590 Nucleotide Incorporation during First-round RT PCR as the Main Contributor of Sequence-based HIV Genotyping Variability B. Sattha, B. Wynhoven, P.R. Harrigan, M.V. O'Shaughnessy, R. Galli* British Columbia Ctr for Excellence in HIV/AIDS, Vancouver, Canada
Background: Many studies have shown a high degree of inter-laboratory variation of sequence-based resistance testing methods attributed to factors such as assay performance characteristics, population diversity, and technical expertise. Here, we assess the sources and magnitude of intra-laboratory variability of a sequence-based HIV genotypic assay.
Methods: Routine HIV drug-resistance testing is conducted using in-house automated nucleic acid extraction, nested RT-PCR, and population-based dideoxynucleotide cycle sequencing with sequence data analysis, covering the entire HIV-1 protease (PR) region and most of reverse transcriptase (RT). We assessed the intra-laboratory reproducibility and relative contributions of different procedural steps towards final sequence variability using a study algorithm consisting of: 1) duplicate assembly of sequence data by different operators; 2) duplicate sequencing reactions (+ step 1); 3) duplicate 2nd round PCR (+1+2); 4) duplicate RT/1st round PCR (+1+2+3). The study was conducted on 46 consecutive plasma samples collected from 39 HIV-1 infected, antiretroviral experienced patients (pts).
Results: Mismatches from a total of 68,862 nucleotides (1,497 bases each from 46 sequences) compared in each algorithm portion were, step 1): 70 (> 99.9% concordance); step 2): 96 (99.9% concordance); step 3): 123 (99.8% concordance); and step 4): 440 (99.4% concordance). Complete and partial mismatches occurred scattered throughout the PR/RT genome segment at > 300 positions. Approximately 75% of the discordances involved mixtures, and A to G, A to R, and G to R mismatches were significantly more common than other mismatched base pairs (p < 0.001). There was a significantly higher proportion of base A (51.4%), 5' to the mismatch than the total prevalence of A (39.9%) in the samples (p < 0.001).
Conclusions: These results confirm that sequence-based genotyping can be a precise and reliable tool for monitoring HIV drug resistance and suggest that efforts to reduce variability should focus on the first RT-PCR step. The data suggest that the composition of External Quality Assessment panels should be based on clinical HIV RNA isolates rather than DNA clones since the RT-PCR step may increase the variability.