Secondary Logo

Journal Logo

BASIC SCIENCE

Minority and majority pretreatment HIV-1 drug resistance associated with failure of first-line nonnucleoside reverse-transcriptase inhibitor antiretroviral therapy in Kenyan women

Milne, Ross S.a; Silverman, Rachel A.b,c; Beck, Ingrid A.a; Mckernan-Mullin, Jennifera; Deng, Wenjied; Sibley, Thomas R.d; Dross, Sandrae; Kiarie, James N.h; Sakr, Samah R.i; Coombs, Robert W.e,f; Chung, Michael H.b,c,f; Frenkel, Lisa M.a,c,e,f,g

Author Information
doi: 10.1097/QAD.0000000000002134

Abstract

Introduction

Access to antiretroviral-therapy (ART) for HIV type 1-infected persons has increased markedly in resource-limited settings (RLS) [1]. Although ART decreases morbidity and mortality [2] and new infections [3], access to ART is associated with increased ‘acquisition’ of drug resistance and transmission of drug resistance [4]. Pre-ART drug resistance is rising in Africa [5–7], especially in young Kenyan women [8]. Increases in virologic failure associated with pre-ART drug resistance [6,9–10] warrant expectant management to maintain ART benefits and to minimize forward transmission of drug-resistant variants.

Peripartum single-dose nevirapine (sdNVP) has frequently been used in resource-limited settings to prevent mother-to-child-transmission (PMTCT). Although moderately effective [11], sdNVP can select drug resistance that impacts subsequent ART efficacy [10,12]. Prepartum zidovudine (ZDV) added to sdNVP [13], and later combined with a 5–7-day postpartum ‘tail’ of ZDV and lamivudine (3TC) [14] to prevent drug-resistance selection in women, dramatically reduced MTCT [15,16], but was complex to administer and contributed to growing rates of pre-ART drug resistance [17]. Furthermore, NVP-resistant viruses demonstrate cross-resistance to other nonnucleoside reverse-transcriptase inhibitors (NNRTIs) including efavirenz (EFV) [18], which became the WHO-preferred first-line ART regimen for individuals at least 2 years of age in 2013, including pregnant women [19].

To evaluate the role of pre-ART drug-resistance in virologic failure of women, adult Kenyan women with and without a history of sdNVP who qualified for initial first-line NNRTI-based-ART were prospectively enrolled and followed for 1 year. Pre-ART drug resistance was identified retrospectively by consensus sequencing, and a relatively low-cost oligonucleotide-ligation-assay (OLA) previously validated to detect transmitted or sdNVP-selected pre-ART drug-resistance associated with failure [6,20]. Our study included primary, secondary, and post hoc hypotheses; primary: pre-ART drug-resistance detected by OLA at four codons associated with resistance to NNRTI/NRTIs would predict virologic failure as well or better than consensus sequencing; secondary #1: sdNVP-experienced women without pre-ART drug-resistance by OLA and consensus sequencing would rapidly select NNRTI/NRTI-associated mutations and experience failure (detected by frequent monitoring of plasma viral load) within a few months of ART initiation; secondary #2: archived mutations not detected as pre-ART drug-resistance by OLA that contribute to failure can be identified by longitudinal OLA genotyping of PBMC; and post hoc #1: sdNVP-experienced women who failed without pre-ART drug-resistance by OLA and consensus sequencing would harbor pre-ART drug-resistant minority variants detectable by next-generation sequencing.

Methods

HIV-infected women at least 18 years old initiating first-line ART entered the study at Coptic Hope Center for Infectious Diseases, an HIV treatment clinic in Nairobi [21] (Fig. 1). ART-experienced, seriously ill, or individuals planning to move within a year study were excluded. Participants provided informed consent, as approved by Human Subjects’ Ethics Boards (Seattle #: 11676 and Kenya #: P209/08/2008). Sociodemographic and clinical data from prior to ART initiation were collected: age, CD4+ T-lymphocyte counts, and prior exposure to peripartum antiretrovirals for PMTCT (by pharmacy records and self-report), including sdNVP, third-trimester ZDV and/or postpartum ZDV with or without 3TC (sdNVP ± ZDV ± 3TC). Women were prescribed first-line ART containing NVP or EFV and ZDV combined with 3TC; EFV was used when NVP was contraindicated or not tolerated. Blood plasma and peripheral blood mononuclear cells (PBMC) were collected prior to ART initiation and every 2 months through month 12 of ART.

F1-2
Fig. 1:
Schema of study of women initiating first-line nonnucleoside reverse transcriptase inhibitor-based antiretroviral therapy (NNRTI-ART) with retrospective assessments of pre-antiretroviral therapy HIV drug resistance (PDR), and virologic outcome at month 12 of study follow-up.Enrolled participants either had no history of antiretroviral treatment or a history of single-dose nevirapine (sdNVP) with or without prepartum zidovudine (ZDV) and/or postpartum ZDV with or without lamivudine (3TC) for prevention of mother-to-child transmission (PMTCT). Specimens were retrospectively assessed for PDR by an oligonucleotide ligation assay (OLA), consensus sequencing and next-generation sequencing using the Illumina Miseq platform. Study outcomes, virologic failure (plasma viral load ≥400 HIV RNA copies/ml) or virologic suppression (plasma viral load <400 copies/ml) are shown, with contributions of participants with PDR vs. wild-type by each method of drug-resistance genotyping shown for each outcome. Diagnostic sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) have been calculated and presented for OLA, consensus sequencing, and next-generation sequencing. Participants lost to follow-up (LTFU) are shown. NNRTI, nonnucleoside reverse transcriptase inhibitor.

Pre-ART drug resistance was assessed in plasma and PBMC by OLA, in plasma and/or PBMC by consensus sequencing [22] and in PBMC by next-generation sequencing using the Illumina MiSeq platform. PBMC DNA and plasma RNA had HIV templates quantified by a CLIA-certified in-house quantitative PCR (qPCR) targeting the HIV gag[23]. An estimated 100–300 amplifiable copies were submitted to OLA designed to detect mutations at pol reverse transcriptase codons 103, 181, 184, and 190. OLA standard curves allowed quantification of mutant frequencies within participants’ viral quasispecies. If multiple mutant codons were detected within an individual's quasispecies, the value from the codon with the highest mutant frequency was considered ‘peak’ and used for analysis. The amplicon from RNA and/or DNA also underwent consensus sequencing. Nucleotide sequences are available in the NCBI Genbank under accession numbers MH509760–MH509936.

Next-generation sequencing, as described [8], evaluated pre-ART drug-resistant minority variants in HIV pol below the limit of detection by OLA (2%) and consensus sequencing (15–25%). Briefly, two regions of pol encoding reverse transcriptase were amplified from PBMC DNA by nested PCR with a high-fidelity enzyme and second-round primers containing Illumina sequencing adapters. These amplicons were purified, pooled, and sequenced bidirectionally over 300 base-pairs on an Illumina Miseq. Raw sequence reads were processed by a custom variant-calling analysis pipeline (https://github.com/MullinsLab/drm-snp-calling), resulting in tables containing amino acid variants and associated frequencies. PCR and sequencing error rates at each base were assessed by an in-house Perl script to estimate genuine pre-ART drug-resistant populations (https://indra.mullins.microbiol.washington.edu/perlscript/docs/parseBlastXML_calcErrRate.html). The average mismatch error rate ranged from 0.58 to 0.68%, suggesting a conservative 1% cut-off frequency. To exclude minority variants resulting from Illumina ‘index hopping,’ all minority variants were evaluated by phylogenetic clustering to participants’ consensus sequences, and nonclustering variants were discarded. Next-generation-sequencing datasets are available in the NCBI Sequence Read Archive under accession number SRP154562.

Pre-ART drug-resistance, hypermutation, and HIV subtypes were defined by the Stanford HIV Database [24]. Next-generation sequencing pre-ART drug-resistant mutants encoded by hypermutation were excluded from analysis. When pre-ART drug resistance was not detected by any assay or conferred a Stanford penalty score less than 10, the participant's HIV was defined as wild-type. Subtypes were determined from consensus sequences, or next-generation-sequencing data when consensus sequences were unavailable.

Plasma viral load (RealTime HIV, Abbott Molecular, Abbott Park, Illinois, USA) at month 12 of ART or final specimen identified participants with virologic failure, defined as at least 400 HIV RNA copies/ml plasma. In participants with failure and/or pre-ART drug-resistance by OLA, longitudinal plasma RNA and PBMC DNA specimens collected at 2-month intervals between months 0 and 12 had plasma viral load measured and OLA performed, respectively, to identify the onset of failure and detect emerging drug resistance. Consensus sequencing of plasma at failure assessed accumulated drug resistance.

Data analysis

Amongst participants stratified by prior antiretroviral experience for PMTCT, Welch two-sample t-tests compared continuous enrollment characteristics (CD4+ cell count, enrollment plasma viral load, the interval between PMTCT, and ART initiation), whereas chi-square tests compared binary variables (pre-ART drug resistance prevalence by OLA). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each genotypic resistance assay using the MedCalc Diagnostic test evaluation calculator for Windows, version 15.0 (MedCalc Software, Ostend, Belgium). Pre-ART risk factors including plasma viral load, CD4+ T-cell count, prior antiretroviral experience for PMTCT, NNRTI (NVP or EFV) used for ART, and pre-ART drug resistance by OLA, consensus sequencing, and next-generation sequencing, were assessed for associations with virologic failure using both univariable and multivariable Cox proportional-hazards regression with robust variance estimates. Pre-ART drug resistance was assessed as both a binary and categorical independent variable, the latter stratified by mutant frequency for pre-ART drug resistance by OLA and next-generation sequencing. Multivariable regression analysis included the covariates plasma viral load, prior antiretroviral experience, pre-ART drug resistance (by OLA, consensus sequencing, or next-generation-sequencing), and NNRTI used for ART. Kaplan–Meier survival analyses show time from ART initiation to failure among all women stratified by prior antiretroviral experience, among women with next-generation sequencing results stratified by pre-ART drug-resistant mutant frequency, and in a second analysis with women with minority variants further stratified by exposure to sdNVP and ZDV with or without 3TC. The survival distributions were compared across these groups using pairwise log-rank tests with Benjamini–Hochberg adjustment for multiple comparisons. All statistical analyses were performed in RStudio (version 1.0.44; R Foundation, Vienna, Austria).

Results

Study population

In total, 176 Kenyan women enrolled and 169 completed the study. Seven (4%) women in total were lost-to-follow-up, including four prior to ART initiation and three during the study (at study months 2, 2, and 4); these were excluded from analyses (Fig. 1). NVP-based and EFV-based ART were prescribed to 144 and 25 women, respectively. The 169 women analyzed were infected with HIV subtypes A (n = 64, 37.9%), CRF01_AE (n = 39, 23.1%), C (n = 14, 8.3%), or D (n = 40, 23.7%). sdNVP was used for PMTCT by 72/169 (43%) women at a median of 424 days (IQR 169–784) prior to ART initiation. Among the sdNVP-experienced women, prepartum ZDV or stavudine (d4T) was taken by 34/72 (47%) women for a median duration of 90 days (IQR 60–90), and postpartum ZDV with or without 3TC was taken by 14 of these women for a median duration of 7 (IQR 7–7) days. sdNVP with or without ZDV with or without 3TC-experienced vs. antiretroviral-naive women had lower enrollment age [mean (95% confidence interval: CI) years, 30.3 (29.1–31.5) vs. 36.9 (35.1–38.7), P < 0.0001] and higher enrollment CD4+ T-cell count [mean (95% CI) cells/μl, 188.8 (168.9–208.7) vs. 159.6 (143.9–175.3), P = 0.02], whereas enrollment plasma viral loads [log10 copies/ml, 4.8 (4.6–5.0) vs. 4.7 (4.4–4.9), P = 0.44] were similar. The interval between PMTCT and ART initiation was greater among those who took sdNVP alone vs. sdNVP and ZDV with and without 3TC [mean (95% CI) days, 635 (510–761) vs. 349 (241–457), P < 0.001].

