title: Sources and magnitude of intralaboratory variability in a sequence-based genotypic assay for human immunodeficiency virus type 1 drug resistance.
abstract: We assessed the intralaboratory reproducibility of a system for sequencing human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) by using replicate subanalyses of 46 plasma samples collected from HIV-1-infected, antiretroviral-experienced patients in order to determine the relative contributions of the different procedural steps to final sequence variability. Complete sequence concordance between duplicates of each sample was 99.4%. Complete and partial mismatches occurred scattered throughout the PR-RT genome segment at >300 positions. Approximately 75% of the discordances involved mixtures, some of which appeared at key resistance sites. Most differences were the result of the first-round RT-PCR procedure. Inter-rater concordance for sequence analysis and assembly was >99.9%. There was no observed correlation between the number or frequency of mismatches and plasma viral loads. A separate longitudinal analysis of a single routine control sample sequenced 103 times over 9 months consistently gave highly reproducible sequences (median percentage of nucleotide discordances, 0.04%; range, 0 to 0.2%). Finally, sequence data from 168 sequential samples collected from 22 patients with long-term, predominantly wild type HIV showed that intrapatient nucleotide concordance with individual index sequences ranged from 96.5 to 100%. Together, these results confirm that sequence-based genotyping can be a precise and reliable tool for monitoring HIV drug resistance, and they suggest that efforts to reduce variability should focus on the first RT-PCR step. Consequently, the data suggest that the composition of external quality assessment panels should be based on clinical HIV isolates rather than DNA clones.
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canada V6Z 1Y6.
authors.forename: R A
authors.initials: RA
authors.lastname: Galli
authors.forename: B
authors.initials: B
authors.lastname: Sattha
authors.forename: B
authors.initials: B
authors.lastname: Wynhoven
authors.forename: M V
authors.initials: MV
authors.lastname: O'Shaughnessy
authors.forename: P R
authors.initials: PR
authors.lastname: Harrigan
mesh_terms: D000595:Amino Acid Sequence; D023241:Antiretroviral Therapy, Highly Active; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007753:Laboratories / Q000592:standards*; D008826:Microbial Sensitivity Tests / Q000379:methods / Q000592:standards; D009154:Mutation; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards*
chemical_list: D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease