Postpartum antiretroviral drug resistance in HIV-1-infected women receiving pregnancy-limited antiretroviral therapy
Paredes, Rogera,b; Cheng, Irenec; Kuritzkes, Daniel Ra; Tuomala, Ruth Ea; for the Women and Infants Transmission Study (WITS) Group
aBrigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
bFundacions irsiCaixa i Lluita contra la SIDA, Universitat Autònoma de Barcelona, Catalonia, Spain
cClinical Trials & Surveys Corp., Baltimore, Maryland, USA.
Received 13 October, 2008
Revised 18 May, 2009
Accepted 18 May, 2009
Correspondence to Dr Ruth E. Tuomala, MD, Division of Maternal-Fetal Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, CWN-3, Boston, MA 02115, USA. E-mail: email@example.com
Background: Pregnancy-limited antiretroviral therapy (PLAT) drastically reduces HIV-1 transmission to the newborn, but may select for antiretroviral drug resistance mutations in mothers.
Methods: We evaluated antiretroviral-naive, HIV-1-infected pregnant women who received PLAT between 1998 and 2005, and had 2-month or 6-month postpartum plasma samples available with HIV-1 RNA levels more than 500 copies/ml. Postpartum drug resistance mutation rates were assessed blindly using population sequencing and allele-specific PCR (ASPCR) of the M184V, K103N and D30N mutations. Factors associated with selection of drug resistance mutations were investigated.
Results: One hundred and forty-six women were included. All women received zidovudine and lamivudine during pregnancy; 76% also received nelfinavir and 8.2% nevirapine. Resistance data were available from 114 women (78%). Postpartum rates of single-class, dual-class, and triple-class resistance were, respectively, 43, 6.1 and 0% (63.2, 10.5 and 1.7% by ASPCR). In women receiving dual or triple PLAT, respectively, postpartum M184V/I rates were 65% (95% by ASPCR) and 28.7% (51.6% by ASPCR), respectively (P < 0.01). Postpartum nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance rates among women receiving nevirapine were 25% for K103N (37.5% by ASPCR) and 12.5% for Y188C. Protease inhibitor resistance rates in women receiving nelfinavir were 1.1% for D30N (1.1% by ASPCR) and 1.1% for L90M. Dual versus triple PLAT and prolonged zidovudine exposure were associated with selection of M184V. Nevirapine use and length of zidovudine and lamivudine exposure were associated with selection of K103N.
Conclusion: PLAT is associated with frequent selection of resistance to drugs with low-genetic barrier. Triple-drug PLAT decreases the odds for M184V selection. Routine postpartum genotypic resistance testing may be useful to guide future treatment decisions in mothers.
Programs to prevent mother-to-child HIV-1 transmission (MTCT) have averted thousands of new HIV-1 infections worldwide. Present rates of vertical transmission in women with sustained access to triple-drug antiretroviral therapy (ART), perinatal zidovudine treatment, elective C-sections, and alternatives to breast-feeding are extremely low [1–5]. Even a simplified approach based on the intrapartum and neonatal administration of a single dose of nevirapine with or without zidovudine, lamivudine or tenofovir has reduced MTCT in resource-poor areas [6,7]. In spite of these successes, preexistence of transmitted drug-resistant viruses [8,9], or selection of antiretroviral drug resistance viruses during MTCT prophylaxis may hinder the long-term efficacy of MTCT preventive programs and severely constrain future treatment options for both mother and child [7,10–15].
A suitable way of investigating how often antiretroviral MTCT prophylaxis selects for drug-resistant HIV-1 in mothers is to assess the postpartum rates of resistant viruses in antiretroviral-naive pregnant women who receive pregnancy-limited antiretroviral therapy (PLAT). Selection of resistant viruses during PLAT, even as minority variants, could impair the virological outcome when women subsequently initiate long-term ART [16,17], particularly if treatment is started within 6–12 months after delivery [18,19].
The present study sought to investigate postpartum drug resistance in antiretroviral-naive women who received PLAT in the United States between 1998 and 2005, using population-based sequencing of plasma virus and allele-specific PCR (ASPCR) testing for the M184V, K103N and D30N mutations, associated with high-level resistance to lamivudine and emtricitabine, nevirapine and efavirenz, and nelfinavir, respectively. We also examined factors associated with an increased risk for selecting resistant viruses during pregnancy.
The study was a substudy utilizing specimens obtained from participants in the Women and Infants Transmission Study (WITS) . The WITS is a multisite observational study designed to examine the impact of HIV infection on HIV-infected women and their infants. WITS sites are located in Brooklyn, New York; New York City, New York; Boston and Worcester, Massachusetts; Houston, Texas; Chicago, Illinois; and Puerto Rico.
Women enrolled in WITS between June 1, 1998 and December 31, 2004, were evaluated for eligibility for this study. Study participants included HIV-1-infected pregnant women who had never received ART before pregnancy, initiated PLAT consisting of zidovudine and lamivudine either solely or in addition to either nevirapine or nelfinavir, stopped therapy postpartum, and had specimens available from 2-month or 6-month postpartum visits during which the presence of plasma virus by RNA-PCR testing had previously been documented. A single laboratory, blinded as to PLAT received by participants, analyzed all specimens. Specimens collected within the first 14 days of ART were also analyzed posthoc, when available. However, as most PLAT initiation occurred prior to WITS enrollment, these specimens were lacking for the majority of women.
Detection of resistance mutations
Population sequencing of plasma viruses
HIV-1 RNA was extracted from 500 μl EDTA-anticoagulated plasma using the QIAamp Viral RNA MiniKit (QIAGEN Sciences, Germantown, Maryland, USA) after centrifugation at 24 000g for 1 h at 4°C. Part of each RNA sample was used for cDNA synthesis immediately after extraction, and the remainder was stored at −80°C.
The extracted RNA was transcribed to cDNA and amplified by PCR in a one-step process (Superscript III One-step RT-PCR with Platinum Taq Kit; Invitrogen Corp., Carlsbad, California, USA) following the manufacturer's instructions. Cycling conditions included an initial cDNA synthesis step at 55°C for 25 min followed by a denaturation step at 94°C for 2 min; 30 cycles of PCR amplification (94°C for 40 s, 60°C for 40 s, 68°C for 1 min and 20 s); and a final 5 min extension step at 68°C. The PCR mix contained 25 μl of 2× Reaction Mix (including 0.4 mmol/l of each dNTP and 3.2 mmol/l of MgCl2), 0.2 mmol/l of each primer OOPF (HXB2: 2211–2232) [5′-GAAGCAGGAGCCGATAGACAAG-3′] and OOR2 (HXB2: 3466-3444) [5′-TTTTCTGCCAGTTCTAGCTCTGC-3′], 15 μl of extracted RNA as template and nuclease-free H2O to a final volume of 50 μl.
The resulting PCR product was purified using the QIAquick PCR Purification Kit (QIAquick PCR Purification Kit; QIAGEN Sciences) and used as the starting template for a 30-cycle nested PCR amplification (High Fidelity Platinum Taq; Invitrogen Corp.) using primers OOPF2 (HXB2: 2218–2241) [5′-GAGCCGATAGACAAGGAACTGTAT-3′] and OOR3 (HXB2: 3457–3432) [5′-AGTTCTAGCTCTGCTTCTTCAGTTAG-3′].
The nested PCR product was purified and sequenced (3730XL DNA Analyzer; Applied Biosystems, Foster City, California, USA). Resistance mutations and polymorphisms were defined according to the International AIDS-Society-USA Panel (Spring 2008 Update) . Standard phylogenetic analyses ruled out sequence contamination.
To quantify the proportion of mutant sequences contained within each specimen, 5 μl of reverse transcriptase-PCR product were added to the real-time PCR together with selective or nonselective primers. When the initial DNA copy number was lower than 106, the nested DNA product was used. PCR conditions have been previously published [8,22].
Mutant-specific primers [(HXB2: 2319–2340] 5′-CTATTAGATACAGGAGCAAATA-3′ for D30N; (HXB2: 3078–3098) 5′- GACATAGTTATCTATCAATICG-3′ for M184V; (HXB22884–2858) 5′-CCCACATCCAGTACTGTTACTGATTGG-3′ for the K103N AAC allele; and (HXB22884–2858) 5′-CCACATCCAGTACTGTTACTGATTCA-3′ for the K103N AAT allele] included the target codon in its 3′ end and an intentional mismatch at positions −3 or −2, relative to the 3′ end. Nonspecific primers [(HXB2: 2319–2339) 5′-CTATTAGATACAGGAGCAGAT-3′ for D30N; (HXB2: 3078–3098) 5-GACATAGTTATCTATCAATAC-3′ for M184V; and (HXB22884–2859) 5′-CCCACATCCAGTACTGTTACTGATTT-3 for both AAC and AAT K103N alleles] were similar to the former, but ended just before the target base pair and did not include intentional mismatches. The antiparallel primer [(HXB2: 2592–2571) 5′-CTGGCTTTAATTTTACTGGTAC-3′ for D30N; (HXB2: 3277–3258) 5′-GGCTGTACTGTCCATTTATC-3′ for M184V; and (HXB22757–2785) 5′-AAATGGAGAAAATTAGTAGATTTCAGAGA-3′ for both AAC and AAT K103N alleles] was common for each pair of mutant-specific and nonspecific reactions.
The different ASPCR assays were performed separately. Within each ASPCR assay, nonselective and selective amplifications were always performed in parallel. All reactions were performed in duplicate, and the mean of the two values was used for calculation. The percentage of viral sequences containing each mutation was calculated as follows: percentage mutant sequences = [(quantity of mutant sequences in the sample)/(quantity of total viral sequences in the sample)] × 100.
