Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine–Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso

Journal of Infectious Diseases, Feb 2015

Background. Methylene blue (MB) has been shown to be safe and effective against falciparum malaria in Africa and to have pronounced gametocytocidal properties. Methods. Three days of treatment with artesunate (AS)-amodiaquine (AQ) combined with MB was compared with AS-AQ treatment in a randomized controlled phase IIb study; the study included 221 children aged 6–59 months with uncomplicated falciparum malaria in Burkina Faso. The primary end point was gametocyte prevalence during follow-up, as determined by microscopy and real-time quantitative nucleic acid sequence-based amplification (QT-NASBA). Results. The gametocyte prevalence of Plasmodium falciparum at baseline was 3.6% (microscopy) and 97% (QT-NASBA). It was significantly lower in the AS-AQ-MB than in the AS-AQ group on day 7 of follow-up (microscopy, 1.2% vs 8.9% [P < .05]; QT-NASBA, 36.7% vs 63.3% [P < .001]). Hemoglobin values were significantly lower in the AS-AQ-MB group than in the AS-AQ group at days 2 and 7 of follow-up. Vomiting of the study medication occurred significantly more frequently in the AS-AQ-MB group. Conclusions. The combination of MB with an artemisinin-based combination therapy has been confirmed to be effective against the gametocytes of P. falciparum. MB-based combinations need to be compared with primaquine-based combinations, preferably using MB in an improved pediatric formulation. Clinical Trials Registration. NCT01407887.

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Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine–Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso

JID Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine- Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso Received 0 accepted 0 2 September 0 electronically published 0 September 0 B. C. 0 M. P. contributed equally to this work. 0 0 Ruprecht-Karls-University Heidelberg , INF 324, 69120 Heidelberg, Germany (olaf. The Journal of Infectious Diseases 1 Department of Medical Microbiology, Radboud University Medical Center , Nijmegen , the Netherlands 2 Institute of Tropical Medicine and International Health , Charité-Universitätsmedizin Berlin , Germany 3 Department of Immunology & Infection, London School of Tropical Medicine and Hygiene , United Kingdom 4 Division of Infectious Diseases and Tropical Medicine, Medical Center 5 Centre de Recherche et de la Formation au Paludisme , Ouagadougou , Burkina Faso 6 Centre de Recherche en Santé de Nouna , Nouna 7 Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University Düsseldorf 8 Institute of Medical Biometry and Informatics 9 Department of Pediatrics and Adolescent Medicine, Medical School, Ulm University 10 German Center for Infection Research , Partner Site Munich 11 Department of Bacteriology, Max von Pettenkofer-Institute, Ludwig Maximilian University of Munich 12 Biochemistry Centre, Ruprecht-Karls-University , Heidelberg 13 Institute of Public Health, Medical School Background. Methylene blue (MB) has been shown to be safe and effective against falciparum malaria in Africa and to have pronounced gametocytocidal properties. Methods. Three days of treatment with artesunate (AS)-amodiaquine (AQ) combined with MB was compared with AS-AQ treatment in a randomized controlled phase IIb study; the study included 221 children aged 6-59 months with uncomplicated falciparum malaria in Burkina Faso. The primary end point was gametocyte prevalence during follow-up, as determined by microscopy and real-time quantitative nucleic acid sequence-based amplification (QT-NASBA). Results. The gametocyte prevalence of Plasmodium falciparum at baseline was 3.6% (microscopy) and 97% (QT-NASBA). It was significantly lower in the AS-AQ-MB than in the AS-AQ group on day 7 of follow-up (microscopy, 1.2% vs 8.9% [P < .05]; QT-NASBA, 36.7% vs 63.3% [P < .001]). Hemoglobin values were significantly lower in the AS-AQ-MB group than in the AS-AQ group at days 2 and 7 of follow-up. Vomiting of the study medication occurred significantly more frequently in the AS-AQ-MB group. Conclusions. The combination of MB with an artemisinin-based combination therapy has been confirmed to be effective against the gametocytes