Background:  Single-dose nevirapine (sdNVP) reduces the risk of HIV vertical transmission, but can cause selection of NVP-resistant HIV in breast milk. Emergence of NVP-resistant HIV in breast milk could potentially compromise efficacy of extended infant NVP regimens for prevention of post-natal HIV transmission. We assessed risk factors for emergence of NVP resistance in breast milk in sdNVP-exposed Ugandan women, and examined persistence of NVP-resistant strains.

Methods:  Breast milk and plasma samples were collected at 4 weeks postpartum from 51 HIV-infected Ugandan women who received sdNVP in an observational study; follow-up samples were available for some women. Viral loads were measured using the Roche AMPLICOR Monitor test kit v1.5. HIV RNA was extracted from breast milk supernatants using the Boom method and was measured using the Roche AMPLICOR Ultrasensitive Monitor test kit. HIV genotyping was performed using the ViroSeq HIV-1 Genotyping System; a nested polymerase chain reaction procedure was used to amplify breast milk samples with <500 copies/mL HIV RNA. HIV subtypes were determined by phylogenetic analysis of pol region sequences. NVP concentrations were determined by liquid chromatography with tandem mass spectroscopy. Statistical significance was tested using Fishers Exact test and the Wilcoxon test with a 2-sided alpha of 0.05.

Results:  Genotyping results were obtained for all 10 breast-milk samples with viral loads >500 copies/mL and for 21 (51.2%) of 41 samples with viral loads <500 copies. Results from 1 woman were excluded because of a possible sample mix-up. HIV subtypes of the 30 paired plasma and breast milk samples were:  15 A, 1 C, 12 D, 2 recombinant. NVP resistance was detected in 12 (40%) of 30 breast milk samples. There was a non-significant association of NVP resistance in breast milk and plasma (P = 0.06). There was no association of HIV resistance in breast milk with median maternal pre-NVP viral load or CD4 cell count, median breast milk viral load at 4 weeks, breast milk sodium >10 mmol/L, HIV subtype, or concentration of NVP in breast milk or plasma. NVP resistance faded from detection in all evaluable breast milk samples by 6 months postpartum.

Conclusions:  NVP resistance was frequently detected in breast milk 4 weeks after sdNVP exposure. We were unable to identify specific factors associated with breast-milk NVP resistance. Using the ViroSeq system, NVP-resistant variants in breast milk faded from detection by 6 months postpartum.