McConnell, Michelle S MD*; Bakaki, Paul MBChB, MMED†; Eure, Chineta MD, MPH‡; Mubiru, Michael†; Bagenda, Danstan PhD†§; Downing, Robert PhD∥; Matovu, Flavia MBChB†; Thigpen, Michael C MD‡; Greenberg, Alan E MD, MPH¶; Fowler, Mary Glenn MD, MPH†
Single-dose nevirapine (SDNVP) given to mothers at labor onset and to their newborns has been shown to be effective in reducing mother-to-child transmission (MTCT) of HIV by nearly half.1 Its low cost, lack of refrigeration requirements, availability from donation programs, ability for self-administration, need for only simplified health care worker training, and sustained efficacy at 18 months postpartum in breast-feeding populations, relative to other short-course perinatal prophylaxis regimens,2 has made it a first-line MTCT prophylaxis regimen in resource-constrained settings when other antiretroviral drugs are not available or where infrastructure does not allow for more complex regimens. Although longer and more complex regimens have shown greater efficacy3-7 in many settings with low rates of antenatal care, limited antenatal HIV testing, or high rates of noninstitutional deliveries, SDNVP currently remains one of the most practical, deliverable, and sustainable options for reducing MTCT of HIV.8-10
A number of studies have demonstrated the detection of genotypic resistance postpartum in 19% to 76% of women after nevirapine (NVP) prophylaxis alone or in combination with other antiretrovirals,11-14 using standard genotypic assays, however. Even higher percentages have been detected in women using ultrasensitive resistant testing techniques.15-17 By 12 months postpartum, however, detectable mutations wane;13 even using ultrasensitive techniques, mutant virus represents a small proportion of the circulating viral genotype and archiving is detected in only a few peripheral blood mononuclear cells (PBMCs).18
As a result of these findings, there are concerns about the possible clinical consequences of viral resistance after receipt of SDNVP, in terms of effectiveness of its prophylactic use in subsequent pregnancies and potential impact on later virologic treatment. A number of studies have assessed treatment response with a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based combination regimen after SDNVP.14,19,20 There has only been 1 other report of the effectiveness of SDNVP in repeat pregnancies, however.21,22
Many HIV-infected women in resource-constrained settings receive MTCT prophylaxis in more than a single pregnancy. Despite the move to implement more efficacious complex regimens to reduce MTCT, NVP resistance continues to be an issue for women who receive less complex PMTCT regimens that include NVP, such as short-course zidovudine plus SDNVP or SDNVP alone. The aim of this study was to assess the effectiveness of SDNVP in repeat pregnancies among women previously exposed to SDNVP when compared with NVP-naive women.
Data from 2 groups of previously pregnant (retrospective group) or pregnant (prospective group) HIV-positive women in Kampala, Uganda were analyzed. All women and infants were followed at the Makerere University-Johns Hopkins University (MUJHU) Research Collaboration affiliated with Mulago Hospital between July 2004 and May 2006. The retrospective mother and infant group included women previously enrolled in the HIV Network for Prevention (HIVNET) 012 trial or prevention of mother-to-child transmission (PMTCT) program who received SDNVP (exposed women), or zidovudine (unexposed women)1 and subsequently had another pregnancy in which they received the SDNVP regimen as part of the routine PMTCT program in Uganda. These women had completed at least 2 pregnancies at the time of study enrollment. The prospective mother and infant group included women who were pregnant at the time of study enrollment but who had previously received SDNVP as part of a study or the routine PMTCT program (exposed women) or who had never before received NVP (unexposed women).
The retrospective study women were identified from the follow-up records of the HIVNET 012 trial, conducted from 1997 to 1999. As follow-up to that trial, the HIVNET 012 research group also temporarily undertook the routine implementation of SDNVP at Mulago Hospital, and some women were noted to have received SDNVP in a subsequent pregnancy through these records. Women who had a subsequent pregnancy were contacted by study health visitors and consented to enroll in the present study to determine the HIV infection outcome of their most recently delivered infant. Study follow-up data collected for this group of mothers and infants included polymerase chain reaction (PCR) testing to confirm the infant's HIV status.
The prospective study women were recruited between 28 and 40 weeks of gestation from the antenatal clinic at Mulago Hospital in Kampala. Women were asked verbally about prior receipt of NVP, and all responses were confirmed with medical records. Women who were uncertain about prior receipt of NVP or were on highly active antiretroviral therapy (HAART) were excluded from the study. Inclusion criteria for prospective women were age ≥18 years, multiparity, and no known contraindication to receipt of NVP. Prospective mothers and their babies were followed for a period of 1 year, with study visits at enrollment; delivery; and 7 days, 6 weeks, 3 months, 6 months, and 12 months postpartum. Maternal clinical and pregnancy data included maternal age and parity; months since last delivery; duration of membrane rupture; mode of delivery; birth outcome, including newborn birth weight; type of infant feeding at each visit; and infant age at weaning.
Maternal laboratory testing, including viral load, was conducted at each visit; CD4 cell counts were only measured at enrollment. For mothers who were still breast-feeding at the 6 months' visit or who had stopped breast-feeding <3 months previously, an optional visit to confirm infant HIV status was done around 9 months. At each visit, the infants were assessed for growth and feeding status. PCR testing was done at 0 to 7 days, 6 weeks, 6 months, and 12 months. Because of the fact that some sampling was not able to be done at birth, samples were collected within 1 week of birth for those newborns not sampled at birth. If the initial PCR assay was positive, the baby was brought back for confirmatory PCR testing as soon as possible.
Infant HIV Infection Status
An infant was classified as HIV-infected based on 2 separate positive PCR specimens, at least 1 of which was done as part of the study. An infant was classified as HIV-uninfected based on at least 1 negative PCR assay done in the study, which was obtained at least 2 months after weaning.
Infection status of the babies was determined at 0 to 7 days, 6 weeks, and 6 or more months of age, based on whether complete cessation of breast-feeding had occurred at least 2 months before the test. Infants were classified as HIV-positive based on 2 separate HIV-positive specimens by PCR and as HIV-negative based on at least 2 negative PCR results and no positive results, with at least 1 PCR test being done at least 2 months after complete cessation of breast-feeding. At 6 weeks of age, HIV-positive infants had birth DNA PCR testing using dried blood spots. If DNA filter paper testing was unsuccessful or if the infant died before the HIV status could be confirmed, RNA PCR testing was done on a stored plasma sample. RNA PCR testing was done for 14 infants. Final HIV infection status and infant HIV-free survival were determined based on the infant's HIV infection and vital status by the age of 12 months.
DNA PCR testing was done for all infants using the Roche Amplicor 1.5 assay (Roche Diagnostics, Branchburg, NJ). CD4 cell testing on mothers was performed with the FACS Calibur (Becton Dickinson Biosciences, San Jose, CA). All infant DNA PCR and maternal CD4 cell testing was performed at the College of American Pathologists (CAP)-certified MUJHU Core Research Laboratory in Kampala, Uganda. Maternal viral load samples were tested at the Centers for Disease Control and Prevention (CDC) in Entebbe, Uganda using the Roche RNA Amplicor 1.5 assay.
Comparison of prospective mother and infant characteristics was done using the Pearson χ2 test or Fisher exact test for categoric variables. Continuous variables were analyzed with the Student t test.
Kaplan-Meier survival analysis and Cox proportional hazards models were performed to determine the primary endpoints of HIV infection and HIV-free survival. For these analyses, the time to the first positive HIV result was used to determine the time to the endpoint for HIV-infected infants and the time to HIV infection and/or death was used to determine HIV-free survival endpoints. HIV-negative infants were censored from the time of the last negative HIV test result or death. Cox models adjusted for breast-feeding duration, baseline maternal viral load, and CD4 cell count were used in determining the hazard of HIV infection. One-tailed tests were used to determine significance, based on the assumption that HIV transmission rates would not be superior in the exposed group. All analyses were performed with SAS version 9.1 (SAS Institute, Cary, NC).
Institutional Review Board (IRB) approval was obtained from all participating sites, including the US CDC and the Uganda Virus Research Institute. Written informed consent was obtained from all study participants before enrollment.
A total of 207 HIV-exposed infants were included in the analysis: 104 infants in the retrospective group (62 [59.6%] exposed to SDNVP and 42 [40.4%] unexposed) and 103 in the prospective group (39 [37.9%] exposed to SDNVP and 64 [62.1%] unexposed).
Among the retrospective group, 62 (45.9%) of 135 of all SDNVP-exposed and 42 (59.1%) of 71 of all SDVNP-unexposed infants from the HIVNET 012 trial were included in this analysis (Fig. 1A). The proportion of infants not included in the analysis because of death was 27 (20.6%) of 135 exposed infants and 2 (2.8%) of 71 unexposed infants (P < 0.001). Equal proportions of exposed and unexposed infants were excluded from the analysis because of other reasons, including study refusal (1.5% and 4.2%, respectively; P = 0.25), moving or loss to follow-up (22.9% and 15.5%, respectively; P = 0.19), and failure of the mother to take SDNVP (10.7% and 18.3%, respectively; P = 0.14).
