Women of child-bearing age comprise approximately half of the estimated 33.3 million people living with HIV-1. Children born to HIV-infected women have an increased risk of adverse birth outcomes, including congenital HIV-1 infection and low birthweight (LBW).1–3 Malaria is also prevalent among pregnant women, and children born to women with malaria also have an increased risk of LBW.4 LBW is defined as “infants born weighing ≤2500 g, regardless of gestational age or cause of LBW,” and it can be caused by preterm delivery (PTD, birth before 37 week's gestation), intrauterine growth restriction, or congenital/genetic abnormalities. LBW is a significant cause of infant morbidity and mortality.5 In 2010, PTD was the second leading cause of child deaths worldwide,6 and Malawi had the highest prevalence of PTD in the world.7
The placenta provides fetal nourishment and serves as a barrier to fetal infection, and deficiencies in placental function are causally linked to obstetric complications.8 Infections that colonize the placenta, such as Plasmodium falciparum, are associated with adverse birth outcomes.9,10 Plasmodium falciparum parasites can be sequestered in the intervillous space of the placenta,11 and placental malaria has been associated with higher HIV-1 concentration in both peripheral and placental plasma.12 Our group13 and others14 have shown that HIV-1 replicates in the placenta, and it has also been shown that HIV-1 infection alters the cytokine profile in the placenta.15–17 Thus, when evaluating risk factors for adverse birth outcomes, potential interactions between malaria and HIV-1 coinfection should be considered.18 The objective of this study was to determine if the severity of HIV-1 infection is associated with LBW or PTD.
We describe a secondary analysis of data collected through The Malaria and HIV-1 in Pregnancy (MHP) prospective cohort study. The primary purpose of the parent study was to determine the association between malaria and HIV-1 mother-to-child transmission.19 From December 2000 to March 2004, women who presented for delivery to the Antenatal Ward at the Queen Elizabeth Central Hospital in Blantyre, Malawi, were screened for eligibility for MHP. Women were ineligible if they were in the active phase of labor, were participating in other research studies, lived outside Blantyre district, were younger than 15 years, were hypertensive, had altered consciousness, delivered a stillbirth, or twins. HIV-infected women and their offspring received nevirapine according to the HIVNET 012 protocol. Only women who delivered at the Queen Elizabeth Central Hospital were included in this study.
Gestational age at delivery was self-reported. Maternal CD4+ T cells were quantified by FACScan, and HIV-1 RNA was quantified using Amplicor HIV-1 Monitor v1.5 (Roche Diagnostics, Branchburg, NJ). Peripheral and placental thick blood films were Giemsa stained and examined for P. falciparum parasites (malaria) by 2 trained microscopists. Thick smears were considered P. falciparum negative if parasites were not observed after counting 200 leukocytes. Full-thickness placenta sections were formalin fixed and paraffin embedded according to the standard procedures. After Giemsa staining, placenta sections were examined for malaria parasites.19 Women were considered syphilis seroreactive if they were both RPR and TPHA positive, as described.20 Anemia was defined as hemoglobin <11 g/dL; “any malaria” was defined as a positive malaria diagnosis on peripheral blood smear or placental blood smear or histology. Infant HIV-1 status at birth was determined by real-time polymerase chain reaction.19 Chorioamnionitis was assessed by histopathologic examination according to the published methods.21
Statistical analysis was performed with STATA (version 10.1) and SAS (version 9.2). Using binomial regression models, we assessed the unadjusted and adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs) of the effect of HIV-1 severity on prevalence of adverse birth outcomes. We characterized HIV-1 severity through 3 separate primary exposure variables: (1) continuous placental HIV-1 viral load, (2) continuous maternal peripheral HIV-1 viral load, and (3) continuous maternal CD4+ T-cell count. We characterized adverse birth outcomes through 2 separate, primary dichotomously-coded outcome variables: LBW and PTD. We separately assessed each exposure–outcome relationship to produce unadjusted and adjusted PRs. We selected binomial regression for multivariable modeling because the prevalence of each outcome was >10%, the cutoff value above which logistic regression is not recommended.22 All three HIV-1 severity variables met the assumption of linearity in the log risk for each outcome. Multivariable models were constructed as follows. Using product–interaction terms, we evaluated whether any of the exposure–outcome associations under investigation varied by maternal malaria status or maternal anemia. A P value for any interaction term of <0.20 was interpreted as evidence of meaningful modification. We developed a starting list of potential confounders of each exposure–outcome association using Directed Acyclic Graphs.23 Variables assessed for possible confounding included those previously associated with either HIV-1 severity or adverse birth outcomes, and included demographic characteristics (age, education, rural/urban residence), clinical factors (intermittent preventative therapy for malaria during pregnancy, maternal syphilis, anemia, chorioamnionitis, primigravid status), and infant factors (gender). To construct the final models, we used a manual, backward elimination, change-in-estimate strategy,24 with a 10% threshold for variable retention. To ease interpretability, any variable surviving backward elimination for any of the models was retained in all multivariable models.
Our primary analyses evaluated the effect of HIV-1 severity separately on prevalence of LBW and PTD. We undertook 3 sensitivity analyses to assess the robustness of our findings. First, because preterm infants will naturally have lower birthweight because of their shorter gestation, we repeated our LBW analyses after removing from the data set all observations with PTD. We then reran our unadjusted and adjusted multivariable models (using the same set of adjustment variables as in the main analysis) to see whether any increase in prevalence of LBW due to increased severity of HIV-1 disease persisted in term infants. Second, because fetal HIV-1 status may be associated with adverse birth outcomes, we repeated both the LWB and PTD analyses after restricting the data set to only children confirmed to be HIV negative at delivery. We again reran the unadjusted and adjusted models to determine whether the associations between HIV-1 disease severity and adverse birth outcomes changed. Third, to further assess potential modification of the exposure–outcome association by malaria status, we examined separately malaria diagnosed by peripheral blood smear versus placental blood smear versus placental histology.
MHP was approved by the College of Medicine Research Committee at the University of Malawi and the Institutional Review Boards of the University of Michigan and the University of North Carolina at Chapel Hill. The analyses presented in this manuscript also received Institutional Review Board approval from the Ohio State University.
Characteristics of HIV-Positive Participants Who Delivered Live, Singleton Infants in the Malaria and HIV-1 in Pregnancy Study, Malawi
Among the HIV-infected women in the parent Malaria and HIV-1 in Pregnancy (MHP) cohort, 809 live singleton deliveries occurred, 80% via vaginal delivery. Participant's median age at enrollment was 24 years (interquartile range: 21–28 years), and the median gravidity was 2 (interquartile range: 1–4 pregnancies) (Table 1). A majority of the participants had attended primary school (91%), were unemployed (75%), and lived in an urban setting (76%). At the time of delivery, 56% of the participants had anemia, 26% had histological evidence of chorioamnionitis, and 7% were syphilis seropositive. One quarter of the participants were primigravid. Most (86%) participants took iron–folate supplements during the pregnancy, and 93% received at least 1 dose of intermittent preventative therapy for malaria during pregnancy for malaria (Table 1). With regard to malaria, 10 of the women were peripheral blood smear positive, 8% were placental blood smear positive, and 18% had histological evidence of malaria in a placental biopsy. Because all HIV-positive women in the MHP study received their HIV-1 diagnosis at enrollment, all participants were antiretroviral treatment naive; 95% of the HIV-infected participants received single-dose nevirapine to prevent HIV-1 mother-to-child transmission.
LBW and PTD Are Common in Malawi
In this cohort of HIV-infected women, 21% of participants delivered a LBW infant and 16% delivered preterm. Age, malaria status, and gravidity were not associated with prevalence of LBW or PTD (Table 2). Anemia was more common among women with infants with LBW (68%) and PTD (68%) compared with women with normal weight term infants (52%). Chorioamnionitis was also more commonly observed in the LBW (33%) and PTD (37%) groups compared with the normal weight term delivery group (23%). Female sex was more prevalent among LBW infants (57%) compared with normal weight term infants (46%). Finally, syphilis seropositivity was more common among mothers of PTD infants (11%) than normal weight term infants (6%) (Table 2).
Placental Viral Load Correlates With Birthweight and Gestational Age
Maternal peripheral HIV-1 viral load was inversely correlated with continuous infant birthweight (correlation coefficient = −0.08, P = 0.06), but not with continuous duration of gestation (correlation coefficient = −0.05, P = 0.5) (Figs. 1A, C). HIV-1 concentration in the placenta was inversely correlated with both birthweight (correlation coefficient = −0.15, P = 0.01) and duration of gestation (correlation coefficient = −0.20, P = 0.0008) (Figs. 1B, D).
The effect of HIV-1 disease severity on prevalence of LBW differed meaningfully by maternal malaria status. Residence, education level, primigravid status, and anemia were retained as confounders in adjusted multivariable models. Among women with malaria, we observed no significant association between any measure of HIV-1 disease severity and prevalence of LBW in unadjusted or adjusted analyses (Table 3). Among malaria-negative women, for all measures of HIV-1 disease severity, more severe HIV-1 disease was significantly associated with increased prevalence of LBW in both unadjusted and adjusted models. The adjusted PR for a 1-log increase in placental HIV-1 viral load was 1.22 (95% CI: 1.00 to 1.48); for a 1-log increase in peripheral HIV-1, viral load was 1.38 (95% CI: 1.08 to 1.77); and for a 100-cell per microliter decrease in CD4+ T cells was 1.12 (95% CI: 1.05 to 1.21) (Table 3).
The effect of HIV-1 disease severity on prevalence of PTD also differed by maternal malaria status (Table 4). Among malaria-positive women, neither placental viral load nor CD4+ T-cell count were significantly associated with PTD in unadjusted or adjusted analyses, but a 1-log increase in peripheral viral load was significantly protective against PTD in adjusted analyses (PR: 0.56, 95% CI: 0.47 to 0.85). Among women without malaria, higher placental viral load and lower CD4+ T-cell count were both significantly associated with increased prevalence of PTD (adjusted PR for a 1-log increase in placental viral load: 1.29, 95% CI: 1.02 to 1.63; adjusted PR for 100-cell per microliter decrease in CD4+ T cells: 1.16, 95% CI: 1.05 to 1.28). Increases in peripheral HIV-1 viral load did not significantly increase prevalence of PTD in malaria-negative women (Table 4).
The Association Between HIV-1 and LBW Remains After Exclusion of PTD and HIV-Infected Infants
When the data set was restricted to include only infants born after 37 weeks' gestation (n = 662), the associations between HIV-1 severity and prevalence of LBW among women with malaria were strengthened, but remained not statistically significant: the adjusted PR for a 1-log10 increase in placental viral load was 1.64 (95% CI: 0.98 to 2.74) compared with 1.26 (95% CI: 0.87 to 1.83) in the primary analysis (see Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A445). The PR for LBW for a 1-log10 increase in peripheral HIV-1 viral load was 1.66 (95% CI: 0.80 to 3.47) compared with 0.88 (95% CI: 0.57 to 1.35) in the primary analysis. However, excluding these observations had almost no affect on the magnitude of the associations between HIV-1 severity and prevalence of LBW in women without malaria. Given the reduced sample size, many of the PRs are no longer statistically significant, but the strength of the associations was essentially unchanged (see Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A445).
When the data set was restricted only to observations with infants known to be HIV negative at birth (n = 686), we observed no meaningful changes in the associations between HIV-1 severity and prevalence of LBW or PTD for malaria-positive or malaria-negative women (see Table S2 and S3, Supplemental Digital Content, http://links.lww.com/QAI/A445). Thus, the observed associations in our primary analysis between HIV-1 severity and LBW or PTD are unlikely to be explained by infant HIV-1 status at birth.
Our primary analysis established that most associations between HIV-1 severity and birth outcomes were meaningfully different by malaria status. Our final sensitivity analysis assessed whether our findings were robust to the method of malaria diagnosis; in other words, was the same pattern observed for women with malaria diagnosed by peripheral blood smear, placental blood smear, and placental histology? For both LBW and PTD, the patterns observed in the primary analysis were repeated in the sensitivity analysis, with no meaningful differences by method of malaria diagnosis (see Table S4 and S5, Supplemental Digital Content, http://links.lww.com/QAI/A445).
In this secondary analysis of a large cohort of HIV-infected pregnant women who delivered a live singleton infant in Malawi between 2000 and 2004, we tested the hypothesis that severity of HIV-1 infection is associated with risk of LBW or PTD. This cohort is unique because all women were infected with HIV-1 subtype C, were antiretroviral treatment naive, were rigorously tested for malaria, and were normotensive. This study was not designed to comprehensively determine significant predictors of LBW or PTD in HIV-infected women; rather, it was designed to characterize the independent associations between 3 specific measures of HIV-1 disease severity, LBW, and PTD, adjusting for factors that confounded or modified those associations. Although our malaria-positive sample size was small, HIV-1 severity in this group appeared not to be associated with adverse birth outcomes. However in malaria-negative women, maternal HIV-1 disease severity was significantly associated with increased prevalence of LBW and PTD. This association persisted in multivariate models adjusted for residence, primigravid status, maternal education, and maternal anemia, and the result was also robust to several sensitivity analyses, including exclusion of PTD, exclusion of infants who became HIV infected in utero, and stratification by mode of malaria diagnosis.
The average peripheral viral load of participants in this study, all of whom were antiretroviral treatment naive, was approximately 32,000 copies per milliliter. The adjusted PR for LBW corresponding to a 3-log increase in viral load among women without malaria is 2.82 (95% CI: 1.32 to 6.07), meaning that women with average viral loads have 3 times the prevalence of LBW as women with an undetectable viral load. Administration of antiretroviral therapy (ART) that reduces viral load to an undetectable limit could lead to extremely significant relative reductions in LBW among malaria-negative Malawian women.
An association between maternal HIV-1 infection and LBW has been previously observed,1,2 including several studies in Malawi.3,18 In a meta-analysis of 6 cohorts of HIV-infected Malawian women, 13% of the infants born to HIV-infected women had LBW, although malaria status was not reported.3 In this study, 21% of the infants born to HIV-infected women were LBW, more than double the 8% prevalence of LBW among HIV-negative women in the MHP cohort (Jess Kwiek, PhD, 2013, unpublished observation). Our findings are consistent with the results of a study from Lusaka, Zambia, which quantified the association between maternal viral load and infant birth outcomes, and found that greater HIV-1 disease severity, characterized as peripheral viral load above 100,000 copies per milliliter, was associated with decreased infant weight and increased infant mortality and morbidity.25 Several US-based studies have also quantified the association between HIV-1 disease severity and LBW (or intrauterine growth restriction). Lambert et al26 found that neither HIV-1 RNA copy number nor CD4+ T-cell counts were associated with LBW, but increasing HIV-1 culture titer was associated with LBW; these authors also observed that timing of ART initiation—before or during pregnancy—was not associated with LBW. Tuomala et al27 compared women who received ART to ART-naive women and found no difference in the prevalence of LBW between the treated and untreated women. Although the authors did not report viral loads, presumably the women on ART had lower viral loads than the untreated women, and thus it can be inferred that viral load was not associated with LBW.
