Agence Nationale De Recherche Sur Le SIDA Research Articles

HIV-1 infection in South Kivu (Democratic Republic of Congo): high genotypic resistance to antiretrovirals.

Panzi General Reference Hospital (HGR Panzi) in the Democratic Republic of Congo follows a large number of patients living with HIV-1 (PLWHIV). Although antiretrovirals (ARVs) are available, HIV-1 viral load (HIV-VL) measurement has only been implemented in the hospital since 2018. No data on ARV resistance levels and ARV dosage in plasma have yet been published for this region. We determined the prevalence of virological failure due to ARV resistance amongst patients and assessed the degree of genotypic resistance of the viral strains. We performed an HIV-VL test and determined dosage of ARVs on samples collected from 205 PLWHIV at HGR Panzi between 2017 and 2018, including 13 ARV-naive patients. Genotypic resistance testing was performed on all samples with detectable HIV-VLs, and interpreted with the Agence Nationale de Recherches sur le Sida (ANRS) 2018 algorithm. Baseline resistance to NNRTIs was found in 2 of the 13 treatment-naive individuals (15%). ARV dosage was non-optimal for 44/192 of treated patients (22.9%), with an HIV-VL ≥1000 IU/mL for 40/192 (20.8%) of them. In particular, treatment-experienced viruses presented resistance to at least one NRTI (52.5%), to at least one NNRTIs (70%) or to at least one PIs (15%). Finally, two samples contained viruses with resistance polymorphism in the integrase gene. The high level of resistance to ARVs observed during this study, mainly due to treatment compliance default, fully justifies the implementation of means for closer patient monitoring. The provision of VL tests and therapeutic education management tools in a PLWHIV follow-up remains an absolute necessity to best adapt the current treatment lines in this region.

Does Changing Antiretroviral Therapy in the First Trimester for Pregnancy-Related Concerns Have an Impact on Viral Suppression?

Objective: To determine whether changing antiretroviral therapy (ART) during pregnancy in order to follow guidelines because of concern about fetal risks led to poorer virological outcomes. Methods: All pregnancies in women with HIV1 infection enrolled in the national multicenter prospective French Perinatal cohort were included between 01/2005 and 12/2015, at 14 gestational weeks or more and if the mother was on ART at conception with a plasma viral load < 50 copies/ml. The reasons for a change in the ART were analyzed according to treatment guidelines at the time of the pregnancy, and defined as for pregnancy-related concerns in the absence of reported maternal intolerance. Virological and pregnancy outcomes were studied by survival analysis and logistic regression adjusted for a propensity score established for each patient according to baseline characteristics. Results: Of 10553 pregnancies in the cohort, 1797 were taking ART at conception and had a viral load <50 copies/mL before 14 weeks' gestation. Of these, 411 changed regimens in the first trimester for pregnancy-related concerns. The proportion of change was statistically higher when initial treatment was clearly contraindicated (OR adjusted: 23.1 [14.0- 38.2]) or was regarded as an alternative option (ORa: 2.2 [1.3-3.7]), as compared to recommended first-line regimens. Treatment changes for pregnancy-related concerns did not lead to poorer virological control, compared to pregnancies without such changes (19.3% vs. 15.6%, HRa: 1.0 [0.7-1.4]). Conclusions: Changing ART early in pregnancy with the goal of improving fetal and pregnancy outcomes did not appear to have a destabilizing effect on viral suppression. Financial Support: This work was supported by the French Agence Nationale de Recherche sur le SIDA (ANRS). Potential Conflicts of Interest: The authors declare no conflicts of interest in relation with this work. Ethics Approval Statement: The French Perinatal Cohort (ANRS EPF CO1/CO11) is a prospective, obervational study ongoing since 1985 in 90 perinatal centers throughout France (8) which includes all pregnancies in women with HIV-1 and/or HIV-2 with their informed consent and ethics committee (Comite de Protection des Personnes) approval.

Ensemble Classifiers for Predicting HIV-1 Resistance from Three Rule-Based Genotypic Resistance Interpretation Systems.

