Study sites. The study was conducted in the western and south-western regions of Burkina Faso, in the health districts of Banfora, Orodara and Gaoua. This part of the country is very humid with rainfall ranging from 1,000 to 1,200 millimetres per year. Malaria is holoendemic with peak transmission from June to October. The main vector is the Anopheles gambiae complex. The entomological inoculation rate (EIR) ranges from 0.91 to 2.35 infective bites/person/day [18, 19]. The health districts of Banfora, Orodara and Gaoua cover an area of 6,266 km2, 8,404 km2 and 7,472 km2 respectively, with a population of 401,381, 259,974 and 251,611[20]. Malaria prevalence and incidence in 2021 were 63.2% and 537.5‰, 57.1% and 541.9‰ and 77% and 860‰, respectively[2]. Seasonal chemoprophylaxis coverage is over 100%. [2]
Study design and time period. The study was conducted using a cross-sectional household survey at the beginning of the rainy season in July 2022, the peak period for malaria transmission. Briefly, a minimum of 190 households with a child aged 6–59 months and/or 190 children aged 5–10 years in each study district were sampled using a standard systematic sampling approach, clustered by sector[21]. A total of 380 children were enrolled in 10 randomly selected villages in each health district. Households were selected if they included children in the selected age group. Written informed consent was required from the child’s parent or legal guardian. This study was part of a larger research project that aimed to investigate the prevalence of asymptomatic malaria using rapid diagnostic tests. Therefore, individuals with known malaria, who were receiving anti-malarial treatment or who had received anti-malarial treatment within one month prior to enrollment were excluded from the study.
Blood collection and processing for malaria screening.
Blood samples were collected aseptically from each participant by finger prick using a sterile disposable lancet by well-trained nurses. The axillary temperature of selected children was also noted. A 50-µL drop of blood sample was then spotted onto a 903TM Whatman TM (LASEC 10530151 Rev.AC, USA) five-spot blood card. Thick and thin blood films were made on the same slide and labelled with a unique code. The blood spots on the filter paper were individually air dried and stored at room temperature in a sealed plastic bag containing a desiccant. All field samples (thick, thin, and dried spots) were then transported to the CNRFP laboratory in Burkina Faso for processing and analysis.
Malaria HRP2 Pf Ag detection
ADX TM Malaria HRP2 Pf Ag were provided by the Programme National de Lutte contre le Paludisme (PNLP) of Burkina Faso. This test was performed according to the manufacturer’s instructions (ADX-004-025 HRP2 Pf Ag, ADVY CHEMICAL PVT.LTD, Inde). The kit was labelled with the appropriate sample code and 5 µl of capillary blood sample was added to the sample well of the test device. Four drops corresponding to approximately 110 µL drops of test buffer solution were added to the buffer well to lyse the cells, release the antigen and facilitate antibody recognition. The RDT test results were then read after 20 minutes and interpreted as negative or positive for Plasmodium falciparum in accordance to the appearance of the visible bands.
Screening by light microscopy. Thin blood smears were fixed in methanol for 30 seconds. The smears were then air dried and stained with 3% Giemsa solution (QCA#990939) for 45 minutes. Thick and thin blood smears were read by two independent level 1 certified malaria microscopists from the Clinical Laboratory Service according to CNRFP standard protocols. Slides were re-read by a third independent microscopist if the two readers disagreed on the species, presence or absence of malaria parasites, or if parasite densities differed by > 25%. The final value for parasite density was the mean of the two closest readings. Parasites were counted on the thick film and the parasite density was calculated using the formula: number of parasites counted x 8000/number of leukocytes counted. If no parasites were detected in at least 200 fields, the slide was considered negative.
Parasite DNA extraction, PCR analysis and DNA sequencing. The filter paper blood samples collected from each individual were stored in individual plastic bags with desiccant. They were protected from light, moisture, and extremes of temperature until analysis. Each dried blood spot was sterile-cut and placed in an Eppendorf tube. Parasite DNA was extracted in a 96-well format as described by Zainabadi and colleagues[22]. The eluted DNA was then quantified by fluorometric quantification (Qubit, Thermo Fisher), adjusted to 20 ng/µL and stored at -20°C until further use. Amplicons from the target regions (exon 2 of the hrp2 and hrp3 genes) were generated using multiplexed nested PCR assays with indexed primers containing specific tags (barcodes). A total of 4 µL of PCR reactions from each sample were pooled (96 samples) to increase the sample volume and to minimise the use of samples for the downstream steps of the protocol. For each pool, amplicons were purified using AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol. This was done to remove dNTPs, salts, primers, and primer dimers. The quality of the purified PCR products was assessed by analyzing eluates containing the purified amplicons on a fragment analyser (Agilent). The concentration of DNA in the pooled fragments was assessed by fluorometric quantification (Qubit, Thermo Fisher). Pooled libraries were denatured with NaOH to a final concentration of 0.1N and diluted with hybridization buffer prior to sequencing. Sequencing was performed with MiSeq v2 reagents using the 300-cycle kit (Illumina) as recommended by the manufacturer. A phred score of 30 was used to demultiplex and quality trim the raw sequences. Primer sequences were trimmed from the 5′ end of the sequences to avoid any bias of the primers in the sequenced fragments. Base calling was performed by comparing the reads to a custom database consisting of the 3D7 reference sequence (v45). Bioinformatic analyses were performed using CLC Genomics Workbench22 (Qiagen). Laboratory reference parasite strains (Dd2 with a single pfhrp2 deletion; 7G8; HB3 with a single pfhrp3 deletion and 3601, a Cambodian culture-adapted parasite) were used as controls to validate each run.
Ethical considerations. The protocol was approved by the Burkina Faso Health Research Ethics Committee and received approval number 2022-05-117. Prior to the start of the study, a community meeting was held in each selected village to discuss the study with community leaders. Individual informed consent was obtained from each head of household during a home visit prior to any study procedure.
Statistical analysis. Sociodemographic characteristics of participants were described using summary statistics. Continuous variables were described using mean (and standard deviation), median (and 95% confidence interval) and range. Continuous variables were compared using ANOVA. Categorical variables were expressed as proportions. A p-value test of < 0.05 was considered statistically significant. Analyses were performed using MedCalc®statistical software version 20.218 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2023).