of P. falciparum. MB-based combinations need to be compared with primaquinebased combinations, preferably using MB in an improved pediatric formulation. Clinical Trials Registration. NCT01407887. - Combination therapy is the current cornerstone of malaria control, with the particular aim to delay and possibly reverse the development of drug resistance [ 1 ]. Artemisinin-based combination therapy (ACT) is highly effective, but artemisinin resistance may be selected in vivo by uncontrolled use of artemisinin monotherapies or may result from combination with ineffective partner drugs [ 2 ]. The artemisinins also show pronounced activity against immature Plasmodium falciparum gametocytes, but they leave mature gametocytes unaffected and therefore do not prevent malaria transmission shortly after treatment [ 3 ]. There are also differences in gametocytocidal activity between the available ACT regimens [ 4 ]. Methylene blue (MB) is a water-soluble dye that has been used for a long time in industry and medicine [ 5 ]. It was historically used to successfully treat malaria [ 6 ], but only observational data supported its efficacy particularly in quinine-refractory cases [ 7 ]. In humans, MB is well absorbed from the gastrointestinal tract, peak plasma concentrations are reached 2 hours after oral administration, and the plasma half-life is about 20 hours [ 8 ]. Renal excretion is the major elimination pathway [ 9 ]. Known adverse effects of MB include bitter taste, green or blue coloration of urine, and mild and self-limiting dysuria [ 5, 10– 14 ]. Moreover, a transient reduction of hemoglobin values in glucose-6-phosphate dehydrogenase (G6PD)–deficient patients but without overt clinical consequences has been observed in clinical trials with some 1000 patients in Burkina Faso [15]. The interest in MB as an antimalarial drug was reactivated when P. falciparum glutathione reductase was identified as a new drug target [ 5 ]. In a series of studies conducted in Burkina Faso, oral MB (4–24 mg/kg/d) was shown to be safe and effective in the treatment of uncomplicated falciparum malaria when combined with other antimalarials [ 10–13 ] but to act more slowly against asexual parasites than artemisinins [14]. Nevertheless, MB has shown a strong effect in terms of P. falciparum gametocyte reduction [ 16 ], matching pharmacokinetics and potential synergy with artemisinin drugs [ 17 ], and a low potential for resistance development [ 8 ]. Consequently, MB has been considered a potentially useful partner drug for existing ACTs, particularly in regions where malaria elimination is the final goal [ 18 ]. We therefore tested the gametocytocidal effect of MB when combined with a common ACT, amodiaquineartesunate (AS-AQ), as well as the efficacy and safety of this triple combination therapy in children with uncomplicated falciparum malaria in Burkina Faso. MATERIALS AND METHODS Study Area The study was conducted from August to October 2011 in the urban research zone of the Centre de Recherche en Santé de Nouna (CRSN) in Burkina Faso [ 19–23 ]. Study Design and Objectives The study was designed as a randomized controlled phase IIb trial with follow-up for 28 days. The study was open label but with blinding for the laboratory technicians and laboratory scientists involved. The primary objective was to assess the reduction of P. falciparum gametocytes among children with uncomplicated malaria after treatment with the triple combination AS-AQ-MB compared with AS-AQ. The secondary objectives were to study the safety and the efficacy against asexual parasites of the 2 regimens. The primary end point of the study was P. falciparum gametocyte prevalence during follow-up, as assessed by microscopy and real-time quantitative nucleic acid sequencebased amplification (QT-NASBA). The secondary end points were P. falciparum gametocyte density during follow-up as assessed by microscopy and QT-NASBA, fever and parasite clearance times, hemoglobin concentrations during follow-up, and rates of adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late treatment failure (LCF), and late parasitological failure (LPF) (both crude and corrected with polymerase chain reaction [PCR] corrected for reinfections) [ 24 ], as well as the incidence of observed and self-reported adverse events (including hemolysis, defined