The study protocol was approved by the Uganda National Council of Science and Technology and the institutional review boards of the University of California, San Francisco, Makerere University, and the U.S. Centers for Disease Control and Prevention. Written informed consent was sought from all participants before being enrolled in the study.

This study was conducted in Tororo, an area of southeastern Uganda with high malaria transmission intensity and an entomological inoculation rate recently estimated to be 125 infectious bites per person per year in 2011–12 (Grant Dorsey, personal communication). Study participants were women giving birth at Tororo District Hospital (TDH), a government hospital that provides antenatal services and free HIV testing to all pregnant women. Using a cross-sectional study design, all pregnant women with singleton births delivering at TDH who were known to be HIV-uninfected were screened for enrollment if they delivered between Monday 8∶00 am through Friday 4∶00 pm from February 28^th through July 4^th, 2011. Women were enrolled if they fulfilled the following selection criteria: 1) SP use documented from antenatal card if attended antenatal care, 2) HIV status known and negative, 3) absence of reported antimalarial therapy other than SP in the previous 1 month, and 4) provision of informed consent. A standardized questionnaire was administered to all enrolled women including review of their government issued antenatal card. Information collected as part of the questionnaire included demographics, previous pregnancies, bednet use, education level, ownership of household items, and the number and timing of doses of SP (for which administration is directly observed in the antenatal clinic) and other medications. Delivery outcomes were assessed and birth weight obtained using a digital scale (Seca, Birmingham, U.K.). Data on gestational age was not collected because information on last menstrual period was often missing and if present thought to be inaccurate. Biological samples collected included maternal finger prick for blood smear and hemoglobin measurement and placental blood and tissue biopsy.

Hemoglobin measurements from maternal blood were made using a portable spectrophotometer (HemoCue, Ängelholm, Sweden). Maternal and placental thick blood smears were stained with 2% Giemsa for 30 minutes and examined for malaria parasites by standard microscopy. Parasite density was estimated by counting the number of asexual parasites per 200 white blood cells and calculating parasites per µL, assuming a white blood cell count of 8,000 cells per µL. A smear was judged to be negative if no parasites were seen after review of 500 high-powered fields. Final microscopy results were based on a rigorous quality control system with re-reading all blood smears by a second microscopist and resolution of any discrepancies by a third microscopist. PCR for the detection of malaria parasites were performed on placental blood stored on filter paper using nested PCR as previously described [12].

Placental biopsies of approximately 1–2 cm×1–2 cm from the maternal side were collected using scissors and placed in 10% neutral buffered formalin. After 24 hours, biopsies were trimmed with a razor blade to 1×1 cm size and formalin replaced with fresh neutral buffered formalin. Following 1–3 months of storage, placental tissue was embedded in paraffin wax, microtome sectioned and stained with 2% Giemsa, and hematoxylin and eosin. Histopathological slides were examined using standard and polarized light microscopy for hemozoin pigment in intervillous fibrin, malaria parasites, and macrophages with hemozoin pigment using standardized criteria as previously described [13].

Data were double entered in Access (Microsoft Corporation, Redmond, Washington, USA), and analyses performed using STATA (Stata Corp., College Station, Texas, USA). The primary exposure variable of interest was IPTp use with SP as indicated on the participant’s antenatal card. Given the distribution of the number of SP doses reported taken, SP usage was dichotomized into <2 doses vs. ≥2 doses of IPTp. Comparisons of characteristics between the two SP usage groups were made using the chi-squared or t-test. Three definitions of placenta malaria were used: 1) any parasitemia by placental blood smear, 2) detection of parasites by PCR, and 3) any evidence of placental malaria by histopathology. Maternal peripheral parasitemia was defined as a positive blood smear and maternal anemia defined as a hemoglobin level <11 gm/dL. Low birth weight was defined as <2500 gm. Univariate and multivariate analyses of associations between SP usage and outcomes of interest were performed using logistic regression. Covariates adjusted for in multivariate analyses included maternal age, gravidity, bednet use, level of education, transmission season, and a wealth index generated using principal component analysis as previously described [14]. Characteristics used to calculate the wealth index included number of people sleeping in room with participant, and household ownership of electricity, television, mobile phone, radio, bicycle, motorcycle, refrigerator and toilet. A p-value of <0.05 was considered statistically significant.

