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Background:  Single-dose nevirapine (sdNVP) selects for drug-resistance mutations in reverse transcriptase (RT) in plasma and breast milk HIV-1 RNA. Population sequencing, sensitive to drug-resistance mutations at > 20% may underestimate their dynamics after sdNVP.

Methods:  HIV-1 RNA from plasma and breast milk, from a woman in HPTN 023 study, quanitified by ultra-sensitive Roche Amplicor 1.5, was subject to population sequencing by Trugene 1.5 (Bayer) and Stanford ABI. Population RT sequences were compared to > 50 RT clones from plasma at 0, 2, 8, and 24 weeks, and right and left breast milk at 2 and 8 weeks, a total of ~ 400 RT clones from 8 samples. Drug-resistance mutations were scored for RT codons 98, 100, 101, 103, 106, 108, 179, 181, 188, 190, 225, 227, and 230. Statistical comparisons of plasma and breast milk drug-resistance mutations were performed as differences in proportions (Proc Freq) in SAS.

Results:  HIV-1 RNA levels in plasma ranged from 4.85 to 3.82 log₁₀ copies/mL at 0 to 24 weeks. Breast milk HIV-1 RNA at 2 weeks from left and right breast milk (1.62 and 1.54 log₁₀ copies/mL) were lower than 8-week values (left breast milk 3.25 and right breast milk 4.85 log₁₀ copies/mL). Phylogenetic analysis showed all sequences (clonal and consensus) were closely related, but not identical subtype C RT. Population sequencing (Trugene) in P identified Y181YC and K103KN at 2 and 8 weeks, respectively. Analysis of ~50 unique RT sequences (clones) from plasma demonstrated drug-resistance mutations in 3 of 64 (5%) at 0 week, 46 of 48 (97%) at 2 weeks, 41 of 50 (80%) at 8 weeks, and 3 of 59 (5%) at 24 weeks (p < 0.01). The proportions of drug-resistance mutations in plasma clones at 2 weeks (40% Y181C, 19% K103N, 27% V106AM, 19% Y188C) was significantly different from week 8 where drug-resistance mutations at 103, 106, 181, 188, and 190 were each ~16% (p < 0.01). Breast milk population sequencing at 2 weeks showed L K103KN, Y181YC, Y188YC vs R K103KN,Y181YC and at 8 weeks; L K103KN, Y188YC and R K103KN, Y188YC. The distribution of clonal drug-resistance mutations between left and right breast milk samples at 2 and 8 weeks, and between plasma and breast milk were significantly different (p < 0.05).

Conclusions:  Rapid selection of NVP-resistance mutations following sdNVP in plasma and breasts milk HIV-1 RNA may be underestimated by population sequencing methods. Both ABI and VGI consensus sequences were insensitive to the diversity found in clones, where a majority of viruses had at least 1 drug-resistance mutations at 2 and 8 weeks, although most of drug-resistance mutations existed in < 20% of clones (below the threshold of detection). Different patterns of drug-resistance mutations in plasma and breast milk clones provide evidence for the independent inter and intra compartmental selection of NVP resistance following sdNVP.

Keywords: Nevirapine; Resistance; HIV-1 Subtype C