as any drop in hemoglobin of >2.5 g/dL within 24 hours) and the acceptance of the different treatment regimens by mothers/caretakers, as determined by a standardized questionnaire on day 14. Study participants with ETF, LCF, or late parasitological failure received rescue treatment with artemether-lumefantrine (Coartem). Study Population The community was informed of the project and invited to attend central locations in the town of Nouna for fever measurement. Children who were eligible for the study (see inclusion criteria) were referred to the Nouna district hospital outpatient clinic for further examinations. Noneligible sick children received free treatment in the hospital. The inclusion criteria were age 6–59 months, weight ≥6 kg, uncomplicated falciparum malaria (axillary temperature ≥37.5°C or a history of fever during the last 24 hours and asexual parasitemia [parasite count, ≥2000/µL and ≤200 000/µL blood]), Burkinabé nationality, permanent residence in the study area, and informed consent of parents/caregivers. Exclusion criteria were signs of severe malaria [ 25 ], moderately severe anemia (hemoglobin, <7 g/dL), any apparent other disease, and treatment with malaria drugs during the preceding week. Study Intervention Study children were randomly assigned to receive either AS-AQ-MB or AS-AQ during a 3-day period. AS-AQ is the official first-line antimalarial used in Burkina Faso. Children in the AS-AQ-MB group received a once-daily tablet of fixeddosed AS-AQ (Coarsucam; Sanofi Aventis), with dosage determined by weight group (6.0–8.9 kg, 25 mg of AS plus 67.5 mg of AQ; 9.0–17.9 kg, 50 mg of AS plus 135 mg of AQ; >17.9 kg, 100 mg AS plus 270 mg of AQ), combined with once-daily MB (15 mg/kg) minitablets in prepackaged sachets according to weight group (6.0–8.9 kg, 100 mg MB; 9.0–12.9 kg, 150 mg MB; 13.0– 16.9 kg, 200 mg MB; >16.9 kg, 250 mg MB). Coarsucam was procured from the quality-controlled stock of the Essential Drug Store, Ministry of Health, Burkina Faso. The MB minitablets were developed at the Düsseldorf University in Germany and produced by the Pharbil Waltrop company in Germany under good manufacturing practice conditions. The formulation is based on the recently introduced principle of orodispersible minitablets, which dissolve rapidly in the oral cavity, preventing aspiration [ 26 ]. The blue biconvex minitablets are 2 mm in diameter and approximately 2 mm thick. They contain 2.0 mg MB as the active pharmaceutical ingredient, mannitol/polyvinylacetate (Ludiflash; BASF) as a ready-to-use filler and binder, sucralose (Merck) as a sweetener, and magnesium stearate (Peter Greven) as a lubricant. Coarsucam tablets were taken with water. The MB minitablets were provided on a spoon with local food to improve the acceptability for children. All study drugs were administered under direct observation by study nurses. In case of vomiting within the first 30 minutes, the treatment was repeated. If vomiting occurred again, the patient was excluded, referred to the pediatrics department of the hospital, and replaced. Children with a temperature ≥38.5°C received a standard dose of paracetamol (acetaminophen) tablets (10 mg/kg) every 6 hours until the fever subsided. Follow-up Children were recruited on day 0 and followed up on days 1, 2, 3, 7, 14, and 28. Parents and caregivers were advised to come back at any time between scheduled visits in case of unforeseen symptoms. Laboratory Examinations A finger-prick blood sample was taken on days 0, 1, 2, 3, 7, 14, and 28, and during unscheduled visits. Malaria parasitemia and Characteristic Male sex, No. (%) Age, median (range), mo Weight, median (range), kg Duration of current disease episode, median (range), d Hemoglobin, median (range), g/dL Temperature, mean (SD), °C P. falciparum asexual parasites/µL Mean (SD) Median (range) P. falciparum gametocytemia prevalence, No. (%) Microscopy QT-NASBA P. falciparum gametocyte density by microscopy, mean (SD), parasites/µL P. falciparum gametocyte density by QT-NASBA, median TTP−1 hemoglobin values were determined using standard CRSN procedures [ 20 ]. In brief, thick and thin blood films were examined by 2 experienced laboratory technicians supervised by one of the investigators (B. C.). Asexual parasites and gametocytes were counted on thick blood films against 200 white blood cells, and parasite density was calculated assuming an average white blood cell count of 10 000/mL. Slides were declared negative if no parasites were seen in 400 fields on the thick film. Hemoglobin concentrations were measured using a HemoCue analyzer (HemoCue). From each blood sample, 2 aliquots (50 µL each) were kept as dried blood spots on filter paper (Whatman 903; Whatman International). They were stored in the laboratory of the CRSN individually sealed in plastic bags with a desiccant. After shipment to the Centre National de Recherche et de Formation sur le Paludisme in Ouagadougou, recrudescence was differentiated from new infection by comparing PCR-generated P. falciparum merozoite surface protein 1 (msp-1) and 2 (msp-2) genotype patterns in matched pairs of isolates obtained on admission and on the day of reappearance of parasitemia, as recommended by the World Health Organization [ 27 ] (see also Supplementary Material Methods). The interpretation of the results was based on the consensus definitions of recrudescence and new infections [ 28 ]. At the Ludwig Maximilian University of Munich, QTNASBA of Pfs25 messenger RNA was performed, as described in detail elsewhere [ 29 ] (see also Supplementary Material Methods). Owing to a transport-related loss of all filter papers taken for QT-NASBA analysis, this analysis could only be performed on 109 backup day 0 and day 7 samples (49 AS-AQ-MB and 60 AS-AQ group). Statistical Analysis Calculations were performed with SAS (version 9.1, SAS Institute Inc., Cary, NC) and Sigma-Stat (version 3.5, Systat Software, San José, CA) statistical software. Two-sided P values are reported throughout. For a detailed description of sample size calculation and statistical tests used, see also the Supplemental Material. Ethical Aspects The study protocol was approved by the ethics committees of Heidelberg University and the CRSN. Parents/caregivers were asked for their written consent after having received detailed information from a study nurse about all known risks and benefits of the study. RESULTS Baseline Data A total of 1029 children were referred from the fever measurement points to the hospital for assessment, and of those 221 (111 AS-AQ-MB, 110 AS-AQ) were included in the study (see also Supplementary Figure 1). Seven children refused to take the first treatment dose (3 AS-AQ-MB, 4 AS-AQ), 18 repeatedly vomited the first treatment dose (15 AS-AQ-MB, 3 AS-AQ), and 3 were erroneously included (1 AS-AQ-MB, 2 AS-AQ). The remaining children (92 AS-AQ-MB, 101 AS-AQ) formed the a priori defined full analysis set for the modified intention-to-treat (mITT) analysis. Three cases of minor protocol violations at enrollment were kept in the per protocol (PP) analysis: 1 child was 63 months old, 1 had a P. falciparum parasite count of 1600/µL, and 1 had a hemoglobin value of 6.2 g/dL. During follow-up, another 10 children were withdrawn from the study, all from the AS-AQ-MB group. The reasons were repeated vomiting after the second treatment dose (7 children), refusal of the second treatment dose (2 children), and protocol violation (1 child). The remaining children (82 AS-AQ-MB, 101 AS-AQ) form the a priori defined analysis set for the PP analysis. The demographic and clinical characteristics of the mITT participants were similar in the 2 study groups (Table 1). The enrollment prevalence of P. falciparum gametocytes was 3.6% by microscopy (AS-AQ-MB, 6.5%; AS-AQ, 1.0%) and 97.2% by QT-NASBA (AS-AQ-MB, 100.0%; AS-AQ, 95.0%). Microscopic gametocyte prevalence (P = .04) and density (P = .03) were significantly higher, whereas the gametocyte prevalence as determined by QT-NASBA was nonsignificantly higher (P = .25) in the AS-AQ-MB group than in the AS-AQ group (Table 1). Follow-up Data Gametocytes The prevalence of microscopically assessed gametocyte substantially increased in both study groups from day 0 to day 1 and continued to do so in the AS-AQ group until day 3. In contrast, gametocyte prevalence decreased continuously in the AS-AQMB group from day 1 onward (Figure 1A; Table 2). By day 7, only 1 patient (1%) in the AS-AQ-MB group had microscopically detectable gametocytes, and none (0%) by day 14 (AS-AQ, 9% and 5% respectively; P < .05 each). Microscopic gametocyte densities in gametocyte-positive individuals did