Vitamin A and zinc supplementation among pregnant women to prevent placental malaria: A randomized, double-blind, placebo-controlled trial in Tanzania

Vitamin A and zinc are important for immune function and may improve host defense against malaria and reduce the risk of adverse pregnancy outcomes. Our objective was to determine whether daily oral supplementation with either or both nutrients starting in the first trimester reduces the risk of placental malaria and adverse pregnancy outcomes. We undertook a randomized, double-blind placebo-controlled trial with a factorial design among 2,500 human immunodeficiency virus-negative primigravid or secundigravid pregnant women in their first trimester of pregnancy in Dar es Salaam, Tanzania. We randomly allocated equal numbers of participants to 2,500 IU of Vitamin A, 25 mg of zinc, both 2,500 IU of Vitamin A and 25 mg of zinc, or a placebo until delivery. A total of 625 participants were allocated to each treatment group. Our primary outcome, placental malaria infection (past or current), was assessed in all randomized participants for whom placental samples were obtained at delivery (N = 1,404), which represents 56% of total participants and 62% of all pregnancies lasting 28 weeks or longer (N = 2,266). Birth outcomes were obtained for 2,434 of the 2,500 randomized participants. Secondary outcomes included small for gestational age (SGA) births and prematurity. All analyses were intent to treat. Those who received zinc had a lower risk of histopathology-positive placental malaria compared with those who did not receive zinc (risk ratio = 0.64, 95% confidence interval = 0.44, 0.91), but neither nutrient had an effect on polymerase chain reaction-positive malaria, SGA, or prematurity. No safety concerns were identified. We recommend additional studies in other geographic locations to confirm these findings. This randomized, double-blind, placebo-controlled trial was implemented at 8 antenatal care clinics in the urban Temeke and Ilala districts of Dar es Salaam, Tanzania. The trial was registered at ClinicalTrials.gov on April 30, 2010, as NCT0111478. This report has been prepared in accordance with Consolidated Standards of Reporting Trials (CONSORT) guidelines. Eligible participants were in their first trimester of pregnancy, primigravida or secundigravida, human immunodeficiency virus (HIV) uninfected, and intending to stay in Dar es Salaam for at least 6 weeks after delivery. Participants were recruited at their first antenatal care visit or through a demographic surveillance system (DSS) that was established in the Temeke District to identify early pregnancies in the population. The DSS consisted of two types of operations: 1) a cross-sectional census that enumerated the age and gender of the residents of all households within the geographic location and 2) repeated visits every 2 months to the households of all nulli- and primiparous reproductive-aged women and ascertainment of pregnancy status through a urine test. All first trimester pregnancies identified though the DSS were referred for enrollment at the nearest study clinic. Although initially only women who were less than 8 weeks’ gestation were considered eligible, the gestational cutoff for eligibility was raised to 13 weeks in December 2010 because of low enrollment due to the challenge of identifying early pregnancies. Gestational age was estimated using the first date of last menstrual period (LMP). Between July 12, 2010, and September 17, 2013, 101,184 women were screened for eligibility. Follow-up was completed in June 2014. HIV testing was performed at the first clinic visit using two rapid assays (Alere Determine Walnut Creek, CA, followed by Uni-Gold HIV 1/2; Trinity Biotech; Wicklow, Ireland). Discrepant results were confirmed using enzyme-linked immunosorbent assay (Enzygnost HIV Integral II; Siemens, Marburg, Germany) at the Muhimbili University of Health and Allied Sciences (MUHAS) clinical research laboratory. All women received pre- and posttest counseling. Those who tested positive were referred for standard prenatal care services including antiretroviral therapy. Women who tested HIV negative and provided written informed consent for participation in the trial were enrolled the same day. Participants were individually randomized in equal numbers to receive single tablets containing one of the following regimens: 1) 2,500 IU vitamin A, 2) 25 mg of zinc (as zinc sulfate), 3) both 2,500 IU vitamin A and 25 mg zinc, or 4) placebo and instructed to take the regimen orally each day until delivery. The tablets were indistinguishable from one another in appearance and taste. Allocation to treatment groups was performed according to a computer-generated randomization sequence using blocks of size 20 by a scientist not involved in the data collection. Neither the participants nor the study personnel had access to the masking information. Each study clinic was issued regimen bottles that were prelabeled according to this sequence by study pharmacists who had no contact with participants. At enrollment, each participant was assigned to the next numbered bottle at their site. The study remained blinded until all trial assessments and database cleaning were completed, at which time study staff analyzing the data were given access to treatment assignments. Enrolled participants completed background and food frequency questionnaires, underwent a full clinical examination, provided a blood sample that was tested for a complete blood count (CBC; Ac⋅T 5diff AL [Beckman Coulter, Miami FL]) at the MUHAS clinical research laboratory, and were given a 45-day supply of study regimen. In accordance with Tanzanian standard of care, participants were also provided with iron (60 mg daily) and folic acid (5 mg daily) supplements and vouchers for insecticide-treated bednets issued through a government program. Participants attended monthly visits at study clinics until delivery. At each visit, participants were administered a health questionnaire, given an obstetric