Preantiretroviral therapy drug resistance by oligonucleotide ligation assay, consensus sequencing and next-generation sequencing

OLA assessed pre-ART drug resistance on an estimated median of 471 [interquartile range (IQR) 121–551] and 131 (IQR 108–195) HIV RNA (quantified by gag qPCR in only sdNVP experienced; otherwise estimated from plasma viral load) and DNA templates (all quantified by gag qPCR), respectively. OLA detected pre-ART drug resistance at codons K103N, Y181C, M184V, and/or G190A in the plasma and/or PBMC samples of 18 of 169 (10.7%) women (Supplemental Table 1, https://links.lww.com/QAD/B441). Pre-ART drug resistance was more prevalent in sdNVP-experienced vs. antiretroviral-naive women: 12 of 72 (16.7%) vs. 6/97 (6.2%), prevalence ratio (95% CI) = 2.69 (1.06–6.84), P = 0.03. Among sdNVP-experienced women, the prevalence of pre-ART drug resistance by OLA was lower in those who received sdNVP and ZDV with and without 3TC (4/34, 11.8%) vs. sdNVP alone (8/38, 21.1%), but this difference was not statistically significant, prevalence ratio (95% CI) = 0.56 (0.18–1.69), P = 0.29.

In women with pre-ART drug-resistance by OLA, the median pre-ART mutant frequency was 100% (IQR 17.5–100) in plasma and 85% (IQR 37–92) in PBMC. The six antiretroviral-naive women with pre-ART drug resistance had a median frequency of 100% (IQR 89–100) in plasma and 81% (IQR 60–88.5) in PBMC and none had peak pre-ART drug resistance at frequencies 2–9%. In contrast, two of 12 (16.7%) sdNVP-experienced women had peak mutants of frequencies 2–9%.

Consensus sequencing detected 22 of 26 (84.6%) pre-ART drug-resistance mutations identified by OLA in 15 of 18 women but missed four mutations with a median OLA frequency of 7% (range 2–14). In the 18 women with pre-ART drug-resistance by OLA, consensus sequencing detected seven additional mutant codons associated with intermediate-to-high-level resistance to NNRTI/NRTIs, including K70R, L74I, A98G, K101E, and T215Y. Consensus sequencing identified one additional participant with intermediate-to-high-level pre-ART drug resistance (M230I), and nine women with polymorphisms conferring potential or low-level resistance.

Next-generation sequencing was performed on pre-ART PBMC DNA, a compartment more likely to harbor archived drug resistance [25], from 162 of 169 (95.9%) women (seven had insufficient specimen/amplification), including all 38 participants that experienced virologic failure. A median of 1147 (IQR 793–1441) HIV DNA templates per specimen were sequenced, as estimated by gag qPCR, producing a median of 129 (IQR 90–190) sequence reads per input viral template. Next-generation sequencing confirmed 25 of 26 (96.2%) drug resistance mutations identified by OLA and 30 of 30 detected by consensus sequencing (Supplemental Table 1, https://links.lww.com/QAD/B441). The single mutant population detected by OLA but not next-generation-sequencing was Y181C at a frequency of 4%. Next-generation sequencing detected 26 minority variants not detected by consensus sequencing or OLA, including six of 26 (26%) at OLA codons: K103N (n = 3), M184V (n = 2), and G190A (n = 1). These 26 minority variants conferred low-to-high level resistance in 22 women, including 14 of 22 (64%) who were wild type by OLA and consensus sequencing. Notably, nine of the latter women were sdNVP-experienced, and six also ZDV with and without 3TC-experienced. The minority variants had a median frequency of 2.0% (IQR 1.3–3.8) within participants’ viral quasispecies. One of these variants exceeded the 1–9% threshold (K70R = 24%) but was considered minority as it was not detected by consensus sequencing.

Preantiretroviral therapy risk factors associated with virologic failure

Among 169 participants, 38 (22.5%) experienced virologic failure by study month 12, including 34 of 144 (24%) prescribed NVP-based and 4/25 (16%) prescribed EFV-based ART (P = 0.34). At study enrollment, women with and without failure were similar in age, plasma viral load, and CD4+ T-cell counts (Table 1). In univariable Cox regression analyses, pre-ART drug resistance in plasma and/or PBMC by OLA, consensus sequencing or next-generation-sequencing were associated with increased risk of failure; hazard ratio (95% CI): 7.11 (3.32–15.23), 7.59 (3.50–16.46), 9.50 (4.95–18.22), respectively, all P < 0.0001. Mutations K103N and/or G190A identified all women with pre-ART drug resistance by OLA that experienced failure. Pre-ART drug resistance by OLA at frequencies at least 10% in plasma and/or PBMC were associated with increased risk of failure compared with wild-type; hazard ratio (95% CI): 9.28 (4.20–20.45), P < 0.0001. Only two participants had pre-ART drug resistance by OLA at peak frequencies between 2 and 9% and neither had failure. In contrast, detection of minority variants by next-generation sequencing (Table 1) with Stanford mutation penalty scores of at least 10 was associated with increased risk of failure compared with wild-type, hazard ratio (95% CI): 7.05 (3.21–15.48), P < 0.0001.

T1-2
Table 1:
Demographic, immunologic, and virologic parameters at enrollment associated with virologic suppression vs. failure (>400 copies/ml) at month 12 of antiretroviral therapy.