In addition to the sensitivity threshold for each ASPCR assay, we calculated a specific detection threshold per each sample, defined as the minimum proportion of variants that could be detected based on the specimen's HIV-1 RNA level (pVL), the volume of plasma used in the RNA extraction (V), the fraction of the RNA elution volume used for cDNA synthesis (fe), and the assumed efficiencies of the RNA extraction (ERNAX) and cDNA synthesis (EcDNA). The sample-specific detection threshold was calculated as 1/NRNA, where NRNA was the number of viral RNA copies that were effectively sampled after RNA extraction and reverse transcription. NRNA was calculated as: NRNA = pVL × V × fe × ERNAX × EcDNA. We assumed an ERNAX of 0.96 and an EcDNA of 0.7 [23,24]. ASPCR values between the ASPCR assay sensitivity threshold and the sample-specific detection threshold were considered undetectable.
Subjects' characteristics and postpartum rates of resistance mutations were described using standard descriptive methods. 95% Confidence intervals (CIs) for resistance mutation rates were generated using the Exact Binominal Test. Variables associated with the selection of M184V, K103N and D30N mutations were investigated using chi-squared, Fisher's exact test or F-test, as needed. Odds of developing M184V or K103N were evaluated using the Generalized Estimating Equations model (SAS procedure GENMOD).
Alpha was set at 0.05 for determining statistical significance in all univariate and multivariate analyses. For M184V and K103N mutations, the multivariate analysis models were built using forward selection technique. The multivariate analysis model only included characteristics that achieved statistical significance in the univariate analysis. All analyses were intention-to-treat.
Performance of the allele-specific PCR assays
Using the mean plus 3 standard deviations of 20 negative control repeats (wild-type laboratory viral constructs), the detection threshold of the ASPCR was calculated at: 0.1, 0.4 and 0.003% for the D30N, M184V and K103N ASPCR assays, respectively. Delta Cts between mutant and wild-type DNA equivalents were more than 10 cycles for the D30N and M184V assays and more than 17 cycles for the K103N assays. Proportion measurements were linear down to at least 1% for the D30N and M184V assays and to 0.01% for the K103N ASPCR assays [17,24].
One thousand, three hundred and twenty-eight women were enrolled in the WITS Study between 1 June 1998 and 31 December 2004; 636 of them were ART-naive prior to current pregnancy; 315 women started and received one of the three targeted regimens (zidovudine and lamivudine; zidovudine and lamivudine and nevirapine; or zidovudine and lamivudine and nelfinavir) during more than 28 days during the index pregnancy. Of them, 146 women stopped therapy postpartum and had plasma specimens available from 2-month or 6-month postpartum visits with HIV-1 RNA levels at least 500 copies/ml, thereby being included in the present study.
The mean age of the 146 women fulfilling the inclusion criteria was 27 years, most were African-American or Hispanic, the majority were CDC disease category A, and almost 30% had used hard drugs and/or had a known history of alcohol use prior to delivery (Table 1). The median gestational age was 29.3 years [interquartile range (IQR: 22.4–36.3 years)]. The median first CD4+ cell count available during pregnancy was 455 cells/μl (IQR: 316–690 cells/μl); being at least 200 cells/μl in more than 90% of women. The median time of blood sample collection was 2.2 months postpartum (IQR: 1.9–4.6 months), with 111 samples (76%) collected at the 2-month postpartum visit [median 2.0 months; (IQR: 1.8–2.5 months)]; the remaining 35 samples (24%) were collected at the 6-month postpartum visit [median 6.2 months (IQR: 5.9–6.9 months)]. Median postpartum CD4+ cell counts were 575 cells/μl (IQR: 397–767), and median postpartum HIV-1 RNA levels were 4780 copies/ml (IQR: 1352–18121 copies/ml). Seventy women (48%) maintained HIV-1 RNA levels less than 400 copies/ml at all timepoints during PLAT after their enrollment in WITS; 40 (27%) had all HIV-1 RNA levels more than 400 copies/ml during PLAT, and 33 (23%) alternated viremic with aviremic periods during PLAT. All women initiated PLAT including zidovudine and lamivudine; 93 (64%) also started nelfinavir and 10 (7%) nevirapine. Some women switched between dual- and triple-PLAT during pregnancy. Of the 146 women, 18 (12%) switched from dual-PLAT to triple-PLAT (1 added nevirapine and 17 added nelfinavir). Another two women switched between triple-PLAT with nevirapine and triple-PLAT with nelfinavir. The duration of exposure to zidovudine, lamivudine, nelfinavir and nevirapine are shown in Table 1.
Postpartum rates of antiretroviral resistance
Postpartum resistance data were available from 114 women (78%). The virological, immunological and clinical characteristics of this subset of women did not differ from those fulfilling the study inclusion criteria. Pretreatment resistance data was available from 25 of these 114 women (22%). Twenty women (18%) remained on dual-PLAT through delivery whereas 94 (82%) received triple-PLAT during pregnancy.
Overall, 49 women (43%) had at least one resistance mutation detected by the population-based sequencing analysis postpartum; seven women (6.1%) had dual-class resistance after delivery. Three (2.6%) had resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors, three (2.6%) had resistance to NRTIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs) and one (0.9%) had resistance to NNRTIs and protease inhibitors. When including the results from the ASPCR analysis for D30N, M184V and K103N mutations, 72 women (63.2%) had at least one resistance mutation detected postpartum, 12 women (10.5%) had dual-class resistance, and 2 (1.7%) women had triple-class resistance after delivery. Five women with dual-class resistance (4.4%) had resistance to NRTIs and protease inhibitors, whereas 7 (6.1%) had resistance to NRTIs and NNRTIs.
Nucleoside reverse transcriptase inhibitor resistance
Using population sequencing of plasma viruses, the M184V/I mutation was detected postpartum in 65% of women receiving dual PLAT throughout pregnancy compared with 28.7% of women treated with three drugs (P = 0.004) (Table 2). Using ASPCR, this mutation was detected in 95% of women who had received dual PLAT and in 51.6% of those having received triple-drug PLAT (P < 0.001). Using ASPCR, the M184V mutation was detected postpartum in two of five (40%) women with M184V present by population sequencing at baseline and in 15 of 20 (75%) women who did not have this mutation before treatment.
Mutations associated with resistance to nucleoside analogues (NAMs) were also more frequent among women exposed only to dual-drug PLAT (M41L, 5%; D67N, 5%; K70R, 10; and T215Y, 5%) than in those treated with three drugs (M41L, 1.1%; D67N, 1.1%; K70R, 1.1%; L210F, 1.1%; K219Q, 1.1%). Such differences were, at most, marginally significant.
Nonnucleoside reverse transcriptase inhibitor resistance
Using population sequencing of plasma viruses, the postpartum rates of NNRTI resistance among the eight women receiving nevirapine were: 25% [95% CI: 3.2–65.1%] for K103N (two cases), and 12.5% [95% CI: 0.3–52.7%] for Y188C (one case). Using ASPCR, the K103N mutation was detected in three out of eight women exposed to nevirapine (37.5% [95 CI: 8.5–75.5%]), including the two cases detected through population sequencing, and in eight out of 106 women not exposed to nevirapine (7.5%) (P = 0.029). Using ASPCR, the K103N mutation was detected postpartum in one woman harboring this mutation before treatment and in two of 24 (8.3%) women who did not have this mutation before starting PLAT.
Protease inhibitor resistance
Using population sequencing, each of the D30N and L90M mutations were detected in one out of 87 women receiving nelfinavir during PLAT [1.1% (95% CI: 0.03–6.24%) for each mutation]. The ASPCR testing confirmed the detection of the D30N mutation in one out of 87 women exposed to nelfinavir [1.1% (95% CI: 0.03–6.24%)] and allowed detection of this mutation in two women out of 27 (7.4%) who did not receive nelfinavir during PLAT. One D30N mutation detected at baseline was not found postpartum.
Factors associated with selection of postpartum antiretroviral resistance
In the univariate analysis (Table 3), postpartum detection of the M184V mutation was more likely in women who had all HIV-1 RNA levels more than 400 copies/ml during PLAT [odds ratio (OR) = 2.65, 95% CI: 1.02–6.87, P = 0.05] relative to those who remaining aviremic through delivery, and in those with longer exposure to zidovudine (OR = 1.25, 95% CI: 1.01–1.54, P = 0.04, per each additional month). The M184V mutation, conversely, was less likely to be observed in women who received triple-drug PLAT including either nelfinavir or nevirapine (OR = 0.06, 95% CI: 0.007–0.44, P = 0.006), relative to dual therapy only; in those treated with triple-drug PLAT including nelfinavir (OR = 0.06, 95% CI: 0.008–0.46, P = 0.007) or nevirapine (OR = 0.04, 95% CI: 0.003–0.48, P = 0.01), relative to dual therapy alone; and in those treated with triple-drug PLAT including nelfinavir, relative to any other regimen (OR = 0.25, 95% CI: 1.01–1.54, P = 0.04). Univariate factors associated with postpartum detection of K103N were nevirapine exposure (OR = 7.35, 95% CI: 1.48–36.5, P = 0.01), and longer exposure to zidovudine (OR = 1.40, 95% CI: 1.02–1.91, P = 0.04) or to the combination of zidovudine and lamivudine (OR = 1.42, 95% CI: 1.04–1.94, P = 0.03).
In the multivariate analysis (Table 4), the variables significantly associated with selection of M184V were exposure to dual versus triple-drug PLAT (OR = 19.64, 95% CI: 2.47–156.25, P < 0.01), and duration of zidovudine exposure (OR = 1.29, 95% CI: 1.03–1.63, P = 0.03, per additional month). Variables associated with selection of K103N were nevirapine use during pregnancy (OR = 9.75, 95% CI: 1.62–58.84, P = 0.01) and length of dual-PLAT zidovudine + lamivudine exposure (OR = 1.46, 95% CI: 1.05–2.02, P = 0.02, per additional month).
