Background:  More than one-third of women who receive intrapartum single-dose nevirapine (SD-NVP) for the prevention of mother-to-child HIV transmission generate virus with resistance mutations (e.g., K103N, Y181C, G190A). HIV drug resistance mutations are conventionally detected by nucleotide sequence analysis which, due to sensitivity limitations, does not reliably detect variants that comprise less than 20% of the sample virus population. Therefore, there is a need for sensitive resistance assays to better detect the emergence of resistance following SD-NVP in order to assess its clinical implications.

Methods:  To identify low-frequency resistant variants, we developed a real-time PCR HIV-1 subtype C assay for the most common NVP mutation, K103N. Plasmid evaluations revealed an assay detection limit of 0.2% 103N in a background of wildtype virus, a sensitivity 100-times greater than conventional sequencing. The real-time assay was used to reassess the emergence of drug resistance in women who received SD-NVP. The study specimens consisted of matched pairs of pre-NVP and post-NVP plasma from 50 women who enrolled in a South African study of prevention of mother-to-child HIV transmission. The post-NVP samples were collected 6-36 weeks postpartum. None of the pre-NVP specimens had evidence of resistance by population-based sequencing; whereas, K103N was detected in 10 of 50 post-NVP samples.

Results:  Using real-time analysis, we found all pre-treatment samples to have undetectable K103N. The assay successfully identified the mutation in all 10 post-NVP samples (100%) that had detectable K103N by conventional sequencing. Of the 40 remaining post-NVP specimens for which no mutation was indicated by sequence analysis, the real-time assay found 16 (40%) positive for K103N at 6 to 36 weeks post-exposure. Clonal sequence analysis confirmed the resistance mutation in 5 of 5 samples selected and provided K103N-positive virus frequencies of 1.1 of 11.1% of the sample virus populations.

Conclusions:  The finding of K103N in an additional 40% of women with previously undetectable resistance suggests that only a minority of women receiving SD-NVP do not develop resistance mutations, and that conventional sequencing substantially underestimates the emergence of resistance. Real-time testing for other NVP-associated mutations may show that the proportion of women with undetectable resistance is even smaller. These data emphasize the importance of assessing the clinical implications of resistant variants.

Keywords: single-dose NVP; treatment implications; resistance detection