Genotypic prediction of human immunodeficiency virus type 1 CRF02-AG tropism.

We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.

Validation of an automated real-time PCR protocol for detection and quantitation of HIV and HCV genomes in semen.

The ability to detect and quantify HIV and HCV genomes is important for checking spermatozoid preparation protocols also known as "sperm washing". But no commercial assay is available. A method was developed for detecting HIV and HCV in semen fractions using the COBAS Ampliprep and COBAS Taqman instruments. It will detect fewer than 200 copies of HIV RNA per ml of semen plasma and fewer than 200 copies/3 x 10(6) semen cells. The sensitivity for HCV is similar at more than 200 IU/ml and below 200 IU/3 x 10(6) semen cells. No inhibitor of PCR amplification was detected. This automated protocol permits a convenient, standardized testing for HIV and HCV in semen. The performance is the same as that of the previous generation of automated assays but the cost and operating time are both reduced.

Assessment of HIV-1 subtyping for Cameroon strains using phylogenetic analysis of pol gene sequences.

Phylogenetic analysis of human immunodeficiency virus type 1 (HIV-1) pol gene is a useful method for subtyping European strains of HIV-1. The suitability of this method for genetically diverse African strains was evaluated by comparing HIV-1 subtyping of Cameroon strains using a long fragment of the pol gene sequence to the findings obtained using env gene sequences. When the pol gene could not be amplified, the reverse transcriptase (RT) or the protease (PR) genes were used. Phylogenetic analysis of the env C2/V3 gene sequences of 60 HIV-1 isolates showed 52 to be subtype A, 2 subtype G, plus one each of subtypes C, F2 and H, with 3 subtypes not determined. A long fragment of the pol gene was amplified successfully and sequenced in 23% of cases. The RT region was amplified for 42% of the samples that could not be typed by analysing the long fragment, and the PR gene was amplified for 40% of them. Thus, 63% of samples were typable. Env and pol gene subtypings were in agreement in 86% of cases. It is concluded that the phylogenetic analysis of pol gene sequences is not a practical method for HIV-1 subtyping in areas of high subtype diversity, despite the good agreement between the env and pol gene subtypings. However, it can be a useful method for HIV-1 subtyping, provided that the gene is amplifiable.

CXCR4-using viruses in plasma and peripheral blood mononuclear cells during primary HIV-1 infection and impact on disease progression.

OBJECTIVE: Cysteine-cysteine receptor 5 (CCR5)-using viruses classically predominate during HIV-1 primary infection but the frequency of cysteine-X-cysteine receptor 4 (CXCR4)-using viruses varies between studies and could be different between plasma and peripheral blood mononuclear cells (PBMCs). Thus, we determined HIV-1 tropism in both these compartments during primary infection and evaluated the impact of CXCR4-using viruses on disease progression. DESIGN: One hundred and thirty-three patients with primary HIV-1 infection were screened for HIV-1 coreceptor usage in plasma and PBMCs using both genotypic and phenotypic methods. The impact of CXCR4-using viruses' transmission on subsequent disease progression was assessed in a case-control study. METHODS: HIV-1 coreceptor usage was determined using a recombinant virus phenotypic entry assay and V3-based genotypic algorithms. We also monitored CD4(+) T-cell count, clinical events and therapeutic intervention. RESULTS: There was 6.4% of CXCR4-using HIV-1 in plasma during primary infection as measured by a phenotypic assay and combined criteria from the 11/25 and net charge genotypic rules. Geno2pheno10 overestimated the prevalence of CXCR4-using viruses (12%). HIV-1 tropism in plasma and PBMCs was 98% concordant. The HIV-1 RNA load and CD4(+) T-cell count during primary infection were not related to virus tropism. Primary infection with CXCR4-using viruses was associated with an accelerated rate of disease progression, estimated by a faster decline of CD4 T-cell count under 350 cells/microl and by a reduced delay in initiating a first antiretroviral treatment. CONCLUSIONS: Plasma or PBMC samples can be used for determining HIV-1 tropism during primary infection. CXCR4-using viruses are rare during primary infection but increase the risk of disease progression.

Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage.

BACKGROUND: Clinical trials of CCR5 antagonists have relied on the phenotypic determination of HIV-1 coreceptor usage. Few phenotypic assays are available, with few data on their concordance, and none has been designed to determine tropism from cell-associated HIV-1 DNA. OBJECTIVES: To assess the performance of the new Toulouse Tropism Test (TTT) phenotypic assay to characterize HIV-1 tropism using blood plasma and peripheral blood mononuclear cells (PBMC). STUDY DESIGN: 434 plasma and 168 PBMC samples were tested with the TTT assay. We determined the correlation between our assay results on plasma samples and those of the commercial Trofile assay. RESULTS: The TTT assay determined the tropism of 97% of samples after successful amplification of the env gene. It performed well on both cell samples and plasma samples with various HIV-1 loads and subtypes. It detected 0.5% of minor CXCR4-using variants in the virus population. The TTT and the Trofile assays were >90% concordant for predicting HIV-1 tropism. CONCLUSION: We have validated a new recombinant virus phenotypic assay for determining HIV-1 tropism using both plasma and cell samples from patients who are candidates for treatment with CCR5 antagonists.

Prediction of HIV type 1 subtype C tropism by genotypic algorithms built from subtype B viruses.

BACKGROUND: Genotypic predictions of HIV-1 tropism could simplify CCR5 antagonist usage. However, the genotypic algorithms built from subtype B viruses could be inadequate for non-B subtypes. We therefore performed paired genotypic and phenotypic determination of subtype C tropism. METHODS: We studied 52 patients recruited in Malawi and 21 patients recruited in France. We directly sequenced the V3 env region and performed a recombinant virus phenotypic entry assay in parallel. RESULTS: The Malawi patients had 29% of CXCR4-using subtype C viruses compared with only 5% in the patients from France. For detecting CXCR4-using subtype C viruses, the genotypic rule combining the amino acids at positions 11/25 and the net charge of V3 was 93.3% sensitive and 96.4% specific. The Geno2pheno tool was 86.7% sensitive and 89.1% specific. The WebPSSM tool with the SI/NSI matrix was 80% sensitive and 98.2% specific in its subtype B version and 93.3% sensitive and 81.8% specific in its subtype C version. CONCLUSIONS: The genotypic determinants of coreceptor usage for HIV-1 subtype C were mainly in V3 and were globally similar to those previously reported for subtype B viruses. The main genotypic algorithms built from subtype B viruses perform well when applied to subtype C viruses.

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism.

OBJECTIVE: Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. DESIGN: Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. METHODS: HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. RESULTS: The 103 patients had a median CD4+ T lymphocyte count of 268 x 10(6)cells/l and nadirs of 98 x 10(6)cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. CONCLUSION: Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

HIV therapy after treatment interruption in patients with multiple failure and more than 200 CD4+ T lymphocyte count.

The aim of the study was to investigate the safety and efficacy of a salvage therapy initiated after interrupting treatment in patients with virological failure and more than 200 CD4(+) T lymphocyte count. In this prospective study, 77 patients who received failing regimens had stopped completely all medication for 3 months before starting an optimised regimen consisting of 3-5 drugs. Patients were tested for HIV resistance before and after treatment interruption. Discontinuation of therapy for 3 months was associated with a median increase in HIV RNA of 1.1 log(10), a median decrease in CD4(+) T cell count of 136 x 10(6)/L and five clinical events related to HIV, but no AIDS-defining event. Eighty-seven percent of patients showed a shift from a drug resistant genotype to a wild-type genotype based on the major resistance mutations. Forty-seven percent of patients with a genotype shift reached fewer than 200 HIV RNA copies/ml of plasma 6 and 12 months after treatment resumption whereas none of those without a genotype shift did so (P = 0.03). However, the genotypic shift was not associated with a sustained virological response by multivariate analysis. The use of a new therapeutic class of compound in the salvage regimen was the only predictor of the sustained virological response. Salvage therapy with 3-5 drugs after interrupting treatment for 3 months can be a safe and effective strategy provided the HIV disease is not too advanced. Randomised trials in this population are needed to assess the clinical benefit of this strategy.

Evolution of human immunodeficiency virus type 1 populations after resumption of therapy following treatment interruption and shift in resistance genotype.

Conventional genotyping of human immunodeficiency virus type 1 often reveals a shift from a drug-resistant genotype to a wild-type genotype after treatment interruption. A real-time polymerase chain reaction-based technique was used to detect minority resistant populations in 13 patients who showed genotype reversion after interruption of treatment for 3 months. Sixty-two percent of patients in whom the V82A and L90M protease mutations were no longer detectable by conventional genotyping still harbored minority resistant variants, in proportions ranging from 0.1% to 21%. None of the patients with these minority resistant variants who received a protease-inhibitor regimen on resumption of therapy had a response to treatment. However, population sequencing and clonal analysis of plasma samples obtained 1-2 months after resumption of treatment revealed the presence of wild-type virus during the initial decline in plasma virus load, which indicates that minority resistant variants were not rapidly selected.

Virological and immunological effects of salvage therapy following treatment interruption and a shift in HIV-1 resistance genotype.

The circulating human immunodeficiency virus type 1 (HIV-1) population of patients in whom many prior therapy regimens have failed often undergo a shift from a drug-resistant virus to a wild-type virus following interruption of treatment. This study analyses the virological and immunological effects of salvage therapy following treatment interruption and a shift from a drug-resistance genotype. Twenty-one HIV-1 infected patients who had genotype reversion by population-based sequencing after 3 months of treatment interruption were given a new salvage regimen consisting of 3-5 drugs selected according to their treatment history. Seven (33%) of 21 patients who had fewer than 200 HIV-1 RNA copies/ml until month 12 were defined as virological responders. Four patients were transient responders and 10 were nonresponders. The virological responders were more frequently CDC group A and had higher CD4 + T lymphocyte counts at the time of treatment resumption. The peripheral blood T CD4 + and T CD8 + lymphocyte populations of the patients declined significantly during treatment interruption. Only virological responders showed significant increases in their CD4 + T lymphocyte count 12 months after treatment resumption and these counts rapidly returned to pre-interruption baseline values in most of these patients. Treatment interruption could be useful for optimising salvage therapy for patients previously given many failing regimens. However, controlled trials are needed to assess the clinical benefit of this strategy.