HIV superinfection occurs when an HIV-infected individual is infected with a new, phylogenetically distinct HIV strain . HIV superinfection has been demonstrated in multiple cohorts around the world, and occurs at varying rates that can be similar to the corresponding primary HIV incidence rate in the population, although this has not been observed in all studies [2–7]. Rates of HIV superinfection appear to be higher in populations at increased risk of primary HIV infection, such as female sex workers . HIV superinfection can cause temporary and sustained increases in viral load, which could increase the risk of HIV transmission to other individuals [4,9,10]. Examining the role of superinfection and sexual transmission in HIV discordant sexual partnerships is difficult because it is often impossible to establish whether the superinfection event occurred prior to the subsequent transmission, or vice versa . In contrast, studying superinfection in the context of mother-to-child transmission (MTCT) of HIV through breastfeeding is simpler because there is no question about the directionality of superinfection and subsequent transmission, and because there are fewer confounding behavioral factors.
The Post-Exposure Prophylaxis of Infants trial in Malawi (PEPI-Malawi, 2004–2009) demonstrated that the risk of MTCT of HIV via breastfeeding was significantly reduced by providing infants an extended regimen of nevirapine or nevirapine plus zidovudine prophylaxis up to age 14 weeks, compared with infants who received a short regimen for prophylaxis (single dose nevirapine with 1 week of zidovudine) . Several studies in sub-Saharan Africa, including Malawi, have demonstrated that postpartum women are at increased risk of HIV infection, particularly at the time when sexual activity is resumed [12–14]. Genital tract infections and risk behaviors were the main factors associated with HIV acquisition postpartum. It is not known whether there is an increased risk of HIV superinfection in HIV-infected women postpartum or whether maternal HIV superinfection increases the risk of MTCT of HIV during breastfeeding.
Women provided written informed consent for participation in the PEPI-Malawi trial (NCT00115648). The study was approved by the institutional review boards in Malawi and the United States. .
A detailed description of the PEPI-Malawi trial has been described previously . Briefly, eligible HIV-infected pregnant women presenting for either antenatal or delivery services in Blantyre, Malawi, were offered participation in the PEPI-Malawi trial. Infants who were HIV uninfected at birth were then randomized to either a control regimen (single-dose nevirapine and 1 week of zidovudine, the standard of care at the time) or the control regimen and one of two extended antiretroviral prophylaxis regimens (daily nevirapine, or daily nevirapine plus zidovudine) for up to 14 weeks of age . Women whose infants tested HIV-negative at 6 weeks of age and HIV-positive by 24 months of age were identified as transmitters. Transmitters were included in the current study if they had plasma samples available at both baseline and at the time of their infant's HIV diagnosis, or at a later time point up to age 24 months (n = 120). Transmitters were included in the analysis if their samples were successfully amplified and analyzed by next-generation sequencing (NGS), for at least one genomic region of both samples screened (n = 91). Transmitters were initially matched as a group on study arm and duration of follow-up to women who did not transmit HIV to their infants (nontransmitters, n = 454). Appropriate baseline and follow-up samples according to length of follow-up were available for 164 nontransmitters. Amplification and NGS of at least one genomic region of both samples was successful for 91 nontransmitters who were included in the current study. HIV viral load and CD4+ cell count testing were performed on baseline samples in the PEPI-Malawi trial .
HIV superinfection analysis
Maternal plasma samples were analyzed using NGS to identify HIV superinfection, as described previously [1,8]. Briefly, HIV RNA was extracted from 140 μl plasma, reverse-transcribed, and amplified using a nested PCR to produce amplicons corresponding to portions of the viral p24 (∼390 basepairs) and gp41 (∼324 basepairs) genomic regions. Samples that were successfully amplified for both study visits (baseline and follow-up) in at least one region were sequenced using the 454 DNA sequencing platform as previously described, with adjustments to use a 2-region format (Roche, Branford, Connecticut, USA) [1,2,8]. Pools of samples were processed using emPCR Amplification Manual-Lib-L-LV – June 2013 (Roche, Branford) using 25% of the recommended amplification primer amount and a 0.2 copy-per-bead ratio .
