title: Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: INSERM, U1043, Toulouse, F-31300 France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Raymond
authors.forename: Patricia
authors.initials: P
authors.lastname: Recordon-Pinson
authors.forename: Adrien
authors.initials: A
authors.lastname: Saliou
authors.forename: Pierre
authors.initials: P
authors.lastname: Delobel
authors.forename: Florence
authors.initials: F
authors.lastname: Nicot
authors.forename: Diane
authors.initials: D
authors.lastname: Descamps
authors.forename: Anne-Geneviève
authors.initials: AG
authors.lastname: Marcelin
authors.forename: Philippe
authors.initials: P
authors.lastname: Flandre
authors.forename: Vincent
authors.initials: V
authors.lastname: Calvez
authors.forename: Bernard
authors.initials: B
authors.lastname: Masquelier
authors.forename: Jacques
authors.initials: J
authors.lastname: Izopet
mesh_terms: D000328:Adult; D005260:Female; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D019718:Receptors, CXCR4 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D056189:Viral Tropism / Q000235:genetics*
chemical_list: D019718:Receptors, CXCR4