Detection of HIV-1 drug resistance in women following administration of a single dose of nevirapine: comparison of plasma RNA to cellular DNA by consensus sequencing and by oligonucleotide ligation assay

Abstract

A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P<0.01). When resistance was detected only by OLA (n=45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n=51) (P<0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.

FIG. 1.

Schema for selection of the sdNVP-treated women with NVP resistance.

FIG. 2.

NVP resistance mutations detected in HIV-1 RNA and DNA from each woman by consensus sequencing (seq) and oligonucleotide ligation assay (OLA). Participants are listed by increasing time interval between sdNVP therapy and initiation of NVP-ART (ART start). Data for each specimen (RNA and DNA) at 10 days (10d) and 6 weeks following sdNVP therapy and just prior to ART start are shown for each woman, with the symbols in the key indicating the resistant codons detected. n/a, not available.

FIG. 3.

Percentages of women with NVP resistance (K103N, Y181C, or G190A) detected in their HIV-1 RNA or DNA by consensus sequencing and OLA.

FIG. 4.

The dynamics of NVP-resistant-HIV-1 selection and decay are compared in the HIV-1 RNA and DNA of subjects known to harbor mutations at each codon. The prevalence of samples with detectable resistance (A to C) and the concentrations (D to F) of NVP-resistant variants as determined by oligonucleotide ligation assay (OLA) at each point in time are shown by codon. For all codons, the prevalence of resistance and the estimated concentrations of resistant viruses peak sooner in RNA but decay more slowly in DNA.