Among retrospective SDNVP-exposed and -unexposed mothers and infants, there were no significant differences in breast-feeding duration and interpregnancy duration. The median durations of breast-feeding in this group were 3.7 (range: 0 to 19) months and 3.9 (range: 0 to 27) months, respectively, among exposed and unexposed women (P = 0.33). Interpregnancy duration was 24 months or greater in 88% of mothers and 12 to 24 months in 12% of mothers.
Among the prospective group, 2 (4.9%) of 41 infants born to women with prior SDNVP exposure and 7 (9.9%) of 71 infants born to women with no prior exposure were not included in the analysis because of stillbirth, consent withdrawal, loss to follow-up, or maternal study ineligibility (P = 0.48; see Fig. 1B).
There were no significant differences in the characteristics of prospective SDNVP-exposed and -unexposed mothers and infants, with the exception of maternal parity (P = 0.03; Table 1). Among NVP-exposed and -unexposed women, respectively, baseline median CD4 cell counts were 470 and 459 cells/mm3 (P = 0.90) and viral loads were 20,200 and 14,100 copies/mL (P = 0.12). Most women in both groups had a vaginal delivery and had newborns weighing >3200 g. The median infant age at the time of weaning was 3 months in both groups. The interpregnancy duration was at least 24 months in 62% of SDNVP-exposed women, 12 to 24 months in 36% of women, and <12 months in 3% of women. The median time between deliveries for all women was 32 months.
HIV transmission rates were compared in the retrospective group for SDNVP-exposed and -unexposed women at enrollment and in the prospective cohort for exposed and unexposed women and babies at 0 to 7 days; 6 weeks; and 3, 6, and 12 months (Table 2). No significant differences were noted in the HIV transmission rates between NVP-exposed and -unexposed women for the retrospective or prospective group. Specifically, in the retrospective group, the infant infection rate was 11.3% for infants of mothers with prior SDNVP exposure and 16.7% for infants of NVP-naive mothers (P = 0.41). For the prospective group, the cumulative HIV infection rates among infants born to NVP-exposed and -unexposed women, respectively, were 17.9% and 18.7% (P = 0.92) at 6 weeks and 20.5% and 18.7% (P = 0.81) at 12 months. The HIV infection and/or infant death rates among infants born to NVP-exposed and -unexposed women, respectively, were 17.9% and 18.7% (P = 0.92) at 6 weeks and 25.6% and 21.9% (P = 0.66) at 12 months.
Transmitting and nontransmitting mothers did have significantly different mean log viral load results (4.80 and 4.05, respectively; P = 0.005). Furthermore, there were 10 women, in the prospective and retrospective groups combined, who received SDNVP in more than 2 pregnancies, with 2 of their most recently delivered infants being HIV infected. There were 6 mothers who received more than a single dose of SDNVP in a pregnancy.
For the prospective group, the Cox multivariate hazard ratio for infant HIV infection among infants born to women with prior SDNVP exposure, compared with women with no prior SDNVP exposure, was 0.39 (95% confidence interval [CI]: 0.07 to 1.98) when adjusted for baseline maternal CD4 cell count, viral load, and breast-feeding duration. Kaplan-Meier HIV infection curves and HIV-free survival curves are presented in Figures 2A and B, respectively. The Kaplan-Meier plots for HIV infection and HIV-free survival show no significant differences in the rates or timing of HIV infection for the infants born to mothers with prior NVP exposure and those born to NVP-naive women (log rank = 0.84 and 0.69, respectively).
This study in Uganda found no significant differences in HIV transmission rates among SDNVP-experienced women who received SDNVP again in a subsequent pregnancy when compared with NVP-unexposed women who received SDNVP for PMTCT for the first time. This held true for the retrospective group, which included women exposed to SDNVP or zidovudine in the HIVNET 012 trial, and the prospective group enrolled from the PMTCT antenatal services at Mulago Hospital in Kampala, Uganda.
The effectiveness of SDNVP in repeat pregnancies is supported by the fact that almost all deliveries of subsequent pregnancies occurred at least a year after exposure to the initial SDNVP. With detection of resistant mutations fading within the first 12 months postpartum13 and archiving of mutant virus in PBMCs at 1 year among a few women with SDNVP exposure,18 the wild-type (NVP-sensitive) virus re-emerges as the predominant quasispecies to which the baby is exposed during delivery.
There are certain caveats to these data. Among the retrospective group, infant HIV status could not be determined for those infants who died or were lost to follow-up before this data collection; thus, the study population is not a complete account of all subsequent births to HIVNET 012-enrolled mothers. Furthermore, the difference in the proportion of SDNVP-exposed (20%) and -unexposed (3%) mothers in the retrospective group whose infants died before this study may have biased our data to a lower transmission rate in the exposed group. Given the high infant mortality rate in Uganda (80 per 1000), however, it is unlikely that all early infant deaths would have been HIV related, and the prospective group corroborates the retrospective findings of no difference in transmission rates between SDNVP-exposed and -unexposed women having subsequent pregnancies. Finally, the study power is limited by the retrospective and prospective group sample sizes, but the similar findings in the 2 study groups support the conclusion that previous SDNVP exposure is not associated with an increased risk of HIV transmission.
Despite the findings from this study in Uganda on the effectiveness of SDNVP for PMTCT in subsequent pregnancies, a number of research questions persist, particularly with regard to the potential impact of postpartum detection of mutant virus on later treatment options.14,19,20 To date, several studies have found that NNRTI-containing treatment regimens may still be effective as first-line therapy in women with prior SDNVP exposure, particularly as 1 study found, if there is a lapse of more than 6 months between SDNVP dosing and treatment initiation.19
There also remain questions about the impact of minority mutant variants archived at low levels after they fade from detection. Ultrasensitive real-time PCR studies have shown persistent, albeit low (1% to 2% of all circulating quasispecies), levels of NVP-resistant mutants in as many as 40% of women who had no detectable resistance by standard sequencing assays in the first few months after delivery,15,16 and NVP-resistant mutants were still detectable as a small percentage of the circulating virus in 22% of women up to 1 year after SDNVP exposure.17 The clinical relevance of this finding, where detectable mutant virus is present at low levels, remains a question in view of the Botswana data, however, which did not demonstrate any evidence of higher rates of virologic failure for women with prior SDNVP exposure who began NVP-based HAART at a time at least 6 months after exposure to SDNVP.19
There is also great interest in how the development of NVP resistance can be reduced. Based on studies in Cote d'Ivoire and South Africa,22-24 the World Health Organization (WHO) recommends a 7-day postpartum “tail” of zidovudine and lamivudine in settings where resources allow.8,9 These types of regimens are not yet feasible in many resource-limited settings, however, and more complex postpartum regimens may be even more difficult to implement widely.
In conclusion, the findings from this Ugandan study regarding the effectiveness of SDNVP in subsequent pregnancies as compared with first pregnancies support international recommendations to offer SDNVP to HIV-infected women in any pregnancy, irrespective of prior use of NVP for PMTCT, when alternative and more complex regimens are not available or cannot be provided.8 At this time, estimates are that <10% of eligible HIV-infected pregnant women in resource-constrained settings receive some antiretroviral prophylaxis or treatment.25 Furthermore, given high fertility rates and limited family planning options, many HIV-infected women in resource-limited settings may have more than 1 pregnancy, regardless of their HIV status.
Although efforts are continuing to reduce unintended pregnancies and to increase HIV-infected women's access to more effective PMTCT interventions in resource-limited settings, SDNVP is anticipated to continue to play an important role in providing an effective and safe intervention that can be delivered in resource-limited settings and that remains effective to reduce the risk of transmission in subsequent pregnancies among HIV-infected women who have already received NVP for PMTCT in a previous pregnancy.
The authors thank Philippa Musoke, Laura Guay, Betty Kagoda, Fiona Kusasira, Dorothy Nakintu, Agnes Ssemuwemba, Florence Nampewo, Frances Nakayiwa, and the core laboratory staff (MUJHU Research Collaboration) for their participation in and support of this study. They also thank the study participants who volunteered for this study.
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22. McIntyre JA, Martinson N, Gray GE, et al. Addition of short course Combivir (CBV) to single dose Viramune (SDNVP) for the prevention of mother-to-child transmission (pMTCT) of HIV-1 can significantly decrease the subsequent development of maternal and pediatric NNRTI resistant virus [abstract TuFo0204]. Presented at: Third International AIDS Society Conference; 2005; Rio de Janeiro.
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25. McIntyre J. Preventing mother-to-child transmission of HIV: successes and challenges. BJOG. 2005;112:1196-1203.
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