Study design and participants
We performed an open comparative intervention study to assess the efficacy of AL and the capillary blood concentrations of lumefantrine in uncomplicated SAM and non-SAM children. The study protocol and procedures have been described elsewhere [19]. The study was conducted in Oulessebougou district hospital, region of Koulikoro, Mali, and the primary healthcare center of Andoume, Maradi city, Niger. In these areas, malaria transmission is hyperendemic with seasonal peaks during the rainy season (between July and November [19]) and AL is recommended as first-line malaria treatment. Each year, during the hunger gap period (generally from June to October), acute malnutrition increases among young children [20, 21]. According to the 2012 Demographic and Health Surveys, the prevalence of global acute malnutrition in the Koulikoro region of Mali (Aug–Sep 2012) and Maradi region of Niger (Jun–Aug 2012) were 8.6 % (95 % confidence interval (CI), 6.7–9.5) and 16.2 % (14.2–18.5), respectively, while those of SAM were 1.8 % (1.0–2.2) and 2.5 % (1.8–3.6), respectively.
Children aged between 6 and 59 months with uncomplicated P. falciparum malaria were eligible if they fulfilled criteria listed in Box 1. After their parent or guardian provided written informed consent, children with weight-for-height z-score < –3 or MUAC < 115 mm were enrolled in the “SAM” group, then two children without SAM were subsequently enrolled in the “non-SAM” group. Children with kwashiorkor or complications requiring hospitalization were excluded as were children with severe stunting (height-for-age z-score < –3).
Procedures
Children were treated with a fixed dose combination of non-dispersible artemether 20 mg-lumefantrine 120 mg (Coartem® Novartis) following the manufacturer weight-based dose recommendation (one tablet per intake for bodyweights < 15 kg; two tablets for those weighing ≥ 15 kg), twice daily for 3 days. The drug was administered under direct observation with a fat intake consisting of milk (one glass, approximately 15 mL), or RUTF (Plumpy’Nut®, one bag of 92 g) in case of SAM. If vomiting occurred within 30 minutes after intake, a second dose was administered. Children vomiting the second dose were given rescue medication (Additional file 1: Table S1) and excluded.
Children were given an insecticide-treated bed net at enrolment. Other treatments included iron and folic acid supplementation, deworming, and for SAM children, RUTF, amoxicillin and others as recommended in national nutritional protocols (Additional file 1: Table S1).
Children were followed for 42 days. Any clinical or laboratory adverse event was reported by the investigator as described elsewhere [19]. Serious adverse events were reviewed by a Data Safety and Monitoring Board.
Laboratory methods
Only capillary blood was collected from finger pricks. SD Bioline® HRP2 RDT (Gyeonggi-do, Republic of Korea) was used for screening of malaria parasitemia. Thick and thin blood films were performed at baseline, at 6, 12, 24, 36, 48, and 72, hours, and at day 7, and then weekly until day 42, or in case of malaria signs. All blood films were read by two microscopists blinded to the other reading, and a third reading was performed in case of discrepancy. Films were read using a 100× objective and considered negative after 200 microscopic fields were assessed. P. falciparum asexual forms were counted on the thick film against at least 200 leukocytes [22]. Parasite density was calculated assuming a leukocyte density of 8000/μL. The presence of gametocytes was assessed.
Hemoglobin concentration was determined using HemoCue HB 301®-Hemoglobin brand device (Ängelholm, Sweden) on days 0 and 28. Anemia was defined as a hemoglobin concentration < 10 g/dL and severe anemia as a concentration < 7 g/dL.
PCR genotyping of malaria parasites collected from filter papers at enrolment and at the day of treatment failure were performed in MRTC laboratory in Bamako by amplification of the merozoite surface protein 2 (MSP-2) gene [23] and the microsatellites CA1 and TA87 [24]. Outcomes were defined as recrudescent if at least one shared allele was found with all three markers tested and as reinfection if day 0 and day of failure alleles were different in any of the three markers tested [25].