A total of 581 women were screened and 565 enrolled (Figure 1). Reasons for exclusion included reported antimalarial use in the last month other than SP (n = 10), antenatal card unavailable if attended antenatal clinic (n = 4), HIV status unknown (n = 1), and refusal to provide informed consent (n = 1). Of the 565 women enrolled, 32 (5.7%) women reported to have not taken any SP doses during pregnancy, 202 (35.8%) reported taking 1 dose of SP, 320 (56.6%) reported taking 2 doses of SP, and 11 (2.0%) reported taking 3 doses of SP (Figure 1). Characteristics of study participants stratified by SP usage are presented in Table 1. Maternal age, gravidity, bednet use the prior evening, and delivery during a high transmission season were similar between those with <2 doses of SP and ≥2 doses of SP. Women who reported taking ≥2 doses of SP were more likely to be in the higher wealth index categories (p = 0.008) and had a trend towards a higher level of education (p = 0.06).

Overall the risk of placental malaria as defined by placental blood smear, placental PCR, and histopathology were 17.5%, 28.1%, and 66.2%, respectively. Among 466 placental blood samples that were negative by blood smear, 62 (13.3%) were positive by PCR suggesting the presence of sub-patent parasitemia. Histopathology was the most sensitive measure of placental malaria with 235 of 404 (58.2%) of samples negative for both placental blood smear and PCR having histopathological evidence of placental malaria, the majority (76.2%) of which only had evidence of malarial pigment indicative of past infection. Associations between SP usage and various measures of placental malaria are presented in Table 2. Compared to <2 doses of SP, ≥2 doses of SP was associated with modest trends towards a lower odds of a positive placental blood smear (OR = 0.75, p = 0.25), positive placental PCR (OR = 0.93, p = 0.71), and placental malaria by histopathology (OR = 0.75, p = 0.16), but none of these associations reached statistical significance after controlling for potential confounders. There was no significant interaction between gravidity and associations between SP usage and various measures of placental malaria. Considering only samples with a positive placental blood smear, there was no difference in the geometric mean parasite densities between those with ≥2 doses of SP compared to those with <2 doses of SP (1253 vs. 1267/µL, p = 0.98).

Associations between the duration since the last reported dose of SP taken and the risk of a positive placental blood smear and placental malaria by histopathology are presented in Figure 2. Women who reported taking SP 3–14 days prior to delivery had a significantly lower risk of a positive placental blood smear compared to those who reported taking their last dose of SP more than 14 days prior to delivery (2.8% vs. 17.8%, p = 0.02). Similar findings were seen when considering maternal peripheral parasitemia (2.8% vs. 19.6%, p = 0.01). There was no association between the timing of the last reported dose of SP taken and the risk of active malaria defined as the presence of parasites using histopathology (p = 0.48).

Considering birth outcomes, 545 infants survived, 13 were stillborn, and 7 died shortly after birth prior to discharge. Overall the risk of LBW was 9.9%. Compared to <2 doses of SP, ≥2 doses of SP was associated with a modest trend towards a lower odds of LBW (OR = 0.63, p = 0.11), but this did not reach statistical significance after controlling for potential confounders (Table 2). When evaluating birth weight as a continuous outcome, there was no significant difference between mean birth weight for women who reported taking <2 doses of SP vs. ≥2 doses of SP after controlling for potential confounders (3066 vs. 3093 gm, p = 0.12). Considering maternal outcomes, the overall risk of peripheral parasitemia and anemia were 19.1% and 43.7%, respectively. Compared to <2 doses of SP, ≥2 doses of SP was not associated with peripheral parasitemia (OR = 0.88, p = 0.60) or maternal anemia (OR = 0.88, p = 0.48) after controlling for potential confounders. When evaluating maternal hemoglobin as a continuous outcome, there was no significant difference between mean hemoglobin for women who reported taking <2 doses of SP vs. ≥2 doses of SP after controlling for potential confounders (11.1 vs. 11.2 gm/dL, p = 0.94). Based on a composite outcome, ≥2 doses of SP was associated with a significantly lower odds (OR = 0.52, p = 0.01) of any of evidence of placental malaria, LBW, maternal parasitemia, or maternal anemia (Table 2). There was no significant interaction between gravidity and associations between SP usage and various birth outcomes.