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We used ultra-deep pyrosequencing and the Toulouse Tropism Test phenotypic assay to determine the prevalence of CXCR4-using viruses in 21 patients with primary HIV-1 infections. We found X4-containing virus populations in 9% of patients by ultra-deep pyrosequencing using position-specific scoring matrices (PSSM(X4/R5)) or geno2pheno(5.75) and in 14% using the combined 11/25 and net charge rule. The phenotypic assay identified 9% of CXCR4-using viruses. This confirms that R5 viruses are predominant in primary HIV-1 infections.

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.

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.

Objective: Reservoirs of HIV-1 are a major obstacle to virus eradication. There is therefore a need to clearly understand the molecular nature of the virus populations that persist in patients with sustained suppression of plasma viraemia on highly active antiretroviral therapy (HAART)., Design: We performed a detailed analysis of the genotypes of HIV-1 quasispecies isolated from highly purified blood cell types taken from three selected patients with sustained undetectable viral loads on HAART for 7 years., Methods: We used polychromatic flow cytometry to sort naive and memory CD4 T cells, CD14 monocytes, and CD56+CD3- natural killer (NK) cells from the total peripheral blood mononuclear cells after 7 years of HAART. Clonal analysis was used to determine coreceptor use and drug-resistance genotypes of HIV-1 quasispecies in the sorted blood cell types., Results: We detected HIV-1 DNA in memory and naive CD4 T cells and in CD14 monocytes, but not in the CD56+CD3- NK cells. Phylogenetic analysis demonstrated that the various blood cells types of two of the three patients harboured genetically distinct HIV-1 quasispecies. Drug-resistance mutations were also distributed differently from one cell type to another. This compartmentalization suggests a minimal virus trafficking between blood cell types during suppressive HAART., Conclusions: We observed a cell-specific compartmentalization of the residual virus populations during prolonged suppressive HAART. The coexistence of numerous HIV-1 quasispecies with different resistance genotypes and coreceptor use in cellular reservoirs may be relevant for future antiretroviral treatment strategies.