Compared with OLA [31.6% (17.5–48.7%)] and consensus sequencing [31.6% (17.5–48.7%)], next-generation sequencing appeared to detect pre-ART drug resistance in women with virologic failure with greater sensitivity [57.9% (40.8–73.7%)] (Fig. 1). However, specificity of next-generation sequencing [91.1% (84.7–95.5%)] was slightly less than OLA [95.4% (90.3–98.3%)] and consensus sequencing [95.9% (90.7–98.7%)]. Overall, OLA, consensus sequencing, and next-generation sequencing had similar positive [66.7% (44.6–83.3%) vs. 70.6% (47.5–86.5%) vs. 66.7% (51.7–78.9%), respectively] and negative [82.8% (79.4–85.7%) vs. 81.8% (73.6–86.4%) vs. 87.6% (82.9–91.2%), respectively] predictive values (Fig. 1).

sdNVP-experienced vs. antiretroviral-naive women had increased risk of virologic failure, but this was not statistically significant, [hazard ratio (95% CI): 1.87 (0.99–3.52), P = 0.06 (Table 1)]. Compared with antiretroviral-naive participants, a statistically significant increase in failure was observed in women who took sdNVP and ZDV with or without 3TC but not in those who took sdNVP alone [hazard ratio (95% CI): 2.43 (1.15–5.14), P = 0.02 and 1.43 (0.64–3.18), P = 0.38, respectively]. However, a comparison of the time interval between PMTCT and ART initiation found no difference in risk of failure between women exposed to sdNVP only vs. sdNVP and ZDV with or without 3TC; hazard ratio (95% CI): 1.00 (1.00–1.00), P = 0.45. Previous sdNVP and ZDV with or without 3TC experience remained associated with failure in multivariable Cox regression analyses that included the covariates: enrollment plasma viral load, NVP or EFV-based ART, and pre-ART drug resistance by OLA [hazard ratio (95% CI), 2.63 (1.26–5.47), P < 0.01] or consensus sequencing [hazard ratio (95% CI) 2.18 (1.04–4.54), P = 0.04]. This indicated potential additional sources of risk of failure in sdNVP and ZDV with or without 3TC-experienced women and precipitated the analysis of minority variants by next-generation sequencing. Indeed, adjusting for pre-ART drug-resistance by next-generation sequencing at frequencies 1–9% and 10–100% attenuated the association between prior sdNVP and ZDV with or without 3TC experience and risk of virologic failure [hazard ratio (95% CI), 1.70 (0.80–3.62), P = 0.17], compared with models adjusting for pre-ART drug resistance by OLA or consensus sequencing.

Kaplan–Meier survival analyses, showing the proportion of participants without virologic failure, are presented for all women stratified by history of antiretrovirals for PMTCT (panel a), for women with next-generation sequencing data available stratified by pre-ART drug-resistance category (panel b), and for women with next-generation sequencing data by pre-ART drug-resistance category with those with minority variants further stratified by exposure to ZDV with or without 3TC for PMTCT (panel c) (Fig. 2). Increased virologic failure was observed in women with a history of sdNVP and ZDV with or without 3TC for PMTCT (panel a, P = 0.05) and with pre-ART drug resistance as both minority variants and majority variants (panel b, both P < 0.0001). Although sample sizes were small, among women with minority variants exposure to ZDV with or without 3TC was associated with increased failure compared with sdNVP only and antiretroviral-naive women, P = 0.01 (panel c).

F2-2
Fig. 2:
Kaplan–Meier survival analysis.Survival probabilities (i.e. proportion without virologic failure) during 12 months of nonnucleoside reverse transcriptase inhibitor-based antiretroviral therapy (NNRTI-ART) of all women stratified by history of antiretroviral treatment for prevention of mother-to-child transmission (PMTCT) (panel a), and by pre-ART drug-resistance category (wild-type, minority variants (MinV), majority variants (MajV)) (panel b). Women with minority variants were further stratified into those with [MinV(+)) and without (MinV(−)] exposure to sdNVP and ZDV with or without 3TC. Benjamini–Hochberg-adjusted P values denote levels of significance for pairwise log-rank comparisons between survival curves: * P = 0.05, ** P = 0.01, *** P < 0.0001. Risk tables denote total number of participants with outcome at each timepoint below survival plots. Panel a: increased virologic failure was observed in women with a history of sdNVP and ZDV with or without 3TC for PMTCT compared with antiretroviral-naive women (P = 0.05). Panel b: next-generation sequencing analyses found that minority variants (MinV) (1–9% of one or more mutant variants within an individual's HIV quasispecies) and majority variants (MajV) (10–100% mutant) were associated with increased virologic failure compared with wild-type (<1% mutant; both P < 0.0001). Panel c: next-generation sequencing analyses found that among women with minority variants, increased virologic failure was associated with sdNVP and ZDV with or without 3TC experience [MinV(+)] compared with antiretroviral-naive and sdNVP-only-experienced women [MinV(−)], (P = 0.01).

Longitudinal oligonucleotide ligation assay for detection of emergent disease resistance in women with failure

To evaluate if ‘archived’ mutations not detected as pre-ART drug resistance by OLA contributed to failure through selection by first-line ART, longitudinal OLA genotyping of PBMC and plasma viral load measurements were performed for 45 women with failure and/or pre-ART drug resistance by OLA (Supplemental Figure 1, https://links.lww.com/QAD/B441). In women with failure but without pre-ART drug-resistance by OLA, longitudinal OLA genotyping of PBMC detected emergent drug resistance in nine of 11 (81.8%) antiretroviral-naive and 12 of 14 (78.6%) sdNVP-experienced women, including all nine sdNVP and ZDV with or without 3TC-experienced women (Supplemental Figure 1, panels b, d and f, https://links.lww.com/QAD/B441). Drug resistance emerged prior to failure in only three women (two antiretroviral-naive and one sdNVP-only-experienced), and at the onset of failure in 10 women. Failure occurred rapidly, by month 6 of ART, in seven of 10 of these women, including one of three antiretroviral-naive and six of seven sdNVP-experienced women. Notably, all six sdNVP-experienced women had peripartum ZDV with or without 3TC. Pre-ART drug resistance composed of minority variants was detected by next-generation sequencing in five of six of these women at both OLA (M184V) and non-OLA codons (D67N, K70R, V108I, K219Q) conferring Stanford penalty scores of at least 10 to NVP, ZDV, and 3TC.

