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.Human immunodeficiency virus type 1 (HIV-1) mutations associated with resistance to currently available antiretroviral drugs occur predominantly in the reverse transcriptase (RT) and protease (PR) regions of the viral genome. Drug-resistant variants are considered one of the most important contributors to antiretroviral drug failure, particularly in treatment-experienced patients. Consequently, commercially available as well as "in-house" molecular sequencing methods have been developed to determine the presence of drug-resistant HIV in blood samples from individual patients. Numerous retrospective and prospective clinical trials (1,3,5,8,12,22,23) have evaluated the potential clinical utility of resistance testing, and in recent years, expert panels have recommended its use to help guide the management of antiretroviral therapy in certain clinical situations (2, 13).Despite these advances, there are currently no specific standards for either genotypic or phenotypic testing for HIV drug resistance. Consequently, aspects such as technical laboratory expertise, assay performance characteristics and quality control, diversity of patient populations, and drug resistance interpretation algorithms may produce variability of resistance testing results. Previous studies (16,17) have shown a high ...

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