Of note, other factors such as presence of resistance mutations before starting PLAT, the first available CD4+ cell count during pregnancy, use of hard drugs before delivery, alcohol use before delivery, ethnicity, or age at delivery, were not associated with the postpartum detection of the M184V or K103N mutations.
This study showed high postpartum antiretroviral resistance rates in antiretroviral-naive women receiving PLAT. The main determinants of resistance selection during pregnancy were the characteristics of the antiretroviral regimen chosen to prevent MTCT.
Virtually all women receiving dual therapy developed the M184V mutation. This mutation confers high-level resistance to lamivudine and emtricitabine [25,26] and is associated with an increased risk of virological failure of treatment combinations including these drugs . Thymidine analogue resistance mutations were detected in few women but were also more frequent in those receiving dual therapy. A high proportion of women receiving triple-drug therapy also selected resistance mutations during pregnancy. Based on allele-specific PCR testing, 50% of women treated with three drugs developed the M184V mutation. Moreover, although few women received nevirapine in this study, almost 40% of these women had nonnucleoside analogue (NNRTI) resistance mutations detected at the postpartum visit. On the contrary, selection of protease inhibitor resistance was rare in women treated with nelfinavir.
Use of dual therapy and duration of zidovudine exposure, which reflects the overall duration of ART, were the only two variables that were independently associated with an increased risk of M184V detection after delivery. Similarly, the postpartum detection of the K103N mutation was independently associated with exposure to nevirapine and duration of exposure to zidovudine and lamivudine. These findings strongly argue against using dual therapy to prevent MTCT whenever triple therapy is available.
The fact that resistance mutations were so frequently detected among women receiving triple therapy contrasts with previous estimates from Latin America and Caribbean countries , and suggests that PLAT was less effective than expected at continuously suppressing HIV-1 RNA levels. In the univariate risk factor analysis, women who had all HIV-1 RNA levels above 400 copies/ml during the study were 2.7 times more likely to have the M184V mutation detected postpartum than those remaining aviremic through delivery. Similarly, individuals receiving nelfinavir were four times less likely to develop postpartum M184V than those not receiving this drug. These findings suggest that women treated with drugs with high genetic barrier to attain resistance are less likely to develop the M184V mutation. None of these variables, however, remained independently associated with risk of postpartum resistance in the multivariate analyses.
The preexistence of primary resistance mutations could also have explained the high frequency of postpartum resistance found in this study. The prevalence of primary resistance has increased during the last decade in pregnant women in the United States . Using allele-specific PCR in women enrolled in WITS with similar characteristics to those included in this study, we previously reported a 9.4% prevalence of primary lamivudine and emtricitabine resistance, and a 6.3% prevalence of nelfinavir resistance between 1998 and 2004 . As most women included in this analysis started ART before WITS enrolment, pretreatment resistance data was only available from one-third of women. Based on this limited number of subjects, we did not observe an association between preexisting resistance and the postpartum selection of M184V or K103N.
We did not evaluate treatment adherence; therefore, we cannot rule out an association between suboptimal adherence and the observed rates of postpartum resistance. Ethnicity and hard drug or alcohol consumption have been previously associated with lower adherence to ART and worse virological outcomes [29–31]; however, these factors were not associated with postpartum resistance in this study.
Finally, altered drug pharmacokinetics due to physiological changes occurring in women during pregnancy could have favored the existence of suboptimal drug levels during pregnancy or prolonged drug elimination in the postpartum period. Exposure to most protease inhibitors, including nelfinavir, is reduced in HIV-1-infected women during pregnancy due to increased intestinal and/or hepatic CYP3A activity [32–36]. Pregnant women, as well, have increased nevirapine clearance and lower plasma concentrations than nonpregnant women, although plasma levels are largely influenced by body weight . Chaix et al.  found that selection of nevirapine-resistance strongly correlated with higher median nevirapine plasma concentration. Prolonged nevirapine elimination after delivery in subjects with higher plasma levels could have allowed viral replication in the presence of suboptimal nevirapine levels after delivery.
In concordance with previous studies [8,17,39], allele-specific PCR increased the frequency of detection of key resistance mutations relative to population sequencing of plasma viruses. Several studies have shown that pretreatment detection of minority NNRTI-resistant variants more than triples the risk of virological failure to subsequent NNRTI-based therapy [17,40]. Therefore, the results of this study have important clinical implications for women receiving PLAT during pregnancy. It is well established that women selecting lamivudine, emtricitabine or NNRTI-resistant mutants during PLAT are at a higher risk of failing subsequent NNRTI-based ART, particularly if treatment is started within 6 to 12 months after delivery [18,19].