Discussion

In this study of Kenyan women, virologic failure within 12 months of initiating first-line NNRTI-based ART, primarily with NVP, was increased in association with pre-ART drug resistance by OLA and consensus sequencing, minority and majority frequency variants by next-generation sequencing, and a history of PMTCT. Consistent with past findings and our primary hypothesis, a more than 10-fold increase in failure was observed in women with pre-ART drug-resistance by OLA; particularly those who had previously taken sdNVP with or without ZDV with or without 3TC for PMTCT [25–28], or if antiretroviral-naive, in those with apparent transmitted drug resistance [6–7]. The risk of failure from pre-ART drug resistance appeared primarily conferred by majority frequency mutations detected similarly in participants’ plasma RNA and/or PBMC DNA HIV, by OLA and consensus sequencing. OLA detection of K103N and/or G190A codons identified all women with pre-ART drug-resistance by OLA who experienced failure, which generally occurred at or prior to month 4 of ART. However, a subset of women with majority K103N achieved and maintained ART suppression, as has been reported [26].

Although evaluation of different participant subsets and amplicons prevented direct statistical comparison of assay sensitivity and specificity, next-generation sequencing detection of pre-ART drug- resistance appeared to be more sensitive, identifying 10 more women with failure than both OLA and consensus sequencing. This potential advantage was offset slightly by a reduced specificity. However, the assays had similar PPVs and NPVs. Notably, none of the assays had sensitivity greater than 58%, with OLA and consensus sequencing sensitive to 32% only. This likely reflects the multiple pathways by which failure may occur, including poor adherence to ART, which was not assessed in this study.

Testing for pre-ART drug-resistance by OLA and consensus sequencing identified individuals at increased risk of failure, as in our previous studies [6,20,22]. However, because we anticipated that neither OLA nor consensus sequencing would detect all pre-ART drug resistance in women who experienced failure, we explored additional strategies to identify at-risk women: frequent plasma viral load monitoring and testing PBMC by OLA at 2-month intervals during ART; and added post hoc testing for pre-ART drug-resistant minority variants by next-generation sequencing.

Longitudinal plasma viral load testing identified viral rebound or failure-to-suppress viral replication within a few months of initiating ART in women with high-frequency pre-ART drug resistance. Among those wild-type by OLA and consensus sequencing, failure among antiretroviral-naive women often occurred after month 6 of ART, consistent with incomplete adherence [29]. In contrast, sdNVP with or without ZDV with or without 3TC-experienced women without pre-ART drug resistance by OLA or consensus sequencing experienced failure-to-suppress or rapid viral rebound, resembling those with high-frequency pre-ART drug resistance, suggesting that frequent plasma viral load monitoring may benefit these women. Emergent drug-resistant variants in most were detected by OLA at or shortly after the onset of failure, and rarely before failure, indicating that OLA of PBMC did not effectively screen for ongoing selection of mutations during ART.

Cox proportional hazards regression and survival analyses indicated that sdNVP and ZDV with or without 3TC experience, but not sdNVP alone, conferred residual risk of failure among women without pre-ART drug resistance by OLA and consensus sequencing. Other studies suggest an interaction between the interval of time between PMTCT and initiation of ART and risk of failure [10,20]. However, we did not observe such an interaction in our analysis, mostly likely because the median time intervals from sdNVP or sdNVP and ZDV with or without 3TC to ART initiation in our subgroups were two-fold to four-fold longer than the 6-month interval previously analyzed. The longer interval in our study may have allowed decay of rare undetected mutant variants to clinically irrelevant levels [26]. Pre-ART drug-resistance genotyping by next-generation sequencing was performed. Next-generation sequencing provides sequence data with a greater sensitivity compared with consensus sequencing and a broader range of codons compared with the OLA. Next-generation sequencing detected pre-ART drug-resistant minority variants associated with failure, especially among women who had taken sdNVP and ZDV with or without 3TC for PMTCT. Notably, failure occurred by month 4 of ART in six of six sdNVP and ZDV with or without 3TC-experienced women with minority variants by next-generation sequencing who were wild-type by OLA and consensus sequencing. Minority variants were detected at codons associated with ZDV, 3TC, or NNRTI resistance, including thymidine analogue mutations (TAMs), suggesting that mutants selected by multiple nonsuppressive antiretrovirals for PMTCT were archived at levels below the limit of detection by consensus sequencing, and rapidly re-selected upon initiation of ART. Persistent minority variants following antiretrovirals for PMTCT have been linked to poor response to ART in women [26,30,31] and infants [32] in other studies, which combined with our observations emphasize the risks of serial antiretrovirals for PMTCT. Although numbers were too small to draw statistical conclusions, antiretroviral-naive women with minority variants in our study did not appear to experience rapid failure, in agreement with some [33–34] but not other reports [35,36], suggesting a complex interaction of multiple factors on this outcome. The median frequency of detected minority variants (2%, range 1–24%), suggests an OLA or allele-specific PCR could assay for clinically relevant mutants. Although ZDV monotherapy has been abandoned, individuals who fail preexposure prophylaxis (PrEP) may harbor similarly relevant minority variants [37–40].

Our and others’ [20,30,41] findings demonstrate that past antiretroviral experience can increase the risk of failure, and thus, should be considered when choosing initial ART regimens. In such antiretroviral-experienced individuals, the use of a sensitive point-mutation or next-generation-sequencing genotypic resistance assays could also provide helpful guidance. Our finding of TAMs among minority variants associated with failure suggests that OLA (or similar point-mutation assays) require timely tailoring for the detection of prevalent disease resistance codons.

