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Background: Malaria in pregnancy has been associated with maternal morbidity, placental malaria, and adverse birth outcomes. However, data are limited on the relationships between longitudinal measures of malaria during pregnancy, measures of placental malaria, and birth outcomes. Methods: This is a nested observational study of data from a randomized controlled trial of intermittent preventive therapy during pregnancy among 282 participants with assessment of placental malaria and delivery outcomes. HIV-uninfected pregnant women were enrolled at 12-20 weeks of gestation. Symptomatic malaria during pregnancy was measured using passive surveillance and monthly detection of asymptomatic parasitaemia using loop-mediated isothermal amplification (LAMP). Placental malaria was defined as either the presence of parasites in placental blood by microscopy, detection of parasites in placental blood by LAMP, or histopathologic evidence of parasites or pigment. Adverse birth outcomes assessed included low birth weight (LBW), preterm birth (PTB), and small for gestational age (SGA) infants. Results: The 282 women were divided into three groups representing increasing malaria burden during pregnancy. Fifty-two (18.4%) had no episodes of symptomatic malaria or asymptomatic parasitaemia during the pregnancy, 157 (55.7%) had low malaria burden (0-1 episodes of symptomatic malaria and < 50% of samples LAMP+), and 73 (25.9%) had high malaria burden during pregnancy (≥ 2 episodes of symptomatic malaria or ≥ 50% of samples LAMP+). Women with high malaria burden had increased risks of placental malaria by blood microscopy and LAMP [aRR 14.2 (1.80-111.6) and 4.06 (1.73-9.51), respectively], compared to the other two groups combined. Compared with women with no malaria exposure during pregnancy, the risk of placental malaria by histopathology was higher among low and high burden groups [aRR = 3.27 (1.32-8.12) and aRR = 7.07 (2.84-17.6), respectively]. Detection of placental parasites by any method was significantly associated with PTB [aRR 5.64 (1.46-21.8)], and with a trend towards increased risk for LBW and SGA irrespective of the level of malaria burden during pregnancy. Conclusion: Higher malaria burden during pregnancy was associated with placental malaria and together with the detection of parasites in the placenta were associated with increased risk for adverse birth outcomes. Trial Registration Current Controlled Trials Identifier NCT02163447
This was a cohort study that utilized data from a randomized controlled trial of IPTp in Tororo, Uganda. Tororo is a rural district in southeastern Uganda with an entomologic inoculation rate estimated at 310 infectious bites per person year in 2012 [11]. From June 2014 to October 2014, 300 pregnant women were enrolled into a three-arm, double-blinded, placebo-controlled trial of sulfadoxine-pyrimethamine (SP) given every 8 weeks versus dihydroartemisinin-piperaquine (DP) given every 8 weeks versus DP given every 4 weeks for IPTp. Details of the parent study have been described elsewhere [12]. Briefly, participants were HIV-negative pregnant women of at least 16 years of age, with an estimated gestational age of 12–20 weeks confirmed by ultrasound. For the present study, all 282 women from this cohort with placental histopathology and known birth outcomes were included. At enrollment, pregnant women underwent a standardized history and physical exam including assessment of wealth status, gravidity, gestational age by ultrasound, and age. Each participant received a long-lasting insecticide-treated bed net (LLIN). Women received all their medical care at a designated study clinic that was open daily. Routine visits were conducted every 4 weeks, including collection of dried blood spots (DBS) for LAMP. Participants with positive LAMP results were not treated for malaria. Women were also encouraged to present to the clinic with any illness. Patients who presented with a documented fever (tympanic temperature ≥ 38.0 °C) or a history of fever in the prior 24 h had peripheral blood collected for a thick blood smear. If the blood smear was positive, the patient was diagnosed with symptomatic malaria and treated with artemether–lumefantrine. At delivery, a standardized assessment was completed including evaluation of infant birth weight and gestational age, and collection of specimens within 1 h of delivery, including placental tissue, placental blood smears, and DBS of placental blood. All women were encouraged to deliver at the hospital adjacent to the study clinic. Women delivering at home were visited by study staff as close to the time of delivery as possible for assessment and sample collection. DBS were tested for the presence of malaria parasites using LAMP as previously described [13, 14]. Formalin-fixed paraffin-embedded placental biopsies were processed in duplicate for histological evidence of placental malaria using a standardized case record form by two independent readers as previously described [12]. A third reader resolved any discrepant results. Blood smears were stained with 2% Giemsa and read by two trained laboratory technicians. Smears were considered negative if no asexual parasites were detected in 100 high-powered fields. A third reviewer settled any discrepant readings. The following demographic data was collected: maternal age, possession of bed net at enrollment, wealth status, gravidity, gestational age, and IPTp treatment arm. Wealth status was categorized into lowest, middle, and highest tertiles designed as a composite variable using ownership of several household items and land. Gravidity was grouped as primigravidas (1st pregnancy) and multigravidas (≥ 2 pregnancies). Gestational age at enrollment was confirmed by ultrasound measurements by fetal biometry at less than 20 weeks’ gestation. IPTp treatment arm was categorized as SP given every 8 weeks, DP given every 8 weeks and DP given every 4 weeks. Malaria in pregnancy was defined as both symptomatic malaria and asymptomatic parasitaemia. Symptomatic malaria was measured using passive surveillance and defined as fever with positive blood smear. Asymptomatic parasitaemia was measured using active surveillance every 4 weeks and defined as molecular detection of malaria parasites from a DBS by LAMP. Measures of placental malaria at the time of delivery included: the detection of malaria parasites in placental blood by both microscopy and LAMP, and histopathologic evidence of placental malaria (parasites and/or pigment) from placental biopsies. Birth outcomes assessed included: LBW (< 2500 g), PTB (< 37 weeks gestational age), and SGA (birth weight < 10th percentile for gestational age according to East African fetal weight standards) [15]. In this study, an East African fetal weight standard was used because international growth standards (such as the Intergrowth-21st and the WHO growth curves) have shown greater variance of estimated fetal weight between countries, especially later in gestation [16, 17]. Utilizing the composite international standards above would have under- (Intergrowth-21st) or over-estimated (WHO) the incidence of SGA compared to the East African standards. There were eight cases of twin gestation; four were monochorionic–diamniotic and four were dichorionic–diamniotic. In these cases, outcomes were considered positive if present in at least one placenta and/or child. Data were double entered into a Microsoft Access database. Data analysis was done using Stata 14 (Stata Corp, College Station TX). For baseline characteristics, comparison of proportions was done using the χ2 test and the one way anova test for normally-distributed continuous variables. Generalized linear Poisson regression models with robust standard errors were used to investigate associations between a categorical measure of malaria burden during pregnancy and measures of placental malaria as well as associations between a composite measure of malaria in pregnancy and adverse birth outcomes. Associations were expressed as relative risks. Multivariate analyses included adjustment for which drug was given for IPTp and gravidity. All p values were two-sided and values of < 0.05 were considered statistically significant. Informed consent was obtained from all study participants. Ethical approval was obtained from the Uganda National Council of Science and Technology, the Makerere University School of Medicine Research and Ethics Committee, the Makerere University School of Biomedical Sciences Research and Ethics Committee, and the University of California, San Francisco, Committee on Human Research.
