Journal of Infectious Diseases Advance Access published July 15, 2014

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1 Journal of Infectious Diseases Advance Access published July 15, 2014 MAJOR ARTICLE Randomized Trial of Artesunate-Amodiaquine, Atovaquone-Proguanil, and Artesunate- Atovaquone-Proguanil for the Treatment of Uncomplicated Falciparum Malaria in Children Rachida Tahar, 1,4 Talleh Almelli, 1 Camille Debue, 1 Vincent Foumane Ngane, 4 Joseph Djaman Allico, 2,5,6 Solange Whegang Youdom, 4 and Leonardo K. Basco 3,4,a 1 Unité Mixte de Recherche 216 Mère et Enfant Face aux Infections Tropicales, Institut de Recherche pour le Développement (IRD), Unité de formation et de recherche de Pharmacie, Paris, 2 Unité de Recherche Neurobiologie et Développement, Unité Propre de Recherche 3294, Centre National de la Recherche Scientifique, Université Paris-Sud XI, Orsay, and 3 Unité de Recherche 198, Unité de Recherche des Maladies Infectieuses et Tropicales Emergentes, IRD, Faculté de Médecine La Timone, Aix-Marseille Université, Marseille, France; 4 Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaoundé, Cameroon; 5 Laboratoire de Pharmacodynamie Biochimique, Unité de Formation et de Recherche Biosciences, Université de Cocody, and 6 Département de Biochimie, Institut Pasteur de Côte d Ivoire, Abidjan, Côte d Ivoire Background. Artemisinin-based combination therapies (ACTs) are recommended for the treatment of acute uncomplicated falciparum malaria in many malaria-endemic countries. Despite the emergence of artemisinin resistance, few alternative non-acts, including atovaquone-proguanil, are currently available. Methods. Plasmodium falciparum infected Cameroonian children 5 years old (n = 338) were randomly assigned to artesunate-amodiaquine, atovaquone-proguanil, or artesunate-atovaquone-proguanil treatment groups and followed for 28 days, according to the standard World Health Organization protocol. In vitro response to atovaquone and cytochrome b sequence of clinical isolates were determined. Results. Eight late failures and 16 failures (8 late and 8 early failures) were observed after artesunateamodiaquine and atovaquone-proguanil therapies, respectively. Most late failures were due to reinfections. Artesunateatovaquone-proguanil was not associated with any failure. After correction by genotyping, per-protocol analysis showed no difference in the efficacy of 3 drugs. However, the proportion of atovaquone-proguanil treated patients with positive smears on day 3 was much higher (36.0%; P <.05) than that of the artesunate-amodiaquine (2.9%) and artesunate-atovaquone-proguanil (1.0%) groups. In vitro response and cytochrome b sequence did not indicate atovaquone resistance. Conclusions. Atovaquone-proguanil was characterized by a slow blood schizontocidal action and resulted in early treatment failure in a few patients. Artesunate-atovaquone-proguanil was a highly effective alternative treatment. Clinical Trials Registration. UMIN Keywords. drug resistance; Plasmodium falciparum; cytochrome b; artemisinin; molecular epidemiology. Received 8 February 2014; accepted 30 May a Present affiliation: Unité de Parasitologie, Institut de Recherche Biomédicale des Armées, Ancienne Base Aérienne 217, Brétigny-sur-Orge, France. Correspondence: Leonardo K. Basco, MD, PhD, Unité de Parasitologie, Institut de Recherche Biomédicale des Armées, Ancienne Base Aérienne 217, Brétignysur-Orge, France (leonardo.basco@ird.fr). The Journal of Infectious Diseases The Author Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com. DOI: /infdis/jiu341 Artemisinin-based combination therapies (ACTs) are the current drugs of choice for the treatment of acute uncomplicated Plasmodium falciparum malaria in many parts of the world where malaria is endemic. Artesunate-amodiaquine (ASAQ) and artemetherlumefantrine have been adopted for first- or secondline treatment in many sub-saharan African countries, including Cameroon. Although these 2 ACTs are highly effective and rapidly acting and resistance has not been Atovaquone-Proguanil Treatment for Malaria JID 1

2 documented in the African continent [1, 2], both of these drug combinations have some limitations. There has been some concern with the wide-scale use of amodiaquine in a highly chloroquine-resistant area because of possible cross-resistance and toxicity related to repeated intake of the drug, and amodiaquine is associated with minor but common adverse effects, such as asthenia and pruritus [3]. Artemether-lumefantrine is well tolerated, but the requirements for twice daily doses for 3 days and coadministration with fatty food to improve lumefantrine absorption may compromise patient compliance. Other ACTs recommended by the World Health Organization (WHO) [1], including artesunate-sulfadoxine-pyrimethamine, artesunatemefloquine, and dihydroartemisinin-piperaquine, are less commonly used in Africa today. Moreover, resistance to artemisinin derivatives is emerging and has become an ominous reality in Southeast Asia [4 6], requiring a continuous effort to develop and evaluate the clinical efficacy of alternative drugs to treat multidrug-resistant malaria. Atovaquone-proguanil (ATPG) is a highly efficacious non- ACT drug for multidrug-resistant P. falciparum. Atovaquone is a ubiquinone analogue that selectively inhibits cytochrome b, reduces mitochondrial membrane potential, and inhibits pyrimidine biosynthesis [7 10]. Proguanil, a biguanide, exerts a synergistic schizontocidal action with atovaquone and enhances atovaquone-mediated collapse of the mitochondrial membrane potential [11, 12]. Earlier studies with ATPG showed its safety, tolerance, and high prophylactic efficacy and cure rate in several epidemiological settings [13 15]. At present, ATPG is one of the drugs of choice for both chemoprophylaxis of nonimmune temporary visitors traveling to areas where multidrug resistance has been reported and treatment of imported cases of acute uncomplicated P. falciparum infections in many industrialized countries where malaria is not endemic [16 19]. In contrast, the use of ATPG by local populations in malaria-endemic countries has been largely limited because of its prohibitive cost and the availability of alternative antimalarial drugs. In the African continent, most of the clinical studies of ATPG were performed in the 1990s, in Kenya, Gabon, Zambia, and Malawi [20 28]. There is little recent information on its efficacy in African children, who constitute the most vulnerable population affected by malaria. Moreover, none of the previous studies of ATPG conducted in Africa involved comparison with an ACT, and artesunate was not coadministered with ATPG. Artesunate-ATPG (AS-ATPG) was found to be safe, well tolerated, and highly effective in Thai subjects, including children <5 years old and pregnant women [29 31]. The aims of the present study were to compare the efficacy of ASAQ, ATPG, and AS-ATPG in Cameroonian children aged 5 years.p. falciparum isolates from patients treated with ATPG or AS-ATPG were analyzed for in vitro response to atovaquone and underwent analysis of DNA sequences of the gene encoding cytochrome b. PATIENTS, MATERIALS, AND METHODS Patients Clinical studies were conducted at Nlongkak Catholic missionary dispensary (Yaoundé, Cameroon) in Malaria transmission occurs throughout the year in Yaoundé. The entomological inoculation rate varies from 3 to 34 infective bites per person per year [32 34]. Symptomatic patients with acute uncomplicated malaria were enrolled, after free and informed consent was received from the parents or legal guardians, if the following inclusion criteria were met: age between 6 months old and 5 years, fever at the time of consultation (measured rectal temperature, 38.0 C), and parasite density of 2000 asexual P. falciparum parasites/µl of blood, without other Plasmodium species [35]. In addition, because of limited data on the safety of ATPG for treatment in children weighing <5 kg [17, 18, 26], a lower limit for body weight was set at 5.0 kg. Patients with symptoms associated with concomitant infectious diseases, severe malnutrition, or any general danger signs of severe and complicated malaria as defined by the WHO were excluded [35]. After informed consent was obtained, capillary blood was collected by finger prick in a capillary tube for hematocrit measurement, and approximately µl of capillary blood was imbibed onto Whatman 3MM filter paper (GE Healthcare Life Sciences, Vélizy-Villacoublay, France) to store parasite DNA. The study protocol was reviewed and approved by the Cameroonian National Ethics Committee and the Cameroonian Ministry of Public Health. Clinical guidelines of the Institut de Recherche pour le Développement (France) were followed in the conduct of this clinical research. Treatment and Follow-Up This was an open-label, randomized study on the efficacy of 3 antimalarial drugs: ASAQ, ATPG, and AS-ATPG. Two subtrials were conducted sequentially. Patients were randomly assigned to one of the 2 treatment groups in each subtrial: ASAQ vs ATPG-1 and ATPG-2 vs AS-ATPG. Noncoformulated ASAQ was used for precise dose calculations based on the individual patient s body weight. Amodiaquine (AQ; Camoquin 200 mg amodiaquine base tablet or 10 mg amodiaquine base/ml syrup; Pfizer Afrique de l Ouest, Dakar, Senegal) was administered at a standard dose of 10 mg base/kg body weight on days 0, 1, and 2. Artesunate (AS; Arinate 100 mg tablets; Dafra Pharma, Oud-Turnhout, Belgium) was administered at a total dose of 12 mg/kg body weight (4 mg/kg body weight on days 0, 1, and 2). ATPG (Malarone pediatric dose formulation, 62.5 mg atovaquone + 25 mg proguanil hydrochloride; GlaxoSmithKline, Bad Oldesloe, Germany) was administered at a once daily dose of 20 mg/kg body weight/day for atovaquone and 8 mg/kg body weight/day for proguanil for 3 days. All antimalarial drugs were administered simultaneously and once daily 2 JID Tahar et al

3 on days 0, 1, and 2. Each dose of antimalarial drug was administered under supervision at the dispensary or during home visits. Paracetamol (10 mg/kg body weight) was administered to all patients 3 times daily for fever and headache. Patients were followedupondays1,2,3,7,14,21,and28[35]. Hematocrit measurement was performed on day 0 and repeated on day 14. Patients who did not respond to the assigned drug were treated with oral quinine (25 mg base/kg body weight/day for 5 days) or artemether-lumefantrine. The primary treatment outcome was determined on day 28 and classified into one of the following categories: early treatment failure, late clinical failure, late parasitological failure, or adequate clinical and parasitological response [35]. Treatment failure refers to one of the following outcomes: early treatment failure, late clinical failure, or late parasitological failure. Adequate clinical and parasitological response is synonymous with treatment success. The secondary outcomes were the proportion of patients who cleared parasitemia on or before day 3 and hematocrit improvement on day 14. In Vitro Assays In vitro drug susceptibility of fresh clinical isolates to atovaquone was determined by the [ 3 H]hypoxanthine incorporation method [36, 37]. A venous blood sample (5 ml) was collected into ethylenediaminetetraacetic acid coated tubes for in vitro assays from patients aged 3 5 years with parasitemia 0.1% and assigned to the ATPG or AS-ATPG group. Polymerase Chain Reaction (PCR) Analysis, Sequencing, and Genotyping Parasite DNA was extracted from filter papers, using the Chelex method [38]. A 565 base pair fragment of the P. falciparum mitochondrial cytochrome b gene was amplified as described in our earlier study [36], and DNA sequence was determined by direct sequencing of the PCR products. Genotypes of paired pretreatment and recurrent parasites on or after day 7 were determined to distinguish between recrudescence (ie, reappearance of the same parasite population as that of pretreatment sample) and reinfection (ie, appearance of different parasite populations). Genotyping was based on 3 polymorphic markers merozoite surface antigen-1 (msa1), merozoite surface antigen-2 (msa2), and glutamine-rich protein (glurp) as recommended by the WHO [39]. Data Analysis Based on the PCR-uncorrected failure rate of 20% after ASAQ treatment observed at the same study site in 2005 [40], ATPG, with or without artesunate, was assumed to reduce the failure rate to <5%. The approximate total sample size for comparing proportions and detecting the difference, with a sided Figure 1. Enrollment and flow of subjects through the study. Abbreviations: ASAQ, artesunate-amodiaquine; AS-ATPG, artesunate-atovaquone-proguanil; ATPG, atovaquone-proguanil; ETF, early treatment failure; LPF, late parasitological failure. Atovaquone-Proguanil Treatment for Malaria JID 3

4 significance level and 80% power, in 2 treatment arms was 150 patients. Continuous variables were compared using the unpaired or paired t test. Proportions were arranged in a 2 2 contingency table and compared using the Fisher exact test. Logistic regression analysis was used to determine the adjusted odds ratios (ORs) for risk factors associated with positive smears on day 3 and to estimate associations between the day 3 secondary outcome measure and pretreatment variables. All statistical tests were 2-sided, and the level of statistical significance was fixed at a P value of <.05. SigmaStat 3.5 (Systat Software, Point Richmond, CA) and Stata 11 (StataCorp, College Station, TX) software were used for these analyses. The primary end points were determined on day 28 and transformed to binary categorical variables. Both intention-totreat (ITT) and per-protocol (PP) analyses of the percentage of ACPR were performed. A fixed-effect model of meta-analysis was used to analyze dichotomous data, calculate the Peto OR, and compare treatment effect [41]. Data were analyzed using the meta-analysis package of software R (version 2.8.1; R Foundation for Statistical Computing) and Review Manager software (Nordic Cochrane Center, Cochrane Collaboration, Copenhagen, Denmark, 2012). Table 1. Kaplan Meier survival analysis was performed to calculate the probability of the time to treatment failure during the 28-day follow-up period. Pairs of survival curves were compared using the log-rank test. Survival curves were plotted andanalyzedusingprism4.0(graphpadsoftware,lajolla, CA) software. Parasite growth of individual clinical isolates in the presence of various concentrations of atovaquone was expressed as the percentage of parasite growth in drug-free control wells and plotted against the logarithmic values of drug concentrations. The 50% inhibitory concentration (IC 50 ), defined as the drug concentration corresponding to 50% of the uptake of [ 3 H]hypoxanthine measured in the drug-free control wells, was determined by a nonlinear regression analysis using Prism 4.0. Data were expressed as geometric mean IC 50 values and 95% confidence intervals (CIs). Recrudescent parasites after ATPG treatment failure have been characterized by Y268S, Y268N, or Y268C substitutions in cytochrome b [42 47]. Moreover, a recrudescent isolate TM93-C1088 obtained from a Thai patient after treatment with atovaquone-pyrimethamine and other P. falciparum clones that have been selected during in vitro culture by stepwise exposure to increasing concentration of atovaquone Baseline Clinical and Parasitological Characteristics by Treatment Group, Intention-to-Treat Analysis Characteristic ASAQ (n = 70) ATPG-1 (n = 69) ATPG-2 (n = 99) AS-ATPG (n = 100) Male sex Age, mo Mean ± SD 35.9 ± ± ± ± 16.4 Range Weight, kg Mean ± SD 14.4 ± ± ± ± 4.1 Range Rectal temperature, C Mean ± SD 38.9 ± ± ± ± 0.8 Range Days since symptom onset, no., 3.8 ± ± ± ± 2.4 mean ± SD Reported self-medication for current febrile episode, patients, % Antimalarial drug a Antipyretic drug Parasitemia, asexual parasites/µl blood Geometric mean (95% CI) ( ) ( ) ( ) ( ) Range Parasitemia > /µL, patients, % Hematocrit, % Mean ± SD 29.0 ± ± ± ± 5.2 Range There was statistically no significant difference (P >.05) in all comparisons between the artesunate-amodiaquine (ASAQ) and atovaquone-proguanil (ATPG-1) treatment groups and between the ATPG-2 and artesunate-atovaquone-proguanil (AS-ATPG) treatment groups. Abbreviation: CI, confidence interval. a A total of 4 patients reported self-medication with amodiaquine monotherapy (n = 3) or oral quinine (n = 1) within 2 weeks before consultation. 4 JID Tahar et al

5 displayed the following amino acid substitutions, either singly or in combination: M133I, L271F, K272R, P275T, G280D, L283I, and V284K [45, 48]. Mutant cytochrome b alleles were defined on the basis of these amino acid changes. RESULTS A total of 338 children were enrolled (Figure 1). The baseline clinical and laboratory characteristics of the ITT population were not different (P >.05) between the study groups (Table 1). A total of 19 study participants had no outcome data: 10 were lost to follow-up, and 9 were excluded after enrollment for protocol violation, persistent vomiting, or withdrawal of consent. Clinical Outcome The assessment of therapeutic efficacy on day 28 showed that in PCR-uncorrected ITT and PP populations, ASAQ was as effective as ATPG-1 but that both drugs were less effective than AS- ATPG (Tables 2 and 3). For the PP population, the cure rate was Table % in the AS-ATPG group (ITT population, 95% cure rate). Compared with the cure rates observed with AS-ATPG, the PCR-uncorrected cure rates were significantly lower in both the ASAQ group (PP population, 88.2%; ITT population, 85.7%; P <.05), the ATPG-1 group (PP population, 85.9%; ITT population, 79.7%; P <.05),andtheATPG-2group(PP population, 92.4%; ITT population, 85.9%; P <.05). The day 28 PCR-uncorrected cure rates in the ASAQ and ATPG-1 groups were not significantly different (OR for ITT data, 0.66 [95% CI, ]; OR for PP data, 0.82 [95% CI, ]; P >.05; Supplementary Figure 1). The log-rank survival analysis showed similar results. There was no statistically significant difference (P =.649) in the PCR-uncorrected survival data for the ASAQ and ATPG-1 groups, whereas the paired survival curves for the ATPG-2 and AS-ATPG groups were significantly different (P =.006; Supplementary Figure 2). After correction by genotyping, ITT analysis of day 28 data showed that the difference in the proportions of ACPR in the ASAQ and ATPG-1 groups was not statistically significant (OR, 0.66 [95% CI, ]; P >.05). PCR adjustment was Primary Outcomes by Treatment Groups, Intention-to-Treat (ITT) and Per-Protocol (PP) Populations Outcome ASAQ ATPG-1 ATPG-2 AS-ATPG ITT population, patients, no PP population, patients, no Excluded from PP analysis on or before day 14, n (%) 2 (2.9) 4 (5.8) 5 (5.0) 4 (4.0) Evaluation on day 14 Early treatment failure 0 6 (8.7; 9.2) 2 (2.0; 2.1) 0 Late clinical failure Late parasitological failure PCR unadjusted 1 (1.4; 1.5) PCR adjusted ACPR PCR unadjusted 67 (95.7; 98.5) 59 (85.5; 90.8) 92 (92.9; 97.9) 96 (96; 100) PCR adjusted 67 (95.7; 100) 59 (85.5; 90.8) 92 (92.9; 97.9) 96 (96; 100) Evaluation on day 28 Excluded from PP analysis after day 14, patients, no. (%) 0 1 (1.4) 2 (2.0) 1 (1.0) Censored due to reinfection, patients, no. (%) 6 (8.6) 3 (4.3) 4 (4.0) 0 Late clinical failure PCR unadjusted 5 (7.1; 7.4) 1 (1.4; 1.6) 0 0 PCR adjusted 2 (2.9; 3.2) Late parasitological failure PCR unadjusted 3 a (4.3; 4.4) 2 (2.9; 3.1) 5 (5.0; 5.4) 0 PCR adjusted (1.0; 1.1) 0 ACPR PCR unadjusted 60 (85.7; 88.2) 55 (79.7; 85.9) 85 (85.9; 92.4) 95 (95; 100) PCR adjusted 60 (85.7; 96.8) 55 (79.7; 90.2) 85 (85.9; 96.6) 95 (95; 100) Data are no. of patients (% of the ITT population; % of the PP population), unless otherwise indicated. All PCR adjustments were performed for late clinical failure and late parasitological failure, in which reappearance of parasitemia occurred between day 7 and day 28. Data were censored if reinfection occurred. Abbreviations: ACPR, adequate clinical and parasitological response; ASAQ, artesunate-amodiaquine; AS-ATPG, artesunate-atovaquone-proguanil; ATPG, atovaquone-proguanil; PCR, polymerase chain reaction. a Includes 1 failure that occurred on day 7. Atovaquone-Proguanil Treatment for Malaria JID 5