Resistance to antiretrovirals (ARVs) is a major problem faced by HIV-infected individuals. Different rule-based algorithms were developed to infer HIV-1 susceptibility to antiretrovirals from genotypic data. However, there is discordance between them, resulting in difficulties for clinical decisions about which treatment to use. Here, we developed ensemble classifiers integrating three interpretation algorithms: Agence Nationale de Recherche sur le SIDA (ANRS), Rega, and the genotypic resistance interpretation system from Stanford HIV Drug Resistance Database (HIVdb). Three approaches were applied to develop a classifier with a single resistance profile: stacked generalization, a simple plurality vote scheme and the selection of the interpretation system with the best performance. The strategies were compared with the Friedman's test and the performance of the classifiers was evaluated using the F-measure, sensitivity and specificity values. We found that the three strategies had similar performances for the selected antiretrovirals. For some cases, the stacking technique with naïve Bayes as the learning algorithm showed a statistically superior F-measure. This study demonstrates that ensemble classifiers can be an alternative tool for clinical decision-making since they provide a single resistance profile from the most commonly used resistance interpretation systems.

Machine Learning to Improve the Effectiveness of ANRS in Predicting HIV Drug Resistance.

ObjectivesHuman immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) is one of the major burdens of disease in developing countries, and the standard-of-care treatment includes prescribing antiretroviral drugs. However, antiretroviral drug resistance is inevitable due to selective pressure associated with the high mutation rate of HIV. Determining antiretroviral resistance can be done by phenotypic laboratory tests or by computer-based interpretation algorithms. Computer-based algorithms have been shown to have many advantages over laboratory tests. The ANRS (Agence Nationale de Recherches sur le SIDA) is regarded as a gold standard in interpreting HIV drug resistance using mutations in genomes. The aim of this study was to improve the prediction of the ANRS gold standard in predicting HIV drug resistance.MethodsA genome sequence and HIV drug resistance measures were obtained from the Stanford HIV database (http://hivdb.stanford.edu/). Feature selection was used to determine the most important mutations associated with resistance prediction. These mutations were added to the ANRS rules, and the difference in the prediction ability was measured.ResultsThis study uncovered important mutations that were not associated with the original ANRS rules. On average, the ANRS algorithm was improved by 79% ± 6.6%. The positive predictive value improved by 28%, and the negative predicative value improved by 10%.ConclusionsThe study shows that there is a significant improvement in the prediction ability of ANRS gold standard.

Performance of Xpert MTB/RIF and Alternative Specimen Collection Methods for the Diagnosis of Tuberculosis in HIV-Infected Children

The diagnosis of tuberculosis in human immunodeficiency virus (HIV)-infected children is challenging. We assessed the performance of alternative specimen collection methods for tuberculosis diagnosis in HIV-infected children using Xpert MTB/RIF (Xpert). HIV-infected children aged ≤13 years with suspected intrathoracic tuberculosis were enrolled in 8 hospitals in Burkina Faso, Cambodia, Cameroon, and Vietnam. Gastric aspirates were taken for children aged <10 years and expectorated sputum samples were taken for children aged ≥10 years (standard samples); nasopharyngeal aspirate and stool were taken for all children, and a string test was performed if the child was aged ≥4 years (alternative samples). All samples were tested with Xpert. The diagnostic accuracy of Xpert for culture-confirmed tuberculosis was analyzed in intention-to-diagnose and per-protocol approaches. Of 281 children enrolled, 272 (96.8%) had ≥1 specimen tested with Xpert (intention-to-diagnose population), and 179 (63.5%) had all samples tested with Xpert (per-protocol population). Tuberculosis was culture-confirmed in 29/272 (10.7%) children. Intention-to-diagnose sensitivities of Xpert performed on all, standard, and alternative samples were 79.3% (95% confidence interval [CI], 60.3-92.0), 72.4% (95% CI, 52.8-87.3), and 75.9% (95% CI, 56.5-89.7), respectively. Specificities were ≥97.5%. Xpert combined on nasopharyngeal aspirate and stool had intention-to-diagnose and per-protocol sensitivities of 75.9% (95% CI, 56.5-89.7) and 75.0% (95% CI, 47.6-92.7), respectively. The combination of nasopharyngeal aspirate and stool sample is a promising alternative to methods usually recommended by national programs. Xpert performed on respiratory and stools samples enables rapid confirmation of tuberculosis diagnosis in HIV-infected children. The ANRS (Agence Nationale de Recherche sur le Sida) 12229 PAANTHER (Pediatric Asian African Network for Tuberculosis and HIV Research) 01 study is registered at ClinicalTrials.gov (NCT01331811).

