The study was conducted from June to December 2017 in Banfora Health District, Cascades region, south-west Burkina Faso (Figure 1), an area of Sudanian savannah covering 6,295 km2 and with an estimated population of 407,073 inhabitants (4). Subsistence farming and animal husbandry are the main activities. Banfora District has intense seasonal malaria transmission over a six-month period following seasonal rains from May to November (20). Plasmodium falciparum accounts for 90% of cases (20). The main malaria vector is Anopheles gambiae sensu stricto, but An. coluzzii is also found (21). A universal coverage (defined as one ITN for every two persons at risk of malaria) campaign in 2016 distributed ITNs with permethrin or deltamethrin (Sumitomo Chemical, Vestergaard and BASF) and therefore no new ITNs were distributed by the study. No indoor residual spraying (IRS) was conducted. Since 2014, children under five years of age in the study area receive seasonal malaria chemoprevention (SMC) during the transmission season (22).
Recruitment of study cohort
Ten villages were randomly selected from a list of villages in the study area following a two-stage process. Firstly, an area spanning the catchment areas of five health centres was chosen. Each health centre had a catchment radius of approximately 10 km. Secondly, two villages at least 3 km apart were chosen at convenience from each catchment area, giving a total of 10 villages. 30 children were randomly selected from each village using the Health and Demographic Surveillance System enumeration list. Children were eligible to participate if they were aged 5-15 years, likely to remain resident in the village for the duration of the study and the caregiver provided informed consent (assent of child if aged 12-15 years). Older children were selected as they have the highest malaria incidence, relatively low immunity and contribute substantially to transmission (23), while children under five years were excluded due to roll-out of SMC. Children were not eligible to participate if they were participating in a malaria clinical trial, or had a contraindication to the artemisinin-combination therapy (ACT), artemether-lumefantrine (AL). At enrolment in June 2017, 300 children provided a blood film. Irrespective of their malaria parasite status, all children received a curative dose of AL (Wellona Pharma Private Limited, Nana Varachha, Surat, India) to clear any existing parasitaemia. Children were revisited 21 days later, at which time two blood slides and a blood spot were taken and examined to ensure parasite clearance. Those with a negative parasite status confirmed by polymerase chain reaction (PCR) were enrolled in the study. Children re-infected in the 21-day period were re-treated with AL and were eligible for enrolment in the study after 28 days once parasite clearance was confirmed using PCR.
Follow-up of study cohort
Symptomatic and asymptomatic Plasmodium infections were recorded using both active and passive detection. Study children were visited at home every two weeks by fieldworkers during the peak transmission season, from July-December 2017. At each visit, fieldworkers measured the child’s axillary temperature and prepared two blood films and a filter paper blood spot. Children with an axillary temperature ≥37.5°C or history of fever in the previous 48 h were advised to visit the local health centre and, if they were a malaria case, treated with AL, following National Malaria Control Programme (NMCP) guidelines (24). If the child was absent at the time of the visit, the fieldworker made one more attempt to locate them the following day, after which the child was recorded as being absent. Caregivers were encouraged by the study team at enrolment and the fortnightly visits to take their child to the nearest government health centre should the child have a fever or feel unwell. Travel and treatment costs for sick children were reimbursed by the study. Study nurses were posted in the five health centres close to the study villages and performed rapid diagnostic tests for malaria (SD BIOLINE Malaria Ag P.f/Pan, Abbott Laboratories, Illinois, USA) in study children presenting with an axillary temperature ≥37.5°C or history of fever within the previous 48 h, and prepared two blood films and a filter paper blood spot. Children diagnosed with clinical malaria received AL (24). Clinical data were recorded on dedicated study logs and transcribed by field workers who visited each nurse weekly.
Thick blood films were stained with Giemsa and examined under 1000-fold magnification by experienced microscopists at the Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Banfora. Parasite counts were recorded per high power field and 100 fields counted before a slide was declared negative. Two blood slides from each subject were read separately by two microscopists. Discrepancies in positive and negative reads and parasite counts differing by more than 10-fold between the two reads were resolved by the supervisor. Filter paper blood samples were analysed for the presence of 18s rRNA gene using PCR (25).
At enrolment, sociodemographic characteristics of the child (age, gender, religion, and ethnicity) and their caregiver (education and occupation) were recorded. Questionnaires recorded the presence or absence of large domestic animals (cattle, goats, sheep, donkeys or horses) within 5 m of each study child’s houses and the materials used to construct the building in which the study child slept including presence of a metal roof, eaves and door and window screening. The head of household completed a questionnaire on their asset ownership, house construction and other variables, following standard procedures used in the Burkina Faso Malaria Indicator Survey (MIS) (26). Houses of study children were geolocated using a global positioning system (GARMIN eTrex 20).
Use of an ITN the previous night by each study child and use of spatial or topical repellents were recorded at enrolment and each active visit. Survivorship and integrity of each study child’s ITN was measured in July, October and December. Each net was recorded as being in use (i.e. hanging over the study child’s sleeping space), in storage, being washed or lost. Loss was categorised as i) net given away voluntarily, ii) net stolen or iii) net destroyed, discarded or used for alternative purposes. Fabric integrity of the net was assessed by counting the number of holes and their size according to WHO guidance (27). A weighted sum of hole counts, the proportionate hole index (pHI), was calculated with a pHI of 0-64 categorised as good, 65-642 as acceptable and 643+ as too torn and non-protective (28). To measure ITN bio-efficacy, 26 bednets were sampled (at least 2 randomly selected nets per village, except for Sitiena village where 1 net was tested) in October 2017 and stored at +4°C. ITNs taken for testing were replaced with new ones. WHO cone bioassays were performed using the pyrethroid-susceptible Kisumu strain of An. gambiae s.l. at the CNRFP insectaries in Banfora using the WHO efficacy requirement of ≥ 80% mortality (29).
Mosquitoes were sampled with CDC light traps, positioned with the light 1 m above the ground at the foot end of the bed of each study child sleeping under an ITN from 19.00 h to 06.00 h, every four weeks from July to December 2017. In addition, each child’s net was systematically searched for mosquitoes between 06.00 and 07.00 h every four weeks using a torch. Mosquitoes were identified morphologically using established keys and female An. gambiae s.l. typed to species using PCR. The presence of sporozoites in An. gambiae s.l. was determined using an enzyme-linked immunosorbent assay (30). Larval surveys to determine the proximity of anopheline larval habitats to study children’s houses were carried out in the vicinity of all 10 villages during September 2017. All types of larval habitats were surveyed including irrigated fields, puddles, muddy foot or hoof prints, streams and ponds.
Phenotypic insecticide resistance was measured using WHO tube tests as per standard procedures (31). Assays were performed with An. gambiae s.l. mosquitoes reared from immatures collected in seven study villages (larvae were not found in the other three villages).
Data management and statistical analysis
Data were collected on Android personal digital assistants programmed using KoboCollect and included drop down boxes and consistency checks to reduce data entry errors. Following cleaning, the dataset was locked and saved in Microsoft Access. An analytical plan was developed prior to data analysis.
The primary outcome was the incidence rate of microscopically confirmed P. falciparum infection during the transmission season, detected using active and passive case detection. PCR-confirmed P. falciparum incidence rate was a secondary outcome. After ACT treatment, further infections were censored for 28 days since infections during this time were most likely due to recrudescent parasites. Time at risk was also censored for time study children spent away from their compound should they be found to be absent at the two-weekly home visits. The entomological inoculation rate (EIR) or estimated number of infectious bites per study child during the transmission season was calculated using the formula EIR=MaSd where Ma is the human biting rate, estimated from the arithmetic mean number of female An. gambiae s.l. caught per light trap night across the six-month transmission season, where S is the proportion of female An. gambiae s.l. found to be sporozoite positive by village and d is the number of days in the transmission season (n). Cone bioassay results for the netting pieces from each sampled net were pooled by village and by net type. QGIS Geographic Information System (QGIS Development Team (2019), Open Source Geospatial Foundation Project) was used to determine distances between the child’s home and aquatic habitats. Distance to the nearest health facility was determined based on the shortest distance to travel by road. Principal component analysis was used to calculate a SES factor score based on asset ownership and household characteristics. SES factor scores (ranging from -1.8 to 3.2) were ranked and households divided into 5 equal wealth quintiles (1 poorest, through to 5, least poor).
Mean values were compared using a t-test and proportions compared using chi-squared tests. Poisson regression models were used to identify risk factors associated with P. falciparum infection incidence rate, adjusting for clustering by village. Risk factors assessed were: child’s age, gender, ethnic group, religion, caregivers education and occupation, wealth quintile and SES factor score, use of ITNs and other personal protection, ITN integrity, number of people sleeping in the room with the study child, housing materials (roof, eaves, wall and floor material, door and window screening), presence of cows, horses, sheep or goats within 5 m of the child’s home, Euclidean distance to the nearest aquatic habitat with larval anophelines, distance by road to the nearest health centre, EIR at village level, and percentage mortality of An. gambiae s.l. mosquitoes from the village when exposed to 0.05% deltamethrin in a WHO tube test. A multi-variate regression model was developed using a forward stepwise approach. Statistical analysis was carried out in Stata 15 (Statacorp, Texas, USA).
Assuming two P. falciparum infections per child during the transmission season, an intraclass correlation coefficient of 0.1 (design effect of 3.4), and a loss to follow up of 10%, the study had greater than 90% power to detect effect sizes of >50% at the 5% level of significance, assuming 50% prevalence of the risk factor of interest in the population using the formula for comparison of two rates (32). The study is reported following STROBE guidelines (33).