Whereas ASPCR is several orders of magnitude more sensitive than viral population sequencing, it has a number of limitations that currently prevent its routine clinical application; that is ASPCR interrogates only one codon per experiment, proportion measurements might be biased in the presence of polymorphisms at primer sites, and minority variant cut-offs that predict ART outcomes with high sensitivity and specificity have not been established.
In spite of the limited numbers in some of the categories assessed and the limited pretreatment resistance data available, our findings suggest that triple-drug therapy should be the preferred MTCT prevention approach to preserve future treatment options for mothers. When possible, antiretroviral regimens to prevent MTCT should include drugs with high genetic barrier. Nelfinavir-based therapy is no longer a preferred regimen for MTCT , but the findings of this study likely apply to other protease inhibitors . In women treated with nevirapine-based regimens the optimal timing of nevirapine interruption and length of continuation of other concomitant agents merits further inquiry to avoid active viral replication in the presence of suboptimal nevirapine levels. All efforts should be undertaken to ensure optimal adherence to ART during pregnancy. Lastly, given that resistance mutations selected during pregnancy will wane after PLAT interruption, performing postpartum genotypic resistance testing within 1 to 2 months after delivery would be highly informative for designing future treatment regimens for women exposed to PLAT and may be useful in guiding the choice of antiretroviral regimen postpartum.
R.P. performed the population and allele-specific resistance testing, drafted the initial manuscript and participated in its discussion and review. I.C. performed the statistical analyses and participated in drafting, discussing and reviewing the manuscript. D.R.K. and R.E.T. conceived and coordinated the study; reviewed the study results and drafted, discussed and reviewed the manuscript. The Women and Infant's Transmission Study group contributed to the study design and logistics and reviewed, discussed and helped in drafting the manuscript.
This work was supported in part by the following US Public Health Service grants from the National Institutes of Health: K24 RR16482, a Virology Support Laboratory contract from the Adult ACTG (U01 AI-38858), and the Harvard Medical School Center for AIDS Research Virology Core (P30 AI60354). R.P. is a recipient of the ‘La Caixa’ Fellowship Grant for Post-Graduate Studies, Caixa d'Estalvis i Pensions de Barcelona, Catalonia, Spain. The Women and Infants Transmission Study Principal investigators, study coordinators, program officers and funding include: Clemente Diaz, Edna Pacheco-Acosta (University of Puerto Rico, San Juan, PR; U01 AI 034858); Ruth Tuomala, Ellen Cooper, Donna Mesthene (Boston/Worcester Site, Boston, MA; 9U01 DA 015054); Phil LaRussa, Alice Higgins (Columbia Presbyterian Hospital, New York, NY; U01 DA 015053); Sheldon Landesman, Herman Mendez, Ava Dennie (State University of New York, Brooklyn, NY; U01 HD 036117); Kenneth Rich, Delmyra Turpin (University of Illinois at Chicago, Chicago, IL; U01 AI 034841); William Shearer, Norma Cooper (Baylor College of Medicine, Houston, TX; U01 HD 041983); Joana Rosario (National Institute of Allergy and Infectious Diseases, Bethesda, MD); Kevin Ryan, (National Institute of Child Health and Human Development, Bethesda, MD); Vincent Smeriglio, Katherine Davenny (National Institute on Drug Abuse, Bethesda, MD); and Bruce Thompson (Clinical Trials & Surveys Corp., Baltimore, MD, N01 AI 085339). The scientific leadership core include: Kenneth Rich (PI), Delmyra Turpin (Study Coordinator) (1 U01 AI 050274-01). Additional support was provided by local Clinical Research Centers as follows: Baylor College of Medicine, Houston,TX; NIH GCRC RR000188; Columbia University, New York, NY; NIH GCRC RR000645.
This work was partially presented at the 15th Conference on Retroviruses and Opportunistic Infections, Boston, Massachusetts, 2008.
1. Townsend CL, Cortina-Borja M, Peckham CS, de Ruiter A, Lyall H, Tookey PA. Low rates of mother-to-child transmission of HIV following effective pregnancy interventions in the United Kingdom and Ireland, 2000–2006. AIDS 2008; 22:973–981.
2. Horvath T, Madi BC, Iuppa IM, Kennedy GE, Rutherford G, Read JS. Interventions for preventing late postnatal mother-to-child transmission of HIV. Cochrane Database Syst Rev
3. Suksomboon N, Poolsup N, Ket-Aim S. Systematic review of the efficacy of antiretroviral therapies for reducing the risk of mother-to-child transmission of HIV infection. J Clin Pharm Ther 2007; 32:293–311.
4. Volmink J, Siegfried NL, van der Merwe L, Brocklehurst P. Antiretrovirals for reducing the risk of mother-to-child transmission of HIV infection. Cochrane Database Syst Rev
5. Read JS, Newell MK. Efficacy and safety of cesarean delivery for prevention of mother-to-child transmission of HIV-1. Cochrane Database Syst Rev
6. 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.
