258 or 80%) had optimistic expectations of the effects of ART on their health status, a sizable minority believed that treatment could cure HIV (44%) and 118 patients (38%) were unaware of possible ART side effects.Of 312 patients in the initial cohort, 219 (70%) were still coming to the pharmacy to refill their medication after 6 months of follow-up, while 51 (17%) were lost, 28 (9%) had died and 14 (4%) were had been transferred to other care centres. The incidence rate of loss to follow-up was 40.1 per 100 persons years (95% CI: 30.5 - 52.8) and mortality rate was 21.2 per 100 persons years (95% CI: 15 - 31). Drop-out occurred at a median of 44 days (IQR: 28 - 80 days) and death occurred at a median of 55 days (IQR: 28 - 81 days) after treatment initiation.3 and 16 patients (22%) with available follow-up CD4 cell count met criteria for immunological treatment failure. Using phone call tracing alongside tracking of patients during regular follow-up visits, we obtained viral load values for 206 patients. Virological treatment failure  at 6 months was experienced by 26 patients, representing 13% of patients with available viral load value and 8% of the entire cohort.The first routinely scheduled follow-up CD4 cell count was performed by only 71 patients (23% of patients still followed-up). The median rise in CD4 cell count from baseline to this point was 117 cells/mmSelf-reported adherence data after one month of therapy were obtained for 238 patients (76%). The majority of respondents (78%) claimed not to have missed a single dose during this period. The proportion of participants that reported full adherence to treatment during the past month decreased from 83% at one month to 57% at 6 months.Adherence based on pharmacy refill charts after 6 months of follow-up could be analysed for 278 participants. Twenty-eight subjects were excluded because of their short follow-up duration (<2 months) and 6 patients had incomplete data on the refill chart Fig . Two-thiThere were significant differences between the ability of different measures of adherence to predict virological treatment failure after 6 months of therapy Table . PharmacNotably, self-reported adherence at 1 month was not significantly associated with virological treatment outcomes.3 were at significantly greater risk of death during the follow-up period . We found only a trend for association of HIV CDC stage with mortality. However, HIV CDC clinical stage at the beginning of treatment significantly predicted (P < 0.001) loss to follow-up: compared with asymptomatic patients CDC stage A, CDC stage B patients  and specially CDC stage C patients  had greater rates of loss to follow-up.Univariate analysis of individual patient factors Table  showed tThere was a non-significant trend to lower rates of interruption of follow-up for women  and for those reporting improving ART side-effects after one month of treatment . None of the socio-economic determinants studied appeared to influence patient retention and mortality: neither age, nor level of education, nor economic situation and social support were significantly associated with loss to follow-up or with death.As patients lost to follow-up may have introduced bias into the analysis, we analysed the association of individual patient characteristics with pharmacy adherence in a sensitivity analysis, using two scenarios, a best-case and a worst-case scenario Table . In the In the multivariate analysis Table , women pDespite substantial improvements in the affordability and availability of ART in recent years, African health-care systems face enormous challenges in the context of exploding demand for HIV care . The maiOur cohort showed a relative low patient retention rate: 70% of the patients who started ART still came to the pharmacy to take their prescription after six months of follow-up, 17% disappeared, 9% died and 4% were referred to other care centres. These results confirm that most losses to follow-up and deaths occur during the initial period after ART initiation.A recent systematic review  of 33 eaPharmacy-refill history gives no description of daily adherence to treatment, because patients may not take all prescribed medications. It could also be considered as a time-consuming monitoring tool for the pharmacy staff, owing to the rapidly growing number of patients in public ART programs. However, this is a simple, inexpensive approach and it was previously reported to be as accurate as CD4 counts for predicting virological response . We founSelf-reported adherence has been described in several studies as a rapid and inexpensive method, albeit subject to social-desirability and recall biases ,27. In oWe examined determinants of adherence in two scenarios to reduce bias due to loss to follow-up. We identified female sex, middle monthly income and less ART related side-effects at one month as predictors of higher pharmacy adherence under both scenarios. Age and HIV clinical CDC stage correlated with pharmacy adherence only under best and worst-case scenarios, respectively. Gender remained of borderline significance after adjusting for potential confounders in the multivariate model.To the best of our knowledge, the