not differ between the 2 treatment groups at baseline or at follow-up dates (data not shown). QT-NASBA results were available only for a limited number of randomly chosen study participants in the AS-AQ (60 paired day 0 and day 7 samples) and AS-AQ-MB (49 samples) arms. The available samples indicated that the effect of AS-AQ-MB on QT-NASBA–detected gametocyte prevalence was even more pronounced (Table 2). On day 7, gametocyte prevalence had decreased from 100.0% (49 of 49 samples) to 36.7% (18 of 49 samples) in the AS-AQ-MB arm compared with 97.2% (57 of 60 samples) to 63.3% (38 of 60 samples) in the AS-AQ arm (P < .001). The reciprocal time to positivity (TTP−1) was determined for QT-NASBA–positive samples of both groups to allow for relative density comparison between the groups. The TTP−1 (indicating gametocyte concentration, calculated only for QT-NASBA–positive samples) decreased from day 0 to day 7 in both the AS-AQ-MB (P < .001) and the AS-AQ (P = .003) arms. On day 7, the median TTP−1 was significantly lower in the AS-AQ-MB than in the AS-AQ arm (P < .001) (Figure 1B; Table 2). Asexual Parasites and Fever Clearance The clearance of P. falciparum asexual parasites was nonsignificantly more rapid (1.82 vs 1.96 days; P = .22) in the AS-AQ-MB than in the AS-AQ group (Table 3). The microscopically determined parasite prevalence and densities were consistently lower during day 1 to day 3 in the AS-AQ-MB group than in the ASAQ group; the difference in parasite density was significant on day 1 (P = .004). Fever disappeared rapidly in both groups, with only 6 febrile patients on day 1 (2 AS-AQ-MB, 4 AS-AQ) and 1 on day 2 (AS-AQ-MB). Asexual Parasite Prevalence and Density Day 0 Prevalence, No. (%) Density, asexual parasites/µL Mean (SD) Median Day 1 Prevalence, No. (%) Density, asexual parasites/µL Mean (SD) Median Day 2 Prevalence, No. (%) Density, asexual parasites/µL Mean (SD) Median Day 3 Prevalence, No. (%) Density, asexual parasites/µL Mean (SD) Median AS-AQ-MB (N = 92) Treatment Efficacy The uncorrected rate of ACPR in mITT analysis was 44% in the AS-AQ-MB and 55% in the AS-AQ group (P = .10). This difference was largely due to an increased rate of ETF (11%; danger sign: persistent vomiting) in the AS-AQ-MB group (Table 4). After correction for reinfections, the rate of ACPR was significantly lower in the AS-AQ-MB group than in the AS-AQ group (71.4% vs 85.1%; P = .024); again, this difference was due to the ETFs in the former group. In the PP analysis, ACPR occurred at a similar rate in the 2 study groups (80.2% vs 85.1%; P = .395). Hemoglobin values decreased from baseline to day 1 in both groups and steadily increased thereafter in the AS-AQ group (Figure 2). Values were significantly lower in the AS-AQ-MB than in the AS-AQ group on day 2 (P = .04) and day 7 (P = .005). There were a total of 6 hemolysis cases (defined as an hemoglobin drop of >2.5 g/dL within 24 hours), with 3 cases in each treatment group. Five hemolysis cases occurred between days 0 and 1, and 1 (AS-AQ group) between days 2 and 3. Hemoglobin values rapidly increased thereafter without therapeutic intervention. Abbreviations: ACPR, adequate clinical and parasitological response; AS-AQ, artesunate-amodiaquine; AS-AQ-MB, AS-AQ combined with methylene blue; ETF, early treatment failure; ITT, intention-to-treat; LCF, late treatment failure; LPF, late parasitological failure. a This group included 92 patients without correction and 82 with correction. Adverse Events There were no deaths and only 1 serious adverse event during the follow-up period. The latter occurred in a 59-month- old child (AS-AQ-MB) who developed malaria and typhoid fever on day 17 and recovered after appropriate treatment. The event was considered unrelated to the study medication. There were no differences in the number and pattern of adverse events between the 2 study groups (Table 5). However, it has to be considered that 25 children vomited the first and second doses of the study medication and were either excluded from the study or judged as having ETF. Of these, significantly more were from the AS-AQ-MB group than from the AS-AQ group (22 of 92 vs 3 of 101; P < .001). Despite these findings, no differences in the pattern of selfreported acceptance of the study medication between groups were observed. Most mothers considered the treatment to be good (AS-AQ-MB, 67%; AS-AQ, 65%) or acceptable (AS-AQMB, 33%; AS-AQ, 35%). DISCUSSION The present data extend our previous findings on the pronounced gametocytocidal effects of MB [ 16 ] by using it in combination with an ACT and using a molecular gametocyte detection tool to determine the effect of MB on submicroscopic gametocyte densities. Posttreatment gametocyte prevalence was significantly lower in patients treated with AS-AQMB than in those treated with AS-AQ. When molecular QT-NASBA gametocyte detection as used in a subset of samples, AS-AQ-MB–treated children had a 2-fold lower gametocyte prevalence and density compared with their AS-AQ–treated peers. Delayed clearance of parasitemia after ACT treatment as observed in Southeast Asia may favor the transmission of artemisinin-resistant malaria [ 30 ]. Of note, studies in Kenya suggested a reduction in ACT efficacy in recent years [ 31 ] and an association between parasite clearance time and transmissibility to mosquitoes [ 30 ]. Against this background, the gametocytocidal effects of antimalarials are likely to be of increasing importance in the near future. Primaquine (PQ) is a gametocytocidal compound with well-known activity against mature P. falciparum AEs During 28-Day Follow-up by Study Group (ITT gametocytes but has been associated with intravascular hemolysis [ 32 ]. The present study shows that MB may be a viable alternative to PQ. Similar to the situation with PQ, the infectivity of gametocytes that persist after MB treatment is currently unclear and needs further investigation [ 33 ]. One of the main limitations of a number of malaria drugs is the risk of hemolysis in G6PD-deficient patients [ 33 ]. The importance of such effects was recently demonstrated by the failure of the large chlorproguanil-dapsone (Lapdap) drug development project [ 34 ]. In the present study, the hematological recovery was less rapid in children treated with AS-AQ-MB than in those treated with AS-AQ, but there were no differences in the number of hemolytic events. This supports the notion of an existing but possibly clinically not relevant hematological risk [ 15 ]. Clinical trials directly comparing MB and PQ are required to finally assess the potential superiority of MB concerning the risk of hemolysis. Addition of a single low dose of PQ (0.25 mg base per kilogram) to ACT as a P. falciparum gametocytocide without requirement for G6PD testing has currently been recommended by the World Health Organization for elimination areas or areas threatened by artemisinin resistance [ 35 ]. It has been shown that MB acts synergistically with artemisinins against the asexual blood stage parasites of P. falciparum [ 17 ]. This in vitro finding was supported by consistent observations of more rapid parasite clearance during clinical trials in Burkina Faso [ 13, 15 ]. Notably, however, the overall efficacy of AS-AQ, with or without MB, was comparatively low at 80%– 85% in PP analysis. Previous studies among children with uncomplicated malaria in the study area yielded PCR-corrected ACPR rates of 82% for AS-AQ in 2006 [13] and 61% for AQ alone in 2005 [ 10 ]. Impaired AQ efficacy in this area of intense chloroquine resistance [ 12 ] may thus partially explain the suboptimal efficacy of the ACT. Our findings of a more rapid clearance of asexual parasites in the AS-AQ-MB group than in the AS-AQ group may therefore need to be interpreted with some caution. Future studies should investigate whether the addition of MB to more efficacious ACTs, such as artemether-lumefantrine or dihydroartemisinin-piperaquine, also increases the parasite clearance rates. A more rapid parasite clearance would probably correspond to a more benign clinical course and a quicker reduction in the source of any new gametocytes, which further supports the idea of adding MB to ACTs [ 18 ]. QT-NASBA revealed much higher gametocyte prevalence than microscopy, which supports similar findings from other African studies wherein gametocyte prevalence in patients ranged from 9% to 25% by microscopy but from 68% to 89% by QTNASBA [ 36–38 ]. Because even low gametocyte densities can result in mosquito infection, molecular methods are more informative than microscopy for determining posttreatment transmission potential, although both assays fall