7. Arrive E, Newell ML, Ekouevi DK, Chaix ML, Thiebaut R, Masquelier B, et al
. Prevalence of resistance to nevirapine in mothers and children after single-dose exposure to prevent vertical transmission of HIV-1: a meta-analysis. Int J Epidemiol 2007; 36:1009–1021.
8. Paredes R, Cheng I, Kuritzkes DR, Tuomala RE. High prevalence of primary lamivudine and nelfinavir resistance in HIV-1-infected pregnant women in the United States, 1998–2004. AIDS 2007; 21:2103–2106.
9. Shah SS, Crane M, Monaghan K, McGowan JP. Genotypic resistance testing in HIV-infected pregnant women in an urban setting. Int J STD AIDS 2004; 15:384–387.
10. Henry M, Thuret I, Solas C, Genot S, Colson P, Tamalet C. Vertical transmission of multidrug-resistant Q151M human immunodeficiency virus type 1 strains. Pediatr Infect Dis J 2008; 27:278–280.
11. Martinson NA, Morris L, Gray G, Moodley D, Pillay V, Cohen S, et al
. Selection and persistence of viral resistance in HIV-infected children after exposure to single-dose nevirapine. J Acquir Immune Defic Syndr 2007; 44:148–153.
12. Lee EJ, Kantor R, Zijenah L, Sheldon W, Emel L, Mateta P, et al
. Breast-milk shedding of drug-resistant HIV-1 subtype C in women exposed to single-dose nevirapine. J Infect Dis 2005; 192:1260–1264.
13. Johnson VA, Petropoulos CJ, Woods CR, Hazelwood JD, Parkin NT, Hamilton CD, Fiscus SA. Vertical transmission of multidrug-resistant human immunodeficiency virus type 1 (HIV-1) and continued evolution of drug resistance in an HIV-1-infected infant. J Infect Dis 2001; 183:1688–1693.
14. 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.
15. Siegrist CA, Yerly S, Kaiser L, Wyler CA, Perrin L. Mother to child transmission of zidovudine-resistant HIV-1. Lancet 1994; 344:1771–1772.
16. 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. N Engl J Med 2004; 351:229–240.
17. Paredes R, Lalama C, Ribaudo H, Schackman B, Shikuma C, Meyer W, et al
. Presence of minor populations of Y181C mutants detected by Allele-Specific PCR (ASPCR) and risk of efavirenz failure in treatment-naïve patients: results of an ACTG 5095 case-cohort study.
In: 15th Conference on Retroviruses and Opportunistic Infections
; Boston; 2008.
18. Coffie PA, Ekouevi DK, Chaix ML, Tonwe-Gold B, Clarisse AB, Becquet R, et al
. Maternal 12-month response to antiretroviral therapy following prevention of mother-to-child transmission of HIV type 1, Ivory Coast, 2003–2006. Clin Infect Dis 2008; 46:611–621.
19. 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.
20. Sheon AR, Fox HE, Rich KC, Stratton P, Diaz C, Tuomala R, et al
. The women and infants transmission study (WITS) of maternal-infant HIV transmission: study design, methods and baseline data. J Womens Health 1996; 5:69–78.
21. Johnson VA, Brun-Vezinet F, Clotet B, Gunthard HF, Kuritzkes DR, Pillay D, et al
. Update of the drug resistance mutations in HIV-1: Spring 2008. Top HIV Med 2008; 16:62–68.
22. Paredes R, Marconi VC, Campbell TB, Kuritzkes DR. Systematic evaluation of allele-specific real-time PCR for the detection of minor HIV-1 variants with pol and env resistance mutations. J Virol Methods 2007; 146:136–146.
23. Lin HJ, Tanwandee T, Hollinger FB. Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products. J Med Virol 1997; 51:56–63.
24. Paredes R, Marconi VC, Campbell TB, Kuritzkes DR. Systematic evaluation of allele-specific real-time PCR for the detection of minor HIV-1 variants with pol and env resistance mutations. J Virol Methods 2007; 146:136–146.
25. Wainberg MASH, Gu Z, Montaner JS, Cooley TP, McCaffrey R, Ruedy J, et al
. Development of HIV-1 resistance to (−)2′-deoxy-3′-thiacytidine in patients with AIDS or advanced AIDS-related complex. AIDS 1995; 9:351–357.
26. Gao Q, Gu Z, Parniak MA, Cameron J, Cammack N, Boucher C, Wainberg MA. The same mutation that encodes low-level human immunodeficiency virus type 1 resistance to 2′,3′-dideoxyinosine and 2′,3′-dideoxycytidine confers high-level resistance to the (−) enantiomer of 2′,3′-dideoxy-3′-thiacytidine. Antimicrob Agents Chemother 1993; 37:1390–1392.