An investigation into the comparison of three human immunodeficiency virus (HIV) drug resistance interpretation algorithms

Human immunodeficiency virus (HIV) drug resistance is caused by mutations in the patient’s human immunodeficiency virus genome that renders antiretroviral (ARV) drugs less effective. Drug resistance not only results in the patient being more vulnerable to opportunistic infections, but also may increase the spread of resistant strains of HIV. Interpretation computer algorithms may be used to determine which ARV drug(s) the patient are resistant to, by analyzing the mutations that occurred in the patient’s HIV genome, instead of using expensive time consuming phenotypic laboratory tests. There are many different interpretation algorithms, but they often provide different resistance measures, even if applied to the same resistance profile. The aim of this study was to compare the latest versions of three HIV drug resistance interpretation algorithms in order to determine the extent of discrepancies between them. 2926 protease and 1981 reverse transcriptase subtype B sequences where obtained from the Stanford HIV-db genotype-phenotype correlation database. These sequences were pre-processed and the latest rules of the ANRS, HIV-db and REGA algorithms applied to them. The results were then compared with each other. These results indicate that although the accuracy of REGA, ANRS and HIV-db are similar, a deeper analysis of the results indicates that the interpretation algorithms are different. There need to be a mechanism of providing a single interpretation for a resistance profile of a genome. This may be created by collating the strengths of each of the interpretation algorithms. Key words: Bioinformatics, human immunodeficiency virus (HIV), REGA, Agence Nationale de Recherches sur le SIDA (ANRS), HIV-db, genotype, interpretation algorithms.

Premature Delivery in HIV-Infected Women Starting Protease Inhibitor Therapy During Pregnancy: Role of the Ritonavir Boost?

The association between combination antiretroviral (cARV) therapy use by human immunodeficiency virus (HIV)-infected women during pregnancy and risk of prematurity is still controversial. We explored this question, focusing on the initiation of ritonavir-boosted protease inhibitors (PIs) during pregnancy, which is now standard care. Trends in prematurity (<37 gestational weeks) were studied among all singleton pregnancies in the Agence Nationale de Recherche sur le SIDA (ANRS) French Perinatal Cohort from 1990 through 2009 (n = 13 271). In-depth analysis was conducted in a more detailed substudy of the cohort, among women starting PI-based ARV therapy during pregnancy (n = 1253). Multivariable analysis adjusted for immunovirological status and known risk factors for prematurity. Prematurity increased from 9.2% during 1990-1993 (no therapy) and 9.6% during 1994-1996 (mostly zidovudine monotherapy) to 12.4% during 1997-1999 (dual-nucleoside analog therapy) and 14.3% during 2005-2009 (routine cARV therapy; P < .01). Prematurity was associated with cARV therapy, compared with zidovudine monotherapy, with an adjusted odds ratio of 1.69 (95% confidence interval [CI], 1.38-2.07; P < .01) when accounting for maternal age, intravenous drug use, geographic origin, and CD4 cell count. During 2005-2009, the prematurity rate was higher with boosted than with nonboosted PI therapy started during pregnancy (14.4% vs 9.1% [P = .05]; adjusted hazard ratio, 2.03 [95% CI, 1.06-3.89; P = .03] in multivariate analysis). The difference concerned mainly induced preterm delivery for maternal or fetal indications (5.6% vs 1.6%; P = .02), The prematurity rate among HIV-infected pregnant women was twice that in the general population in France; this was not entirely explained by sociodemographic characteristics. Prematurity was independently associated with cARV therapy and, particularly, with the initiation of ritonavir-boosted PI therapy during pregnancy.

