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Single-dose tenofovir and emtricitabine for reduction of viral resistance to NNRTIs in women given intrapartum nevirapine for perinatal HIV prevention: an open-label randomised trial
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The Lancet Early Online Publication, 7 November 2007
Dr Benjamin H Chi MD a b , Moses Sinkala MBChB b c, Felistas Mbewe BSc a, Ronald A Cantrell MPH a b, Gina Kruse MSc a, Namwinga Chintu MBChB a, Grace M Aldrovandi MD d, Elizabeth M Stringer MD a b, Chipepo Kankasa MD e, Jeffrey T Safrit PhD f and Prof Jeffrey SA Stringer MD a b
a. Centre for Infectious Disease Research in Zambia, Lusaka, Zambia
b. Schools of Medicine and Public Health, University of Alabama, Birmingham, AL, USA
c. Catholic Medical Missions Board, Lusaka, Zambia
d. Saban Research Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
e. University Teaching Hospital, Lusaka, Zambia
f. Elizabeth Glaser Pediatric AIDS Foundation, Santa Monica, CA, USA
Summary
Background
Intrapartum and neonatal single-dose nevirapine are essential components of perinatal HIV prevention in resource-constrained settings, but can induce resistance to other non-nucleoside reverse transcriptase inhibitor drugs. We aimed to investigate whether this complication would be reduced with a single peripartum intervention of tenofovir and emtricitabine.
Methods
We randomly assigned 400 HIV-infected pregnant women who sought care at two public-sector primary health facilities in Lusaka, Zambia. One was excluded, 200 were assigned to receive a single oral dose of 300 mg tenofovir disoproxil fumarate with 200 mg emtricitabine under direct observation, and 199 to receive no study drug. Short-course zidovudine and intrapartum nevirapine were offered to all HIV-infected women, according to the local standard of care. Women who met national criteria for antiretroviral therapy were referred for care and not enrolled. Our primary study outcome was resistance to non-nucleoside reverse transcriptase inhibitors at 6 weeks after delivery. We used standard population sequencing to determine HIV genotypes. Analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00204308.
Findings
Of the 200 women who were randomly assigned to the intervention, 14 were lost to follow-up or withdrew from the study, two did not take study drug according to protocol, and one specimen was lost; 23 of 199 controls were lost to follow-up or withdrew from the study, and three specimens were lost. Women given the intervention were 53% less likely than controls to have a mutation that conferred resistance to non-nucleoside reverse transcriptase inhibitors at 6 weeks after delivery (20/173 [12%] vs 41/166 [25%]; risk ratio [RR] 0·47, 95% CI 0·29-0·76). We noted postpartum anaemia, the most common serious adverse event in mothers, in four women in each group. 20 of 198 (10%) infants in the intervention group and 23 of 199 (12%) controls had a serious adverse event, mostly due to septicaemia (n=22) or pneumonia (n=8); these events did not differ between groups, and none were judged to be caused by the study intervention.
Interpretation
A single dose of tenofovir and emtricitabine at delivery reduced resistance to non-nucleoside reverse transcriptase inhibitors at 6 weeks after delivery by half; therefore this treatment should be considered as an adjuvant to intrapartum nevirapine.
Introduction
Intrapartum and neonatal single-dose nevirapine are essential components of perinatal HIV prevention in many resource-constrained settings.1-6 However, in the weeks after ingestion, 20% to 69% of women have been shown to develop HIV resistance to the non-nucleoside reverse transcriptase inhibitor class of drugs.7-9 Emergence of resistance to this class of antiretroviral drugs has also been recorded when single-dose nevirapine was used in conjunction with other agents that are used to prevent perinatal HIV transmission, such as zidovudine10,11 and a combination of zidovudine and lamivudine.12 The long-term effect of these transient resistance mutations on later antiretroviral therapy (ART) is not completely understood, especially when ART is initiated more than 6 months after maternal exposure to nevirapine.10,13-15
The antiretroviral drugs tenofovir disoproxil fumarate and emtricitabine are in common use worldwide. Both drugs have long intracellular half-lives (50 h for tenofovir16 and 39 h for emtricitabine17) and are categorised as "probably safe" for use in pregnancy by the US Food and Drug Administration, on the basis of safety in animals. We aimed to investigate whether the addition of the tenofovir and emtricitabine combination to intrapartum nevirapine would reduce the emergence of resistance to non-nucleoside reverse transcriptase inhibitors in the weeks after ingestion. We reasoned that a single adjunctive dose of tenofovir and emtricitabine in labour, if proven effective, could reduce the adverse consequences of intrapartum nevirapine without compromising the regimen's well known feasibility and cost-effectiveness.
Results
We enrolled 627 women. 227 were not eligible, either because their babies were delivered before arrival at the centre or at different facilities; they were referred for obstetric complications; they withdrew consent; or they had not yet given birth when the randomisation period was over (figure 1). We randomly assigned 397 women between March 16, 2005, and Feb 13, 2007. Three others were excluded from the analysis: two because the study drug was not dispensed according to protocol by delivery ward staff, and a third because a randomisation envelope was incorrectly opened and not replaced. Table 1 shows antenatal, medical, and delivery characteristics. Overall, 322 of 397 women (81%) initiated short-course zidovudine in the antenatal period. Cord-plasma specimens were available for nevirapine drug analysis from 386 of 397 (97%) women who had been randomly assigned. From detectable drug concentrations in these specimens, we confirmed that 315 (82%) had ingested intrapartum nevirapine. This proportion did not differ between randomised groups. Of the 198 women in the intervention arm who received tenofovir and emtricitabine according to protocol, the mean time from witnessed ingestion until delivery was 3·5 (SD 4·5) h.
In patients who were given intrapartum single-dose tenofovir and emtricitabine, the maternal plasma viral load was lower after delivery than in controls. At 2 weeks postpartum, 116 of 183 women (63%) in the intervention group had undetectable viral load (ie, fewer than 400 copies per mL), compared with 93 of 177 (53%) controls (p=0·043). Of those with detectable virus at 2 weeks, the mean log viral load was also lower (3·49 vs 3·83; p=0·004). By 6 weeks after delivery, this difference between the intervention and control groups had disappeared, both by the proportion in which viral load was suppressed (10/183 [5%] vs 18/173 [10%], p=0·114) and by mean concentration in those with detectable viral load (4·40 vs 4·37; p=0·733).
Women who had been randomly assigned to receive intrapartum tenofovir and emtricitabine were much less likely to develop resistance to non-nucleoside reverse transcriptase inhibitor drugs at 2 weeks after childbirth (RR 0·27; 95% CI 0·11-0·66), at 6 weeks (0·47; 0·29-0·76), and cumulatively (0·44; 0·28-0·69). We noted similar results when we limited the analysis to specimens with greater than 2000 viral copies per mL, although the protective effect of tenofovir and emtricitabine was attenuated somewhat at the 2-week secondary outcome (table 2). In a secondary analysis, in which we stratified the study population according to maternal viral load at delivery (the time of nevirapine exposure), the protective effect of tenofovir and emtricitabine was most pronounced in women whose delivery viral load was greater than 10,000 copies per ml.
Of 397 infants born to mothers in the intervention and control groups, 355 (89%) remained in the study at 6 weeks of life. Three fetuses (1%) died before delivery, nine infants (2%) died in the first 6 weeks of life, and 30 (8%) were lost to follow-up. The overall rate of perinatal HIV transmission was similar in the intervention group (10/180 [5·6%]) and the control group (14/175 [8·0%]; p=0·403). This finding was consistent for intrauterine transmission (8/180 [4·4%] vs 10/175 [5·7%]; p=0·635) and intrapartum/early postpartum transmission (2/127 [1·6%] vs 4/165 [2·4%]; p=0·440) (table 4).
At 2 weeks after delivery, we recorded no differences in mean serum creatinine (53·77 vs 53·83; p=0·968), alanine aminotransferase (18·3 vs 20·4 U/L; p=0·643), or haemoglobin (122 vs 118 g/L; p=0·106). Seven of 198 women (4%) in the intervention group had serious adverse events, compared with nine of 199 (5%) controls (p=0·800). Four women in each group had postpartum anaemia, which was the most common serious maternal event. 20 of 198 (10%) infants in the intervention group and 23 of 199 (12%) controls had a serious adverse event. Common causes of infant morbidity were septicaemia (n=22) and pneumonia (n=8), again with no difference between groups. None of the adverse events were judged to be caused by the study intervention.