In 2017, the WHO included ART with dolutegravir (DTG) in first-line combination recommendations. Although antiretroviral-naive individuals given DTG-based ART in randomized clinical trials rarely had drug resistance at virologic failure, DTG-monotherapy selects drug resistance [42–44]. Whether DTG will suppress HIV replication in individuals with pre-ART drug-resistance or who are experiencing failure of first-line NNRTI-based ART remains uncertain. The high prevalence of the drug-resistant mutants observed at failure in our study, including M184V and TAMs, strongly suggests potential benefits from pre-ART drug-resistance testing to guide the choice of antiretroviral and prevent de facto DTG monotherapy, especially among those with sequential antiretroviral experience for PMTCT or PrEP. Additionally, past antiretroviral use may inform selection of alternate ART options for successful viral suppression.

This study has several important limitations. First, and most salient, the participants were not randomly assigned to antiretrovirals for PMTCT. Thus, risk factors other than previous antiretroviral, such as adherence and access to care may be unmeasured contributors to virologic failure, a possibility that is further suggested by the high rate of failure among antiretroviral-naive women relative to our previous study [7]. Second, small sample sizes reduced our ability to discern more nuanced associations between certain risk factors and failure; specifically, more precise comparisons of failure rates and PMTCT-associated minority variants between antiretroviral-naive, sdNVP-experienced and sdNVP and ZDV with or without 3TC-experienced women. Third, the read length of the Illumina platform necessitated sequencing two separate regions, preventing linkage analyses of resistance mutations across these two amplicons on virologic outcome. Importantly, PCR primers for next-generation sequencing were different from those used for consensus sequencing and OLA, and represent a potential source of bias across assays. Fourth, next-generation sequencing of PBMC DNA precluded the use of ‘primer ID’ methodology [45]. Thus, although the number of viral DNA templates sequenced was estimated using gag qPCR, the next-generation-sequencing mutant frequencies presented were not direct measurements from the participants’ HIV quasispecies. Similarly, the input number of viral templates into OLA, consensus sequencing, and next-generation sequencing for a given participant likely differed, and could have resulted in variable mutant genotypes and frequencies across the three assays, particularly for minority variants. Fifth, nevirapine has been replaced by efavirenz in NNRTI-based ART, and recent studies suggest that pre-ART drug resistance to multiple antiretroviral classes, but not single NNRTI mutations increase the risk of failure with EFV-based ART [46,47].

In conclusion, we examined several strategies to identify women at high risk for virologic failure of first-line NNRTI (primarily NVP)-based ART, including diagnosis of pre-ART drug resistance by multiple genotyping assays and early detection of emergent drug resistance and failure. OLA and consensus sequencing had similar sensitivity in identifying individuals with pre-ART drug resistance associated with failure. Testing for pre-ART drug-resistant minority variants by next-generation sequencing and consideration of antiretroviral histories for past use of ZDV and sdNVP for PMTCT identified additional women with failure not captured by OLA or consensus sequencing. Testing for detectable viral load soon after the initiation of ART identified women with apparent re-selection of pre-ART drug-resistant minority variants. Given the residual risk for virologic failure conferred by nonsuppressive antiretrovirals for PMTCT, our findings endorse evaluating pre-ART drug resistance in ZDV and sdNVP-experienced women of childbearing age who may be restricted from initiating DTG-based ART and treated instead with EFV-based ART [48]. In addition, it is uncertain whether DTG-based ART will suppress viral replication in those with drug resistance because of past PMTCT, failed PrEP or virologic failure during NNRTI-based ART, warranting further studies among such individuals.

Acknowledgements

The authors acknowledge the commitment of the women participants, study nurses, contributions of Steven Bii, visiting scientist Segundo Leon, and valuable advice from Joseph Fitzgibbon, PhD. Sequence analysis was performed with assistance of the Molecular Profiling and Computational Biology core of the UW CFAR (P30 AI027757).

Authorship statement: I.A.B., S.D., J.N.K., S.R.S., M.H.C., R.W.C., and L.M.F. conceived and designed the study; R.S.M, R.A.S., J.M., W.D., and T.R.S. performed data collection and analysis; R.S.M., R.A.S., and L.M.F., wrote the article. None of the authors declare a conflict of interest in this study, and all have reviewed and approved this manuscript.

Funding: This work was supported by grants from the National Institutes of Health awards (R01 AI058723, R01 AI100037 to L.M.F.), including an American Recovery and Reinvestment Act supplement (R01 AI058723). Support of the study was also provided by the Clinical Retrovirology (P30 AI027757 to R.W.C.) core of the University of Washington Center for AIDS Research. The Coptic Hope Center for Infectious Diseases is supported by the President's Emergency Plan for AIDS Relief through a cooperative agreement (U62/CCU024512 to M.H.C.) from the Centers for Disease Control and Prevention.

Conflicts of interest

There are no conflicts of interest.

These data were presented, in part, at the 2014 International Workshop on Antiviral Drug Resistance held June 3–7, 2014 in Berlin, Germany.