Comparison of resistance mutation patterns in historical plasma HIV RNA genotypes with those in current proviral HIV DNA genotypes among extensively treated patients with suppressed replication

Heavily treatment-experienced patients with good virological control could be at risk of virological failure on switching to a new regimen if pre-existing drug resistance is not taken into account. We examined whether genotyping based on cellular HIV-1 DNA during controlled viraemia identifies resistance mutations detected in plasma HIV-1 RNA during treatment with previous antiretroviral regimens. All 169 patients enrolled in the Agence Nationale de Recherche sur le SIDA (ANRS) 138-intEgrase inhibitor MK_0518 to Avoid Subcutaneous Injections of EnfuviRtide (EASIER) trial had already received three antiretroviral drug classes [nucleoside reverse transcriptase inhibitor (NRTI), nonnucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitor (PI)] and had plasma HIV-1 RNA<400 copies/ml at baseline. The results of previous resistance genotyping of plasma HIV-1 RNA in individual patients were compared with those of resistance genotyping of whole-blood HIV-1 DNA at randomization. A median of 4 plasma RNA genotypes were available for the 169 patients. The median numbers of resistance mutations in HIV-1 RNA and DNA were, respectively, 5 and 4 for NRTIs, 2 and 1 for NNRTIs, and 10 and 8 for PIs. The difference was significant for all three drug classes (P=0.001). Resistance to at least one antiretroviral drug was detected exclusively in HIV-1 RNA or in DNA in 63% and 13% of patients for NRTI, 47% and 1% of patients for NNRTI, and 50% and 7% of patients for PI, respectively. This study shows that, among highly treatment-experienced patients on effective highly active antiretroviral therapy, resistance genotyping of HIV-1 DNA detects fewer resistance mutations than previous analyses of HIV-1 RNA. These results have implications for patient management and for the design of switch studies.

Is long‐term virological response related to CCR5 Δ32 deletion in HIV‐1‐infected patients started on highly active antiretroviral therapy?

The aim of the study was to determine whether the chemokine (C-C motif) receptor 5 (CCR5) Delta32 deletion is associated with long-term response to combination antiretroviral treatment (cART) in HIV-1-infected patients. The genetic substudy of the Agence Nationale de Recherche sur le SIDA (ANRS) CO8 APROCO-COPILOTE cohort included 609 patients who started protease inhibitor-containing cART in 1997-1999. Patients were considered to have a sustained virological response if all plasma HIV RNA measurements in the period considered were <500 HIV-1 RNA copies/ml, allowing for a single blip. Virological response was compared between patients heterozygous for CCR5 Delta32 (Delta32/wt) and wild-type patients (wt/wt) from month 4 to year 3 and from month 4 to year 5. Logistic regression analysis was used to adjust for baseline demographical data, HIV RNA, CD4 cell count, antiretroviral exposure status, time spent on antiretroviral therapy at years 3 and 5 and adherence to treatment (month 4 to year 3 or 5). A sustained virological response was more frequent in Delta32/wt than in wt/wt patients from month 4 to year 3, with 66%vs. 52% of patients, respectively, showing a sustained response (P=0.02); after adjustment for potential confounders, the association of Delta32 with a sustained response was nearly significant (P=0.07). A sustained virological response was also more frequent in Delta32/wt patients up to year 5, with 48% showing a sustained response vs. 35% of wt/wt patients (P=0.01); after adjustment, Delta32 remained significantly associated with a sustained virological response up to year 5 (P=0.04). There was no association with CD4 response. The Delta32 deletion in Delta32/wt patients is associated with a beneficial virological response to cART in the long term. Whether this association is a direct effect of the Delta32 deletion remains unclear and requires confirmation in further observational studies.

Role of Uncontrolled HIV RNA Level and Immunodeficiency in the Occurrence of Malignancy in HIV‐Infected Patients during the Combination Antiretroviral Therapy Era: Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort

Human immunodeficiency virus (HIV)-infected patients are at higher risk of malignancies. In addition to traditional determinants, a specific deleterious effect of HIV and immunodeficiency is speculated. We aimed at studying the association between immunological and virological characteristics of HIV-infected patients in care and the risk of acquired immunodeficiency syndrome (AIDS)-defining and non-AIDS-defining malignancies. Patients consecutively enrolled in the hospital-based Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort were included if the duration of follow-up was >3 months during the period 1998-2006. Multivariate modeling used an extended Cox proportional hazards model for time-dependent covariates and delayed entry. The 4194 patients included in the study developed 251 first malignancies during 22,389 person-years. A higher incidence of AIDS-defining malignancies (107 cases) was independently associated with (1) both longer and current exposures to a plasma HIV RNA level >500 copies/mL (hazard ratio [HR], 1.27 per year [P<.001] and 3.30 [P<.001], respectively) and (2) both longer and current exposure to a CD4(+) cell count <200 cells/mm(3) (HR, 1.36 per year [P<.001] and 6.33 [P<.001], respectively). A higher incidence of non-AIDS-defining malignancies (144 cases) was independently associated with longer and current exposure to a CD4(+) cell count <500 cells/mm(3) (HR, 1.13 per year [P=.01] and 2.07 [P<.001], respectively) and male sex (HR, 1.69; P=.02) but not with plasma HIV RNA level (P=.49 and P=.10 for cumulative and current exposures, respectively). Uncontrolled plasma HIV RNA level was independently associated with a higher likelihood of developing AIDS-defining malignancies, whereas immunosuppression was associated with a higher risk of developing any type of malignancies. Antiretroviral treatment should aim at reaching and maintaining a CD4(+) count >500 cells/mm(3) to prevent the occurrence of malignancy, this should be integrated to malignancy-prevention policies.

Prediction of the virological response to etravirine in clinical practice: Comparison of three genotype algorithms

The current Agence Nationale de Recherche sur le SIDA (ANRS)/International AIDS Society (IAS) algorithm predicts resistance to etravirine for viruses harboring >/=3 mutations from a list of 13 reverse transcriptase (RT) mutations. Two weighted algorithms, best correlated with fold changes to etravirine, have been described recently. A retrospective virological analysis of a major French city HIV sequences database was undertaken to assess the proportion of etravirine resistant viruses according to these three algorithms and the correlations between them. Two thousand six hundred eighty RT sequences were analyzed, including 749 from naive patients and 926 from patients previously treated with non-nucleoside reverse transcriptase inhibitor (NNRTI). Combinations of mutations associated with etravirine resistance according to the three algorithms were found in 0%, 2.3%, and 3.6% of naive patients, and in 2.4%, 20.4%, and 19.3% of patients previously treated with NNRTIs. Concordance between the algorithms was weak (2 x 2 Kendall's tau: 0.787, 0.395, and 0.584). Most of the discordance was due to the differential weights attributed to Y181C/V, L100I, and K101P in the two weighted algorithms. It is concluded that the current ANRS/ IAS algorithm probably underestimates the proportion of viruses partially resistant to etravirine in NNRTI-experienced patients. Improvements in algorithms are needed to take into account the partial resistance associated with some mutation patterns, and should include either additional mutations to the current list and/or differential weights for specific mutations. Surveys of naive patients should be conducted to estimate the risk of primary resistance to etravirine in a minority of cases.

Genotypic Resistance Analysis of the Virological Response to Fosamprenavir-Ritonavir in Protease Inhibitor-Experienced Patients in CONTEXT and TRIAD Clinical Trials

The aim of this study was to identify human immunodeficiency virus (HIV) protease mutations associated with virological response (VR) to fosamprenavir-ritonavir (FPV/r) in 113 protease inhibitor (PI)-experienced patients randomized in both CONTEXT and TRIAD clinical trials and receiving the same dose (700/100 mg twice daily) of FPV/r. The impact of each protease mutation on the VR to FPV/r, defined as the decrease in HIV RNA at week 12, was investigated with nonparametric analyses. A step-by-step procedure was done using a Jonckheere-Terpstra (JT) test that retains the group of mutations most strongly associated with the VR. Mutations at the following 14 codons were associated with a reduced VR to FPV/r: 10, 15, 33, 46, 54, 60, 62, 63, 72, 73, 82, 84, 89, and 90. The JT procedure led to selecting the CONTEXT/TRIAD genotypic set of mutations, I15V, M46I/L, I54L/M/V, D60E, L63P/T, and I84V, as providing the strongest association with the VR (P = 1.45 x 10(-11)). In the nine patients with zero mutations within this set, the median decrease in HIV RNA was -2.63 log copies/ml, and was -2.22 (n = 45), -1.50 (n = 26), -0.58 (n = 23), -0.47 (n = 6), -0.13 (n = 3), and 0.04 (n = 1) log copies/ml in those with one, two, three, four, five, and six mutations, respectively. This study identified six mutations associated with VR to FPV/r. Some of these mutations are shared with the current FPV/r Agence Nationale de Recherches sur le SIDA (ANRS) resistance score, which has been cross-validated in the CONTEXT/TRIAD data set, suggesting that the current ANRS FPV/r score is a useful tool for the prediction of VR to FPV/r in PI-experienced patients.

