Study site and population
Samples for the present study were collected in Bama, Burkina Faso, a rural area located in the district of Dandé in the Kou Valley. Bama is a rice-growing area of 1200 hectares located 30 km from Bobo-Dioulasso in the southwest of Burkina Faso. The rainy season in this area extends from June to October and the dry season from November to May. The Kou Valley is a permanent source of irrigation water with two rice crops per year from July to November and from January to May. Malaria transmission is perennial with a peak during the rainy season when the density of An. gambiae is very high with an annual entomological inoculation rate of up to 200 infective bites/person/night . The medical center in Bama covers 6 villages, with an estimated population of 23,153 individuals in 2016. The target population for our study was an estimated 4,200 children aged 3-59 months eligible for SMC.
SMC delivery in Bama, Burkina Faso
SMC is distributed by a team of community health workers (CHW) during the rainy season corresponding to the high malaria transmission period. In 2017, SMC was distributed four times, from the end of July to the end of October, and each distribution cycle lasted 4-5 days with an interval of one month between administration. Drugs were delivered to eligible children using a door-to-door strategy. A complete treatment course of SMC includes a single dose of SP and three daily doses of AQ; the CHWs provide the first dose to each child under directly observed treatment (DOT). Drugs are crushed and mixed with sugary water to improve uptake, and, in our study, children were observed for 30 minutes following ingestion. In all situations where the child vomited or regurgitated the drug within 30 minutes, a second treatment was administered. After administration of the first dose of AQ and SP, the CHWs explained to parents/guardians how to administer the second and third doses of AQ to the child at home and informed them about possible side effects.
Participant selection and inclusion
Two weeks’ prior the delivery of the first round of SMC, 124 randomly selected households were visited by the study team. EPI-style random walk method was utilized to randomly select households with children under 5 years of age in a two-step process which involved first selecting a starting point and secondly selecting households from that point onward. The EPI method was appropriate due to lack of local census data and boundary maps. Only one child was selected per household; if multiple children met the inclusion criteria, one child was randomly selected. Households with children aged 3 to 59 months were selected to participate in the study after the parents/guardians gave informed consent. Unoccupied houses were visited a total of three times. The first malaria prevalence survey was conducted in late July 2017, within the period of distribution of the first round of SMC. When logistically feasible, CHWs were accompanied by the study team and blood smears and dried blood spots (DBS) were collected by finger-prick. Otherwise, houses were visited +/- 2 days of the 1st day of SMC administration. The team returned to collect blood samples for pharmacokinetic analyses (see Quantification of DEAQ below) 7–8 days post- SMC administration. For the second malaria prevalence survey in late August 2017, one week before the second round of SMC, the study team visited the same households and collected information on any symptoms related to malaria, along with smears and DBS from the same children, if available for resampling.
Preparation of blood smears and microscopy examination
Thick and thin blood films obtained from children by finger prick were air dried, stained in Giemsa 2% for 30 min, and examined by light microscopy fitted with 100x oil immersion lens in the laboratory of IRSS. A smear was considered negative after examination of at least 100 fields. Parasite density was calculated by counting the number of asexual parasites per 200 leukocytes, assuming a leukocyte count of 8,000/μL. For all positive smears, parasite species were determined using the corresponding thin smear on the same slide. Each smear was read by two experienced microscopists. Results were considered discordant if one result was negative and the other positive for malaria infection. Discordant results were resolved by a third reader.
DNA extraction and Polymerase Chain Reaction (PCR)
Parasite DNA was extracted from DBS using chelex-100. Plasmodium species were subsequently detected by nested PCR as previously described. Species were determined by amplification of 18s RNA using nested PCR with secondary primers specific to the species Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax. For quality control, a template free control was used in all reactions and genomic DNA from laboratory strains (www.beiressource.com) were used as positive control for respective species. All positive samples by microscopy and negative by PCR were reanalyzed by PCR before confirmation of the microscopy results.
Quantification of Desethylamodiaquine (DEAQ)
A total of 200 µL of capillary whole blood was collected in EDTA-containing microtubes on day 7. Samples were centrifuged at 2000 × g for 10 minutes and the plasma was then transferred to cryovials and stored at -80°C. DEAQ plasma concentrations were quantified by reverse phase liquid chromatography and detection with an AB Sciex API 5000 tandem mass spectrometer (LC-MS-MS) in turbo-ion spray-positive mode by NorthEast BioLab (Hamden, CT). 2H5N-Desethylamodiaquine was used as an internal standard and the total assay coefficient of variation were < 7%. The lower limit of quantification (LLOQ) of DEAQ was 0.34 ng/mL.
Genotyping for two nonsynonymous CYP2C8 variants was performed on DNA extracted from DBS using the QIAamp DNA Mini Kit (Qiagen). CYP2C8*2 (805A>T) and CYP2C8*3 (416G>A and 1196A>G) alleles were determined using a TaqMan Drug Metabolism Genotyping Assay (assay ID C_30634034_10 and C_25625794_10, respectively). PCR was performed in a 25 µL reaction with 12.5 µL of TaqMan Universal PCR Master Mix, 1.25 µL of the drug Metabolism Genotyping Assay Mix, and at least 3 ng of DNA template, per the manufacturer’s instructions. Quality control was maintained with no template controls with every reaction, along with previously determined wild type, heterozygous, or homozygous CYP2C8*2 or *3 allele positive controls (from K. Kidd Lab, Yale University).
Genotyping of Drug Resistance Markers
Genotyping was performed on parasite DNA extracted from DBS using the QIAamp DNA Mini Kit (Qiagen). Nested PCR was performed to amplify amodiaquine resistance-associated alleles for pfcrt and pfmdr1. Amplicons were used for a ligase detection reaction (LDR) which contained an allele-specific and conserved sequence primer. Allele-specific primers contained a 5’ nucleotide sequence unique to a MagPlex bead tagged with a complementary sequence. The 3’ end of the allele-specific primer corresponded to a particular drug resistance polymorphism: pfcrt K76T or pfmdr1 N86Y. Conserved sequence primers were modified by 5’ phosphorylation and 3’ biotinylation. After primer ligation, LDR reaction products were hybridized to their respective MagPlex beads in 1.5 TMAC buffer (3 M tetramethylammonium chloride, 50 mM Tris-HCl pH 8, 3 mM EDTA, and 0.1% N-lauroylsarcosine sodium). Fluorescent labeling was performed with a 1:50 dilution of streptavidin-R-phycoerythrin. Drug resistance polymorphisms were determined by measuring fluorescent intensity using xPonent software (Luminex) on a Bio-Plex 200 instrument (Bio-Rad). Genotyping was attempted for SP-associated mutations, but due to a low success rate, data is not included.
Data were collected with Excel and analyzed by R version 3.4.0 (2017-04-21). Chi-square was used to compare proportions and a p value of <0.05 was considered statistically significant.