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Lead and malaria both present significant health risks to children in Sub-Saharan Africa. Previous studies have shown that high blood lead levels in children act as a protective factor against subsequent malaria incidence. The main objective of this study was to investigate associations between blood lead level and malaria outcomes prospectively in Beninese children from 12 to 24 months of age. Two-hundred and four children were assessed for lead at 12 months and closely followed until 24 months for malaria; when symptoms and parasite density were also recorded. Univariate and multivariate negative binomial and linear regression models tested associations between blood lead level quartile and total episodes of malaria (total symptomatic and asymptomatic episodes) and parasite density, respectively. Median blood lead level among children measured at 12 months was 56.50 (4.81–578) μg/ L. During the 12-month follow-up, 172 (84.31%) children had at least one malaria episode. Univariate and multivariate negative binomial and linear regressions did not reveal significant associations between blood lead level quartile and malaria outcomes. Iron deficiency was not found to be an effect modifier. Results from this prospective child-cohort study investigating associations between blood lead level and malaria did not confirm results from previous cross-sectional studies. Further research is needed to further explore this relationship and other co-morbidities due to malaria and lead.
This was an observational, prospective cohort study following children born to women enrolled in a randomized clinical trial (Malaria in Pregnancy Preventive Alternative Drugs, MiPPAD, {“type”:”clinical-trial”,”attrs”:{“text”:”NCT00811421″,”term_id”:”NCT00811421″}}NCT00811421) investigating two intervention therapies for malaria during pregnancy in the semi-rural area of Allada, 40 kilometers north of Cotonou, in Benin, Sub-Saharan Africa. Study protocol and inclusion criteria of participants for the clinical trial is explained elsewhere [15]. All singleton children born to mothers from the MiPPAD trial were invited to participate in a subsequent cross-sectional study (TOVI) at 12 months of age [16]. A sub-cohort of these children was then followed closely to measure malaria incidence and parasite density between 12 to 24 months of age within the TOLIMMUNPAL study. Participating children in our analyses included those who met the following inclusion criteria: had mothers who met the original inclusion criteria for the MiPPAD clinical trial, were assessed for blood lead level within the TOVI study between 10 to 13 months of age, and were followed for malaria incidence within the TOLIMMUNPAL study from 12 to 24 months of age. Blood lead level, the primary exposure of interest in this study, was measured once in children ages 10–13 months within the TOVI study. Eight mL of venous blood was collected from each child and 4mL put into a tube with dipotassium EDTA. An aliquot of EDTA blood was diluted 20-fold in ammonia 0.5% v/v and 0.1% v/v surfactant Triton-X and analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Sciex Elan DRC II ICP-MS instrument) at the Centre de Toxicologie, Institut National de Santé Publique du Québec (Québec, Canada). The limit of detection for blood lead analysis was 0.2 μg/L [9]. Blood lead level was analyzed in quartiles due to the non-linear relationship between lead and malaria and in order to better interpret results. There were two primary outcomes of interest within this study, malaria and parasite density in children. Malaria was diagnosed in children from 12 to 24 months of age through a positive thick blood smear test and/or rapid diagnostic test (RDT) within the TOLIMMUNPAL study. A positive thick blood smear test, the gold standard of malaria diagnosis, involves the examination of blood samples using microscopy in order to diagnosis the presence of parasites [17,18]. Pan/Pf RDT (Parascreen), able to give results within 15 minutes of administration, identifies the presence of Plasmodium-specific histidine-rich protein-2 produced in the infected human blood [19]. Each month during the 12-month follow-up, participating children were visited by a nurse, who performed a thick blood smear test to diagnose malaria, regardless of symptoms, and an RDT if they presented with fever (temperature equal to or greater than 37.5°C). A nurse also visited children at home every 15 days, during which time a thick blood smear test and an RDT were administered if children had fever or history of fever in the previous 24 hours. Additionally, children and their families had access to free emergency care and were able to attend the clinic if symptoms occurred. During these visits, a thick blood smear test and an RDT were administered. Symptomatic malaria was defined as a positive thick blood smear and/or RDT and the presence of fever within three days of diagnosis. Parasite density (in parasites/μL) was measured in children positively diagnosed with malaria by a thick blood smear test, through the use of a multiplication factor applied to the average parasitemia/field [14]. Malaria was analyzed as the total number of malaria episodes, total number of symptomatic episodes, and total number of asymptomatic episodes during the 12-month follow-up. Parasite density was log-transformed and analyzed as mean logarithm parasite density per child. Potential confounders included in adjusted models were socioeconomic status (SES), maternal education, iron deficiency, mosquito-net use, malaria status before 12 months, maternity ward location, and environmental risk of infection. Information regarding maternal education, mosquito-net use, and maternity ward were collected via questionnaire given to mothers during follow-up. Serum ferritin concentrations were measured in children using an AxSym Immuno-Assay Analyzer (Abbott Laboratories, Abbott Park, IL) with a sample of 500 mL of serum. Iron deficiency was defined as a serum ferritin concentration less than 12 μg/L or as serum ferritin concentration of 12 to 70 μg/L in the presence of inflammation (CRP concentration > 5mg/L) [20]. In addition to being considered as a potential confounding factor, iron deficiency was also tested as an effect modifier. Malaria status before 12 months was defined as having at least one malaria episode before the lead assessment at 12 months of age. TOLIMMUNPAL researchers collected information on mosquito density, as well as environmental (rainfall, soil type, nearby water sources, vegetation index) and biological data (number of inhabitants per room in household, use of bed nets and/or insecticides) in order to calculate a time- and space-dependent environmental risk of infection quantifying each child’s exposure to malaria vectors using a predictive model [21]. Univariate analyses of associations between the primary exposure of interest, blood lead level, the primary outcomes of interest, total malaria episodes and mean logarithm parasite density, and potential confounders were performed. Confounders found to be associated to the primary exposure and/or outcomes (p<0.20) were kept in final, adjusted models. Multivariate negative binomial regression models were run to examine associations between blood lead level and number of malaria episodes, including number of symptomatic and asymptomatic malaria episodes separately. Linear regression models tested associations between blood lead level and mean logarithm parasite density measured in children. Additionally, characteristics of children lost to follow-up (i.e. not present at 24 months) were compared to those present for the entire follow-up using t-tests, Wilcoxon-rank sum tests, and Fisher exact tests when appropriate. Two sensitivity analyses were conducted: (1) regression analyses after removal of children potentially exposed to lead through contaminated paint chips, and (2) regression analyses including only malaria episodes diagnosed within first 6 months after lead assessment, due to the 30-day half life of lead. Statistical analyses were completed using STATA 14.2 (StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP) for Windows. All studies from which data were used for the purpose of this study were approved by the following bodies: the Hospital Clinic of Barcelona (Spain), the Comité Consultatif de Déontologie et d’Éthique of the Institut de Recherche pour le Développement (France) [15], the University of Abomey-Calavi in Benin and New York University in the United States (IRB#09–1253) [9], and the Beninese Ethical Committee of the Faculté des Sciences de la Santé (FSS).
