Asymptomatic Plasmodium infection and cognition among primary schoolchildren in a high malaria transmission setting in Uganda

Asymptomatic parasitemia is common among schoolchildren living in areas of high malaria transmission, yet little is known about its effect on cognitive function in these settings. To investigate associations between asymptomatic parasitemia, anemia, and cognition among primary schoolchildren living in a high malaria transmission setting, we studied 740 children enrolled in a clinical trial in Tororo, Uganda. Parasitemia, measured by thick blood smears, was present in 30% of the children. Infected children had lower test scores for abstract reasoning (adjusted mean difference [AMD] -0.6, 95% confidence interval [CI] -1.01 to -0.21) and sustained attention (AMD -1.6 95% CI -2.40 to -0.81) compared with uninfected children. There was also evidence for a dose-response relationship between parasite density and scores for sustained attention. No associations were observed between anemia and either test of cognition. Schoolchildren in high transmission settings may experience cognitive benefits, from interventions aimed at reducing the prevalence of asymptomatic parasitemia. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

The study was conducted in February 2011 in Mulanda primary school located in Mulanda sub-county, Tororo District, eastern Uganda. This area is mainly dry savannah grassland interrupted by bare rocky outcrops and lower lying swamps, although natural vegetation has mostly been replaced by cultivated crops. Tororo District is characterized by a high intensity of malaria transmission,14 with an estimated entomological inoculation rate of 562 infective bites per person per year.15 Transmission is bimodal, with peaks associated with the two rainy seasons. Malaria in the area is predominantly caused by Plasmodium falciparum3 and Anopheles gambiae s.s., and to a lesser extent, Anopheles funestus are the main vectors.15 In the 5 years before this study, malaria control in Tororo District was typically limited to the promotion of IPT during pregnancy, distribution of insecticide-treated nets (ITNs) through antenatal care services and malaria case management with artemisinin-based combined therapy. In January 2011, however, a mass community-based free ITN distribution campaign was conducted throughout the sub-county. Mulanda sub-county has eight primary schools, all of which are under the government supported universal primary education scheme. Mulanda primary school was purposively selected for the study because of its large student population (1,320 students) and close proximity (approximately 500 meters) to the main public health facility in the area (Mulanda Health Center IV). This study reports baseline data collected as part of a randomized placebo-controlled trial investigating the impact of IPT on malaria morbidity and cognitive function in Ugandan schoolchildren (Clinicaltrials.gov identifier {“type”:”clinical-trial”,”attrs”:{“text”:”NCT01231880″,”term_id”:”NCT01231880″}}NCT01231880). All children with parental consent were screened for eligibility to join the study. Children were excluded if they had any of the following: 1) known allergy or prior adverse reaction to artemisinin-based regimens; 2) history of menarche; 3) fever (axillary temperature ≥ 37.5°C) or history of fever in previous 24 hours; 4) evidence of severe malaria or danger signs; or 5) ongoing antimalarial treatment. At enrollment, a standardized questionnaire was administered by study personnel to children fulfilling the selection criteria to record data on socio- demographics, bed-net ownership, and bed-net use. A focused physical examination was conducted that included a general and abdominal examination as well as measurement of temperature and weight. A finger-prick blood sample was obtained for thick and thin blood smears to assess for Plasmodium infection, hemoglobin estimation, and for filter paper storage. Stool samples were also collected. All blood smears were labeled and air dried at the school and subsequently stained with 2% Giemsa for 30 minutes at the health facility at the end of each day. Parasite densities were determined from thick blood smears by counting the number of asexual parasites per 200 white blood cells (or per 500 if the count was < 10 parasites/200 white cells), assuming a white blood cell count of 8,000/mL. A smear was considered negative after reviewing 100 high-powered fields. Asymptomatic malaria was defined as a positive blood smear for Plasmodium parasites with no associated clinical symptoms. All positive thick blood smears had corresponding thin smears viewed for species identification. Gametocytaemia was also determined from thick blood smears. Two independent microscopists read the slides, with a third microscopist resolving discrepant results. Hemoglobin concentration was assessed using a portable hemoglobinometer (HemoCue Ltd., Angelholm, Sweden) and estimated to an accuracy of 1 g/dL. Stool samples were examined microscopically for the eggs of intestinal nematodes and Schistosoma mansoni (the sole cause of schistosomiasis in the study area), using the Kato-Katz technique. Two aspects of cognition were assessed, including sustained attention assessed by a code transmission test and abstract reasoning tested using Ravens matrices. These two tests have been adapted for an African setting and used in a number of previous studies.7,16 The test for sustained