The resulting sequencing reads were analyzed and similar sequences were combined into a single consensus sequence. Consensus sequences that encompassed a cluster of at least 10 individual near-identical sequence reads were determined [1,2]. In order to remove any residual contaminating sequences, a representative sequence from all distinct viral populations for each sample run in a given NGS-sequencing plate were combined in a neighbor-joining tree, and any microcontamination or spillover sequences that localize with another unrelated sample were removed. The clean sequences from the women's two sample time points were analyzed in a neighbor-joining tree with a variety of HIV reference sequences containing representative sequences of the major African subtypes (n = 23 for p24 and n = 25 for gp41) as well as a collection of sequences from Malawi (n = 13 for p24, and n = 30 for gp41).
HIV superinfection was defined when the follow-up sample yielded two or more distinct consensus sequences forming a phylogenetic cluster that was distinct from the consensus sequences in the baseline sample, and were of adequate genetic distance from the baseline sequences to rule out evolutionary drift . Genetic distance cut-off for HIV superinfection was at a rate of at least 0.59% per year for the p24 region, or at least 0.98% per year for the gp41 region, which is equal to the mean plus twice the standard deviation of the intraperson viral divergence from a previous analysis . Any superinfection events were reamplified and sequenced in a separate reaction to confirm superinfection . The consensus sequences for gp41 and p24 are available upon request (firstname.lastname@example.org).
Differences between women in the transmitter and nontransmitter groups were compared using Fisher's exact test for categorical variables. For continuous variables, medians and interquartile ranges (IQR) were reported and nonparametric tests were used for comparisons as data were not normally distributed (Wilcoxon rank-sum test/Kruskal–Wallis test). Similar methods were used to compare characteristics across three groups of nontransmitters to assess for possible bias as follows: all nontransmitters, nontransmitters selected for sequencing, nontransmitters with samples and NGS results available for analysis. The association between HIV superinfection and transmission risk was examined by multiple logistic regression adjusted for maternal age, baseline log10 viral load, and baseline CD4+ cell count. Rates of superinfection were calculated as confirmed superinfection events divided by person-years of follow-up. Incidence rate ratios were estimated using Stata 11 (StataCorp, College Station, Texas, USA). Risk factors for HIV superinfection were examined by logistic regression; PEPI study arm, baseline CD4+ cell count, and breastfeeding duration were included in the multivariate model.
Characteristics of the 91 nontransmitter women included in this analysis were not significantly different from those of all other nontransmitting women enrolled in the PEPI-Malawi trial (Table 1). Nontransmitters analyzed for HIV superinfection had significantly higher baseline CD4+ cell counts (P = 0.001), lower baseline HIV viral loads (P < 0.0001), and slightly shorter breastfeeding duration than transmitters (n = 91, P = 0.048, Table 1). Transmitters and nontransmitters included in the analysis did not differ significantly in age or time to postpartum resumption of sexual activity (Table 1).
Five cases of superinfection were identified in women from the transmitter group compared with five in the nontransmitter group (P = 1.0, Table 1, Fig. 1, and supplementary Figure 1). Three of the 10 superinfection events involved complete replacement of the original strain by the new superinfecting strain . The rates of superinfection in the transmitters and nontransmitters were 4.7/100 person-years (95% confidence interval [CI] = 1.5–11.0) and 4.4/100 person-years (95% CI = 1.4–10.3) respectively (incidence rate ratios = 1.1, 95% CI = 0.3–4.7; P = 0.92). As part of the original trial, women were asked whether they had a sexual partner (excluding husband) since their last visit, and whether they were using condoms with that partner. Of the women examined here, 180 reported no extra sexual partners, with two women having missing data; therefore, no association was found between extra partners and risk of superinfection.