Evolution of Plasma Hepatitis C Virus Load in Patients Coinfected by HIV and Hepatitis C Virus Started on a Protease Inhibitor-Containing Antiretroviral Regimen, Agence Nationale de Recherches sur le SIDA CO8 APROCO-COPILOTE Cohort

Evolution of Plasma Hepatitis C Virus Load in Patients Coinfected by HIV and Hepatitis C Virus Started on a Protease Inhibitor-Containing Antiretroviral Regimen, Agence Nationale de Recherches sur le SIDA CO8 APROCO-COPILOTE Cohort

Expected response to protease inhibitors of HIV-1 non-B subtype viruses according to resistance algorithms

The expected effectiveness of protease inhibitors was assessed according to the Agence Nationale de Recherches sur le SIDA (ANRS), Rega and Stanford 2007 resistance algorithms in 93 and 87 antiretroviral therapy-naive patients, respectively, infected with B and non-B subtype viruses. Either B or non-B subtypes were considered fully susceptible to protease inhibitors, except to tipranavir/ritonavir, for which the 2007 ANRS algorithm scored non-B subtypes as naturally resistant when this algorithm was extended to these subtypes.

Editorial review: Male circumcision, gender and HIV prevention in sub-Saharan Africa: a (social science) research agenda

Sub-Saharan Africa is the part of the world which is the most affected by the HIV and AIDS pandemic with 24.5 million people infected by the virus that causes AIDS. Adult HIV prevalence in southern Africa is estimated at 16% at 6% in East Africa and at 4.5% in West and Central Africa. Ecological studies in sub-Saharan Africa have suggested a geographical association between areas of higher prevalence of HIV and lower prevalence of male circumcision (MC) (Drain Halperin Hughes Klausner & Bailey 2006). An initial short-term randomised controlled study on male circumcision led by the Agence nationale de recherche sur le sida (ANRS) at Orange Farm in South Africa revealed a reduction of 60%-75% in the risk of female to male transmission of HIV-1 in circumcised men. These studies which were supported by the National Institutes of Health (NIH) were conducted in Kisumu in Kenya and in Rakai in Uganda. They demonstrated a risk reduction of around 58% and 53% respectively. On the 28th of March 2007based on these studies the WHO and UNAIDS issued a statement endorsing male circumcision (MC) as an additional strategy in HIV prevention particularly in high HIV prevalence and low male circumcision countries. (excerpt)

Examination of real-time PCR for HIV-1 RNA and DNA quantitation in patients infected with HIV-1 BF intersubtype recombinant variants

The impact of HIV-1 genetic diversity on the performance of laboratory testing is an issue that has to be monitored continuously. An “in-house” real-time PCR assay was developed by the Agence Nationale de Recherche sur le SIDA (ANRS) in France for viral load (VL) quantitation based on the amplification of the HIV-1 long terminal repeat (LTR) region. This technology has not been used in Argentina yet and considering the HIV-1 diversity in the country, a comparative analysis of this assay was undertaken versus the Versant HIV-1 RNA 3.0 Assay (b-DNA). The performance was assessed on 30 drug-naïve HIV-1 infected patients who were characterized previously by phylogenetic analysis of the pol and vpu gene. The results showed that there is a significant linear correlation between values of transformed viral load logarithms measured by both, bDNA and real-time PCR assay and that this assay can be used to quantify viral load in samples from BF-infected patients with the same accuracy and reliability as for B subtype samples. The use of “in-house” real-time PCR to measure DNA in PBMCs correlated strongly with the HIV-1 RNA levels in all specimens.