examination, and provided with a 45-day supply of regimen. Study staff collected used pill bottles at each visit and counted remaining pills. At 20 and 30 weeks’ gestation, all participants were provided with intermittent preventive treatment in pregnancy malaria prophylaxis in the form of 1,500 mg sulfadoxine and 75 mg pyrimethamine per standard of care in Tanzania. Women were screened by clinic staff for malaria symptoms and tested for malaria parasites as needed. Incident cases were managed according to the Tanzanian Ministry of Health and Social Welfare guidelines.21 Considerable effort was made to reduce loss to follow-up including limiting enrollment to women who intended to deliver within the study area, and close follow-up of all enrolled women, especially in the last weeks of pregnancy. Nonetheless, some participants later left Dar es Salaam for cultural reasons (e.g., at the request of their mothers or in-laws) or delivered at a non-study facility. Birth outcomes were obtained for 2,434 of the 2,500 randomized participants. On-call midwives documented birth outcomes, obtained blood samples for a CBC, and collected, examined, and sampled placentas following a standardized protocol after onsite training by a placental pathologist. Participants also attended a 6-week postpartum clinic visit during which they were administered questionnaires pertaining to their health and their infants’ health and both underwent a physical examination. Placental malaria was evaluated using both histopathology and polymerase chain reaction (PCR). Tissue from fresh sampled placentas was divided for both uses. Histopathologic infections were defined as the presence of malaria pigment or parasitized erythrocytes on the slides. Any evidence of placental malaria, whether acute, chronic, or past, was scored as positive. For histopathologic examination, tissue from a central parenchymal section of the placenta was formalin fixed, embedded, sectioned, and stained using hematoxylin and eosin stain. Slides were examined using light microscopy and under polarized light for the presence of hemozoin and parasitized erythrocytes. A single placental histopathologist (Drucilla Roberts), who was masked to treatment groups and clinical history, classified all slides. Diagnoses were externally confirmed in a subset of 100 slides. Samples from 1,361 of the 1,404 obtained placentas (97%) underwent histopathologic examination. For the nucleic acid studies, tissue was stabilized in RNAlater (Qiagen, Hilden, Germany) and homogenized. Genomic DNA was extracted using DNeasy (Qiagen). Taqman® qRT-PCR (Thermo Fisher Scientific, Waltham, MA) was used for amplification using published primer and probe sets (Plasmodium falciparum-specific22 and general Plasmodium 18S rRNA genes23). The tissue was then tested for PCR inhibitors. Positive and negative controls were included on each plate for quality assurance. PCR-detected infections were defined as the detection of Plasmodium DNA through PCR amplification. Samples from 1,158 of the 1,404 obtained placentas (82%) underwent nucleic acid studies. A number of secondary outcomes were also assessed. These included maternal hemoglobin, infant birthweight, low birthweight (< 2,500 g), very low birthweight (< 2,000 g), small for gestational age (SGA; below 10th percentile for gestational age births, based on the Alexander growth standard24 and the INTERGROWTH-21st standard),25 placental weight, maternal hospitalizations during pregnancy, maternal death, fetal loss (miscarriage and stillbirth), preterm birth (< 37 and < 34 weeks gestational age), perinatal death (> 28 weeks of gestation to 7 days after birth), child death (delivery until 6 weeks postpartum), and prevalence of maternal anemia (hemoglobin [Hb] < 11g/dL) and severe maternal anemia (Hb < 8.5 g/dL). A sample size of 2,500 was determined to provide at least 80% power at a 5% significance level to detect a 35% effect of each intervention on placental malaria risk assuming at least a 12% prevalence of placental malaria, a 30% effect of the other intervention, and a 10% loss to follow-up. All analyses followed the intent-to-treat principle. χ2 tests of independence or Fisher's exact tests and Wilcoxon rank-sum test were used to compare study outcomes between those who received vitamin A or placebo and zinc or placebo. Twin pregnancies were analyzed as a single outcome. The final birthweight for twin pregnancies was the average of the two twin birthweights. If either of the twins was stillborn, low birthweight, or SGA, the pregnancy was classified as such. Exclusion of twins from the birthweight analyses in a post hoc sensitivity analysis did not change our findings. The presence of interaction between vitamin A and zinc was evaluated by comparing nested log-binomial models with and without cross product terms for the two nutrients using the likelihood ratio test. Potential selection bias due to incomplete placenta collection was evaluated using marginal structural models (MSMs) weighted by the inverse probability of having a placental sample collected.26 Weights were calculated as the inverse of predicted values from a logistic regression model of inclusion in the primary endpoint analyses containing the following variables: treatment arm, baseline maternal characteristics, household characteristics, compliance with the regimen, and pairwise interactions between treatment and all maternal and household characteristics. The effect of supplementation was then estimated using weighted log-binomial models with empirical standard errors.27 All P values were two-sided. SAS version 9.2 (SAS Institute Inc., Cary, NC) was used for all analyses. The Harvard TH Chan School of Public Health Human Subjects Committee, the MUHAS Senate Research and Publications Committee, and Tanzania's National Institute for Medical Research granted institutional review board approval for the study. The Tanzania Food and Drugs Authority approved the use of the supplements. Study progress was monitored by a Data safety monitoring board annually.