attention was adapted from the Tests of Everyday Attention for Children (TEA-Ch)17 and was administered in groups of 15 or less in the local language, Japhadhola, which is also the language of school instruction for children in classes 1–3 in Mulanda sub-county. The test involved listening to a tape and identifying different numbers (code transmission). During the code transmission task, a list of digits was read out aloud at the speed of one per second. Children were required to listen out for a code—“the number 5 repeated twice consecutively”—and then record the two numbers that preceded the code. The test was administered at school over the weekend when there were no active classes at the school to reduce on external interference. Only children invited for the test were present at school on the testing days. Before the test, children were given three warm-up activities to familiarize them with the tape recorder, as well as assess their ability to count and write numbers. During the test of abstract reasoning, a set of geometric figures with a missing pattern was presented to the children and they were asked to identify the missing item that completes a pattern from a group of almost identical alternatives.18 All data were double-entered and cross-checked in a bespoke Microsoft access database (Microsoft Corp., Seattle, WA). Consistency checks were performed and all discrepancies and queries verified against original paper forms. Plasmodium infection was defined on the basis of expert microscopy results and the proportion of children with asymptomatic Plasmodium parasitemia was calculated as the number of children with any parasites (irrespective of species) on thick smear divided by the total number of children enrolled. Parasite density was categorized as above and below 1,000 parasites/μL. Anemia was defined using the World Health Organization (WHO) age-specific thresholds for hemoglobin (< 11.5 g/dL for children 6 to < 12 years of age and < 12.0 g/dL for those 12–14 years of age).19,20 The anthropometric index z-score weight-for-age was calculated using the egen Stata function for standardizing anthropometric measures in children and adolescents.21 Children were classified as underweight if they were less than two standard deviations below the reference mean. A household wealth index was created using principal component analysis of data on household possessions, utilities, and housing construction for each student.22 Households were ranked according to their distribution along the index, which was then divided into quartiles and classified as an ordinal variable for use in multivariate analysis models. Information on maternal education was also included in the analysis, because of its key influence on children's cognition. All statistical analyses were carried out using Stata version 12.0 software (STATA Corporation, College Station, TX). The outcomes of interest were Plasmodium infection and scores in the code transmission and Raven's matrices tests. Ninety-five percent binomial confidence intervals (CI) were estimated for proportions and standard deviations presented for means. Univariable associations between Plasmodium infection and potential risk factors were assessed using logistic regression and all variables showing an association at a 20% significance level were included into a multivariable logistic regression model. Logical model building using both forward and backward elimination was used to generate minimum adequate model using a 5% significance level; however, bed-net use, socioeconomic group, and anemia were retained as fixed terms in the model regardless of statistical significance because of their known association to Plasmodium infection among school-aged children.3,23 To investigate the association between asymptomatic Plasmodium infection and cognitive function, two sets of analyses, one for abstract reasoning (score 0–20) and the second for sustained attention (score 0–20) were undertaken. The effect of explanatory variables was quantified by mean differences in test performance scores using univariable and multivariable linear regression. Case re-sampling bootstrapping was used to account for non-normality of the scores. Variables identified as significant (P < 0.2) in univariate analysis were considered for multivariable analysis. Logical model building using both forward and backward elimination was used to generate minimum adequate models; however, helminth infection, maternal education, and anemia were retained as fixed terms in all models regardless of statistical significance because of their known effect on cognition.24,25 Interaction was assessed on the basis of likelihood ratio test and included in the final model if P ≤ 0.05. Sensitivity analysis explored the influence of parasite density on cognition, with densities categorized as uninfected, infected with 1–999 parasites/μL, and infected with ≥ 1000 parasites/μL. The study protocol was approved by the Makerere University School of Medicine Research and Ethics Committee (#2010-016) and the Uganda National Council of Science and Technology (#HS 865). Before the start of the study, investigators met with elected government representatives and community leaders to inform them of the study and explain the methodology. Written informed consent was obtained from the parents/guardians of all the children included in the study and written assent was obtained for children 8 years of age and above.

N/A

• Dima Health