title: Sensitivity and specificity of the ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for detection of HIV-1 drug resistance mutations by use of an ABI PRISM 3100 genetic analyzer.
abstract: The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.
authors.affiliation: The Johns Hopkins Medical Institutions, Ross Building 646, 720 Rutland Ave., Baltimore, MD 21205, USA. seshlem@jhmi.edu
authors.forename: Susan H
authors.initials: SH
authors.lastname: Eshleman
authors.forename: Gillian
authors.initials: G
authors.lastname: Crutcher
authors.forename: Olga
authors.initials: O
authors.lastname: Petrauskene
authors.forename: Kevin
authors.initials: K
authors.lastname: Kunstman
authors.forename: Shawn P
authors.initials: SP
authors.lastname: Cunningham
authors.forename: Christina
authors.initials: C
authors.lastname: Trevino
authors.forename: Cheryl
authors.initials: C
authors.lastname: Davis
authors.forename: John
authors.initials: J
authors.lastname: Kennedy
authors.forename: Jeff
authors.initials: J
authors.lastname: Fairman
authors.forename: Brian
authors.initials: B
authors.lastname: Foley
authors.forename: Joann
authors.initials: J
authors.lastname: Kop
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
chemical_list: D019380:Anti-HIV Agents; D011933:Reagent Kits, Diagnostic; D054303:HIV Reverse Transcriptase; D016333:HIV Protease