Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism.

OBJECTIVES: To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. PATIENTS AND METHODS: The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay.
RESULTS: Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%.
CONCLUSIONS: The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism.