Epidemiology and Social Science
More than 1 million children have been infected with HIV-1 worldwide. In developing countries, approximately 15% to 30% of infants born to HIV-1-infected women are infected with the virus during pregnancy and another 10% to 20% are infected through breast milk in the absence of intervention.1 Nevirapine (NVP) is a potent HIV-1 reverse transcriptase (RT) inhibitor. Results from the Ugandan HIV Network for Prevention Trials (HIVNET) 012 study demonstrate that a single-dose regimen of NVP given to the mother at the onset of labor and to the infant within 72 hours of birth can dramatically reduce the rate of mother-to-child transmission (MTCT).2,3 The safety and efficacy of NVP prophylaxis in 1- or 2-dose regimens has been confirmed in other studies.4-6 The HIVNET 012 regimen is endorsed by the World Health Organization and is being implemented in resource-limited settings around the world.
Diverse strains of HIV-1 are found around the world. Nine HIV-1 subtypes and 15 circulating recombinant forms (CRFs) account for most HIV-1 strains. Some data suggest that different HIV-1 subtypes may differ in their efficiency of MTCT.7-10 In Uganda, subtypes A and D account for most HIV-1 infections and are present at similar rates.11,12 In this report, we compare the rates of HIV-1 MTCT in Ugandan women in the HIVNET 012 cohort who had subtype A versus D HIV-1 infection.
Study Visits and Results From HIV Network for Prevention Trials 012
The HIVNET 012 study protocol was reviewed and approved by institutional review boards in Uganda and the United States, and informed consent was obtained from all women before enrollment. Women were antiretroviral drug naive before NVP administration and did not receive antiretroviral therapy after the single dose of NVP, consistent with the standard of care in Uganda. Detailed methods and results of HIVNET 012 are presented elsewhere,2,3 including methods for determining infant infection status, baseline HIV-1 RNA levels, and baseline CD4 cell counts.
HIV-1 Genotyping and Subtyping
HIV-1 genotyping was performed using the ViroSeq HIV-1 Genotyping System (ViroSeq; Celera Diagnostics, Alameda, CA).13 Samples collected before administration of NVP, 7 days after administration of NVP, or 6 to 8 weeks after administration of NVP were used for analysis. The resulting pol region nucleotide sequences included the region encoding protease amino acids 1 through 99 and RT amino acids 1 through 324. Those sequences were used for phylogenetic determination of HIV-1 subtype, as previously described.13
A Kaplan-Meier analysis was performed to estimate the rate of MTCT in women with subtype A versus D at birth and at 8 weeks and 18 months postpartum. A second Kaplan-Meier analysis was performed to estimate the rate of MTCT among women with those subtypes whose infants were infected after 8 weeks of age. A Cox proportional hazards model was used to explore the association between subtype and HIV-1 transmission, adjusting for maternal baseline CD4 cell count and viral load. All statistical analyses were conducted using SAS (version 8.2).
Identification of Women With Subtype A and D HIV-1 Infection
In a previous study, HIV-1 subtyping was performed for 279 of the 306 women who received NVP in HIVNET 012 using plasma samples collected 6 to 8 weeks after single-dose NVP administration (all available 6- to 8-week samples).13 Subtyping was performed for 21 additional women using plasma samples collected before or 7 days after administration of single-dose NVP. The 300 women analyzed included 158 with subtype A, 105 with subtype D, 7 with subtype C, and 30 with intersubtype recombinant HIV-1. This subset of women included all 47 of the women whose infants were diagnosed with HIV-1 infection in HIVNET 012 (all transmitters). Among the 263 women who had subtype A or D, 3 were missing end point data. This included 2 women whose infants died before blood samples were obtained and 1 woman where the infant's father refused further study participation. Among the remaining 260 women with subtype A or D, there were a total of 38 infant infections (20 with subtype A and 18 with subtype D).
Transmission Status of Women With Subtype A Versus D
The relation of HIV-1 subtype and MTCT was evaluated by comparing transmission rates among 156 women with subtype A and 104 women with subtype D. The numbers of women with subtype C (n = 7) or intersubtype recombinant HIV-1 (n = 30) were too small for meaningful statistical analysis. In HIVNET 012, infants were evaluated for evidence of HIV-1 infection at birth (1-3 days) and at 6 to 8 weeks, 14 to 16 weeks, 12 months, and 18 months of age. HIV-1 infection was diagnosed by 18 months of age in 38 infants whose mothers had subtype A or D infection. Seventeen (45%) of those infants were diagnosed with HIV-1 infection at birth, and 21 (55%) were diagnosed with HIV-1 infection by 6 to 8 weeks of age. HIV-1 infection was not diagnosed in infants of the remaining 222 women; however, some of the infants of those women died or were lost to follow-up. The number of infants diagnosed with HIV-1 infection at each of the 3 time points was similar among women with subtype A versus D HIV-1 (not shown).
The cumulative transmission rate in women with subtype A versus D was analyzed further using Kaplan-Meier methods (Table 1). Overall, there was no significant difference in the probability of transmission between the 2 subtypes; however, there was a tendency for women with subtype A HIV-1 infection to transmit less (Fig. 1). The 18-month transmission rate was 13.2% for women with subtype A and 18.3% for women with subtype D (log-rank P = 0.34).
In HIVNET 012, 98% of women breastfed their infants. In the NVP arm of the trial, 11 infants whose mothers had subtype A or D were diagnosed with HIV-1 infection after 6 to 8 weeks of age, presumably by breastfeeding. Five of those infants were born to women with subtype A, and 6 were born to women with subtype D. A second Kaplan-Meier analysis was performed to estimate the rate of late transmission among women with subtype A versus D (Table 2). The cumulative rate of late MTCT was 3.8% for women with subtype A (95% confidence interval [CI]: 0.5%-7.1%) compared with 7.6% for women with subtype D (95% CI: 1.6%-13.6%) (P = 0.27). Although there was still a trend toward a higher transmission rate among women with subtype D, the 95% CIs were wide and overlapping. This may reflect the small number of women with late MTCT in this cohort. When interpreting this analysis, one should also consider that infants who died before 6 to 8 weeks of age (not included in this analysis) may have been more likely to be infected with HIV-1.
Relation of Mother-to-Child Transmission to HIV-1 Viral Load and Maternal CD4 Cell Count
Differences in MTCT rates were evaluated further in a Cox proportional hazards model, including maternal baseline (pre-NVP) log10 HIV-1 RNA, maternal baseline CD4 cell count, and subtype as predictors of transmission. In this model, HIV-1 subtype was not a significant predictor of transmission. The relative risk (RR) for transmission (D vs. A) was 1.1 (95% CI: 0.8-1.5; P = 0.52). A high baseline viral load was an independent predictor of transmission (RR = 1.75 per increase in 1 log10 HIV-1 RNA [95% CI: 1.1-2.9; P = 0.03]). In the larger HIVNET 012 cohort of more than 600 women (NVP and zidovudine arms combined), an association was observed between baseline CD4 cell count and MTCT.3 In this subset of 260 women, a low baseline CD4 cell count was not a significant predictor of transmission (RR = 1.2 per decrease of 100 cells [95% CI: 0.99-1.4; P = 0.07]). The lack of an association between baseline CD4 cell count and MTCT in this subset of 260 women (including only women in the NVP arm with subtypes A and D) may reflect the fact that fewer women were studied and that the number of transmitters in the NVP arm of HIVNET 012 was relatively low (ie, this study may be underpowered to detect this association).
Results presented in this report show a tendency for a higher rate of MTCT among women with subtype D versus A. This difference was not statistically significant, however. This may reflect the relatively small sample size of the study or the relatively low transmission rate of MTCT among women who received single-dose NVP prophylaxis. In previous reports, we found a higher rate of NVP resistance among women with subtype D versus A 6 to 8 weeks after delivery (35.7% for subtype D vs. 19% for subtype A; P = 0.0035).13 A higher rate of NVP resistance in women with subtype D postpartum may not have significantly influenced the rate of MTCT in this cohort, however, because most of the HIV-1-infected infants in the NVP arm of HIVNET 012 were diagnosed with HIV-1 infection by 6 to 8 weeks of age (almost half at the time of birth). This suggests that most cases of MTCT occurred in utero or intrapartum before NVP was administered to the mother or infant or before there was an opportunity for selection of NVP-resistant variants. We cannot exclude the possibility that NVP exerts a differential effect on viral infectivity, replication capacity, or transmissibility in subtype A versus D, however, and that such an effect could contribute to a higher rate of HIV-1 MTCT among the subset of infants with subtype D who are infected after NVP dosing. Large clinical trials comparing the rate of MTCT among women with different HIV-1 subtypes would be needed to determine whether the efficacy of the HIVNET 012 regimen is influenced by HIV-1 subtype.
The current study determined HIV-1 subtypes based on pol region sequences. This may not reflect the subtype in other regions of the genome, because intersubtype recombination is common in regions like Uganda, where multiple subtypes cocirculate. In a previous study, 7 (26%) of 27 Ugandan HIV-1 strains had evidence of intersubtype recombination between the gp41 env, p24 gag, and protease regions.14
Other studies have examined the impact of the env V3 subtype on MTCT. In a study of 100 Tanzanian infants, most infants had a single subtype in the env and gag regions (A, C, or D). Thirty-seven infants had recombinant subtypes (A/D or C/D), however. All those 37 infants had subtype A or C in the V3 region. That study suggested that recombinant viruses with subtype D V3 regions may be less fit for transmission to infants compared with recombinant viruses with subtype A or C V3 regions.7 A second study of 51 women from Tanzania suggested that women with subtype D V3 regions were less likely to transmit HIV-1 to their infants than women with subtype A, subtype C, or intersubtype recombinant HIV-1.8 We previously characterized the HIV-1 V3 region from 11 Ugandan women who transmitted HIV-1 to their infants.15 We found approximately equal numbers of women with subtype A and D V3 regions. Vertical transmission of HIV-1 with subtype D V3 regions was directly confirmed by analysis of HIV-1 V3 regions from the infected infants. Furthermore, 2 women who were dually infected with subtype A and D HIV-1 transmitted subtype D HIV-1 to their infants.15 Although those data confirmed that HIV-1 with subtype D V3 regions can be transmitted, differences in the relative transmissibility of HIV-1 with subtype A versus D V3 regions were not evaluated in that study because of the small sample size.
The impact of subtype in the env gp41, gag p24, env C2V3C3, and long terminal repeat (LTR) regions has also been examined. In 1 study, the env gp41 and gag p24 regions were subtyped in samples from 414 Kenyan women. The rate of MTCT was higher for women with subtype D versus A in gp41/p24. Discordant subtypes were found in 25.9% of the women and were associated with a higher rate of MTCT. Women with gp41/p24 subtype combinations of D/D, D/A, and A/D had an increased risk of MTCT compared with women with subtype A/A infection after adjusting for viral load and other factors.9 In contrast to the studies of the V3 region Tanzanian women and infants,7,8 this study suggested that subtype D in gp41 or p24 favored MTCT. Another study compared the LTR subtypes and MTCT rates among 45 Tanzanian women whose infants were infected with HIV-1 and a control group of 45 women with uninfected infants. The 2 groups were matched in terms of the age of the infant at sampling and the calendar year of enrollment. Viruses with subtype A, subtype C, or intersubtype recombinant LTRs were more likely to be transmitted than viruses with subtype D LTRs. The observed effect of subtype was independent of maternal baseline CD4 cell count.10 A smaller study of 31 Tanzanian women analyzed the LTR and C2V3C3 regions. Eight infants were infected with HIV-1. No significant difference was observed in the rate of MTCT among women with A, C, D, or recombinant strains.16 Those studies did not involve antiretroviral drug prophylaxis.
The studies cited previously provide some evidence that HIV-1 subtype may influence the rate of MTCT; however, not all studies show an effect. Further studies are needed comparing MTCT rates in larger cohorts of women with different HIV-1 subtypes. Studies are also needed to define which regions of HIV-1 are most important and to examine variables such as the timing of transmission and the use of antiretroviral drug prophylaxis.
The authors acknowledge the assistance of Melissa Allen (protocol specialist, Family Health International) and thank Estelle Piwowar-Manning, Constance Ducar, and the laboratory staff in Uganda for assistance with sample processing. The authors thank Thomas Flemming (University of Washington) for critical review of the manuscript.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV-1; nevirapine; subtype; mother-to-child transmission; Uganda