The aim of this study was to compare sensitivity thresholds of two pre-screening methods - the heteroduplex mobility assay (HMA) and the presence of ambiguity codes in population-based sequences - applied for detection of HIV-1 superinfection. HIV-1 env C2-C4 PCR products generated from 48 serum samples isolated from 24 HIV-1 positive and therapy-naïve homosexual men at seroconversion and at approximately 1 year thereafter were subjected to HMA and population sequencing. Clonal sequence analysis was used to determine the sensitivity of each method to detect sequence variability. Results from HMA were compared to pairwise genetic distance of clonal sequences; heteroduplexes resulted from as little as 1.4% pairwise distance between two sequences and were detected even when only 1.5% of the pairwise distance comparisons exceeded this distance threshold. By contrast, the ambiguity code approach using population-based sequencing detected only 20.1% of existing sequence variation and was less sensitive to minority populations <or=20%, resulting in an underestimation of HIV-1 diversity. Thus, HMA was found to be more sensitive for detection of sequence variations than the ambiguity code approach, suggesting that HMA would be a more appropriate method to pre-screen for HIV-1 superinfection.
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