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The evaluation of immune-based approaches to achieve an antiretroviral therapy free remission of HIV infection requires proven efficacy through antiretroviral therapy interruption placebo-controlled trials. This approach is not without risk to participants and innovative trial designs need to be developed that minimise the number of participants treated with placebo and ineffective candidates. Multi-arm, multi-stage (MAMS) trial designs can be used in this context to accelerate the development of an immune-based therapeutic agent for HIV cure. Issues related to implementing a MAMS design within the planned EHVA T01 trial are considered here. EHVA T01 is a multicentre, MAMS, double-blind, phase 1 and 2 trial that aims to evaluate the effect of immune interventions on viral control in HIV-1 infected participants following analytic treatment interruption. The application of a MAMS design increases the likelihood that the EHVA T01 trial will identify a successful treatment and minimises the number of participants undergoing analytical treatment interruptions who have been treated with futile agents. The use of a MAMS design is a promising strategy to evaluate complex immune-based approaches aimed at curing HIV-infection, particularly relevant to the pipeline with multiple agents requiring examination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: This article deals with the attempt to join HIV and geriatric care management in the 2017 edition of the Italian guidelines for the use of antiretrovirals and the diagnostic-clinical management of HIV-1 infected persons., Methods: The outlined recommendations are based on evidence from randomized clinical trials and observational studies published in peer-reviewed journals and/or presented at international scientific conferences in recent years. The principles of starting antiretroviral therapy in elderly patients and the viro-immunological goals are the same as in the general HIV population. However, there are some specificities to consider, related to the host as well as the therapy itself. HIV care in elderly patients must shift from a combined AntiRetroviral Therapy specific approach to a more comprehensive management, and from a disease-based model (list of co-morbidities) to a multi-morbidity and frailty standpoint. The implementation of a geriatric approach, based on the Comprehensive Geriatric Assessment, is essential and consists of a broader evaluation of health status. This multidimensional and multidisciplinary evaluation is focused on the development of a tailored intervention plan. Polypharmacy is a frequent condition in the older population and an independent risk factor for negative health-related outcomes. This can be overcome with a multidisciplinary and cooperative approach involving HIV specialists, geriatricians and primary care physicians., Conclusion: The inclusion of geriatric care becomes necessary due to the novel needs of an evolving patient population. It is important to underline that the HIV specialist will continue to lead multidimensional interventions and optimize quality of care for HIV-positive people.

Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro . However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4 + cell count and CD4 + /CD8 + cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.

Problem: Data on soluble CD14 (sCD14) during pregnancy and lactation are scarce. We assessed the levels of sCD14 in plasma and breastmilk of Malawian HIV-positive women and evaluated the possible association with morbidity and mortality in the HIV-exposed children., Method of Study: One hundred and forty-nine mother/child pairs were studied. Women received antiretroviral therapy from 26 weeks of gestation to at least 6 months of exclusive breastfeeding. sCD14 concentrations were determined using an enzyme-linked immunosorbent assay., Results: sCD14 levels measured at 26 weeks of pregnancy (median: 1418 ng/mL, IQR: 1086-1757) were inversely correlated to maternal CD4+ cell count (r = -.283, P = .001) and to neonatal birthweight (r = -.233, P = .008). At 6 months, sCD14 plasma levels were significantly higher compared to baseline (1993 ng/mL, IQR: 1482-2604, P < .001), and breastmilk sCD14 levels (7668 ng/mL, IQR: 5495-10207) were 4-fold higher than in plasma (although the concentrations in the two compartments were not correlated). No association was found between sCD14 levels in plasma or breastmilk and morbidity or mortality in children., Conclusion: Higher sCD14 levels in HIV-positive women were associated with a more compromised maternal immunological status and to a lower neonatal birthweight, but not to poorer clinical outcomes in the HIV-exposed children., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The objective of this study was to determine the virological response and the possible emergence of drug resistance at 1 and 2 years postpartum in HIV-positive pregnant women enrolled under the Option B approach and meeting the criteria for treatment. In the study, women with baseline CD4(+) <350/mm(3) received a combination of stavudine, lamivudine, and nevirapine during pregnancy (from week 25 of gestation) and continued it indefinitely after delivery. HIV-RNA was measured at 12 and 24 months postpartum. Drug resistance mutations were assessed in those with HIV-RNA >50 copies/ml. Baseline resistance mutations were assessed in the entire cohort. A total of 107 women were studied. At baseline, resistance mutations were seen in 6.6% of the women. At 12 months, 26.7% of the women had >50 copies/ml and among them 12.9% had virological failure (HIV-RNA >1,000 copies/ml). At 24 months, detectable HIV-RNA was seen in 28.3% of the women and virological failure in 10.1% of the women. Resistance mutations (mainly non-nucleoside reverse transcriptase inhibitors mutations) were seen in 40% of the women with detectable HIV-RNA. Baseline mutations did not correlate with virological failure or the emergence of resistance at later time points. Virological failure 2 years postpartum and emergence of resistance were rare in this cohort of HIV-infected women. These findings are reassuring in the light of the new strategies for the prevention of mother-to-child HIV transmission, recommending life-long antiretroviral therapy administration.

Background: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members., Results: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses., Conclusions: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

