Infant sex modifies associations between placental malaria and risk of malaria in infancy

Background: Placental malaria (PM) has been associated with a higher risk of malaria during infancy. However, it is unclear whether this association is causal, and is modified by infant sex, and whether intermittent preventive treatment in pregnancy (IPTp) can reduce infant malaria by preventing PM. Methods: Data from a birth cohort of 656 infants born to HIV-uninfected mothers randomised to IPTp with dihydroartemisinin–piperaquine (DP) or Sulfadoxine–pyrimethamine (SP) was analysed. PM was categorized as no PM, active PM (presence of parasites), mild-moderate past PM (> 0–20% high powered fields [HPFs] with pigment), or severe past PM (> 20% HPFs with pigment). The association between PM and incidence of malaria in infants stratified by infant sex was examined. Causal mediation analysis was used to test whether IPTp can impact infant malaria incidence via preventing PM. Results: There were 1088 malaria episodes diagnosed among infants during 596.6 person years of follow-up. Compared to infants born to mothers with no PM, the incidence of malaria was higher among infants born to mothers with active PM (adjusted incidence rate ratio [aIRR] 1.30, 95% CI 1.00–1.71, p = 0.05) and those born to mothers with severe past PM (aIRR 1.28, 95% CI 0.89–1.83, p = 0.18), but the differences were not statistically significant. However, when stratifying by infant sex, compared to no PM, severe past PM was associated a higher malaria incidence in male (aIRR 2.17, 95% CI 1.45–3.25, p < 0.001), but not female infants (aIRR 0.74, 95% CI 0.46–1.20, p = 0.22). There were no significant associations between active PM or mild-moderate past PM and malaria incidence in male or female infants. Male infants born to mothers given IPTp with DP had significantly less malaria in infancy than males born to mothers given SP, and 89.7% of this effect was mediated through prevention of PM. Conclusion: PM may have more severe consequences for male infants, and interventions which reduce PM could mitigate these sex-specific adverse outcomes. More research is needed to better understand this sex-bias between PM and infant malaria risk. Trial registration ClinicalTrials.gov, NCT02793622. Registered 8 June 2016, https://clinicaltrials.gov/ct2/show/NCT02793622. Data were collected from a birth cohort of infants born to HIV-uninfected pregnant women enrolled in a randomised controlled trial of monthly IPTp with DP vs SP (Trial registration, ClinicalTrials.gov; {"type":"clinical-trial","attrs":{"text":"NCT02793622","term_id":"NCT02793622"}}NCT02793622) conducted in Busia district, Uganda, an area of perennial high malaria transmission intensity. Details of the study have been previously reported [5, 16, 17]. Pregnant women were enrolled at 12-20 weeks of gestation and followed through delivery. At delivery, placental blood and tissue samples were collected. Following delivery, all live births were followed up to 12 months of age. Mothers were encouraged to bring their infants to a dedicated study clinic open every day for all their medical care. Routine assessments were conducted every 4 weeks for clinical assessment and collection of blood smears for the detection of parasites by microscopy. Infants presenting with a history of fever in the past 24 h or a documented tympanic temperature ≥ 38.0 °C had a thick blood smear collected for detection of malaria parasites and those diagnosed with malaria were treated according to the Uganda Ministry of Health guidelines. Non-malarial illnesses were treated according to the integrated management of childhood illnesses guidelines. At 12, 28, and 52 weeks of age, blood was collected for haemoglobin measurement. Thick blood smears were stained with 2% Giemsa and read by microscopists [5]. Haemoglobin measurements were made using a spectrophotometer (Hemocue, Angelholm, Sweden). Malaria parasites were detected in placental blood by microscopy and loop-mediated isothermal amplification (LAMP) [18]. Placental biopsy specimens were embedded in paraffin wax, sectioned using a rotary microtome, fixed on glass slides, and dehydrated in sequential ethanol baths [19]. Separate slides were stained in 0.1% haematoxylin and 1% eosin for 5 and 1 min, respectively, or in 2% Giemsa for 30 min and examined for presence of intervillous parasite-infected erythrocytes and malaria pigment by two independent readers. The proportion of high-power fields (HPF) with malaria pigment deposition in fibrin was analysed as described [20]. The primary outcome was the incidence of malaria from birth to 12 months of age. An incident episode of malaria was defined as the presence of fever (history of fever in the past 24 h or a tympanic temperature ≥ 38·0 °C) with a positive thick blood smear not preceded by another malaria episode in the last 14 days. Secondary outcomes included time to first episode of malaria; incidence of complicated malaria (malaria with danger signs or meeting standardized criteria for severe malaria), all-cause hospitalizations; and non-malarial febrile illnesses; prevalence of malaria parasitaemia during routine visits and anaemia (haemoglobin < 10 g/dL); and infant mortality. Data were double entered and verified in Microsoft Access, and statistical analyses conducted using Stata (14.2) including all live births with placental histology results. Follow-up began at birth and ended at 12 months of age or premature study withdrawal. The primary exposure variable, PM, was categorized as follows: no PM (absence of parasites or pigment); active PM (parasites detected in placental blood or tissue by microscopy LAMP or histology, with or without pigment [9, 21]); or past PM (presence of pigment, without parasites). Relationships between past PM and infant malaria incidence were initially evaluated by considering the proportion of HPF with pigment as a continuous variable. Given the non-linear nature of this relationship, past PM was further characterized into 2 groups based on best fit associations with the outcome variable: mild-moderate past PM (> 0–20% HPFs with pigment without parasites); or severe past PM (> 20% HPFs with pigment without parasites). Analyses were stratified by infant sex a priori. Associations between PM and the incidence of malaria were performed using negative binomial regression and adjusted for maternal parasitaemia status at enrollment, IPTp arm, gravidity, housing construction type, and clustering for twin gestation. The cumulative risk of any first episode of malaria was compared using a Cox proportional hazards model. For secondary outcomes, incident and repeated prevalence measures were compared using negative binomial regression model and generalized estimating equations with robust standard errors, respectively. Mediation analysis, using inverse odds weighting (IOW) [22], was used to estimate what proportion of the reported effect between maternal IPTp regimen and malaria incidence in infants [16] was mediated through preventing PM (Additional file 1). In brief, three models were used to conduct IOW mediation analyses. The first model used logistic regression to model treatment given mediator (PM) and mediator-outcome confounders. Predicted probabilities obtained from this model were then used to calculate treatment IOWs for each mother-infant pair. The second and third models used negative binomial regression to model the outcome given treatment with and without weights, respectively. The treatment coefficient from the model with weights estimated the direct effect, which was then subtracted from the treatment coefficient of the model without weights (total effect) to estimate the mediated effect. Bias-corrected 95% confidence intervals (CIs) were computed using bootstrapping. The proportion mediated by PM was calculated by dividing the mediated effect by the total effect. In all analyses, p-values of < 0·05 were considered statistically significant.