References

1. WHO| HIV/AIDS Data and Statistics: estimated numbers of people receiving antiretroviral therapy globally and by WHO Region and percentage coverage globally, 2000–2015. In: WHO Online; 2017. Chart of HIV-infected persons.
2. Palella FJ Jr, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. New Engl J Med 1998; 338:853–860.
3. Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, Kumarasamy N, et al. Prevention of HIV-1 infection with early antiretroviral therapy. New Engl J Med 2011; 365:493–505.
4. Panichsillapakit T, Smith DM, Wertheim JO, Richman DD, Little SJ, Mehta SR. Prevalence of transmitted HIV drug resistance among recently infected persons in San Diego, CA 1996–2013. J Acquir Immune Defic Syndr 2016; 71:228–236.
5. Gupta RK, Jordan MR, Sultan BJ, Hill A, Davis DHJ, Gregson J, et al. Global trends in antiretroviral resistance in treatment-naive individuals with HIV after rollout of antiretroviral treatment in resource-limited settings: a global collaborative study and meta-regression analysis. Lancet 2012; 380:1250–1258.
6. Chung MH, Beck IA, Dross S, Tapia K, Kiarie JN, Richardson BA, et al. Oligonucleotide ligation assay detects HIV drug resistance associated with virologic failure among antiretroviral-naive adults in Kenya. J Acquir Immune Defic Syndr 2014; 67:246–253.
7. Chung MH, Silverman R, Beck IA, Yatich N, Dross S, McKernan-Mullin J, et al. Increasing HIV-1 pretreatment drug resistance among antiretroviral-naive adults initiating treatment between 2006 and 2014 in Nairobi, Kenya. AIDS 2016; 30:1680–1682.
8. Silverman RA, Beck IA, Kiptinness C, Levine M, Milne R, McGrath CJ, et al. Prevalence of preantiretroviral-treatment drug resistance by gender, age, and other factors in HIV-infected individuals initiating therapy in Kenya, 2013–2014. J Infect Dis 2017; 216:1569–1578.
9. Hamers RL, Schuurman R, Sigaloff KCE, Wallis CL, Kityo C, Siwale M, et al. Effect of pretreatment HIV-1 drug resistance on immunological, virological, and drug-resistance outcomes of first-line antiretroviral treatment in sub-Saharan Africa: a multicentre cohort study. Lancet Infect Dis 2012; 12:307–317.
10. Lockman S, Shapiro RL, Smeaton LM, Wester C, Thior I, Stevens L, et al. Response to antiretroviral therapy after a single, peripartum dose of nevirapine. N Engl J Med 2007; 356:135–147.
11. Guay LA, Musoke P, Fleming T, Bagenda D, Allen M, Nakabiito C, et al. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial. Lancet 1999; 354:795–802.
12. Jourdain G, Ngo-Giang-Huong N, Le Coeur S, Bowonwatanuwong C, Kantipong P, Leechanachai P, et al. Intrapartum exposure to nevirapine and subsequent maternal responses to nevirapine-based antiretroviral therapy. New Engl J Med 2004; 351:229–240.
13. Lallemant M, Jourdain G, Le Coeur S, Mary JY, Ngo-Giang-Huong N, Koetsawang S, et al. Single-dose perinatal nevirapine plus standard zidovudine to prevent mother-to-child transmission of HIV-1 in Thailand. N Engl J Med 2004; 351:217–228.
14. McIntyre JA, Hopley M, Moodley D, Eklund M, Gray GE, Hall DB, et al. Efficacy of short-course AZT plus 3TC to reduce nevirapine resistance in the prevention of mother-to-child HIV transmission: a randomized clinical trial. PLoS Med 2009; 6:e1000172.
15. Micek MA, Blanco AJ, Carlsson J, Beck IA, Dross S, Matunha L, et al. Effects of short-course zidovudine on the selection of nevirapine-resistant HIV-1 in women taking single-dose nevirapine. J Infect Dis 2012; 205:1811–1815.
16. Fowler MG, Qin M, Fiscus SA, Currier JS, Flynn PM, Chipato T, et al. Mofenson LM; IMPAACT 1077BF/1077FF PROMISE Study Team. Benefits and risks of antiretroviral therapy for perinatal HIV prevention. New Engl J Med 2016; 375:1726–1737.
17. Hauser A, Sewangi J, Mbezi P, Dugange F, Lau I, Ziske J, et al. Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission. PloS One 2012; 7:e32055.
18. Melikian GL, Rhee SY, Varghese V, Porter D, White K, Taylor J, et al. Nonnucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance: implications for preclinical evaluation of novel NNRTIs and clinical genotypic resistance testing. J Antimicrob Chemother 2014; 69:12–20.
19. World Health Organization. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Geneva: WHO; 2017.
20. Jourdain G, Wagner TA, Ngo-Giang-Huong N, Sirirungsi W, Klinbuayaem V, Fregonese F, et al. Association between detection of HIV-1 DNA resistance mutations by a sensitive assay at initiation of antiretroviral therapy and virologic failure. Clin Infect Dis 2010; 50:1397–1404.
21. Chung MH, Drake AL, Richardson BA, Reddy A, Thiga J, Sakr SR, et al. Impact of prior HAART use on clinical outcomes in a large Kenyan HIV treatment program. Curr HIV Res 2009; 7:441–446.
22. Beck IA, Deng W, Payant R, Hall R, Bumgarner RE, Mullins JI, et al. Validation of an oligonucleotide ligation assay for quantification of human immunodeficiency virus type 1 drug-resistant mutants by use of massively parallel sequencing. J Clin Microbiol 2014; 52:2320–2327.
23. Gantt S, Shetty AK, Seidel KD, Matasa K, Musingwini G, Woelk G, et al. Laboratory indicators of mastitis are not associated with elevated HIV-1 DNA loads or predictive of HIV-1 RNA loads in breast milk. J Infect Dis 2007; 196:570–576.
24. Liu TF, Shafer RW. Web resources for HIV type 1 genotypic-resistance test interpretation. Clin Infect Dis 2006; 42:1608–1618.
25. Wagner TA, Kress CM, Beck I, Techapornroong M, Wittayapraparat P, Tansuphasawasdikul S, et al. 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. J Clin Microbiol 2010; 48:1555–1561.
