Background: The HIVNET 012 trial in Uganda demonstrated that single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. NVP resistance (NVPR) can emerge in women and infants following single-dose NVP. HIV-1 genotyping can be used to detect NVPR mutations. However, simpler assays would facilitate evaluation of NVPR in women and infants receiving this regimen. Because NVPR mutations have been characterized predominantly in subtype B HIV-1, non-sequencing-based assays for NVPR may be particularly useful for characterizing NVPR in regions where non-B subtypes are prevalent.

Methods: We evaluated a rapid assay (Amp-RT) for NVPR by testing samples from women enrolled in HIVNET 012. This assay measures reverse transcriptase (RT) enzymatic activity and NVPR directly in plasma. We tested 29 plasma samples from 17 women, including pre- and post-NVP samples. Samples were genotyped using the Applied Biosystems ViroSeq HIV-1 Genotyping System. Seventeen (17) samples had no detectable NVPR mutations (WT) and 12 samples had minor variants with NVPR mutations. The subtypes of the samples were 7A, 1C, 7D, and 2 A/D recombinant, and the viral loads ranged from 6,131–631,200 copies/ml. Samples were analyzed in a blinded fashion using 100 microliters of plasma.

Results: Results were obtained for 26 (90%) of the 29 samples, including 16 WT samples and 10 samples with minor NVPR variants (6 with K103N, 3 with K103N+Y181C, and 1 with K103N+G190A). The other 3 samples had undetectable RT activity. The Amp-RT assay detected NVPR in 6 (60%) of the 10 samples with minor NVPR variants. Thirteen (13; 81%) of the 16 WT samples were susceptible to NVP in the Amp-RT assay, and 3 had reduced susceptibility. Two (2) of the samples with reduced susceptibility were pre- and post-NVP samples from the same woman who was antiretroviral drug naïve prior to NVP administration. The pre-NVP sample had a lower level of NVPR than the post-NVP sample.

Conclusions: The lack of phenotypic NVPR in 4 samples with NVPR mutations most likely reflects the low levels of mutant HIV-1 in those samples. The finding of reduced NVP susceptibility in 3 WT samples suggests that mutations other than those defined in subtype B may cause NVPR in other subtypes. Further studies are needed to characterize the full genetic correlates of NVPR in non-subtype B HIV-1 and to assess the utility of biochemical testing of NVPR in resource-poor settings.