title: A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene.
abstract: Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naive and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naive samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36-45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.
authors.affiliation: Rega Institute for Medical Research and University Hospitals Leuven, Microbiology and Immunology, Clinical and Epidemiological Virology, AIDS Reference Laboratory, Minderbroedersstraat 10, Leuven 3000, Belgium. kristel.vanlaethem@uz.kuleuven.ac.be
authors.forename: Kristel
authors.initials: K
authors.lastname: Van Laethem
authors.forename: Yoeri
authors.initials: Y
authors.lastname: Schrooten
authors.forename: Philippe
authors.initials: P
authors.lastname: Lemey
authors.forename: Eric
authors.initials: E
authors.lastname: Van Wijngaerden
authors.forename: Stéphane
authors.initials: S
authors.lastname: De Wit
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
authors.forename: Anne-Mieke
authors.initials: AM
authors.lastname: Vandamme
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics*; D015752:Genes, env / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000235:genetics; D015497:HIV-1 / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments