Antiretroviral drugs for prevention of mother-to-child transmission (PMTCT) of HIV-1, as recommended by the WHO, have led to decreased rates of MTCT in resource-limited regions of the world. Single-dose nevirapine (sdNVP), administered to mothers during labor and infants at delivery, was recommended due to its moderate efficacy, ease of administration, and affordability. However, due to strong evidence that resistance mutations selected following sdNVP negatively impact future combination antiretroviral therapy (cART) in both mothers and infected infants, in 2010, the WHO recommended that mothers with CD4+ T-lymphocyte counts above 350/μl start either zidovudine (ZDV) monotherapy (option A) or triple cART (option B), beginning as early as 14 weeks of gestation . The higher cost of cART compared to ZDV and resource restrictions led many nations that previously relied on sdNVP to adopt option A, despite the fact that in most women, ZDV monotherapy does not completely suppress HIV replication. Implementation studies suggest that cART for all HIV-infected pregnant women increases cost-effectiveness and associated health benefits , leading the WHO to suggest cART for all pregnancies in July 2013 .
Zidovudine monotherapy was used for PMTCT early in the HIV pandemic, and studies from this period demonstrated that resistance mutations were detected infrequently . In these studies, however, ZDV treatment averaged only 10 weeks, and resistance detection relied on consensus sequencing, which has poor sensitivity when mutations are present at less than 20–50% of the viral population [5,6]. Studies evaluating longer periods of ZDV monotherapy or using more sensitive assays (techniques generating multiple-parallel sequences or oligonucleotide ligation assay) have reported a wide range of detected resistance (0–30%) [7,8].
If longer periods of ZDV monotherapy for PMTCT selects resistance mutations at higher rates, and these persist in the viral population, ZDV resistance could negatively impact future treatment, particularly as ZDV mutations impart cross-resistance to other nucleoside reverse transcription inhibitors (NRTIs).
To better assess the selection of resistance following ZDV monotherapy for PMTCT, we performed 454-pyrosequencing, defining the prevalence of resistance mutations across a cohort of women, and the proportion of those mutations in each woman's viral population at the time of delivery and at discontinuation of ZDV. For those with resistance mutations detected, samples at 24 weeks postpartum were evaluated for evidence of persistence or decay of resistance.
Participants in the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Network P1032 study, conducted from June 2006 through June 2008 in Thailand, were pregnant, antiretroviral drug-naive, HIV-1 CRF01_AE-infected, receiving ZDV monotherapy + sdNVP at delivery . They were randomized to one of three postnatal antiretroviral drug tails [arm A: ZDV/didanosine (ddI)/lopinavir/ritonavir (LPV/r) for 7 days; arm B: ZDV/ddI for 30 days; or arm C: ZDV/ddI/LPV/r for 30 days]. The objective of the P1032 study was to compare various postpartum antiretroviral drugs for the reduction of incident NVP-resistance mutations versus no postpartum antiretroviral drug. Women with plasma HIV-1 RNA greater than 500 copies/ml at the time of delivery and adequate available specimens for 454-pyrosequencing were selected for this study.
Plasma viral load was quantified by the Amplicor HIV-1 Monitor test, v1.5 (Roche Molecular Systems, Branchburg New Jersey, USA), with a lower limit of quantification of 50 copies/ml. Plasma samples from delivery were analyzed, as well as plasma from the date of ZDV discontinuation in those women from arm B (prolonged postnatal ZDV exposure), and peripheral blood mononuclear cell (PBMC) DNA from 24 weeks postpartum for those women who were found to have resistance mutations, due to longer persistence in DNA compared to RNA, once selected [10,11]. Clinical data collected included age, plasma viral load, and CD4+ cell count at enrollment, duration of ZDV monotherapy, and plasma viral load at delivery (Table 1).
RNA was extracted from 1000 μl of plasma using NucliSENSE miniMAG kit (Biomerieux, Durham, North Carolina, USA), and reverse-transcribed using random hexamers with the Blue Print 1st Strand cDNA synthesis kit (Takara Bio Inc., Shiga, Japan). DNA was extracted from whole blood collected at 24 weeks postpartum using silica extraction . Real-time PCR of the HIV-1 long terminal repeat (LTR) region was performed on each sample in duplicate to quantify the amplifiable viral cDNA or DNA templates  (Supplemental table).
Amplification of HIV-1 pol region that encodes reverse transcriptase (RT) was performed using FastStart High Fidelity polymerase (Roche Diagnostics, Mannheim, Germany), with an input of 1000 amplifiable viral cDNA/DNA templates per participant. Nested PCR amplified two regions encoding RT using CRF_AE-specific first and second-round primers. Each second-round primer included pyrosequencing adapters for later emulsion PCR, and a multiplex identifier (MID) allowing 14 samples to be sequenced in the same pool (Supplemental table).
Following PCR amplification, each sample was purified using the High Pure PCR purification kit (Roche Applied Science, Mannheim, Germany), quantified with the Quant-iT PicoGreen dsDNA Reagent (Invitrogen, Life Technologies, Carlsbad, California, USA), then diluted to 1 × 109 molecules/μl. Equal volumes of amplicons sequenced together were pooled, and each pool diluted to 1 × 107 molecules/μl.
Clonal amplification on beads (emulsion PCR) of each pool used reagents that allowed bidirectional sequencing (454 Life Sciences; Roche Diagnostics Corporation, Branford, Connecticut, USA). Two-million enriched beads were loaded per region of a PicoTiter plate divided by a gasket for sequencing by GS FLX Titanium System following the manufacturer's instructions (454 Life Sciences). An HIV-1 plasmid was amplified and sequenced with each plate as control for PCR and pyrosequencing error rates at codons of interest.
The 454-sequencing reads containing ambiguous bases (<100 bp), or with average quality score less than 25 were not analyzed. Remaining reads were mapped to a reference HIV-1 sequence using the BLAST algorithm, aligned using the Needleman-Wunsch algorithm, and manually refined .
The 454-pyrosequences from each participant were evaluated for ZDV-resistance mutations encoding M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E. If resistance was detected, the proportion of mutant variant was determined based on forward, reverse, and overall nucleotide frequencies at each site across the refined alignments.
The P values were calculated using the two-sample Wilcoxon test, with two-sided P values less than 0.05 considered statistically significant.
The 50 participants in this study had a median age of 27.5 years, CD4+ T-lymphocytes of 424/μl, and plasma HIV-1 RNA of 12 464 copies/ml at study entry and 7250 copies/ml at delivery. The median duration of prepartum ZDV therapy was 10.7 weeks (Table 1). The distribution of participants across the study arms was relatively balanced, with 15 from arm A, 17 from arm B, and 18 from arm C.
HIV-1 zidovudine resistance by 454-pyrosequencing
Analysis of the plasmid control demonstrated an error rate of less than 0.5% at each codon of interest. Combined with analysis of approximately 1000 viral templates from each participant, a conservative limit of 1% mutant was used for analyses of ZDV-resistance mutations.
Plasma samples from the day of delivery revealed ZDV-resistance mutations at levels at least 1% in 7 women [14%, 95% confidence interval (CI) 6.6–26.5%]. Five women had the K70R mutation, three had the D67N mutation, and one had the M41L mutation. No L210W, T215Y/F, K219Q/E, or intermediate T215 mutations were detected. One woman had three resistance mutations (M41L, D67N, and K70R), whereas all others had one mutation each (Table 2). At delivery, the proportion of mutant in the viral population ranged from 1.1 to 24.5%, with only one codon near the sensitivity of consensus sequencing (K70R at 24.5%). Among 17 women who received 30 days of ZDV/ddI as a postpartum tail, three had mutations detected at discontinuation of ZDV: two had new resistance mutations detected (participants 6 and 8 – the latter with no mutants detected at delivery) and the third (participant 2) had increase in the concentration of mutants (Table 2). Testing at 24 weeks postpartum detected ZDV resistance in the PBMC of only one of the eight participants with mutations – K70R in participant 2 at 1.6% (Table 2).
A comparison of women with and those without mutations at delivery revealed no statistically significant differences in age (P = 0.5), plasma HIV-1 RNA load or CD4+ T-lymphocyte counts at entry (P = 0.72 and 0.31, respectively), plasma HIV-1 RNA load at delivery (P = 0.16), or duration of ZDV monotherapy before delivery (P = 0.51) (Table 1).
In our cohort of women receiving a median of 10.7 weeks of ZDV monotherapy for PMTCT, 14% of them had ZDV resistance detectable by pyrosequencing at delivery. Among the subset of 17 who received 30 days of ZDV/ddI postpartum, one additional woman had selection of a ZDV mutation, and the mutant population increased in size in two others. Although the duration of ZDV varied widely, it was not statistically different between those with and without resistance at delivery, possibly due to the small sample size. ZDV resistance detected following PMTCT was mostly at levels below the threshold detectable by genotypic consensus sequencing. The impact of these minority ZDV-resistant variants on the efficacy of first-line cART is still uncertain. Given that PMTCT programs in some countries have relied on option A (ZDV monotherapy), it is important to assess the selection of ZDV-associated mutations and the risk of both majority and minority ZDV-resistant populations on virologic failure of future cART, as has been described with non-nucleoside reverse transcription inhibitor (NNRTI) mutations following use of sdNVP [15,16].
Most detected ZDV mutations faded below the limit of detection in PBMCs by 24 weeks postpartum, likely due to decay of recently infected cells and poor replication capacity compared to the wild-type variants . The decrease to levels below detection in the peripheral blood does not guarantee that the HIV-1 reservoir is free of replication-competent mutants. Rather, women may be at increased risk of virologic failure for a long time as observed with prolonged selection during virologic failure , or for a limited time as with sdNVP .
A limitation of our study is the absence of genotypes before ZDV to compare to resistance profiles after ZDV. Given that a single HIV variant has been observed to found most infections and that the women were antiretroviral drug-naive, it is unlikely that the ZDV resistance detected represents transmitted drug resistance. Another limitation is that despite an additional 30 days of ZDV in 17 women, its administration in combination with sdNVP at delivery and ddI adds to the genetic barrier for selection of resistance and precludes interpreting the postpartum data, as reflective of option A. Furthermore, enrollment in our cohort occurred when cART was recommended for women with CD4+ less than 250 cells/μl. Three women with mutations in our study would qualify for cART under the 2010 guidelines, which was recommended for women with CD4+ cell counts 350 cells/μl or less.
In our study, 14% of women who received ZDV monotherapy for a median of 10.7 weeks had resistance mutations detected at delivery, albeit at a low proportion of the HIV population in most women. Although the significance of these resistance mutations on later cART is uncertain, recent findings that cART is superior to ZDV in PMTCT makes rapid implementation of cART in all programs the most circumspect approach .
The present study was supported by NIH funds through a supplement (LMF) to IMPAACT (U01 AI068632), a fellowship (SCO) award (T32 HD07233), the 1032 study team in Chiang Mai (U01 AI41089), the Molecular Profiling and Computational Biology Core of the Seattle Centers for AIDS Research (P30 AI 027757), and the IMPAACT Statistical and Data Management Center (U01 AI068616). Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) under Award Numbers UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) and UM1AI106716 (IMPAACT LC), with co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the National Institute of Mental Health (NIMH).
The authors would like to thank Rob Hall for his assistance in pyrosequencing subject samples at the Bumgarner laboratory.
Conflicts of interest
The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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