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ABSTRACTWe compared the ability of the LigAmp assay and an ASPCR assay to detect and quantify K103N-containing HIV variants in samples from 63 women who received single-dose nevirapine in a clinical trial. Samples were first analyzed with the ViroSeq HIV Genotyping system, and ViroSeq PCR products were used as templates for the LigAmp and ASPCR assays. A cutoff of 0.5 K103N for detection of K103N was used for both assays. Results for the percentage K103N were similar for the two assays (R2 0.92). Forty-six samples (73.0) were positive for K103N by both assays and 13 samples (20.6) were negative by both assays. Four samples (6.3) were positive by ASPCR only. No samples were positive by LigAmp only. Eight discordant samples were analyzed in more detail. Sequence polymorphisms near oligonucleotide binding sites provided a possible explanation for the discordance in four of eight samples. The percentage K103N was also determined by analyzing 40 HIV clones from each of these eight samples, using a combined amplification/sequencing method (AmpliSeq). The percentage K103N determined by clonal analysis was consistent with the LigAmp result for five of eight samples, and was consistent with the ASPCR result for three of eight samples. Among 320 clones analyzed, we identified eight different codons at position 103 (mean 3.8 codons/sample), which encoded six different amino acids, illustrating the extensive genetic diversity in HIV. Further studies are needed to compare performance of assays for detection and quantification of HIV drug resistance mutations in clinical samples. [ABSTRACT FROM AUTHOR]

K103N is frequently detected in HIV-infected women after single dose (SD) nevirapine (NVP). K103N-containing variants were detected more frequently by the ViroSeq HIV-1 Genotyping System in women with subtype C (69.2) than subtypes A (19.4, p< 0.0001) or D (36.1, p< 0.0001). K103N-containing variants were also detected more frequently and at higher levels in women with subtype C by the LigAmp assay. In this report, we analyzed samples collected prior to or within hours after SD NVP administration from antiretroviral drug-naive African women with subtypes A, C, and D. Only 1254 samples had an NVP resistance mutation detected with the ViroSeq system, and only 4236 samples had K103N detected at < 0.5 with the LigAmp assay [2110 (1.8) with subtype A, 146 (2.2) with subtype C, and 180 (1.3) with subtype D] (p 0.92). We did not detect significant differences in the pre-NVP frequency of NVP resistance mutations or the pre-NVP levels of K103N-containing variants in women with subtypes A, C, and D that explain the dramatic subtype-based differences in emergence of HIV-1 variants with these mutations after SD NVP exposure. [ABSTRACT FROM AUTHOR]