Mitochondrial dysfunction following perinatal exposure to nucleoside analogues

Mitochondrial dysfunction following perinatal exposure to nucleoside analogues

Compartment-specific HIV-1 resting T-cell reservoirs

A proof-of-concept study recently assessed the efficiency of a standard clinical dose of valproic acid in order selectively to induce the expression of HIV-1 latent proviral genome, and potentially to deplete HIV from the blood resting CD4 T-cell reservoir [1]. The efficacy of this purge of the CD4 T-cell reservoir by valproic acid has been hypothesized to be tissue compartment specific [2]. We conducted a study on HIV-1 T-cell reservoirs in the blood and breast milk of nine HIV-1-infected women. Using an exquisitely sensitive HIV-1 antigen enzyme-linked immunospot (ELISPOT) assay combined with the quantitation of HIV-1 DNA by real time polymerase chain reaction (see Fig. 1), we were able to demonstrate a 17 times higher capacity of HIV-1 latently infected CD4 T cells to enter the replicative cycle after ex-vivo activation, in breast milk compared with blood [3]. The study is important not only for understanding the possible mechanisms of HIV-1 transmission by breastfeeding, but also for anticipating the feasibility of an HIV cure by treatments aimed at depleting the HIV-1 proviral reservoirs.Fig. 1: Characterization of resting T-cell reservoir in blood and breast milk. (a) Isolation of resting CD4 T cells from blood and breast milk samples. (b) Quantification of the HIV-1 DNA and enumeration of HIV-1 antigen-secreting cells after CD4 T-cell polyclonal activation. ELISPOT, Enzyme-linked immunospot; PCR, polymerase chain reaction.First, our HIV-1 antigen ELISPOT assay is at least as sensitive and considerably less time consuming and labour intensive than the limiting-dilution culture of resting CD4 T cells after ex-vivo activation, as used in the study by Lehrman et al. [1]. Major points concerning our approach consist of the combination of: (i) an efficient method of resting CD4 T-cell isolation; (ii) the conservation of CD4 T-cell functions; and (iii) an exquisitely sensitive ELISPOT assay (with the capacity to detect a single HIV-1 antigen-producing cell among 1 × 106 CD4 T lymphocytes) [4]. As it can be applied to other compartments than blood, the HIV-1 antigen ELISPOT assay may become the technique of choice for further studies on the compartmentalization of the HIV-1 resting CD4 T-cell reservoirs. Second, our study demonstrates a strong compartmentalization of the HIV-1 latently infected CD4 T-cell reservoir at least in the mammary gland, because HIV-1-infected resting CD4 T cells can replicate HIV-1 after ex-vivo activation more extensively in breast milk than in blood. Compartmentalization of the viral reservoir is not an academic fantasy. For example, a strong genotypic and phenotypic compartmentalization of cell-free as well as cell-associated HIV-1 has been observed in the blood and seminal reservoirs after an intensification and stimulatory HIV-1 eradication protocol [5]. As suggested by Routy [2], the enumeration and assessment of functional capacities of resting CD4 T cells to enter a replicative cycle in peripheral blood may not necessarily reflect what happens in other anatomical or tissue compartments, such as seminal fluid, cervicovaginal secretions, central nervous system or breast milk. In our ultimate goal of finding a cure for HIV, this compartmentalization should be taken into account in further clinical trials on valproic acid or related compounds aimed at purging the HIV-1 proviral reservoir. The authors declare that they have no conflict of interest. Sponsorship: This work was supported by the Agence Nationale de Recherches sur le SIDA (ANRS), as part of the project ANRS 1271 (contract 2004-010).

Prevalence of Transmitted HIV-1 Drug Resistance and the Role of Resistance Algorithms

To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individual's nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.

HIV Preventive Vaccine Research at the ANRS: The Lipopeptide Vaccine Approach

The HIV (human immunodeficiency virus)/AIDS epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the ANRS (Agence Nationale de Recherches sur le SIDA) has been committed to an original programme combining basic science and clinical research. The HIV preventive vaccine research programme includes upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. In 2004, most researchers believed that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, 15 phase I and II clinical trials have been established with the aim of evaluating the safety of candidate vaccines and their capacity to induce cellular immune responses. The candidate vaccines tested were recombinant canarypox viruses (ALVAC) containing sequences coding for certain viral proteins, utilised alone or combined with other immunogens (whole or truncated envelope proteins). An original strategy, based on the use of lipopeptides, is also under development. These vaccines comprise synthetic fragments of HIV proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches have within a short time allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.