current study is the first to demonstrate that income may not be linearly associated with adherence: patients with monthly middle income had greater pharmacy adherence rates than both the poorest and the richest participants. A recently published meta-analysis  examinedWith the exception of a trend towards greater loss rates among men, we failed to demonstrate any other social or demographic association with loss to follow-up. The same gender association with both death and loss to follow-up was recently reported from a study in Malawi .It is unclear how far our results can be generalised to other countries and healthcare systems. Compared with reports from patients starting ART in other treatment programmes in lower-income countries , our cohThe percentage of male patients in our cohort reflects the gender distribution of HIV prevalence in Cameroon, which indicates that women's access to health care for HIV is improving. We also observed discrepancies between biological (CD4 counts) and clinical (CDC stages) levels of disease in our participants: Only 14% of our patients started therapy at CDC clinical stage C, despite a median CD4 cell count of 107 cells/μl. The simplest explanation for this is an underestimation of CD4 cell count at the Day Hospital Laboratory. This conclusion is supported by our observation of a large variability of baseline CD4 cell counts for patients who were analysed at close intervals at different laboratories (data not shown). A lack of association between immunological and virological outcomes have been found in similar settings  suggestiOnly 15 patients in our study (5%) lived more than 4 hours of travel from the Day Hospital, which may not be representative of patients in ART programmes at other hospitals. A survey of patients initiating ART from 2002 to 2005 in Limbe Provincial Hospital, the only ART clinic serving the Southwest Province of Cameroon at the time of the study, showed that 44.3% of patients were living more than 40 km by inaccessible road from the clinic . As treaOnly 312 out of 440 originally eligible patients were included in our study cohort. Such high attrition rates before initiating ART treatment are a main source of selection bias in studies of retention rates in Africa. Data from different cohorts in other lower-income settings [Our study focused on the first six months of follow-up after ART initiation, a period previously characterized by high attrition ,18,19,32Although there is no 'gold standard' for the assessment of medication adherence, pharmacy-refill adherence or other easily accessible methods should be considered as an alternative to CD4 count monitoring for identification of patients at risk of virological failure, especially in low-income countries. It represents a simple, inexpensive and accurate method that correlates with virological response to treatment. Data from pharmacy refill charts should be made available to health-care workers to help identifying patients at greatest risk of treatment failure.It is still difficult to pinpoint determinants of non-adherence to ART in lower-income countries; for example, our study indicates than the role of economic status is more complicated than may previously have been thought. Preventing treatment discontinuation by enhancing adherence counselling for a higher-risk population may not be effective: all previous studies failed to clearly demonstrate a specific group that would benefit from such intervention. Developing strategies should rather focus on improving adherence follow-up by simple and inexpensive measurement.Finally, more studies in resource-limited countries are urgently needed to understand the underlying reasons for late initiation of ART and for high attrition rates before initiating ART, which account for a large number of early losses to follow-up and deaths in lower-income countries.AIDS: Acquired immune deficiency syndrome; ART: Anti retroviral treatment; CDC: Centers for disease control and prevention; CI: Confidence interval; HIV: Human immune deficiency virus; IQR: Inter quartile range; NNRTI: Non- Nucleoside reverse transcriptase inhibitor; NRTI: Nucleoside reverse transcriptase inhibitor; OR: Odds ratio; P: P value for statistical significance; PLHIV: People living with human immune deficiency virus; SD: Standard deviation; WHO: World Health Organization; YCH: Yaoundé Central Hospital.The authors declare that they have no competing interests.All authors read and approved the final manuscript. MR conceived the study, raised funds, participated in the design and coordination of the study, collected and cleaned data, interpreted data, drafted the manuscript. BES contributed to the design and coordination of the study, contributed to data entering and cleaning, participated to interpreted and helped in drafting and revising the manuscript. NE contributed to data cleaning, performed statistic analysis and interpretation, and helped in revising the manuscript. PN conceived the study, participated in the design and coordination of the study, carried out data collection, coordinated laboratory sample processing, contributed to manuscript revision. All authors read and approved the final manuscript.


