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Session 78 Poster Session Diagnostic Techniques: Viral Sequencing Technologies Session Time: 4:30-6:30 pm Room 4E-F

595-T. Reproducibility and Accuracy of a Novel Kit-Based Genotyping Assay for HIV-1 Drug Resistance R. M. Grant*1, R. Lloyd2, V. A. Johnson3, J. L. Sullivan4, R. Swanstrom5, M. Holodniy6, C. Reid2, G. Morgan2, and D. Winslow2. 1Gladstone Inst. of Virology and Immunology and Univ. of California, San Francisco; 2Visible Genetics, Toronto, ON, Canada; 3VA Med. Ctr. and Univ. of Alabama, Birmingham; 4Univ. of Massachusetts, Worchester; 5Univ. of North Carolina, Chapel Hill; and 6VA Med. Ctr., Palo Alto, CA Background: HIV genotyping as a measure of drug resistance has been demonstrated to improve virological outcomes in clinical trials, but international proficiency testing has revealed inconsistent results across testing sites. Kit-based assays may improve assay performance including consistency. Methods: The TRUGENE HIV-1 Genotyping Kit was used to determine the sequence of HIV-1 protease and reverse transcriptase from plasma in a blinded study. Results were compared with a consensus sequence determined by analysis of 16-20 clones from each specimen using non-kit primers. According to the a priori analysis plan, bases were designated as being part of the consensus sequence if they were found in >=30% of these sequences. Only exact matches (including mixtures) were counted as correct. Results: 18 panels of 18 coded samples each (including 3 negative controls per panel) were assayed in 6 independent laboratories by 9 technicians, over 2 days, using 3 kit-lots, representing 324 independent blinded assays. The accuracy of base and codon identification was 98.7% (range 98.4%-98.9%) overall, and 97.7% (97.0%-98.1%) for 54 sites associated with drug resistance. There were no significant differences in accuracy between laboratories, technologists, kit lots, or days. At codons associated with drug resistance, detection of mutations was less accurate than detection of non-mutated codons (76% vs 99%, respectively). Mutations present in less than 30% of the virus population were commonly detected, and including these as matches improved mutation detection from 76% to 93%. The few codons that were discrepant with the consensus sequence were identified consistently across laboratory sites. Conclusions: The TRUGENE HIV-1 Genotyping Kit is associated with high accuracy and consistent performance across laboratory sites, technicians, days, and kit-lots.

©2002 9th Conference on Retroviruses and Opportunistic Infections