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Journal of Clinical Microbiology, July 2003, p. 2900-2907, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2900-2907.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sources and Magnitude of Intralaboratory Variability in a Sequence-Based Genotypic Assay for Human Immunodeficiency Virus Type 1 Drug Resistance

R. A. Galli, B. Sattha, B. Wynhoven, M. V. O'Shaughnessy, and P. R. Harrigan^*

BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canada V6Z 1Y6

Received 25 September 2002/ Returned for modification 4 March 2003/ Accepted 28 March 2003

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.

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* Corresponding author. Mailing address: BC Centre for Excellence in HIV/AIDS, 613-1081 Burrard St., Vancouver, British Columbia, Canada V6Z 1Y6. Phone: (604) 806-8281. Fax: (604) 806-8464. E-mail: lab{at}hivnet.ubc.ca.

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Journal of Clinical Microbiology, July 2003, p. 2900-2907, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2900-2907.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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