It has been suggested that the stability of the CD4 cell reservoir could be related to the continuous replenishment from plasma-residual HIV-1. Intensification with an entry inhibitor could help eliminate the detectable levels of ongoing viral replication and accelerate the decay of the reservoir by inhibiting the entry of the virus into the cells.3 and CCR5-tropism. Latently infected resting CD4 cells were determined at baseline, week 12 and week 24, with further analyses planned at week 36 and week 48. Highly purified resting CD4 cells were isolated using magnetic beads rendering ~20 million and plated in replicate dilutions and stimulated in a limiting dilution culture assay, with irradiated PBMCs from a seronegative donor. Cells containing HIV-replication-competent were quantified by HIV-1 p24 Antigen Assay (results in infective units per million).The intensification study with an entry inhibitor (maraviroc-MVC) included patients with stable antiretroviral therapy (ART) with three or more drugs, viral load (VL) of <50 copies/ml for two or more years, CD4 count of >350 cells/mm3 [IQR 547-793]. At baseline, the reservoir could be quantified in six out of nine patients. One patient has left the study, and the other eight patients continue with VL of <50 copies/ml and median CD4 count of 680 cells/mm3 [IQR 601-910]. After 24 weeks of intensification with MVC, when compared with baseline, a persistent decrease in the latent reservoir has been observed in three patients, no change  in two patients, and an increase, after an initial decrease, in the remaining three patients.Nine patients have been included. Median time of HIV-1 diagnosis was 103 months [IQR 58-240], ART was administered for a median of 75 months [IQR 38-144], and the median baseline CD4 count was 711 cells/mmIntensification with MVC seems to have a uniform, significant initial impact (measured at 12 weeks) on the HIV-1 latent reservoir that continues to be observed in most patients after 24 weeks.


Guidelines established for the treatment of HIV-1 infection and genotype interpretation do not apply for HIV-2. Data about antiretroviral (ARV) drug efficacy and resistance mutations is scarce.Clinical data about HIV-2 infected patients in Belgium and Luxembourg were collected and the effect of ARV therapy on plasma viral load and CD4 counts were analysed. Viral RNA encoding for protease (PR) and reverse transcriptase (RT) from ARV-naïve and treated patients were sequenced.Sixty-five HIV-2 infected patients were included in this cohort. Twenty patients were treated with 25 different ARV combinations in a total of 34 regimens and six months after the start of ARV therapy, only one third achieved viral load suppression. All of these successful regimens bar one contained protease inhibitors (PIs). Mean CD4 gains in the group of viral load suppressors and the group of patients treated with PI-containing regimens were respectively significantly higher than in the group of non-suppressors and the group of PI-sparing regimens. The most frequent mutations selected under therapy (compared to HIV-2 ROD) were V71I, L90M and I89V within PR. Within RT, they were M184V, Q151M, V111I and K65R. All of these mutations, except K65R and M184V, were also found in variable proportions in ARV-naïve patients.Despite a high rate of ARV treatment failure, better virological and immunological results were achieved with PI-containing regimens. The analysis of polymorphic positions and HIV-2 specific mutations selected during therapy showed for the first time that transmission of drug resistant viruses has occurred in Belgium and Luxembourg. The high heterogeneity in ARV combinations reflects a lack of guidelines for the treatment of HIV-2 infection. Comparein vivo . In vitrme range .The majority of HIV-2 infected persons live in West African countries , where HAlthough viral evolution occurs slowly in HIV-2 infection , the appAn anonymous data sheet was sent to the clinicians in charge of HIV-2 infected patients using the Belgian AIDS Reference Laboratories network and the Laboratoire de Rétrovirologie in Luxembourg. Patients were identified by a code. Patients with an untypeable HIV status in immunoblot or presumably co-infected with HIV-1 and HIV-2 were excluded. In a first round, epidemiological and clinical data were collected as well as the availability of retrospective samples. In a second round, frozen plasma or serum samples stored in the normal diagnosis and follow-up settings, were obtained for the sequencing of PR and RT coding regions at different points during the treatment.Two different methods were used for plasma viral load assessment.PLUS SYBR Green I, 100 μl kit  on a Lightcycler 2.0 platform. Each capillary contained 48 μl PCR grade water, 6 μl of each primer , 20 μl of Master Mix provided in the kit and 20 μl cDNA. Each run consisted of forty cycles of amplification (95°C 15 s – 60°C 40 s – 72°C 30 s) followed by a melting curve analysis.On the one hand, HIV-2 plasma viral load was measured by a real-time PCR in-house assay on a Lightcycler platform . The asset al. [On the other hand, a semi-quantitative assay was used as described by Van Kerckhoven et al.  with HIVet al. . The latet al. [RT-PCR and inner PCR were performed as described by Colson et al.  and PCR PCR products were then purified using the QIAquick PCR purification kit . Twenty nanograms of each PCR product were sequenced with the BigDye Terminator v1.1 cycle sequencing kit . Primers used for sequencing of PCR products obtained by the first protocol were H2Mp3  and JR 4Statistical analysis was performed using the JMP software version 6 . The significant effect of ARV therapy on plasma viral load was assessed using a Kaplan-Meyer survival curve in order to consider if virological failure was the outcome at three different time reference points after therapy initiation. To interpret the impact of ARV therapy on CD4 count evolution, data was first standardised to obtain CD4 values at the same reference points for every therapy, using a linear regression model to interpolate values between existing measurements. In a first model, delta CD4 values were calculated from treatment initiation at intervals of three months. Two different parameters were tested: the therapy (PI-containing or not) and virological failure. Analysis of the variance between groups was done using a Tukey test. In a second model, the raw CD4 data on a time scale between the two different therapy groups was considered and response specification was assessed by constructing linear combinations with a Manova fit model.EF611309 to EF611338: the first 23 were ARV-naïve (from EF611309 to EF611331) and the last 7 were treated patients.Earliest samples from 30 patients were submitted to GenBank and received accession numbers from This evaluation was approved by the ethical committee of the Medical Faculty of the Université Catholique de Louvain.Sixty-five patients were included in our cohort and data is summarised in Table As different methods were used for the viral load determination, and in the absence of a gold standard, we chose qualitative criteria to evaluate the efficacy of therapy. Either the regimen successfully suppressed viral replication and plasma viral load became undetectable, or replication was not suppressed and this was defined as virological failure. The threshold used was the detection limit of the least sensitive method. All therapies were started in the presence of a detectable viral load and baseline viral load values are given in Table 3 (ranging from 10 to 737). In the group of patients who had never been treated, data from 10 patients with baseline CD4 counts between the same ranges were considered, with a mean at the first visit of 402 cells/mm3 (193–660). Over one year, the mean loss was 16 CD4/mm3.Information on CD4 counts evolution was gathered for 25 therapies. Three suboptimal therapies were excluded from the statistical analysis (Table 3 after 12 months. Differences in treatment had a significant influence on CD4 gain (p = 0.003). A mean gain of 106 CD4/mm3/year was observed in the PI-group while a mean loss of 25 CD4/mm3/year was obtained in the PI-sparing group. A second test was performed to establish the immunological response based on raw CD4 data using a Manova test. After 12 months of treatment, a gain of 96 CD4/mm3 in the PI-group and a loss of 11 CD4/mm3 in the PI-sparing group had been recorded. For the PI-group, this gain was already observed after 3 months of therapy and was maintained afterwards while the decrease observed for the PI-sparing group did not significant differ from zero.The CD4 gain after therapy initiation was plotted on a time scale. The PI-containing and PI-sparing groups, as well as the viral load suppressor and non-suppressor groups, were compared Table . A Tukey3. The same tendencies were observed for respectively 4, 7 and 11 different treatments but they had poor statistical relevance, especially for the groups higher than 200 CD4/mm3.Finally, baseline CD4 counts were stratified in three groups, more than 400, between 400 and 200 and less than 200 cells/mmTable In the PR, the most frequent mutation was V71I: it showed 3 times under NFV therapy. This mutation was associated once with V62A and L99F, once with T56A and V62M and in both cases with a plasma viral load higher than 3 log copies per ml. In the third case V71I was detected as part of a combination with V71T before viral load suppression. The I89V and L90M mutations were each detected twice, the latter under an ATV/r containing regimen and in a FPV/r containing regimen in association with I54M and I89V mutations.In the RT, the most frequent mutation selected on therapy was M184V. It appeared in eight different treatment failures with different regimens all containing 3TC, which was also the most prescribed ARV drug.The Q151M mutation was found in viruses from 4 patients with different regimens. Firstly, in association with I10V, K35T, K82R, Y115F and M184V in patient A who had a history of AZT, 3TC, d4T and ABC exposure. Secondly, Q151M appeared in association with the M184V mutation under AZT/3TC pressure (patient C). Thirdly, in patient G, Q151M was observed in association with M184V and F214L under AZT/3TC/ABC pressure. The further continuation of this regimen subsequently resulted in the appearance of K65R, N69S, V83I, I90V and V111I. Lastly, and again under AZT/3TC/ABC pressure (patient I), the Q151M appeared on a M184V background after the disappearance of K70N and with the concomitant presence of K35R, K64R and K82R.The K65R mutation was selected 3 times on therapy and the only drug in common for the 3 regimens was ABC.Fig. In