Background: Standard first-line antiretroviral therapy for HIV-1 infection includes two nucleoside or nucleotide reverse transcriptase inhibitors (NtRTIs), but these drugs have limitations. We assessed the 96 week efficacy and safety of an NtRTI-sparing regimen., Methods: Between August, 2010, and September, 2011, we enrolled treatment-naive adults into this randomised, open-label, non-inferiority trial in treatment-naive adults in 15 European countries. The composite primary outcome was change to randomised treatment before week 32 because of insufficient virological response, no virological response by week 32, HIV-1 RNA concentration 50 copies per mL or higher at any time after week 32; death from any cause; any new or recurrent AIDS event; or any serious non-AIDS event. Patients were randomised in a 1:1 ratio to receive oral treatment with 400 mg raltegravir twice daily plus 800 mg darunavir and 100 mg ritonavir once daily (NtRTI-sparing regimen) or tenofovir-emtricitabine in a 245 mg and 200 mg fixed-dose combination once daily, plus 800 mg darunavir and 100 mg ritonavir once daily (standard regimen). This trial was registered with ClinicalTrials.gov, number NCT01066962., Findings: Of 805 patients enrolled, 401 received the NtRTI-sparing regimen and 404 the standard regimen, with median follow-up of 123 weeks (IQR 112-133). Treatment failure was seen in 77 (19%) in the NtRTI-sparing group and 61 (15%) in the standard group. Kaplan-Meier estimated proportions of treatment failure by week 96 were 17·8% and 13·8%, respectively (difference 4·0%, 95% CI -0·8 to 8·8). The frequency of serious or treatment-modifying adverse events were similar (10·2 vs 8·3 per 100 person-years and 3·9 vs 4·2 per 100 person-years, respectively)., Interpretation: Our NtRTI-sparing regimen was non-inferior to standard treatment and represents a treatment option for patients with CD4 cell counts higher than 200 cells per μL., Funding: European Union Sixth Framework Programme, Inserm-ANRS, Gilead Sciences, Janssen Pharmaceuticals, Merck Laboratories., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We used high-resolution phylogenetic methods in the context of mother-to-child transmission to obtain information on the timing of the infection and on the transmission network. A total of 33 pol sequences (from maternal peripheral blood, from breast milk, and from plasma of children) belonging to five cases of HIV infant transmission were studied. Using time-scaled phylogeny we were able to estimate that in two cases the transmission occurred after the recommended duration of breastfeeding, supporting a longer, not reported, duration of breastfeeding as a significant factor associated with HIV infant acquisition in this cohort. Among the postnatal infections we were also able to demonstrate that the cell-free virus in breast milk was the most likely population associated with the event of transmission. Our study showed that a coalescent-based model within a Bayesian statistical framework can provide important information that can contribute to optimizing preventive strategies.

The purpose of this investigation was to determine the impact on human immunodeficiency virus (HIV) tropism of uncontrolled virus exposure during 2 years of intermittent highly active antiretroviral therapy (HAART). The Istituto Superiore di Sanità-Pulsed Antiretroviral Therapy (ISS-PART) randomized study compared the outcome of 2 years of structured treatment interruptions (STIs) versus standard continuous treatment in first-line HAART responder subjects. The STI schedule consisted of five STIs of 1, 1, 2, 2, and 3 months, respectively, separated by four periods of 3-month therapy. In the present study, coreceptor tropism was assessed in 12 patients of the STI arm at different time points over a period of 2 years. Tropism was determined on DNA and RNA by V3 loop region sequencing. The Geno2pheno algorithm (false-positive rate, FPR: 20%) was used for data interpretation. At baseline, 9/12 subjects (75.0%) had CCR5-tropic viruses in their HIV. Three had a CXCR4-tropic virus. Ten patients maintained the same coreceptor in DNA after 2 years, whereas in two patients, a shift occurred (one R5-X4, one X4-R5). In a patient with an R5 virus at baseline, a transient change to X4 tropism was seen in the rebounding virus during STI. Changes in tropism were not associated with the amplitude and duration of virus exposure during STIs, residual viremia at baseline, or the development of resistance mutations in the RT region. Our preliminary results suggest that viral replication, observed after short periods of treatment interruption, is not enough to drive the evolution of HIV tropism.

Antiretroviral therapy for HIV infection needs lifelong access and strict adherence to regimens that are both expensive and associated with toxic effects. A curative intervention will be needed to fully stop the epidemic. The failure to eradicate HIV infection during long-term antiretroviral therapy shows the intrinsic stability of the viral genome in latently infected CD4T cells and other cells, and possibly a sustained low-level viral replication. Heterogeneity in latently infected cell populations and homoeostatic proliferation of infected cells might affect the dynamics of virus production and persistence. Despite potent antiretroviral therapy, chronic immune activation, inflammation, and immune dysfunction persist, and are likely to have important effects on the size and distribution of the viral reservoir. The inability of the immune system to recognise cells harbouring latent virus and to eliminate cells actively producing virus is the biggest challenge to finding a cure. We look at new approaches to unravelling the complex virus-host interactions that lead to persistent infection and latency, and discuss the rationale for combination of novel treatment strategies with available antiretroviral treatment options to cure HIV., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We evaluated 70 HIV-infected pregnant women with CD4⁺ cell count >350 cells per cubic millimeter who received zidovudine, lamivudine, and nevirapine from week 25 of gestation until 6 months after delivery and a 3-week tail of zidovudine and lamivudine at the moment of drug discontinuation. Forty days after the interruption of all drugs resistance mutations were present in 5 of 70 (7.1%) women. Two of them had the same mutation archived in baseline HIV DNA. The other 3 women had, at least once, detectable viral load and presence of mutations during treatment. Overall, the risk of developing resistance mutations in compliant women was low.

The aim of this study was to assess the correlation between the estimated adherence of HIV-1 infected patients with antiretroviral (ARV) therapy failure and drug-resistant mutations. We studied 40 patients with virological and immunological ARV-therapy failure. In order to assess the adherence of patients we used the SERAD questionnaire. Genomic sequencing of the HIV-1 pol gene was performed. 100% adherence was reported by 27 patients (67.5%) (adherent patients). Multivariate analysis showed that only baseline and nadir CD4+ counts maintained a significant correlation with the adherence. For PR and NNRTI mutations, we did not find any difference between the two groups of patients. Baseline NRTI mutations were higher in adherent patients than in non-adherent patients (p<0.05). No differences were found between plasma mutations and PBMC mutations. The authors conclude that genotypic resistance mutations were found in the majority of patients with ARV-therapy failure despite a good self-reported adherence to therapy. Adequate adherence to therapy is not the only key factor in viral suppression.

Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: The use of simplified methods for viral load determination could greatly increase access to treatment monitoring of HIV patients in resource-limited countries., Objective: The aim of the present study was to optimize and evaluate the performance of the Roche COBAS Taqman assay in HIV-RNA quantification from dried blood spots (DBS) and dried plasma spots (DPS)., Study Design: EDTA blood samples from 108 HIV-infected women were used to prepare 129 DBS and 76 DPS on Whatman 903 card. DBS and DPS were stored at -20 degrees C. HIV-1 RNA was extracted from DBS/DPS using the MiniMAG system (bioMerieux). Amplification and detection were performed using the Roche COBAS TaqMan assay. Plasma viral load results were used as standard., Results: There was a high correlation between measures of viral load in plasma and in DBS/DPS (r=0.96 and 0.85 respectively, P<0.001). Overall, viral load values in DBS and DPS tended to be lower than in plasma with mean (SD) differences of 0.32 log(0.22) for DBS and of 0.35 (0.33) for DPS. Detection rates were 96.4% for DBS and 96.1% for DPS in samples with corresponding plasma values >3.0 log copies/ml. Samples with HIV-RNA below 50 copies/ml were correctly identified in 18/19 DBS and in 7/7 DPS., Conclusions: Both DBS and DPS provided results highly correlated to the plasma values. High detection rate was obtained with both DBS and DPS when HIV-RNA was >3.0 log copies/ml. Our results support the use of DBS/DPS to detect virologic failure in resource-limited settings., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Background: Recent data have shown that plasma levels of lipopolysaccharide (LPS) are a quantitative indicator of microbial translocation in HIV infected individuals., Objectives: To assess the impact of residual viral replication on plasma LPS in HAART-treated HIV+ subjects with <50copies/ml HIV-1 RNA and to evaluate LPS changes during repeated HAART interruptions not exceeding 2-month duration., Study Design: LPS was measured in 44 HIV+ subjects at T0 (during HAART) and at day 15 of the first and fourth HAART interruption. Ten uninfected, healthy donors were studied as well. Residual plasma HIV-1 RNA was measured at T0 by an ultra-ultrasensitive method with limit of detection of 2.5copies HIV-1 RNA/ml. Subjects with less than 2.5copies/ml (fully suppressed - FS) were compared to those with 2.5-50copies/ml (partially suppressed - PS)., Results: At T0, plasma LPS levels were comparable in FS and uninfected subjects, whereas in PS they were higher than in uninfected subjects (p=0.049). After 4 HAART interruptions, they did not change significantly. However, LPS values were lower in FS than in PS (p=0.020). An inverse correlation was found between CD4 and LPS levels (p=0.044) in PS group only., Conclusions: A reduced degree of microbial translocation was seen in subjects with a more complete suppression of viral replication. Repeated HAART interruptions had no significant impact on plasma LPS levels.

Objectives: The aim of the study was to determine the modifications of the mutational archive in proviral HIV-1 DNA occurring during 24 months of intermittent or continuous highly active antiretroviral therapy (HAART)., Methods: The study population included subjects enrolled in the Istituto Superiore di Sanità Pulsed Antiretroviral Therapy (ISS PART) clinical trial. All of these patients were on first-line HAART and had plasma HIV-1 RNA below 50 HIV-1 RNA copies/mL. A genotypic resistance test was performed on HIV-1 DNA extracted from peripheral blood mononuclear cells (PBMC) at baseline and after 24 months of follow-up. Resistance-associated mutations (RAMs) were defined according to the International AIDS Society (IAS) USA classification., Results: Sixty-nine subjects were included in the study [36 enrolled in arm A of the ISS PART (continuous HAART) and 33 enrolled in arm B (intermittent HAART)]. No major modifications of the mutational archive were found in either group after 24 months of follow-up, in terms of both the proportion of subjects with mutations and the total number of mutations., Conclusions: In this patient population, the mutational archive in HIV-1 DNA extracted from PBMC was stable for 24 months, irrespective of HAART modality, whether continuous or intermittent.

We analyzed the sequences of the HIV viral populations obtained from plasma, cell-free breast milk, and breast milk cells of HAART-treated (23) and untreated (30) HIV-infected women to obtain information about the origin of the breast milk virus. Sequence analyses of viruses were performed using the TruGene HIV-1 assay. Direct sequences of the reverse transcriptase (RT) and protease (PR) genes were analyzed using the Phylip 3.68 suite of sequence analysis program and pairwise evolutionary distances were calculated with the Kimura two parameter model for estimation of distances. We found that the genetic distances between the plasma and the cell-free breast milk viruses and between the cell-free and cell-associated breast milk viruses for RT were higher in HAART-receiving women than in untreated women, suggesting viral evolution under selective drug pressure in breast milk. Our data support the hypothesis of the presence of an actively replicating viral population in the breast milk compartment, distinct from that present in plasma.

We analyzed single nucleotide polymorphisms in the 5'-untranslated region (-44C/G and -52G/A) of the beta-defensin-1 gene in 78 Mozambican HIV-1-infected mothers. We observed significantly lower levels of HIV-1 RNA in breast milk, but not in plasma, in women with the -52GG genotype versus women with the -52GA and -52AA genotypes, supporting the hypothesis that different expression of beta-defensins could have an impact on viral replication in breast milk.

Background: Few data have been reported on the dynamics of HIV-1 DNA during intermittent highly active antiretroviral therapy (HAART). In this study, we measured cell-associated HIV-1 DNA and provirus-infected cells during the Istituto Superiore di Sanità-Pulsed Antiretroviral Therapy (ISS-PART) clinical trial., Methods: HIV-1 DNA was measured by real-time polymerase chain reaction (PCR) in the peripheral blood mononuclear cells (PBMCs) of 37 subjects enrolled in the ISS-PART, a randomized clinical trial comparing 24 months of intermittent (arm B) versus continuous (arm A) HAART in chronic HIV infection. In 14 subjects, the number of provirus-infected cells was also measured at baseline and at month 24., Results: At baseline, the number of HIV-1 DNA copies/10(6) PBMCs was similar in arm B (mean +/- SD: 121 +/- 172, median = 35) and arm A (mean +/- SD: 107 +/- 153, median = 10) (P = not significant [n.s.]). No significant variations occurred over time; at 24 months, the HIV-1 DNA level was 77 +/- 28 (median = 30) copies/10(6) PBMCs in arm B and 166 +/- 321 copies/10(6) PBMCs (median = 10) in arm A (P = n.s.). At baseline, the provirus-infected cell counts were 85 +/- 98 (median = 50) cells/10(6) PBMCs in arm B and 92 +/- 113 (median = 50) cells/10(6) PBMCs in arm A (P = n.s.), with no variations at 24 months., Conclusions: These findings suggest that the intermittent schedule of the ISS-PART has no major impact on viral reservoirs, at least in a midterm follow-up.

Objectives: The aim of the study was to compare incidence rates (IRs) of AIDS/death in patients with and without treatment interruption (TI) of combination antiretroviral therapy (cART) for periods of 3 months or more for different categories of CD4 cell count and viral load, and to determine risk factors for clinical progression to AIDS/death., Methods: Patients starting cART with a CD4 cell count and a viral load available within 6 months of starting cART were included in the study. The IR and risk factors of TI were determined. We assessed the incidence rate ratios (IRRs) for TI and AIDS/death events using Poisson regression models., Results: Of 3811 patients included in the study, 26% were ART-naïve prior to cART. The median date of starting cART was July 1997, the median CD4 cell count was 226 cells/microL and the median viral load was 4.36 log(10) HIV-1 RNA copies/mL. We observed 1243 interruptions and 403 AIDS-events/deaths. The IR of AIDS/death was higher in patients with lower CD4 cell counts or higher viral loads, regardless of TI. After adjusting for baseline factors, the IR of AIDS/death was significantly higher in the TI group than in the non-TI group [IRR 2.63; 95% confidence interval (CI) 2.01-3.44; P<0.0001]; this could be explained by current CD4 cell counts and viral loads, as the CD4 cell count- and viral load-adjusted IRR was 1.14 (95% CI 0.86-1.51; P=0.37). Within the TI group, patients with a current CD4 cell count of <200 cells/microL had a 3-fold higher risk of AIDS/death than those with a CD4 cell count of 200-350 cells/microL, whereas patients with a current CD4 cell count of >350 cells/microL had a 4-fold lower risk of disease progression., Conclusions: TI is common in clinical practice. The risk of AIDS/death increased more than 2-fold for patients stopping all cART regimen drugs for 3 months or more. Among patients experiencing a TI, those with low CD4 cell counts, high viral loads or prior AIDS had an increased risk of AIDS/death. Hence, TI should be discouraged and closely monitored if it occurs.

The simultaneous expression of 19 apoptosis-related genes was analyzed by RNA-protection assay in peripheral blood mononuclear cells of HIV-infected patients before and during successful antiretroviral therapy (ART). After 12 months of therapy, the expression of the pro-apoptotic genes FAS, FAS-L, FAF-1, FADD, CASPASE-8, DR3, TRAIL, TNFR-1, TRADD, and BAX was significantly downregulated with respect to time 0, while that of BCL-2, BCL-XL, and MCL-1 was significantly upregulated. The data suggest that inhibition of cell death in HIV-positive patients under successful therapy is the result of a complex network of multifactor signaling, correlated with both death and survival of lymphocytes.

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.

Background: Combination antiretroviral therapy (cART) may vary in ability to suppress viral load and increase CD4+ T-cell count in people infected with different HIV-1 subtypes, possibly due to differences in resistance development. Antiretroviral drugs have predominantly been developed in Western Europe/North America on the basis of the most prevalent subtype, B. However, non-B subtypes are increasingly spreading worldwide., Objective: To compare virological and immunological response to cART between patients infected with B and non-B subtypes across Europe., Design: EuroSIDA prospective, observational cohort with 11,928 HIV-1-infected patients., Methods: Response to cART was analysed in patients with subtypes determined pre-cART, via multivariable logistic regression on the first measurements 6-12 months after starting cART. A virological response was defined as a viral load <500 copies/mi and immunological response as a CD4+ T-cell count increase of > or =100 cells/mm(3)., Results: Forty-five percent of patients were antiretroviral naive at initiation of cART. Virological suppression was achieved by 58% of 689 subtype-B-infected patients and 66% of 102 non-B-infected patients (P=0.159). After adjustment for potential confounders, there was no significant difference in odds of achieving virological suppression (non-B compared with B; odds ratio [OR]: 1.05, 95% confidence interval [CI]: 0.58-1.93, P=0.866). An immunological response was achieved by 43% of 753 B-infected patients and 48% of 114 non-B-infected patients (P=0.334). After adjustment, there was no significant difference in odds of an immunological response (OR: 1.17, 95% CI: 0.73-1.87, P=0.524)., Conclusions: There was no evidence of significant differences in virological or immunological response to cART between patients infected with HIV-1 B and non-B subtypes.

Objective: To determine factors associated with < 2.5 copies/ml plasma HIV RNA in subjects treated with highly active antiretroviral therapy (HAART) and with viraemia < 50 copies/ml., Design: Cross-sectional analysis of 84 HIV-positive patients taking HAART with plasma HIV RNA < 50 copies/ml for at least 6 months and no history of virological failure., Methods: Current HAART therapy was based on a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 66%, a protease inhibitor in 26% and nucleoside reverse transcriptase inhibitors in 7%. Viraemia levels were measured using a modified ultrasensitive Roche Amplicor HIV-1 Monitor test able to quantify plasma HIV RNA to a lower limit of 2.5 copies /ml; proviral DNA was measured with a real-time polymerase chain reaction assay. Analysis of variance and multiple logistic regression analysis were utilized to test for associations between residual replication and other variables., Results: Residual HIV viraemia > 2.5 copies/ml was found in 50% of subjects; 94% of subjects had detectable proviral DNA (>or= 20 copies/10(6) peripheral blood mononuclear cells) and 21% had archived mutations. Usage of a NNRTI-based HAART was the only independent predictor of viral suppression below the cut-off value of the modified ultrasensitive assay., Conclusions: In our population, NNRTI-based HAART seems to have a stronger impact on residual replication than protease inhibitor-based HAART. This finding may be considered in therapeutic decisions such as the choice of initial HAART regimen and the interruption or simplification of treatment.

Background: Whether hepatitis B (HBV) coinfection affects outcome in HIV-1-infected patients remains unclear., Objective: To assess the prevalence of HBV (assessed as HBsAg) coinfection and its possible impact on progression to AIDS, all-cause deaths, liver-related deaths and response to highly active antiretroviral therapy (HAART) in the EuroSIDA cohort., Methods: Data on 9802 patients in 72 European HIV centres were analysed. Incidence rates of AIDS, global mortality and liver-related mortality, time to 25% CD4 cell count increase and time to viral load < 400 copies/ml after starting HAART were calculated and compared between HBsAg-positive and HBsAg-negative patients., Results: HBsAg was found in 498 (8.7%) patients. The incidence of new AIDS diagnosis was similar in HBsAg-positive and HBsAg-negative patients (3.3 and 3.4/100 person-years, respectively) even after adjustment for potential confounders: the incidence rate ratio (IRR) was 0.94 [95% confidence interval (CI), 0.74-1.19; P = 0.61]. The incidences of all-cause and liver-related mortalities were significantly higher in HBsAg-positive subjects (3.7 and 0.7/100 person-years, respectively) compared with HBsAg-negative subjects (2.6 and 0.2/100 person-years, respectively). The adjusted IRR values were 1.53 for global (95% CI, 1.23-1.90; P = 0.0001) and 3.58 for liver-related (95% CI, 2.09-6.16; P < 0.0001) mortality. HBsAg status did not influence viral or immunological responses among the 1679 patients starting HAART., Conclusions: The prevalence of HBV coinfection was 9% in the EuroSIDA cohort. Chronic HBV infection significantly increased liver-related mortality in HIV-1-infected patients but did not impact on progression to AIDS or on viral and immunological responses to HAART.

Objective: To assess gender differences in the long-term clinical, virological and immunological outcomes during highly active antiretroviral therapy (HAART)., Methods: This longitudinal observational multicentre study followed 2460 HIV-infected patients who had begun a protease inhibitor-based regimen for a median period of 43 months. Outcome measures were virological suppression (< 500 copies/ml), confirmed virological rebound after suppression, and death or new AIDS-defining illness (ADI)., Results: At baseline, 690 female patients (28.0%) had significantly lower age, higher prevalence of heterosexual contact and lower prevalence of intravenous drug use as risk factors for HIV infection compared with males. Furthermore, females had a lower number of AIDS-defining illnesses, higher CD4 cell counts and lower viral loads. No gender differences were reported in terms of proportion of patients achieving viral suppression or exhibiting rebound after achieving viral suppression. Female patients experienced reduced clinical progression during follow-up compared with males (P = 0.008) by Kaplan-Meier analysis; however this difference was not significant in an adjusted analysis. In a multivariate model, the interaction between gender and risk factor for HIV or viral load showed that female drug users and female patients with a baseline HIV RNA viral load of 10(4)-10(5) copies/ml had a favourable clinical outcome compared with males (P = 0.035 and P = 0.015, respectively)., Conclusion: No differences were found between genders in terms of virological and immunological outcomes during long-term HAART. Nevertheless, a lower risk of clinical progression was reported among female patients with intermediate baseline viral load than in males.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Background: Factors that determine the immunological response to highly active antiretroviral therapy (HAART) are poorly defined., Objective: Our aim was to investigate predictors of immunological failure after initial CD4(+) response., Methods: Data were from EuroSIDA, a prospective, international, observational human immunodeficiency virus (HIV) type 1 cohort., Results: Of 2347 patients with an increase in CD4(+) cell count >or=100 cells/microL within 6-12 months of the initiation of HAART, 550 (23%) subsequently experienced immunological failure (CD4(+) count less than or equal to the pre-HAART value). The incidence of failure was 11.6 incidences/100 person-years of follow-up (95% confidence interval [CI], 10.2-13.4) during the first 12 months and decreased significantly over time (P<.0001). Independent predictors of immunological failure were pre-HAART CD4(+) cell count (per 50% higher; relative hazard [RH], 2.05; 95% CI, 1.83-2.31; P<.0001), time-updated virus load (per 1 log(10) higher; RH, 1.77; 95% CI, 1.64-1.92; P<.0001), and HIV-1 risk behavior (P=.047 for a global comparison of risk groups)., Conclusion: The risk of immunological failure in patients with an immunological response to HAART diminishes with a longer time receiving treatment and is associated with pretreatment CD4(+) cell count, ongoing viral replication, and intravenous drug use.

Background: Most of the studies evaluating rash in HIV-positive patients have focused on nonnucleoside reverse transcriptase inhibitors (NNRTI), particularly nevirapine, and little is known about the occurrence of rash and the risk factors for its development in patients receiving regimens not based on NNRTI., Methods: We evaluated all cases of rash observed during a 48-week randomized multicentre trial in 1251 nucleoside-experienced patients who started treatment with protease inhibitors (ritonavir or indinavir) at CD4 counts below 50 cells/microL. Incidence rates for rash were calculated according to gender, clinical status, age, use of highly active antiretroviral therapy (HAART), Pneumocystis carinii pneumonia (PCP) prophylaxis and use of individual antiretroviral drugs at enrollment. Differences between groups defined according to the above characteristics were tested for statistical significance using the log-rank test in a Kaplan-Meier survival analysis. All factors that gave results in the univariate analyses below the significance level of 0.05 were included in a multivariate analysis using a Cox regression model., Results: During a follow-up period of 9690 person-months, 66 patients (5.3%) developed rash (0.68 events/100 person-months). In the univariate analyses, risk of rash did not differ with trial treatment (indinavir or ritonavir), clinical status, PCP prophylaxis, or age. During follow-up, rash was observed in 7.5% of enrolled women and in 4.5% of enrolled men (P=0.03). Serious rash occurred in 4.5% of enrolled women and in 1.6% of enrolled men (P=0.003). Use of HAART (P<0.001) and inclusion of zidovudine and of zalcitabine in the prescribed regimen (P=0.02) appeared to be associated with a lower risk of rash. In the multivariate analysis, the variables that remained significantly predictive of rash were gender (risk for women compared to men: 1.65, 95% confidence interval (CI): 1.00-2.72, P=0.048) and use of a non-HAART regimen (risk for non-HAART patients compared to HAART: 2.73, 95% CI: 1.49-5.02, P=0.001)., Conclusions: In our study, about 5% of HIV-positive patients who started treatment with protease inhibitors at very low CD4 counts developed rash, generally in the first few weeks after treatment. Risk was significantly higher in women and in patients who did not receive a HAART regimen. Our data indicate that women have a higher risk of rash than men, also with regimens that do not include NNRTI.

Background: It is unclear whether therapy for human immunodeficiency virus type 1 (HIV-1) should be initiated with a four-drug or two sequential three-drug regimens., Methods: In this multicenter trial we compared initial therapy involving four-drug regimens containing efavirenz and nelfinavir in combination with either didanosine and stavudine or zidovudine and lamivudine with therapy involving two consecutive three-drug regimens the first of which contained either efavirenz or nelfinavir., Results: A total of 980 subjects were followed for a median of 2.3 years. There was no significant difference in the occurrence of regimen failures between the group that received the four-drug regimen containing didanosine, stavudine, nelfinavir, and efavirenz and the groups that received the three-drug regimens beginning with didanosine, stavudine, and nelfinavir (hazard ratio for regimen failure, 1.24) or didanosine, stavudine, and efavirenz (hazard ratio, 1.01). There was no significant difference between the group that received the four-drug regimen containing zidovudine, lamivudine, nelfinavir, and efavirenz and the groups that received the three-drug regimens beginning with zidovudine, lamivudine, and nelfinavir (hazard ratio, 1.06) or zidovudine, lamivudine, and efavirenz (hazard ratio, 1.45). A four-drug regimen was associated with a longer time to the first regimen failure than the three-drug regimens containing didanosine, stavudine, and nelfinavir (hazard ratio for a first regimen failure, 0.55); didanosine, stavudine, and efavirenz (hazard ratio, 0.63); or zidovudine, lamivudine, and nelfinavir (hazard ratio, 0.49), but not the three-drug regimen containing zidovudine, lamivudine, and efavirenz (hazard ratio, 1.21)., Conclusions: There was no significant difference in the duration of successful HIV-1 treatment between a single four-drug regimen and two consecutive three-drug regimens. Among these treatment strategies, initiating therapy with the three-drug regimen of zidovudine, lamivudine, and efavirenz is the optimal choice., (Copyright 2003 Massachusetts Medical Society)

Background: The optimal sequencing of antiretroviral regimens for the treatment of infection with human immunodeficiency virus type 1 (HIV-1) is unknown. We compared several different antiretroviral treatment strategies., Methods: This multicenter, randomized, partially double-blind trial used a factorial design to compare pairs of sequential three-drug regimens, starting with a regimen including zidovudine and lamivudine or a regimen including didanosine and stavudine in combination with either nelfinavir or efavirenz. The primary end point was the length of time to the failure of the second three-drug regimen., Results: A total of 620 subjects who had not previously received antiretroviral therapy were followed for a median of 2.3 years. Starting with a three-drug regimen containing efavirenz combined with zidovudine and lamivudine (but not efavirenz combined with didanosine and stavudine) appeared to delay the failure of the second regimen, as compared with starting with a regimen containing nelfinavir (hazard ratio for failure of the second regimen, 0.71; 95 percent confidence interval, 0.48 to 1.06), as well as to delay the second virologic failure (hazard ratio, 0.56; 95 percent confidence interval, 0.29 to 1.09), and significantly delayed the failure of the first regimen (hazard ratio, 0.39) and the first virologic failure (hazard ratio, 0.34). Starting with zidovudine and lamivudine combined with efavirenz (but not zidovudine and lamivudine combined with nelfinavir) appeared to delay the failure of the second regimen, as compared with starting with didanosine and stavudine (hazard ratio, 0.68), and significantly delayed both the first and the second virologic failures (hazard ratio for the first virologic failure, 0.39; hazard ratio for the second virologic failure, 0.47), as well as the failure of the first regimen (hazard ratio, 0.35). The initial use of zidovudine, lamivudine, and efavirenz resulted in a shorter time to viral suppression., Conclusions: The efficacy of antiretroviral drugs depends on how they are combined. The combination of zidovudine, lamivudine, and efavirenz is superior to the other antiretroviral regimens used as initial therapy in this study., (Copyright 2003 Massachusetts Medical Society)

Objective: The development of drugs that can be used as topical microbicides is currently recognized as a priority area of research., Design: A preclinical evaluation of the potential effectiveness of TMC120, a non-nucleoside reverse transcriptase inhibitor (NNRTI), as a topical microbicide to prevent vaginal HIV-1 transmission in a humanized severe combined immunodeficient (hu-SCID) mouse model., Methods: Reconstituted mice received an intravaginal application of a TMC120-containing gel 20 min prior to a non-invasive vaginal challenge with cell-associated HIV. The possible cytotoxic effect of TMC120-containing-gel on lymphocytes was assessed and their in vivo migration was followed using fluorescently labelled human lymphocytes. Systemic infection was monitored by p24 antigen detection in culture supernatant from cocultured intraperitoneal cells using antigen capture enzyme-linked immunosorbent assay test and by the presence of integrated proviral HIV-1 DNA in DNA extracted from spleen cells. In vivo migration of labelled lymphocytes was examined by analysis of cells isolated from regional lymph nodes., Results: In this model, systemic infection was successfully inhibited by the presence of TMC120-containing gel at vaginal level. The in vivo migration of human lymphocytes from the vagina to regional lymph nodes, following the deposition of TMC120-containing gel, excluded the possibility that inhibition of systemic infection was a result of NNRTI toxicity., Conclusions: Vaginal transmission of HIV was successfully prevented by the application of a gel formulation containing TMC120. This is the first evidence of the in vivo effectiveness of a microbicide preparation containing an NNRTI against cell-associated HIV.

Two zidovudine/lamivudine regimens for the prevention of HIV perinatal transmission were studied for the selection of resistance mutations. The M184V mutation was detected one week after delivery in six out of fifty women (12%) who received the regimen prepartum, intrapartum and postpartum, and was no longer present 3 months later. No nucleoside reverse transcriptase inhibitor resistance-associated mutations were detected in 50 women who received zidovudine/lamivudine intrapartum and postpartum only. No association with the risk of perinatal transmission was found.

Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4(+) T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4(+) cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4(+) cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4(+) cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (<50 copies/ml), whereas in patients with HIV RNA loads of >500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups.

Objectives: To describe the prevalence and risk factors of poor CD4 count rise despite a good virological response on highly active antiretroviral treatment (HAART)., Methods: The patients from the EuroSIDA study who started HAART with a baseline CD4 count of <350 cells/microL and where all viral load (pVL) measures remained below 500 HIV-1 RNA copies/mL between 6 and 12 months after the start of HAART were included. The risk factors for poor CD4 count rise were analyzed by multiple regression., Results: Seven hundred and eighty patients were included. A low CD4 count response was observed in 225 patients (29%). The risk factors for this condition were older age, lower CD4 count at baseline, higher increase from the nadir to baseline CD4 count and lower pVL at baseline. Patients taking > or =one drug from each of the three antiviral classes were more likely to have a good CD4 response but a minority of the study participants was taking this treatment regimen (3.1%) and the confidence interval was large., Conclusions: A poor immune reconstitution despite a good virological control is frequent after initiation of HAART among patients with a baseline CD4 count of <350 cells/microL. The underlying mechanisms leading to this condition seems mainly driven by the age and the baseline immunological and virological status of the patients.

In 32 patients for whom highly active antiretroviral therapy was failing, a good agreement between drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) was found (k = 0.85). The mutations with the lowest agreement were 20R, 63P, and 84V in the protease gene and 184V in the reverse transcriptase gene. In eight patients, primary drug resistance mutations were detected only in PBMCs.

The fitness of human immunodeficiency virus (HIV) in vivo depends on the interaction of a multitude of viral and host factors. The aim of this study was to analyze the biological phenotype and the intrinsic capacity of the HIV isolates with drug-resistance mutations to replicate efficiently in the absence of drugs. An open label multicenter cross-sectional study was undertaken on 28 HIV-infected patients failing antiretroviral treatment. The subjects were studied for CD4+ cell count, HIV viral load, syncytium-inducing phenotype, genotypic drug-resistance assay, and replication capacity of HIV isolates assessed by co-culture assay. All HIV isolates showed a decreased replication capacity compared with wild-type strains. The lowest replication capacity was detected in HIV strains with more than five drug-resistance mutations. The highest replication capacity was observed in strains carrying the K103N and Y181C primary mutations that emerged after treatment with non-nucleoside analogue inhibitors. Isolates with R5 biological phenotype had a higher number of resistant mutations than X4 isolates (P = 0.004). Particularly, the R5 phenotype was detected in all 6 isolates with more than 14 drug-resistance mutations. Patients with R5 strains had plasma viral load similar to patients with X4 strains, but marginally higher CD4+ cell counts, and their HIV isolates had significantly lower replication capacity of HIV isolates (P = 0.008). No patient carrying HIV with a maintained replication capacity had a viral load less than 30,000 copies/ml. In patients failing HAART, the detection of HIV isolates with the R5 biological phenotype correlates with CD4+ cell count, an impaired replication capacity, and a high number of drug-resistance mutations., (Copyright 2003 Wiley-Liss, Inc.)

Objective: To study virologic and immunologic factors associated with discordant treatment response in HIV-infected patients receiving highly active antiretroviral therapy (HAART)., Design: Study participants included a total of 27 patients: (a) 10 discordant patients (mean CD4+ cell count, 396.1 x 10 cells/l; mean HIV-RNA, 5.4 log copies/ml); (b) seven responder patients (mean CD4+ cell count, 997.5 x 10 cells/l); and (c) 10 failing patients (mean CD4+ cell count 66.5 x 10 cells/l; mean HIV-RNA, 5.4 log copies/ml).(10) (10), Methods: The HIV-1 isolation rate and biological phenotype, drug resistance genotypic mutations of HIV-1 strains, recall and HIV-1-specific antigen lymphocyte proliferation (LP), and interleukin (IL)-15 production were studied., Results: Virus isolation was obtained in 30% of discordant patients, and in 100% of failing patients. A higher replication constant was reported in discordant patients. No difference in the number of drug resistance mutations and biological phenotypes of HIV-1 was found in discordant patients with respect to failing patients. Discordant patients developed positive LP responses to and HIV-1 p24. LP in response to, HIV-1 p24 and gp160 was positive in responder patients. No significant LP was found in failing patients. Increased levels of IL-15 after stimulation with lipopolysaccaride (LPS) and were found in both discordant patients and responder patients. Conversely, a strong reduction of IL-15 levels was observed in failing patients., Conclusion: The present results suggest that decreased virus isolation rate, restoration of both lymphocyte proliferation and IL-15 production are factors involved in the discordant antiretroviral therapy response of HIV-infected patients.

This study prospectively assessed the impact of treatment modality, virus load, and CD4 cell count of <50 cells/mm(3) on human immunodeficiency virus disease progression. The incidence rate of new AIDS disease or death was 54.8 (95% confidence interval, 48.7-59.9) per 100 person-years of follow-up. Independent predictors related to progression were latest CD4 cell count (relative risk [RR], 0.84/10 mm(3) higher; P<.0001), latest hemoglobin level (RR, 0.79/g/L higher; P<.0001), Pneumocystis carinii pneumonia prophylaxis (RR, 0.49; P<.0001), latest body mass index (RR, 0.93/kg/m(2) higher; P=.002), latest virus load (RR, 1.11/log(10) higher; P=.03), and intensity of treatment (RR, 1.82, P=.004; RR 2.27, P<.0001; RR 2.46, P=.0001; RR 2.33 P<.0006; 5.10, P<.0001, respectively, for 4, 3, 2, 1, or no drugs vs. >or=5 drugs). Although reverse causality cannot be excluded, more intense antiviral treatment appears to decrease the risk of progression in immunocompromised patients.

Objective: To develop an animal model of vaginal transmission of HIV-1 for the evaluation of vaginal microbicides., Design: Vaginal infection was performed in SCID mice reconstituted with 4 x 107 human peripheral blood lymphocytes (hu-PBL) by non-invasive vaginal administration. The hu-PBL were previously infected in vitro with a non-syncytium (NSI) strain of HIV-1 (SF162) (hu-PBL-SCID). Lymphocyte migration in vivo was examined using fluorescently labelled human lymphocytes., Methods: The percentage of CD4 T cells, plasma viral load and p24 antigen were evaluated using fluorescent activated cell sorting (FACS), the Amplicor HIV-1 monitor kit and enzyme-linked immunosorbent assay, respectively. Polymerase chain reaction (PCR) analysis was performed on DNA extracted from spleen and lymph nodes. For in vivo migration of labelled lymphocytes, the mice were sacrificed after 4, 24 and 48 h; vaginae and local lymph nodes were removed, snap frozen with OCT, sectioned and examined by fluorescent microscopy and FACS., Results: HIV transmission was established using virus-infected cells inoculated vaginally, as shown by FACS, HIV viral load, p24 and PCR results. Labelled cells were easily located within the vaginal tissues after 4 h. However, few or no cells could be identified after 24 or 48 h at the vaginal level, whereas labelled cells could be detected at the level of regional lymph nodes., Conclusions: Because of its simplicity and practical features compared with other animal models, the vaginal HIV-infected hu-SCID mouse model may prove useful to test the activity of compounds against cell-associated HIV and, possibly, other sexually transmitted diseases.

Objective: To explore relationships between exposure to nevirapine and the virological response in HIV-1-infected individuals participating in the INCAS trial., Methods: The elimination rate constant of plasma HIV-1 RNA (k) was calculated during the first 2 weeks of treatment with nevirapine, zidovudine and didanosine in 51 antiretroviral-naive HIV-1-infected patients. The relationships between the value of k, the time to reach an undetectable HIV-1 RNA concentration in plasma (< 20 copies/ml) and the success of therapy after 52 weeks of treatment as dependent variables and the exposure to nevirapine, baseline HIV-1 RNA and baseline CD4 cell count as independent variables, were explored using linear regression analyses, proportional hazard models and logistic analyses, respectively., Results: The value of k for HIV-1 RNA in plasma was positively and significantly associated with the mean plasma nevirapine concentration during the first 2 weeks of therapy (P = 0.011) and the baseline HIV-1 RNA (P = 0.008). Patients with a higher exposure to nevirapine reached undetectable levels of HIV-1 RNA in plasma more rapidly (P = 0.03). From 12 weeks on, the median nevirapine plasma concentration was significantly correlated with success of therapy after 52 weeks (P < 0.02)., Conclusions: A high exposure to nevirapine (in a twice daily regimen) is significantly associated with improved virological response in the short as well as in the long term. These findings suggest that optimization of nevirapine concentration might be used as a tool to improve virological outcome in (antiretroviral-naive) patients treated with nevirapine.

ISS-IP1, a multicenter, randomized, 48-week open trial, was designed to compare the introduction of ritonavir or indinavir in patients with previous nucleoside experience and CD4+ cell counts below 50/mm3. Concomitant antiretroviral treatment with nucleoside analogs was allowed. Primary efficacy measures were survival and time to a new AIDS-defining event or death, analyzed through the whole period of observation by the intention-to-treat approach. Primary toxicity measures were time to treatment discontinuation and adverse events, grade at least 3/serious, analyzed by an on-treatment approach. Evaluation-of efficacy also included CD4+ cell and RNA response. The trial enrolled 1251 patients in 5 months. At baseline, mean CD4+ cell count was about 20 cells/mm3 and mean HIV RNA copy number was 4.9 log10/ml in both groups. Overall, 402 patients in the ritonavir group and 250 patients in the indinavir group permanently discontinued the assigned treatment (relative risk, 1.96; 95% CI, 1.68-2.30; p = 0.0001), with most of this difference dependent on a higher number of discontinuation for adverse events in the ritonavir group. After a mean follow-up of 307 days (ritonavir, 304; indinavir, 309), 124 deaths (ritonavir, 61; indinavir, 63; relative risk, 0.96; 95% CI, 0.67-1.36; p = 0.80) and 330 new AIDS-defining events (ritonavir, 170; indinavir, 160; relative risk, 1.05; 95% CI, 0.85-1.31; p = 0.60) were observed. CD4+ cell counts increased in both groups in patients still receiving treatment, with about 100 cells gained by week 24 and 150 cells gained by week 48. Body weight also increased over time in both groups. Analysis of RNA response showed a decrease of 1.5 log10 or higher in both treatment groups. Overall, 400 patients in the ritonavir group and 338 patients in the indinavir group developed at least one grade 3/serious new adverse event during follow-up (relative risk, 1.48; 95% CI, 1.28-1.72; p = 0.0001). Favorable CD4+ cell and RNA responses at 24 and 48 weeks were observed in both groups of patients remaining on treatment. Indinavir showed slightly better effects in sustaining RNA, CD4+ cell, and body weight responses. Ritonavir and indinavir results were comparable in terms of clinical outcome (survival and AIDS-defining events).

Some studies on untreated patients have shown a general correlation between plasma HIV copy number and plasma infectivity in in vitro models. Recent observations also indicate that HIV-RNA level is an important predictor of perinatal transmission and may also have a role in heterosexual transmission. To further analyse the correlation between HIV viral load and plasma infectivity, we studied the relationship between HIV-1 plasma copy number and plasma infectivity prior to and during treatment with antiretroviral combination regimens in HIV-1 infected adults. Plasma infectivity was assessed in vitro by coculture of plasma from HIV-positive patients with PHA-stimulated fresh PBMC from uninfected donors. A positive plasma isolation, in almost all cases (43/45) and irrespective of treatment status, was associated with an HIV viral load higher than 100000 copies per ml, with higher plasma HIV-1 RNA values in isolation-positive samples compared with isolation-negative samples (median values, 710000 vs. 37500 copies per ml, respectively). SI and NSI strains had similarly high viral load values (470000 vs. 790000 copies per ml), but CD4 counts were lower in the SI phenotype group. Our data indicate that low levels of viral load are only exceptionally associated with isolation from plasma in the in vitro model we used. This observation confirms indirectly the presence of an association between viral load and infectivity. The requisite of a high plasma viral load in order to obtain infectivity (i.e. positivity of HIV isolation from plasma) also seems maintained under antiretroviral treatment, adding confidence in the conclusion that reductions in viral load translate into reduction of plasma infectivity. Due to the extreme complexity of factors determining transmission, a very prudent interpretation of the results is essential when information from experimental studies has to be transferred to clinical situations requiring assessment of risks or clinical decisions.