26. Coovadia A, Hunt G, Abrams EJ, Sherman G, Meyers T, Barry G, et al. Persistent minority K103N mutations among women exposed to single-dose nevirapine and virologic response to nonnucleoside reverse-transcriptase inhibitor–based therapy. Clin Infect Dis 2009; 48:462–472.
27. Eshleman SH, Mracna M, Guay LA, Deseyve M, Cunningham S, Mirochnick M, et al. Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012). AIDS 2001; 15:1951–1957.
28. Flys T, Nissley DV, Claasen CW, Jones D, Shi C, Guay LA, et al. Sensitive drug-resistance assays reveal long-term persistence of HIV-1 variants with the K103N nevirapine (NVP) resistance mutation in some women and infants after the administration of single-dose NVP: HIVNET 012. J Infect Dis 2005; 192:24–29.
29. Harrigan PR, Hogg RS, Dong WWY, Yip B, Wynhoven B, Woodward J, et al. Predictors of HIV drug-resistance mutations in a large antiretroviral-naive cohort initiating triple antiretroviral therapy. J Infect Dis 2005; 191:339–347.
30. Boltz VF, Zheng Y, Lockman S, Hong F, Halvas EK, McIntyre J, et al. Role of low-frequency HIV-1 variants in failure of nevirapine-containing antiviral therapy in women previously exposed to single-dose nevirapine. Proc Natl Acad Sci U S A 2011; 108:9202–9207.
31. Rowley CF, Boutwell CL, Lee EJ, MacLeod IJ, Ribaudo HJ, Essex M, et al. Ultrasensitive detection of minor drug-resistant variants for HIV after nevirapine exposure using allele-specific PCR: clinical significance. AIDS Res Hum Retroviruses 2010; 26:293–300.
32. MacLeod IJ, Rowley CF, Thior I, Wester C, Makhema J, Essex M, et al. Minor resistant variants in nevirapine-exposed infants may predict virologic failure on nevirapine-containing ART. J Clin Virol 2010; 48:162–167.
33. Boltz VF, Bao Y, Lockman S, Halvas EK, Kearney MF, McIntyre JA, et al. Low-frequency nevirapine (NVP)-resistant HIV-1 variants are not associated with failure of antiretroviral therapy in women without prior exposure to single-dose NVP. J Infect Dis 2014; 209:703–710.
34. Metzner KJ, Rauch P, Braun P, Knechten H, Ehret R, Korn K, et al. Prevalence of key resistance mutations K65R, K103N, and M184V as minority HIV-1 variants in chronically HIV-1 infected, treatment-naive patients. J Clin Virol 2011; 50:156–161.
35. Li JZ, Paredes R, Ribaudo HJ, Svarovskaia ES, Metzner KJ, Kozal MJ, et al. Low-frequency HIV-1 drug resistance mutations and risk of NNRTI-based antiretroviral treatment failure: a systematic review and pooled analysis. JAM#A 2011; 305:1327–1335.
36. Johnson JA, Li J-F, Wei X, Lipscomb J, Irlbeck D, Craig C, et al. Minority HIV-1 drug resistance mutations are present in antiretroviral treatment–naïve populations and associate with reduced treatment efficacy. PLoS Med 2008; 5:e158.
37. Lehman DA, Baeten JM, McCoy CO, Weis JF, Peterson D, Mbara G, et al. Partners PrEP Study Team. Risk of drug resistance among persons acquiring HIV within a randomized clinical trial of single-or dual-agent preexposure prophylaxis. J Infect Dis 2015; 211:1211–1218.
38. Grant RM, Liegler T, Defechereux P, Kashuba ADM, Taylor D, Abdel-Mohsen M, et al. Drug resistance and plasma viral RNA level after ineffective use of oral preexposure prophylaxis in women. AIDS 2015; 29:331–337.
39. Hurt CB, Eron JJ Jr, Cohen MS. Preexposure prophylaxis and antiretroviral resistance: HIV prevention at a cost?. Clin Infect Dis 2011; 53:1265–1270.
40. Sivay MV, Li M, Piwowar-Manning E, Zhang Y, Hudelson SE, Marzinke MA, et al. HPTN 067/ADAPT Study Team. Characterization of HIV seroconverters in a TDF/FTC PrEP study: HPTN 067/ADAPT. J Acquir Immune Defic Syndr 2017; 75:271–279.
41. Huntington S, Thorne C, Newell M, Anderson J, Taylor G, Pillay D, et al. Outcomes after 2 years on antiretroviral therapy (ART): comparison of women who were ART-naive and women who previously used short-course ART in pregnancy. 15th European AIDS Conference. Barcelona; 2015.
42. Brenner BG, Thomas R, Blanco JL, Ibanescu R-I, Oliveira M, Mesplede T, et al. Development of a G118R mutation in HIV-1 integrase following a switch to dolutegravir monotherapy leading to cross-resistance to integrase inhibitors. J Antimicrob Chemother 2016; 71:1948–1953.
43. Wijting I, Rokx C, Boucher C, van Kampen J, Pas S, de Vries-Sluijs T, et al. Dolutegravir as maintenance monotherapy for HIV (DOMONO): a phase 2, randomised noninferiority trial. Lancet HIV 2017; 4:e547–e554.
44. Trevillyan JM, Hoy JF. Dolutegravir monotherapy as maintenance ART bites the dust. Lancet HIV 2017; 4:e531–e532.
45. Jabara CB, Jones CD, Roach J, Anderson JA, Swanstrom R. Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc Natl Acad Sci U S A 2011; 108:20166–20171.
46. Beck IA, Levine M, Milne R, So I, Andersen N, Watling M, et al. Impact of pretreatment HIV-drug resistance on virologic outcome of first-line ART. [Abstract 490]24th Conference on retroviruses and opportunistic infections (CROI) 13-16 February. 2017.
47. Derache A, Iwuji CC, Baisley K, Danaviah S, Marcelin AG, Calvez V, et al. Impact of next generation sequencing defined HIV pretreatment drug resistance on virological outcomes in the ANRS 12249 treatment as prevention trial. LID - 10.1093/cid/ciy881 (doi). [1537-6591 (Electronic)].
48. WHO. Updated recommendations on first-line and second-line antiretroviral regimens and post-exposure prophylaxis and recommendations on early infant diagnosis of HIV. Geneva: WHO; 2018.
Keywords:

antiretroviral therapy; HIV drug resistance; nevirapine; prevention of mother-to-child transmission; quasispecies; women

Supplemental Digital Content

Copyright © 2019 Wolters Kluwer Health, Inc.