A Radiolabeled Oligonucleotide Ligation Assay Demonstrates the High Frequency of Nevirapine Resistance Mutations in HIV Type 1 Quasispecies of NVP-Treated and Untreated Mother–Infant Pairs from Uganda

Abstract

This study explores the levels of NVP and AZT resistance mutations in untreated, NVP- or AZT-treated mother-infant pairs in Uganda. PCR-amplified reverse transcriptase (RT) gene fragments derived from PBMC samples of 85 mothers (10 AZT treated, 35 NVP treated, and 40 untreated) and their 52 infected infants (5 AZT, 9 NVP, and 38 untreated) were classified as subtype A (59%), D (29%), C (3%), and recombinant forms (9%) by population sequencing. Only 16% of the NVP-treated infected mothers and infants harbored either the K103N or the Y181C at 6 weeks postdelivery. The majority of these samples (n = 107) were then analyzed using a radiolabeled oligonucleotide ligation assay (OLA) specific for K70R, K103N, and Y181C, using nonstandard bases to accommodate sequence heterogeneity. By OLA, 43% of the NVP-treated group had K103N and/or Y181C mutations in their HIV-1 population, using >0.6% cutoff based on a comparative clonal analysis of clinical isolates. Surprisingly, an equal fraction of the untreated and NVP-treated mother-infant group had the K103N mutation in their HIV-1 population in the range of 0.6-5%. These findings suggest a relatively high frequency of K103N mutation in the drug-naive, subtype A and D infected Ugandan population as compared to the very low frequency of the Y181C and K70R mutation (<0.6%). The prevalence of the K103N mutations may be related to its low fitness cost and high genetic stability. The persistence of these mutations may reduce the effectiveness of subsequent NVP use in treatment or prevention of perinatal transmission.

Selection of a Simian-Human Immunodeficiency Virus Strain Resistant to a Vaginal Microbicide in Macaques

“…A modified, radiolabeled OLA (35) (Fig. 2a) was utilized to determine the prevalence of the K315R and N640D mutations in the (i) inoculating SHIV SF162-p3 population, (ii) virus populations from three macaque m584 samples (Fig.…”

“…Diversity in the transferred virus population may pose problems, as illustrated in prevention of mother-to-infant transmission. Although nevirapine is ϳ80% effective in blocking perinatal HIV-1 transmission (33), at least 50% of infants harbor nevirapine-resistant virus within 48 h of delivery and nevirapine treatment (3,35). Given the relative homogeneity of the inoculating SHIV in macaque studies versus inoculating HIV-1 in human transmissions, current macaque microbicide models may underestimate the possible selection of drug-resistant HIV-1 from the inoculating SHIV.…”

HIV-1 drug resistance at antiretroviral treatment initiation in children previously exposed to single-dose nevirapine

“…Several more sensitive assays to detect minority variants have been developed, including real-time allele-specific PCR (AS-PCR), point mutation assays (LigAmp), oligonucleotide ligation assays (OLA) and pyrosequencing [7, 8, 11, 14, 17-20]. When these methodologies are used a larger proportion of children are found to harbor mutations [7, 8, 11, 14, 17-20]. However, the small numbers of children tested and the limited examination of later time points precludes a confident estimate of the proportion of children whose treatment may be compromised by these selected variants.…”

Intrapartum Tenofovir and Emtricitabine Reduces Low-Concentration Drug Resistance Selected by Single-Dose Nevirapine for Perinatal HIV Prevention

“…Because this technique detects mutations in HIV variants comprising >25-50% of the circulating viral population, 8 the true incidence of viral drug resistance may be underestimated. [9][10][11][12] To determine more fully the impact of TDF=FTC on the selection of NNRTI resistance mutations, we evaluated minority populations of drug-resistant viruses using specimens from a previously reported clinical trial.…”