Maternal HIV superinfection did not increase the odds of MTCT of HIV via breastfeeding when controlling for maternal age, baseline CD4+ cell count, and baseline viral load (adjusted odds ratio = 2.32, 95% CI = 0.47–11.47; P = 0.30, Table 2). In univariate and adjusted models, longer breastfeeding duration was borderline significantly associated with a lower risk of HIV superinfection after controlling for baseline viral load and PEPI study arm (P = 0.05, Table 3).
HIV superinfection has been observed in multiple populations and settings, and is associated with underlying HIV risk in the same populations . Postpartum women in Malawi have been shown to be at increased risk for primary HIV infection . The rate of HIV superinfection observed among HIV-infected women in the PEPI-Malawi trial corroborates this risk of exposure and vulnerability to HIV primary infection in the postpartum period. The study also demonstrated a significant association of maternal viral load with MTCT of HIV, which is a well established risk factor. Longer breastfeeding duration was associated with decreased odds of superinfection, although the mechanism for this relationship is not clear. A previous analysis of this population found that women who breastfed had a 48% lower likelihood of resuming sexual activity early, which may be associated with the lower risk of superinfection . Additionally, in at least four of the transmission events, the mothers reported cessation of breastfeeding prior to the period when their child was infected. It should be noted that this study examined viral populations from the mothers only, and did not address what viral strain transmitted to their infants.
One limitation of this study was that the transmitter group was selected for analysis based on the assumption that MTCT would be associated with superinfection. Although this association was not seen in this population, inclusion of a significant proportion of transmitters in this study (50% of the women assessed for superinfection) may have biased the overall rate of superinfection. Another limitation of this study is that for five of the women who appeared to be superinfected, a sample available post-superinfection was not possible because their superinfection event was detected in samples collected at their last study visit. Additionally, this study was a secondary analysis of a well studied cohort of patients, and therefore in the other five cases later samples were not readily available. These facts limited the ability to verify the superinfection events in a second sample post-superinfection, and mean that in the cases of full replacements, a sample mix-up cannot be ruled out. However, all superinfection events were reamplified to confirm superinfection, which should help address this concern. The limited sample volume for these samples also did not allow performing HIV viral load analysis on the follow-up samples, although the enrollment viral load was previously measured as part of the original trial.
Case studies, as well as a cohort study of Kenyan female bar workers, have demonstrated that HIV superinfection is associated with both transient and persistent increases in HIV viral load, one of the main drivers of HIV transmission [4,9,10,17,18]. It is unknown what role HIV superinfection has on MTCT or sexual transmission, but given the effect it has on viral load, it is possible that superinfection could temporarily increase the infectiousness of the individual. However, it is difficult to establish the directionality and relationship of superinfection and transmission events in the setting of sexual transmission [4,7]. This issue is not relevant in MTCT of HIV, as transmission only occurs vertically. In this study, although the rate of superinfection was significant in women after delivery, superinfection was not associated with an increased risk of MTCT of HIV through breastfeeding. It should be pointed out that this study might have been underpowered to detect this association.
The current best clinical practice in Africa for prevention of MTCT of HIV is to provide all HIV-infected mothers with HAART during pregnancy and to continue antiretroviral therapy for the rest of their lives (i.e. Option B+) . Continued provision of antiretroviral therapy after delivery should reduce the risk of HIV superinfection. The high rates of superinfection observed in this study support the use of Option B+ moving forward, as it is likely to reduce the risk of MTCT and potential adverse effects of HIV superinfection, such as higher viral loads.
The authors would like to thank the study participants and staff in Malawi.
Source of funding: This study was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by grants UM1-AI068613 and 1R01-AI087139 (S.H.E., principal investigator). The P.E.P.I. trial was supported by a cooperative agreement (5-U50-PS022061–05; award U50-CC0222061) from the Centers for Disease Control and Prevention and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. ClinicalTrials.gov number, NCT00115648.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2015 Wolters Kluwer Health, Inc.
breastfeeding; HIV; mother-to-child transmission; superinfection