Study setting and design
An in vivo open-label study to test the efficacy and tolerance of ASAQ and AL for treatment of laboratory-confirmed uncomplicated P. falciparum malaria among Malagasy children was conducted in two sites in Madagascar. Study methods were based on the standard WHO protocol . Cases were recruited during May–September 2018 in two basic health centers (Centre de Santé de Base, CSB) serving the communes of Ankazomborona, in Marovoay District in the northwest, and Matanga, in Vangaindrano District in the southeast (Fig. 1). These CSBs were selected to represent different climatic zones of the country; Ankazomborona is in the tropical zone and Matanga is in the equatorial zone. Both communes are in districts with moderate malaria transmission, defined as 50–100 cases per 1,000 population per year ; in 2016, malaria parasite prevalence by microscopy among children 6–59 months of age was 9.0% and 8.8% in the zones encompassing Ankazomborona and Matanga, respectively . Children in each site were randomly assigned to receive either ASAQ (Winthrop®, Sanofi-Aventis, France) or AL (Coartem®, Novartis, Basel, Switzerland) and treated according to WHO/NMCP recommendations. To ensure study quality, staff from the US President’s Malaria Initiative (PMI) visited each site to observe study procedures and data management and provide feedback and support.
Primary and secondary endpoints and sample size
The primary endpoints were 28-day uncorrected and PCR-corrected efficacy. Sample size calculations were based on this endpoint and powered assuming a 5% failure rate with a 5% margin of error and 95% confidence level. Assuming loss to follow-up of 15%, a minimum of 86 patients per arm were needed at each site, for a total of 344 patients across the two sites. Secondary endpoints included early therapeutic failures, day three parasite clearance rate, late clinical and parasitological failures, and the presence of single nucleotide polymorphisms (SNPs) associated with antimalarial resistance or decreased response in the pfk13, pfmdr-1, and pfcrt genes.
Study population and participant enrollment
All children aged six months through 14 years presenting to the CSB for evaluation of febrile illness were referred to the TES team, who explained the study to parents. A rapid diagnostic test (RDT) (SD Bioline Malaria Ag Pf/Pan, Standard Diagnostics, Inc.) was administered according to the manufacturer’s instructions and thick and thin slides for microscopy were prepared. All children with a positive RDT for P. falciparum that was confirmed by microscopy to be P. falciparum monoinfection with a density of 1,000—100,000 parasites/µl of blood were invited to participate. Children with a negative RDT, and those without parental consent or assent (for those aged seven through 14 years), were referred to the CSB clinician for care. After consent, a questionnaire was administered to eligible children or parents to collect demographic and clinical information and a physical exam was performed by the study physician. Pregnancy and lactation were assessed for all females ≥ 12 years of age; for those who had attained menarche and missed a menstrual period, a pregnancy test was done. Pregnant or lactating females were excluded and referred to the CSB clinician for care. A second finger stick was done to measure hemoglobin concentration (Hemocue®, HB 201+, Angelholm, Sweden); children with hemoglobin concentration < 8 grams/dl were excluded and referred to the CSB clinician. Capillary blood for dried blood spots (DBS) was also collected on Whatman 903 filter papers (GE Healthcare Life Sciences, PA, USA) at this time and used for molecular analyses. Additional criteria for enrollment included weight ≥ 5 kg, ability to take oral medication, and plans to remain in the study area for the following 28 days. Exclusion criteria included: signs of severe disease (i.e., prostration, change in mental status, convulsions, respiratory distress, persistent vomiting, hemoglobinuria, jaundice, hemorrhagic shock); underlying chronic illness or signs of severe malnutrition; reported allergy to one of the ACTs; having taken an antimalarial medication within the previous 30 days; having taken a medication which could interfere with one of the study medications; participation in another clinical study; and residence > 15 kilometers from the CSB. Enrolled children were randomly assigned to treatment with either AL or ASAQ using block randomization, by age group (6–59 months, 5–9 years, 10–14 years), and a one-to-one ratio for the two medications. Once a child was enrolled, study staff selected the top envelope for that child’s age group which held the treatment indication.
Participant treatment, monitoring, and follow-up
Children were treated by study personnel on days 0, 1 and 2 with either ASAQ or AL according to WHO/NMCP recommendations. All doses of medication were administered with water only and were directly observed by study personnel. Clinical and parasitological response to treatment and screening for adverse drug events occurred on days 1, 2, 3, 7, 14, 21, and 28; parents were instructed to bring their child to the health center if symptoms occurred between scheduled visits. Blood samples, for microscopy and preparation of DBS, were collected from a finger stick at every visit, including unscheduled visits. Participants were withdrawn from the study and referred for care if they developed signs of severe disease or if an adverse event required discontinuing study treatment.
