Background:  Single-dose nevirapine (NVP) to prevent mother-to-child HIV-1 transmission selects NVP-resistant variants in 30 to 50% of mothers as determined by standard genotyping (population sequencing). It is not known how long these resistant variants persist because standard genotyping does not reliably detect variants comprising < 25% of the virus population. To address this question, we used an allele-specific RT-PCR assay that quantifies non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants at frequencies < 0.1% to test longitudinal samples from HIV-1 subtype-C-infected women who received single-dose NVP.

Methods:  Follow-up plasma samples from 17 women participating in the South African mother-to-child HIV-1 transmission trial were tested by allele-specific RT-PCR for 103N and 181C. HIV-1 RNA was extracted from plasma, converted to cDNA, and the target sequence region was amplified and quantified by real-time PCR. About 10⁷ copies/reaction were used as template for a second round of real-time PCR, using primers that discriminate between the wild type and mutant alleles (AAT or AAC for 103N and TGT for 181C). To confirm amplification specificity, PCR products were subjected to thermal denaturation analysis.

Results:  Patient samples were separated into 2 groups based on detection of NNRTI-resistance mutations by standard genotyping. Group 1 (8 women) had NNRTI-resistance mutations detected at 6 weeks and 6 months (all 103N) but not at 12 months after single-dose NVP. Group 2 (9 women) had NNRTI-resistance mutations detected at 6 weeks (8 with K103N and 2 of these 8 with Y181C) but not at 6 months after single-dose NVP. In the 12-month follow-up samples from group 1 (negative by standard genotype), allele-specific RT-PCR detected 103N or 181C mutants in 7 of 8 (88%) samples with frequencies ranging from 0.25% to 16%. Similarly, in the 6 month samples from group 2 (negative by standard genotype), 103N mutants were detected in 7 of 9 (78%) samples with frequencies ranging from 0.9% to 10%. Fewer of these were still positive at 12 months.

Conclusions:  NNRTI-resistant variants selected by single-dose NVP can still be detected by allele-specific RT-PCR in the majority of women 6 months after standard genotyping becomes negative. The frequency of NNRTI-resistant mutants declines with time after single-dose NVP but can remain above pretreatment levels for at least 1 year.

Keywords: persistent resistance; single-dose nevirapine; NNRTI resistance