Emergence and persistence of minor drug-resistant HIV-1 variants in Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine (NVP) single-dose is still a widely used antiretroviral prophylaxis for the prevention of vertical HIV-1 transmission in resource-limited settings. However, the main disadvantage of the Non-nucleoside Reverse Transcriptase Inhibitor (NNRTI) NVP is the rapid selection of NVP-resistant virus with negative implications for subsequent NNRTI-based long-term antiretroviral therapy (ART). Here, we analysed the emergence of drug-resistant HIV-1 including minor variants in the early phase after NVP single-dose prophylaxis and the persistence of drug-resistant virus over time. METHODS AND FINDINGS: NVP-resistant HIV-1 harbouring the K103N and/or Y181C resistance mutations in the HIV-1 reverse transcriptase gene was measured from 1 week up to 18 months after NVP single-dose prophylaxis in 29 Ugandan women using allele-specific PCR assays capable of detecting drug-resistant variants representing less than 1% of the whole viral population. In total, drug-resistant HIV-1 was identified in 18/29 (62%) women; rates increased from 18% to 38% and 44% at week 1, 2, 6, respectively, and decreased to 18%, 25%, 13% and 4% at month 3, 6, 12 and 18, respectively. The proportion of NVP-resistant virus of the total viral population was significantly higher in women infected with subtype D (median 40.5%) as compared to subtype A (median 1.3%; p = 0.032, Mann-Whitney U test). 33% of resistant virus was not detectable at week 2 but was for the first time measurable 6-12 weeks after NVP single-dose prophylaxis. Three (10%) women harboured resistant virus in proportions >10% still at month 6. CONCLUSIONS: Current WHO guidelines recommend an additional postnatal intake of AZT and 3TC for one week to avoid NVP resistance formation. Our findings indicate that a 1-week medication might be too short to impede the emergence of NVP resistance in a substantial proportion of women. Furthermore, subsequent NNRTI-based ART should not be started earlier than 12 months after NVP single-dose prophylaxis.

Minor drug-resistant HIV type-1 variants in breast milk and plasma of HIV type-1-infected Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine single-dose (NVP-SD) reduces mother-to-child transmission of HIV type-1 (HIV-1), but frequently induces resistance mutations in the HIV-1 genome. Little is known about drug-resistant HIV-1 variants in the breast milk of women who have taken NVP-SD. METHODS: Blood and breast milk samples of 39 HIV-1-infected Ugandan women were taken 6-12 weeks after NVP-SD intake. Samples were analysed by population sequencing and allele-specific real-time PCR (AS-PCR) with detection limits for NVP-resistant HIV-1 variants (K103N and Y181C) of < 1% of the total viral population. RESULTS: AS-PCR results for both plasma and breast milk were obtained for 19 women who constituted the final study group (HIV-1 subtype frequencies were A1 n = 11, D n = 5, G n = 2 and C n = 1). A total of 7 (37%) and 10 (53%) women carried NVP-resistant virus in breast milk and plasma, respectively. Overall, 71% (5/7) women with NVP-resistant HIV-1 in breast milk displayed >1 drug-resistant variant. Resistance in breast milk was higher at week 6 (6/13 samples [46%]) compared with week 12 (1/6 samples [17%]). In total, 10 drug-resistant populations harbouring the K103N and/or Y181C mutation were detected in the 19 breast milk samples; 7 (70%) were caused by resistant minorities (< 5% of the total HIV-1 population). In the four women with drug-resistant virus in both plasma and breast milk, the mutation patterns differed between the two compartments. CONCLUSIONS: Minor populations of drug-resistant HIV-1 were frequently found in breast milk of Ugandan women after exposure to NVP-SD. Further studies need to explore the role of minor drug-resistant variants in the postnatal transmission of (resistant) HIV-1.

Detection and quantification of minor human immunodeficiency virus type 1 variants harboring K103N and Y181C resistance mutations in subtype A and D isolates by allele-specific real-time PCR.

Nevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.

Persistence of nevirapine in breast milk and plasma of mothers and their children after single-dose administration.

OBJECTIVES: Nevirapine is widely used in the developing world for the prevention of mother-to-child transmission (PMTCT) of HIV. A single mutation in the HIV genome is sufficient to lead to significant nevirapine resistance. Persistence of low-level drug concentrations in body compartments can foster resistance formation. In this study, concentration-time courses of nevirapine after single-dose administration were analysed over an extended post-partum period. PATIENTS AND METHODS: Breast milk and plasma samples of 62 HIV-positive Ugandan mother-child pairs who had received single-dose nevirapine were collected at delivery and 1, 2 and 6 weeks post-partum. Nevirapine concentrations were quantified by LC/tandem-mass-spectrometry using a quantification limit of 15 ng/mL, and a population pharmacokinetic (PK) analysis was performed. RESULTS: Concentration-time profiles in breast milk, maternal plasma and child plasma showed similar shapes. At week 1, median nevirapine concentrations were 164 ng/mL in maternal plasma, 114 ng/mL in breast milk and 183 ng/mL in child plasma. The population PK model predicted nevirapine concentrations>10 ng/mL (IC50 for nevirapine) for 13 days in breast milk, 14 days in maternal plasma and 18 days in child plasma in 80% of the samples. CONCLUSIONS: Nevirapine concentrations were present for 2-3 weeks in the three compartments. The concentrations are probably sufficiently high to protect most breastfed children from HIV transmission during the first 2 weeks. The long presence of slowly decreasing levels of nevirapine is likely to induce resistance formation. Post-natal addition of antiretrovirals for 1 week only, as recommended in the current PMTCT guidelines, will not suffice to avoid nevirapine resistance formation.