short of permitting definitive statements on the transmission-blocking potential of antimalarial drugs that requires mosquito feeding assays [ 38 ]. The finding that some drugs, including MB, may have differential activity against male and female gametocytes [ 39 ], further highlights the value of confirming the infectivity of gametocytes that persist after treatment. Mosquito feeding assays are available only at a limited number of African research centers. Although we did not perform feeding assays and had limited QT-NASBA gametocyte observations owing to an unforeseen loss of FP samples, we consider our conclusions on the enhanced gametocyte clearance of the triple combination therapy justified, given that these are based on both microscopic and QT-NASA data and that the MB combination was superior at all time points beyond day 3 after initiation of treatment. The relatively low efficacy of the AS-AQ combination against asexual parasites may have influenced gametocyte dynamics during follow-up [ 30 ], although our last time point of gametocyte detection by QT-NASBA (day 7) is likely to reflect the effect of MB on gametocyte clearance rather than preventing de novo gametocyte development, which may take 10–15 days from persisting asexual parasites [ 40 ]. Oral MB is known to have a strong bitter taste, which may affect compliance, particularly in the treatment of children. Compared with aqueous MB solutions, MB minitablets are much more acceptable for oral intake together with semisolid or liquid food, but a slightly bitter taste from released MB remains. This is shown by the fact that in this study significantly more children of the MB group than in the ACT control group repeatedly vomited the medication and had to be excluded from the trial or were judged as having ETF. It is very likely that the observed vomiting is linked to MB taste attributes only and not to the ingestion of solid drug carriers as it has been demonstrated for children in the targeted age group [ 41, 42 ]. The World Health Organization recommended this “multiparticulate” dosage form for the use in hot and humid climates to improve drug stability and acceptability and also to reduce transport and storage costs [43]. In conclusion, MB combined with an ACT could function as a useful combination for the treatment of falciparum malaria in elimination programs. Before such a triple therapy is implemented, the safety and efficacy of this new MB formulation needs to be compared with other gametocytocidal drugs in head-to-head clinical trials, and the dosage may need to be optimized. Minitablets are a promising new pediatric formulation for MB but may need to be coated for further taste masking to improve compliance. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Notes Acknowledgments. We thank Provepharm Company (Marseille) for the special provision conditions concerning the MB raw material and David Poluda for helping with the QT-NASBA analysis. Author contributions. P. E. M., J. B., R. H. S., F. P. M., C. D., T. B., and O. M. designed the study. B. C., M. P., N. B. R., A. W., and S. B. S. were responsible for the laboratory work. M. B., P. E. M., E. N., and A. S. were responsible for the clinical work. C. K. and M. K. were responsible for data management and analysis. J. B. developed the MB mini tablets and organized the production. O. M. wrote the first draft of the paper. All authors contributed to the interpretation of the data and to writing the manuscript. Disclaimer. Sponsors had no influence on the study. Financial support. This work was supported by the German Science Foundation (Sonderforschungsbereich 544, project A8) and were supported by the Bill and Melinda Gates Foundation (grant OPP1034789 to C. D. and T. B.). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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Boubacar Coulibaly, Michael Pritsch, Mamadou Bountogo, Peter E. Meissner, Eric Nebié, Christina Klose, Meinhard Kieser, Nicole Berens-Riha, Andreas Wieser, Sodiomon B. Sirima, Jörg Breitkreutz, R. Heiner Schirmer, Ali Sié, Frank P. Mockenhaupt, Chris Drakeley, Teun Bousema, Olaf Müller. Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine–Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso, Journal of Infectious Diseases, 2015, 689-697, DOI: 10.1093/infdis/jiu540