This study was conducted in two of the four sentinel sites across the four natural regions of the country: Maferinyah health center in Forecariah District in Lower-Guinea and Ley-Sare health center in Labé District in Middle-Guinea. Antimalarial resistance surveillance in Guinea rotates between four sentinel sites and the 2016 round occurred in Maferinyah and Labé. Maferinyah is a hyperendemic area with high rainfall (6 to 10 months), and the principal vector of malaria is Anopheles gambiae ss. Labe is a mesoendemic area with low rainfall, with An funestus and An. melas as primary vectors .
Study design, period and population
This was an open-label two-arm randomized controlled trial assessing the therapeutic efficacy of two antimalarial treatments: ASAQ and AL among children aged 6 to 59 months with uncomplicated P. falciparum malaria. Participants meeting study inclusion criteria were allocated to either group in a 1:1 ratio, stratified by study site. The allocation was restricted with randomly varying block sizes of 4-6 and was concealed through sealed opaque white envelopes.
A non-probability sampling methodology was used to select patients presenting at the two study sites. A minimum of 100 patients per treatment arm were required giving a total of 200 children per site. The sample size was determined based on a precision of ± 5% for the proportion of clinical and parasitological cure at Day 28 (D28) after correction by PCR assuming a cure rate of 95% and a loss to follow-up of 25%.
Patients were screened and included according to the WHO standard protocol related to the treatment of uncomplicated P. falciparum malaria (2009) . Briefly, children aged 6-59 months, inclusive, with axillary temperature ≥ 37.5°C or history of fever in last 24 hours and microscopy-confirmed P. falciparum monoinfection with parasitemia between 2,000 and 200,000 p/uL without signs of severe malaria and available for the full period of follow up were invited to participate.
Patients meeting the inclusion criteria were treated with either ASAQ or AL based on randomization on site by an authorized member of the research team and they were given the drug according to their weight and age. The treatment duration was three consecutive days (D0, D1 and D2) with oral intake under medical observation once daily for ASAQ and twice daily for AL. The entire dose was repeated if vomiting occurred within 30 minutes after intake of the antimalarial and half the dose if vomiting occurred between the first 30 minutes and 1 hour after intake. Drugs such as vitamin C , vitamin B12 , retinol supplementation [25,26], that may influence antimalarial drug activity were avoided during prescription of concomitant drugs. Similarly, antibiotics such as cotrimoxazole, macrolides, tetracycline and doxycycline were avoided during the period of follow up.
Patients were actively followed for 28 days for both arms from the first day of treatment D0 till D28 with scheduled visits on D1, D2, D3, D7, D14, D21 and other days of unscheduled visits. Patients were systematically assessed on D0 for splenomegaly according to the Hackett classification that has been used in previous studies [27,28]. Clinical and parasitological assessments were performed and dried blood spots were collected on all days of follow-up with the following exceptions: microscopy was only performed on D1 in the presence of signs of severe malaria and no dried blood spots were collected on D1-D2. Recurrent infections were assessed by the field medical doctor of the site and treated with quinine (injection/intravenous) or artesunate injection based on clinical presentation as per the Guinean National Malaria Control Program protocol.
The on-site research team was available to ensure 24-hour passive monitoring for patients. During enrollment and scheduled visits, parents/guardians were informed and encouraged to bring back their children to the health centers or call the field medical doctor whenever their children felt unwell without waiting for scheduled visits. Patients who did not show up for their scheduled visits by mid-day were first called and asked to come to the health center and then actively searched for by a community health worker. If a patient had travelled and could not be traced for scheduled follow-up, he or she was classified as lost to follow-up.
Data were collected using a standardized case report form. Data variables included sociodemographic characteristics (age, sex), clinical characteristics (weight, body temperature, splenomegaly) and laboratory results (malaria microscopy and hemoglobin).
Parasitemia and hemoglobin
Malaria microscopy was carried out using 10% Giemsa staining of thick and thin smears according to a standard operating procedure on D0 and every day of follow-up except D1. The parasite density from the thick smear was determined according to the following formula: Parasitaemia per microliter = (number of asexual parasites divided by 200 or 500 counted leukocytes) multiplied by 8000. A slide was classified negative when the entire examination of the thick smear revealed no asexual form of Plasmodium. The presence of gametocytes of P. falciparum (sexual forms) was determined over 1000 leucocytes instead of 200 or 500 for the asexual forms. For quality control, each slide was read by two microscopists and, if results differed by more than 30%, were re-examined by a third microscopist, with the final parasite density calculated based on the two closest results. Hemoglobin was measured using HemoCue machines (AB Leo Diagnostics, Helsinborg, Sweden).
Dried blood spots were collected on Whatman 903 filter paper on D0, D3, D7, D14, D21, D28 and at any unscheduled visits. Fragment lengths of seven neutral microsatellite markers (Supplemental Table S1) were used to compare genotypes on D0 and day of failure for patients with recurrent parasitemia using a Bayesian classifier for molecular correction . In brief, the Bayesian algorithm uses allele frequencies to calculate the posterior probability of recrudescence for each recurrent parasitemia. Patients with a posterior probability of recrudescence greater than 0.5 were considered as recrudescences in the analysis.
Additionally, all D0 samples and Day of Failure samples from late treatment failures were systematically amplified and sequenced for pfk13 and pfmdr1 resistance genes following previously described methodologies . Molecular analyses were performed in collaboration with the U.S. Centers for Disease Control and Prevention (CDC) laboratories in Atlanta, USA as part of the PMI-supported Antimalarial Resistance Monitoring in Africa (PARMA) Network .
The primary endpoints were adequate clinical and parasitological response (ACPR), early treatment failure (ETF), and late treatment failure (LTF) in accordance with the WHO in vivo guidelines. Secondary endpoints of therapeutic efficacy included the proportion of patients with negative slides at D3.
Adverse events that occurred were reported on specific forms and classified according to their severity and their assessed relationship to the study. Serious or unexpected side effects were reported to the principal investigator, the sponsor, the study coordinator and the Guinean Ethics Committee on Health Research.
Data from the standardized forms were double-entered into Microsoft Access version 2010 (Microsoft Corporation, Redmond, WA) and then exported into STATA 14 software (Stata Corporation, College Station, TX, USA) for analysis. Primary endpoints findings were tabulated, and the primary outcome was reported as the Kaplan-Meier estimate of the corrected efficacy at D28 by study site and drug.