Pharmacokinetics
A population-based sparse sampling approach was used to limit the number of PK samples required per child and concerned 150 SAM and 150 non-SAM children [26]. For each child, five capillary blood samples (50 μL spotted on filter paper) were collected; first, at 6, 12, 24, 36, or 48 hours (randomly allocated), second at 60 hours, third at 72 hours, fourth at day 7, and fifth at day 14 or day 21 (randomly allocated) post treatment initiation. Lumefantrine concentrations were measured at the Division of Clinical Pharmacology, University of Cape Town, using liquid chromatography tandem mass spectrometry as described previously [19].
Outcomes
The primary outcome was the proportion of patients having an adequate clinical and parasitological response (ACPR) on day 28, after PCR correction.
Secondary outcomes were the proportions of PCR-corrected ACPR on day 42, non PCR-corrected ACPR, early therapeutic failure, late clinical failure, late parasitological failure on days 28 and 42 [8], proportion of reinfection and recrudescence, gametocyte carriage, hematological recovery as witnessed by hemoglobin change between baseline and day 28, and parasite clearance slope half-life.
The main PK outcome was lumefantrine concentration on day 7 since it is strongly correlated with the overall drug exposure in the terminal phase and therefore considered a good predictor of therapeutic response [27]. Secondary PK outcomes were measured lumefantrine concentrations at 60 and 72 hours post treatment initiation. Population-based PK modelling will be reported elsewhere.
Statistical analysis
Unbalanced groups with the non-SAM/SAM ratio set to two was chosen both for ethical and practical reasons, because, for a fixed number of SAM children, twice the number of non-SAM allowed obtaining a higher power than a balanced design. A total of 540 children (180 SAM and 360 non-SAM) allowed detection of a minimum difference of 8 % (87 % ACPR in SAM vs. 95 % in non-SAM children), with a power of 80 %, two-sided significance level of 5 %, and taking into account up to 15 % dropouts. We planned to enroll two thirds of the sample in Mali during the 2013 and 2014 malaria seasons, and one third in Niger during the 2014 malaria season.
Study data were double entered using REDCap electronic data capture tools hosted at Epicentre [28], and analysis was performed with STATA 13, StataCorp®, College Station, TX, USA.
Analyses of treatment response were performed on two different populations: (1) modified intention-to-treat (mITT) population that included all enrolled patients with parasitological confirmation of mono-infection with P. falciparum with density > 1000/μL at screening, who took at least one dose of study drug; and (2) per protocol population including all patients who were part of the mITT and who completed the 3-day treatment course, did not experience major deviation, nor premature discontinuation before day 28 for other reason than failure. Safety analysis was performed in all patients who had received at least one dose of the study drug.
Comparisons of the main treatment outcomes (PCR-uncorrected and corrected ACPR, reinfection) were performed using two analysis methods: Kaplan–Meier analysis comparing the cumulative success rates and allowing to account for censored data, and simple comparison of proportions. The 95 % CIs were estimated using either Wald CI (for Kaplan–Meier estimators) or binomial exact CI (for proportions). Log-rank test for equality of survivor functions was used for comparison of survival curves. Comparisons of proportions were done using a χ2 or Fisher exact test.
For other outcomes (hematological recovery, gametocyte carriage, parasite clearance slope half-life), comparisons were performed between the SAM and non-SAM groups using a Student or Wilcoxon test for continuous variables and a χ2 or Fisher exact test for categorical variables. To calculate the parasite clearance slope half-life, the log-transformed parasite counts over time were modelled using the Parasite Clearance Estimator Tool developed by the WWARN [29].
Cox multivariable modelling investigated the effect of SAM and other cofactors (study site, baseline parasite density, child’s age, and all covariates with a statistically significant difference at baseline between the SAM and non-SAM groups) on malaria-free survival.
Finally, we compared lumefantrine concentration at 60 and 72 hours and at day 7 between groups using Wilcoxon rank-sum test, and we investigated if a lower day 7 lumefantrine concentration was associated with the risk of malaria infection using Cox modelling as described above.
Each adverse event was coded to a “Preferred Term” using the Medical Dictionary for Regulatory Activities, version 11 [30]. Then, the number and percentage of patients with at least one adverse event of the following categories were provided: those leading to treatment discontinuation, serious adverse events, and most common adverse events (≥5 %, regardless of the treatment group).
All analyses described above were also conducted after adjusting for study site, and site by site where the sample size allowed.