the RT, the most polymorphic positions were 5, 10, 11, 28, 64, 82, 111, 121, 123, 126, 162, 167, 176, 180, 200, 227, 228 and 251, with 7 or more variants out of 23 sequences from different naïve patients. Note that the Q151M mutation was also found in a virus from a recently diagnosed naïve patient originating from Belgium. Phylogenetic analysis of the RT sequences (not shown) showed that this virus was related to another one also harbouring the Q151M mutation. The patients affected were the only 2 homosexual men of this group. Furthermore, positions 10, 35, 64, 69, 82, 83, 90 and 111 were found in variable proportions in naïve patients  declare that they have no competing interests.JR coordinated the data and samples collections from the different Belgian centres, analysed the clinical data and the genotypes, and drafted the manuscript. FR coordinated the study in Luxembourg. ATV and CL performed the molecular biology assays and aligned the sequences. KF, FE, DP, CF, KVL, MLD and DV all contributed to the data and samples collections in their own HIV centre. JCS and PG participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To assess the long-term response to add-on quetiapine therapy in patients with bipolar I disorder who were not adequately responding to standard medications.Outpatients with bipolar I disorder (DSM-IV-TR) responding inadequately to standard treatment were observed before and after the addition of quetiapine. Symptom severity was evaluated using the Clinical Global Impressions scale for Bipolar Disorder (CGI-BP) each month. Relapses included hospitalization, treatment in a day hospital or clinic, scores ≥ 1 point higher than previous CGI-BP scores and/or upward titration of quetiapine or other medications.prior to quetiapine therapy was 3.4 for all episodes , 6.7 for depressive episodes , and 2.5 for manic or mixed episodes .Sixty-one patients (age range of 18–68 years) were observed prospectively for an average of 7.5 months (range 3–18 months) prior to addition of quetiapine and subsequently followed for an average of 15.7 months (range 6–42 months). The final mean quetiapine dose was 537.1 ± 91.7 mg/d. Prior to quetiapine addition, an annual relapse rate of 2.09 episodes was recorded, relating to 0.94 depressive and 1.15 manic or mixed episodes. Following quetiapine addition, annual relapse rates were reduced to 0.61 episodes, representing 0.14 depressive and 0.46 manic or mixed episodes. Compared with the period of add-on quetiapine treatment, the relative risk of relapse This naturalistic follow-up study provides preliminary evidence for the efficacy of long-term add-on quetiapine treatment in the prevention of relapses of manic or mixed and depressive episodes of bipolar I disorder, and particularly in the prevention of depressive episodes. Bipolar I disorder is a severe and chronic illness characterized by episodes of mania and depression . Two majTo assess the long-term response to add-on quetiapine therapy in patients with bipolar I disorder who were not adequately responding to standard medications.An open-label study of patients with bipolar I disorder inadequately responsive to ongoing medications who were prospectively observed for 3–18 months before receiving add-on quetiapine treatment for 6–42 months.Adult outpatients with bipolar I disorder (based on DSM-IV-TR)  who had Quetiapine was added to ongoing medication at an initial dose of 25 mg/d for the first 2 days, increased to 50 mg/d for the next 2 days, and then increased by 50 mg increments every 2 days until a clinical response was observed (up to a maximum dose of 800 mg/d). This dose was then maintained throughout the remainder of the study.Prospective evaluations were made at least once every 2 months and no fewer than 8 times per year. Clinical response was evaluated using the CGI-BP scale [Mean CGI-BP scores were compared by one-way analysis of variance (ANOVA) for repeated measures. Confidence intervals for comparing the relative risks of relapse were calculated using the simplified method of Miettinen .Of the 61 patients, 41% were male (mean age 41.4 ± 8.2 years) and 59% were female (mean age 47.2 ± 16.9 years). Patients were prospectively observed for 3–18 months (average 7.5 months) before quetiapine therapy was added. Patients' ongoing medications are listed in Table The overall relapse rate decreased following the addition of quetiapine Table . When anP < 0.001; Table Mean change in CGI-BP score showed a significant improvement in symptoms from baseline at 6, 12, 18, and 24 months (Side effects during quetiapine combination therapy Table  were genIn patients with bipolar I disorder who had shown inadequate responses to prior standard therapy, relapse rates and symptoms were significantly improved with 6 months of add-on quetiapine therapy. These improvements were maintained in 14 patients treated for 24 months. Add-on quetiapine therapy was well tolerated, with no incidences of tardive dyskinesia reported following addition of quetiapine and only 4 patients reporting mild EPS. This naturalistic follow-up study demonstrates the efficacy of quetiapine in the prevention of relapses of manic and depressive episodes of bipolar I disorder in the long term, and particularly in the prevention of depressive episodes, which is consistent with our earlier findings  and with