Study participants were classified as early treatment failures in the following situations: signs of severe malaria in the presence of parasitemia up to day three; day two parasitemia higher than at day zero; day three parasitemia above 25% of day-zero parasitemia in the absence of fever; parasitemia detected on day three in the presence of fever. Participants were classified as late treatment failures if they had not demonstrated early treatment failure and had at least one blood slide positive for asexual P. falciparum parasites after day three. Patients negative for malaria on day 28 and not having previously met any treatment failure criteria, were classified as adequate clinical and parasitological response (ACPR). Participants with early or late treatment failure were discontinued and referred for care.
Microscopy slides were prepared at the study sites according to WHO guidelines; the first slide was prepared by staining with 10% Giemsa to facilitate diagnosis and treatment. The slides were read on site by WHO certified level 1 or 2 microscopists to confirm monoinfection and calculate parasite density. Parasite density, expressed as the number of asexual parasites per µl of blood, was calculated by dividing the number of asexual parasites by the number of white blood cells and then multiplying by an assumed white blood cell density of 8000 per µl. Slides were considered negative if no parasites were seen after examination of 200 oil-immersion fields in a thick blood film. Slides were read by two microscopists; for parasite density, the mean of the two readers was reported. Discrepant results were resolved by a third certified reader. For parasite density discrepancies ≥ 25%, the mean value from all three readers was reported. To ensure microscopy quality, 20% of slides were randomly selected for re-reading after the study by NMCP microscopists, who were blinded to the original result.
DNA extraction and molecular analysis
Collected DBS were transported to the NMCP malaria reference laboratory for molecular analysis. Parasite DNA was extracted from DBS with QIAamp 96 DNA Blood kit (No. 51306) according to the manufacturer’s instructions (Quiagen Inc., Hilden, Germany).
Additional molecular analyses were performed by Madagascar NMCP staff at the U.S. Centers for Disease Control and Prevention Malaria Laboratory in Atlanta, GA, as part of the technical training objective of the PMI-sponsored Antimalarial Resistance Monitoring in Africa (PARMA) network . Investigation of polymorphisms in codons 389—638 of the pfk13 propeller domain, codons 86, 184, 1034, 1042, and 1246 of pfmdr1, and codons 72—76 of pfcrt were done using Sanger sequencing  on 85 day 0 and 18 day of recurrent parasitemia samples. The analysis of SNPs was done using the Geneious software package (Biomatters Inc., San Francisco, CA) utilizing the 3D7 pfk13 (PF3D7_1343700), pfmdr1 (PF3D7_0523000) and pfcrt sequences (PF3D7_0709000) as references. Raw sequence reads were cleaned using default settings and reads with high-quality scores (> 30%) were further analyzed.
Differentiation between recrudescence and reinfection
PCR correction, to differentiate recrudescence from reinfection in those with a late treatment failure, was achieved by comparing seven neutral microsatellite genotypes (TA1 on chromosome 6, Poly-α on chromosome 4, PfPK2 on chromosome 12, 2490 on chromosome 10, C2M34-313 on chromosome 2, C2M69-383 on chromosome 3, and TA109 on chromosome 6) in the paired pre-treatment and post-treatment samples using previously described methods [21, 22]. The sizes of the amplification products were determined by capillary electrophoresis on an Applied Biosystems 3130 xl sequencer (Applied Biosystems, Foster City CA). A previously validated Bayesian algorithm was used to generate a posterior probability of recrudescence for each late treatment failure .
Data were recorded in the field into WHO-standard templates  and later double entered into an Excel database at the NMCP’s reference laboratory. Statistical analyses were performed using R version 4.0.1 (R Foundation for Statistical Computing, Vienna, Austria).
Uncorrected and PCR-corrected per protocol efficacy for each site and drug was calculated by dividing the number of participants classified as ACPR over all participants reaching a study outcome. The sum of posterior probabilities of recrudescence were used to calculate the total number of recrudescent infections for the PCR-corrected analyses. Reinfections were removed from the calculations of PCR-corrected per protocol efficacy. For Kaplan-Meier cumulative efficacy estimates, participants lost to follow-up or excluded were included until the last day of follow-up in uncorrected and PCR-corrected analysis. Posterior sampling was used to generate the PCR-corrected Kaplan-Meier estimates and 95% confidence intervals using the posterior probabilities of recrudescence.
The study was reviewed and approved by the institutional Ethics Committee of Biomedical Research of the Ministry of Public Health of Madagascar. The U.S. CDC’s Center for Global Health Office of the Associate Director for Science determined CDC staff to be non-engaged in this research study (CDC human subjects 2016-012a). Local leaders and community members were informed of the study through meetings and radio broadcasts. Participants and parents were informed about the objectives of the project, benefits, and risks associated with participation in the study; signed informed consent was obtained from parents before enrollment and children aged 7—14 provided verbal assent.