The relationship between severity of HIV-1 infection and PTD differed slightly from the LBW results. Among women without malaria, decreasing CD4 T-cell count, and increasing placental viral load, but not increasing peripheral viral load, was associated with increased prevalence of PTD. Unexpectedly, among women with malaria, increasing peripheral viral load was protective against PTD. Although the basis for this observed association is currently unknown, 1 study suggests that HIV-1 infection can suppress the innate immune response to P. falciparum,28 which has the potential to modulate inflammation and onset of delivery.29,30 Additional analyses are required to determine if this observation is biologically relevant or if it reflects an uncharacterized bias.
There are several limitations to our findings. We cannot determine if the observed associations between severity of HIV-1 infection and LBW are a direct effect of HIV-1 or if the association is secondary to HIV-associated nutritional deficiency; the sample size for the malaria-positive stratum was smaller than the sample size for the malaria-negative stratum; neither prepregnancy body mass index nor other measures of nutritional status were available for analysis; gestational ages were self-reported, and no other measures of gestational age (eg, ultrasonographic fetal measurements31) were available; the results are generalizable only to ART-naive HIV-infected women, with normal blood pressure, who delivered in a hospital. Finally, cigarette smoking, a known LBW and PTD risk factor, was not assessed; however, estimates suggest that <10% of the adult population in Malawi smoke cigarettes.32
Normal fetal growth represents a finely tuned immune state requiring a temporary immune tolerance to the fetal-placental unit while still enabling fetal protection from pathogens. Placental inflammation, which could result from a coinfection, can disrupt this tenuous immunological balance and cause LBW. However, clinical data from the MHP cohort suggest that coinfection with malaria or syphilis is not driving HIV-associated LBW. Furthermore, although chorioamnionitis was associated with PTD and LBW, it was not a confounder of the HIV-1 severity associations: to be a confounder, a variable must be associated with both the exposure (HIV-1 severity) and the outcome (LBW or PTD). The mean placental viral load among women with chorioamnionitis was 3.84 log10 copies per milliliter compared with 3.73 log10 copies per milliliter among women without chorioamnionitis (P = 0.5); the mean log-transformed peripheral viral load among women with chorioamnionitis was 4.43 log10 copies per milliliter compared with 4.39 log10 copies per milliliter among women without chorioamnionitis (P = 0.6), and the mean CD4+ T-cell count among women with chorioamnionitis was 380 cells per microliter compared with 386 cells per microliter among women without chorioamnionitis (P = 0.9). Additional studies to determine the prevalence of other pathogenic microbes in this cohort would address the possibility that these results are confounded by a heretofore undetected coinfection.
Following the paradigm of HIV-associated disruption of the blood–brain barrier33 and HIV-mediated impairment of the mucosal epithelial barrier,34 a working hypothesis posits that HIV-1 replication impairs placental function and causes LBW. HIV-1 can replicate in the placenta and it has been detected in placental cells in vivo,35 and data from several groups indicate that HIV-1 can infect ex vivo placental explant cultures.36,37 Our previous work has shown that the placenta represents a unique and hospitable niche for HIV-1 replication; specifically, we observed that the concentration of HIV-1 in the placenta was different than the concentration of HIV-1 in peripheral blood.13 In addition to the pathogenic effects on HIV-infected cells, it is also plausible that cellular exposure to HIV-1 could be pathogenic and disrupt placental function.
In summary, indicators of severe maternal HIV-1 infection, defined as high placental or peripheral viral load or a low CD4+ T-cell count, were associated in a dose-dependent manner with increased prevalence of LBW. This association was only observed in malaria-negative women, and it was not a secondary effect of PTD or in utero HIV-1 infection. The mechanism of HIV-associated LBW remains unknown.
The authors are grateful for the participation of the Malawian women and their newborns and the Malaria and HIV-1 in Pregnancy Cohort Staff.
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