27. Duran AS, Losso MH, Salomon H, Harris DR, Pampuro S, Soto-Ramirez LE, et al
. Drug resistance among HIV-infected pregnant women receiving antiretrovirals for prophylaxis. AIDS 2007; 21:199–205.
28. Welles SL, Bauer GR, LaRussa PS, Colgrove RC, Pitt J. Time trends for HIV-1 antiretroviral resistance among antiretroviral-experienced and naive pregnant women in New York City during 1991 to early 2001. J Acquir Immune Defic Syndr 2007; 44:329–335.
29. Cohn SE, Umbleja T, Mrus J, Bardeguez AD, Andersen JW, Chesney MA. Prior illicit drug use and missed prenatal vitamins predict nonadherence to antiretroviral therapy in pregnancy: adherence analysis A5084. AIDS Patient Care STDS 2008; 22:29–40.
30. Mellins CA, Chu C, Malee K, Allison S, Smith R, Harris L, et al
. Adherence to antiretroviral treatment among pregnant and postpartum HIV-infected women. AIDS Care 2008; 20:958–968.
31. Schackman BR, Ribaudo HJ, Krambrink A, Hughes V, Kuritzkes DR, Gulick RM. Racial differences in virologic failure associated with adherence and quality of life on efavirenz-containing regimens for initial HIV therapy: results of ACTG A5095. J Acquir Immune Defic Syndr 2007; 46:547–554.
32. Bryson YJ, Mirochnick M, Stek A, Mofenson LM, Connor J, Capparelli E, et al
. Pharmacokinetics and safety of nelfinavir when used in combination with zidovudine and lamivudine in HIV-infected pregnant women: Pediatric AIDS Clinical Trials Group (PACTG) Protocol 353. HIV Clin Trials 2008; 9:115–125.
33. Unadkat JD, Wara DW, Hughes MD, Mathias AA, Holland DT, Paul ME, et al
. Pharmacokinetics and safety of indinavir in human immunodeficiency virus-infected pregnant women. Antimicrob Agents Chemother 2007; 51:783–786.
34. Nellen JF, Schillevoort I, Wit FW, Bergshoeff AS, Godfried MH, Boer K, et al
. Nelfinavir plasma concentrations are low during pregnancy. Clin Infect Dis 2004; 39:736–740.
35. Mathias AA, Maggio-Price L, Lai Y, Gupta A, Unadkat JD. Changes in pharmacokinetics of anti-HIV protease inhibitors during pregnancy: the role of CYP3A and P-glycoprotein. J Pharmacol Exp Ther 2006; 316:1202–1209.
36. Stek AM, Mirochnick M, Capparelli E, Best BM, Hu C, Burchett SK, et al
. Reduced lopinavir exposure during pregnancy. AIDS 2006; 20:1931–1939.
37. von Hentig N, Carlebach A, Gute P, Knecht G, Klauke S, Rohrbacher M, et al
. A comparison of the steady-state pharmacokinetics of nevirapine in men, nonpregnant women and women in late pregnancy. Br J Clin Pharmacol 2006; 62:552–559.
38. Chaix ML, Ekouevi DK, Peytavin G, Rouet F, Tonwe-Gold B, Viho I, et al
. Impact of nevirapine (NVP) plasma concentration on selection of resistant virus in mothers who received single-dose NVP to prevent perinatal human immunodeficiency virus type 1 transmission and persistence of resistant virus in their infected children. Antimicrob Agents Chemother 2007; 51:896–901.
39. Metzner KJ, Rauch P, Walter H, Boesecke C, Zollner B, Jessen H, et al
. Detection of minor populations of drug-resistant HIV-1 in acute seroconverters. AIDS 2005; 19:1819–1825.
40. Simen BB, Huppler Hullsiek K, Novak RM, MacArthur RD, Baxter JD, Huang C, et al. Prevalence of Low abundance drug resistant variants by ultra-deep sequencing in chronically HIV-infected antiretroviral (ARV) naive patients and the impact on virologic outcomes.
In: 16th International HIV Drug Resistance Workshop
; Barbados, West Indies; June 12–16, 2007. 2007.
41. Perinatal HIV Guidelines Working Group. Public Health Service Task Force recommendations for use of antiretroviral drugs in pregnant HIV-infected women for maternal health and interventions to reduce perinatal HIV transmission in the United States
. November 2, 2007. 1–96. http://aidsinfo.nih.gov/ContentFiles/PerinatalGL.pdf
. [Accessed: July 28th, 2008]
This article has been cited 2 time(s).
Clinical Infectious DiseasesHigh Genetic Barrier Antiretroviral Drugs in Human Immunodeficiency Virus-Positive PregnancyClinical Infectious Diseases
Journal of Infectious DiseasesImpact of Antiretroviral Drugs in Pregnant Women and Their Children in Africa: HIV Resistance and Treatment OutcomesJournal of Infectious Diseases
allele-specific PCR; HIV-1; minor variants; mother-to-child transmission; pregnancy; resistance
© 2010 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.