Discussion
Addition of a single dose of combined tenofovir and emtricitabine to the antiretroviral prophylaxis regimen of short-course zidovudine and intrapartum nevirapine effectively reduced the frequency of postpartum resistance to non-nucleoside reverse transcriptase inhibitors. The intervention was associated with a 73% reduction at 2 weeks and a 53% reduction at 6 weeks after nevirapine ingestion. In our setting, where most women elected to use short-course zidovudine along with intrapartum nevirapine, tenofovir and emtricitabine did not reduce perinatal HIV transmission, compared with controls (5·6% vs 8·0%; p=0·403).
Used alone or in combination, nevirapine has been an essential component of perinatal HIV prevention programmes in settings with high HIV burdens and severe resource constraints. Since 2001, for example, more than 50 000 doses of the drug have been given to HIV-infected pregnant mothers in the Lusaka, Zambia public-health sector, and this figure is representative of nevirapine use in other large programmes in the region.31 However, evidence is mounting that use of nevirapine might compromise the treatment response in some women when they later start ART with non-nucleoside reverse transcriptase inhibitors. This risk seems to be related to the length of time between nevirapine exposure and initiation of ART. Studies in sub-Saharan Africa suggest that recent exposure to single-dose nevirapine (ie, less than 6 months) might be associated with risk for virological and clinical treatment failure.13,14 If more than 6 months elapse between nevirapine use and ART initiation, the risk of compromised virological outcomes seems to fall to baseline.13,15 Nevertheless, nevirapine-induced mutations that cause resistance to non-nucleoside reverse transcriptase inhibitors are a concern in resource-constrained settings, where drugs such as nevirapine and efavirenz are common components of first-line antiretroviral regimens,32 and options for second-line treatment are limited.
Use of short-course adjunctive antiretroviral drugs to reduce resistance to non-nucleoside reverse transcriptase inhibitor after intrapartum use of nevirapine has been investigated by others. In a randomised trial in South Africa, the frequency of mutations that cause resistance to non-nucleoside reverse transcriptase inhibitors after intrapartum use of nevirapine was reduced from 57% in controls to 13% in women who received a 4-day course of zidovudine and lamivudine, and to 9% in those who received a 7-day course of zidovudine and lamivudine.33 Similar reductions have been seen in the setting of short-course antenatal zidovudine and lamivudine and intrapartum nevirapine. When zidovudine and lamivudine was started from 32 weeks gestation onward and continued for 3 days after nevirapine ingestion, the frequency of non-nucleoside reverse transcriptase inhibitor mutations declined from 33% (in historical controls) to 1%.12 Unfortunately, use of zidovudine and lamivudine in this way induced viral resistance to lamivudine, which was then associated with treatment failure in women who started ART with lamivudine.34
The primary advantage of our study intervention is its simplicity. We reasoned that a single-dose approach would lead to greater adherence and acceptability than longer regimens,35 and would thus be appropriate for settings where intrapartum nevirapine is prescribed. Despite its proven efficacy, however, the regimen might need further optimisation. Addition of a second dose of tenofovir and emtricitabine-either at discharge from the labour ward or in the days after delivery-might further reduce mutations that confer resistance to non-nucleoside reverse transcriptase inhibitors and also preserve the simplicity of the regimen.
We classified specimens with viral load of less than 2000 copies per mL as non-resistant in our primary analysis. Because resistance to non-nucleoside reverse transcriptase inhibitors develops despite high viral replication, and because plasma viral load was much lower with the tenofovir and emtricitabine intervention than in controls, exclusion of specimens under this viraemia threshold would result in an incorrectly low assessment of the efficacy of the intervention. On the other hand, some of the specimens with fewer than 2000 copies of virus per mL could potentially contain HIV that is resistant to non-nucleoside reverse transcriptase inhibitors, but could be incorrectly categorised because the overall plasma viral burden is too low to allow sufficient amplification for genotyping. By this argument, these same specimens would become detectable by population genotyping at a later point, once concentrations of antiretroviral drugs had waned and plasma viral concentrations increased. To include specimens with viral load of fewer than 2000 copies per mL, and to classify them as non-resistant would thus underestimate the emergence of resistance to non-nucleoside reverse transcriptase inhibitors. Because of this dilemma, and to facilitate direct comparison of our results with those of other studies, we have presented the data both ways (ie, with and without specimens with fewer than 2000 viral copies per mL). However, our main finding-a reduction in non-nucleoside reverse transcriptase inhibitor resistance at 6 weeks-did not differ with these methods of analysis.
We did not detect HIV mutations associated with tenofovir and emtricitabine resistance in the intervention group. This result is especially relevant now that both drugs have been incorporated into first-line treatment in many African countries, including Zambia.36 The frequency of K103N mutations in our study increased from 2 weeks to 6 weeks postpartum, as has been described in other regional studies;37,38 however, the proportion with Y181C/I mutations remained stable. None of the women allocated to the intervention group developed a mutation at codon 181. We cannot explain this result.
Our results for rates of mother-to-child HIV transmission with short-course zidovudine and intrapartum nevirapine were higher than the 2% reported in clinical trials,39 but similar to the rates reported in cohorts in Cote d'Ivoire and Botswana.11,40 This finding could be attributed to the nearly universal practice of breastfeeding in African settings, or to regional differences in HIV subtypes. In our study, most transmission (18 of 24 cases) occurred during the intrauterine period, because women in our study started antenatal zidovudine at late gestation, on average. Although the intervention was not associated with significant differences in perinatal HIV transmission, our study was not sufficiently powered to assess this outcome. Future clinical trials should investigate perinatal HIV transmission as a primary outcome.
We recognise several limitations of our study. First, the HIV genotyping assays that we used could only detect viral mutations that are found in at least 20% of the overall HIV population.23 We therefore need to investigate the efficacy of single-dose tenofovir with emtricitabine in HIV minority populations.9,41,42 Second, our eligibility criteria for maternal HIV status might have introduced selection biases, since we excluded individuals at greatest risk for developing resistance-those with advanced HIV disease (and thus higher concentrations of circulating virus). Third, high uptake of antenatal zidovudine contributed to the low overall viral burden in study participants, which could have reduced the frequency of resistance to non-nucleoside reverse transcriptase inhibitors. Because reductions in resistance were greatest in women with the highest viral burden, the effect of the intervention on drug resistance might in fact be higher in settings where ART (or short-course zidovudine) is poorly accessible. Fourth, because resistance data for HIV-infected infants were not available,7,43-45 we could not assess the effect of our intervention on resistance to non-nucleoside reverse transcriptase inhibitor drugs in newborns exposed to intrapartum and neonatal nevirapine. Last, we did not assess the effect of tenofovir and emtricitabine on neonatal development (specifically, toxic effects on bone) because we did not think it was possible, given the single-dose regimen and our available sample size, to make statistically meaningful comparisons. Future studies should also assess infants exposed to tenofovir and emtricitabine during pregnancy for a longer period.
We showed that a single dose of tenofovir and emtricitabine-taken with antepartum zidovudine and intrapartum nevirapine-can substantially reduce non-nucleoside reverse transcriptase inhibitor resistance mutations at 2 weeks and 6 weeks after ingestion. Despite its effectiveness, this intervention might need modification to achieve the optimum protective effect. Nevertheless, it is an important adjunct to regimens that incorporate intrapartum nevirapine and should be considered in settings where drug combinations to be taken over several days might be impractical for patients or for the local health infrastructure.
Procedures
We tested plasma HIV viral load with commercial tests (COBAS Ampliprep and COBAS Amplicor HIV-1 Monitor, version 1.5, standard format, Roche Molecular Systems, Branchburg, NJ, USA). We did HIV genotyping on specimens with HIV viral load of more than 2000 opies per mL in accordance with the manufacturer's instructions. We extracted viral RNA from plasma with a commercial kit (Qiagen, Chatsworth, CA, USA). We amplified and bidirectionally sequenced the pol gene,21 and assembled and edited sequences with a bioinformatics program (Sequencher, Gene Codes, Ann Arbor, MI, USA). Sequences were then analysed against an HIV drug-resistance database.22
We judged that mutations were present if they were detected alone or in combination with wild-type sequences (mixtures). Samples that did not amplify or were of poor quality were reamplified and sequenced with a genotyping system (ViroSeq HIV-1 Genotyping, Celera Diagnostics, Alameda CA, USA). These assays were based on standard thresholds for detection, which generally only detect a mutant viral subpopulation of greater than 20%.23 Lab personnel were unaware of each participant's randomly allocated treatment.
We categorised mutations of the reverse transcriptase gene that conferred drug resistance based on the recommendations of the International AIDS Society-USA Drug Resistance Mutations Group.24 Resistance to non-nucleoside reverse transcriptase inhibitors was assumed if we identified the mutations L100I, K103N, V106A/M, V108I, Y181C/I, Y188C/L/H, G190A, P225H, or P236L. We also categorised specimens as resistant towards tenofovir (K65R, K70E), emtricitabine and lamivudine (K65R, M184V), or zidovudine (M41L, D67N, K70R, L210W, T215Y/F, K219Q/E).
We collected dried-blood spot specimens from infants on filter paper and tested them with a commercial test (Amplicor HIV-1 DNA, Version 1·5, Roche Molecular Systems, Branchburg, NJ, USA). Two consecutive DNA PCR tests were required for diagnosis of HIV. Transmission was regarded as intrauterine if results at birth and 6 weeks were both positive, and as intrapartum or early postpartum if an infant tested HIV-negative at birth, but positive at 6 weeks (with a confirmatory positive result at least 4 weeks later).25
Because high rates of non-adherence to nevirapine treatment had been previously reported in our setting,26,27 we tested for drug concentrations in cord plasma as a marker for nevirapine ingestion.28 Plasma concentrations were quantified by tandem mass spectrometry using a linear mass spectrometer (API 4000, Applied Biosystems, Foster City, CA, USA); we used high-performance liquid chromatography (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) according to a published method.29 The lower limit of quantification for nevirapine was 0·01 _g/mL.
The primary outcome was maternal resistance to non-nucleoside reverse transcriptase inhibitor drugs at 6 weeks postpartum. Secondary outcomes were maternal non-nucleoside reverse transcriptase inhibitor drug resistance at 2 weeks postpartum, other maternal drug resistance (specifically to tenofovir, emtricitabine, or zidovudine) at 2 and 6 weeks postpartum, perinatal HIV transmission rates, and drug safety.
We assessed maternal haematological, hepatic, and renal function at 2 weeks after delivery, and trained study staff to monitor and report adverse clinical events for both mothers and infants. An adverse event was regarded as serious if it was fatal, life-threatening, required admission to hospital, or resulted in persistent or substantial disability. Events classified as grade 3 or above (or grade 2B or above in the case of rash) according to tables of toxic effects from the US National Institutes of Health's Division of AIDS30 were regarded as serious. Adverse events were reviewed by the chair of an independent project oversight committee within 2 days of the initial report. This committee also reviewed all adverse events twice a year.
Statistical analysis
We calculated our target sample size of 400 women to provide 80% power to detect a large postulated effect size that we believed would be clinically relevant: a three-fold reduction in non-nucleoside reverse transcriptase inhibitor resistance-from 15% to 5%-at 6 weeks postpartum, with Fisher's exact test (two-sided _=0·05). We accounted for losses to follow-up and sub-optimal adherence to nevirapine treatment. We did analyses for the per-protocol group.
Maternal non-nucleoside reverse transcriptase inhibitor resistance was assessed by sequencing the reverse transcriptase gene from maternal plasma specimens obtained at 6 weeks (primary outcome) and 2 weeks (secondary outcome) after childbirth. Specimens that were not genotyped because HIV viral load was less than 2000 copies per mL were classified as non-resistant in the primary analysis. In a complementary analysis, we assessed only specimens with viral load of more than 2000 copies per mL. We compared intrauterine mother-to-child HIV transmission rates with those during and after childbirth and with overall rates according to study group.
We analysed categorical variables with Fisher's exact test. Two-tailed t tests were used for normally distributed continuous variables, and Wilcoxon rank-sum tests to compare medians of variables that were not normally distributed. Viral load measurements were log10 transformed. We used SAS for analyses (version 9·1, SAS Institute, Cary, NC, USA). This study is registered with ClinicalTrials.gov, number NCT00204308.
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