6 Table 3. Comparison of the Primary Outcome, by the Peto Odds Ratio (OR) Method PCR-Unadjusted Outcome PCR-Adjusted Outcome Day, Comparator Treatment Per Protocol Intention to Treat Per Protocol Intention to Treat Peto OR (95% CI) P Peto OR (95% CI) P Peto OR (95% CI) P Peto OR (95% CI) P Day 14 ASAQ ATPG ( ) ( ) ( ) ( ).040 ATPG-2 AS-ATPG 7.63 ( ) ( ) ( ) ( ).34 ASAQ AS-ATPG ( ) ( ) ( ).93 Day 28 ASAQ ATPG ( ) ( ) ( ) ( ).35 ATPG-2 AS-ATPG 8.17 ( ) ( ) ( ) ( ).029 ASAQ AS-ATPG ( ) ( ) ( ) ( ).036 PCR-unadjusted day 14 outcomes were not statistically significant in the intention-to-treat and per-protocol populations. Direct comparison of per-protocol populations on day 14 between the artesunate-amodiaquine (ASAQ) and artesunate-atovaquone-proguanil (AS-ATPG) treatment arms was not done because of 100% efficacy. Statistical significance was defined as a P value of <.05. Abbreviations: ATPG, atovaquone-proguanil; CI, confidence interval; PCR, polymerase chain reaction. not required for the AS-ATPG group because of the absence of treatment failure. In the ITT population, AS-ATPG was significantly more effective than ATPG-2 (OR, 2.87 [95% CI, ]; P <.05) and ASAQ (OR, 3.15 [95% CI, ]; P <.05). However, in the PP analysis of PCR-corrected data, the differences in the efficacy of 3 drugs were not statistically significant (P >.05; Supplementary Figure 1). This observation was due to the fact that most treatment failures in the ASAQ and ATPG groups were caused by reinfections that led to censored data. Results of the fixed-effect model were supported by the log-rank survival analysis, which did not show any Table 4. Outcome Secondary Treatment Outcome in the Per-Protocol Population statistically significant difference in the PCR-corrected survival curves for the ASAQ and ATPG-1 groups (P =.254) and for the ATPG-2 and AS-ATPG groups (P =.077; Supplementary Figure 2). Secondary Outcomes A comparable proportion (P >.05) of patients assigned to different treatment arms presented with an initial parasitemia of > asexual parasites/µl of blood (Table 4 and Supplementary Materials). On day 3, only 2 of 68 patients (2.9%; PP population) treated with ASAQ were afebrile but still had Treatment Group ASAQ ATPG-1 ATPG-2 AS-ATPG Patients on days 2 and 3, no Parasite clearance Positive smear on day 2 a 13 (19.1) 51 (78.5) 70 (72.9) 8 (8.2) Positive smear on day 3 a 2 (2.9) 25 (37.9) 33 (34.4) 1 (1.0) Fever clearance Fever on day 1 1 (1.5) 5 (7.7) 8 (8.3) 2 (2.0) Fever on day 2 1 (1.5) 2 (3.1) 6 (6.2) 0 Fever on day 3 1 (1.5) 0 2 (2.1) 1 (1.0) Hematocrit Level on day 14 %, mean±sd 34.8 ± ± ± ± 3.8 Increase from days 0 to 14, % b Data are no. (%) of patients, unless otherwise indicated. Abbreviations: ASAQ, artesunate-amodiaquine; AS-ATPG, artesunate-atovaquone-proguanil; ATPG, atovaquone-proguanil. a Statistically significant difference (P <.05) in the proportions of patients with parasite-positive smears in the ATPG-1 and ATPG-2 groups. b In each treatment group, the mean increase in hematocrit on day 14, compared with the pretreatment values, was statistically significant (P <.0001). 6 JID Tahar et al

7 parasite-positive smears. Among 98 patients (PP population) treated with AS-ATPG and followed up on day 3, only 1 child still had a parasite-positive smear on day 3, but this patient was afebrile from day 1 onward. These observations suggest that these 2 ACTs rapidly clear fever and parasitemia in a large majority of patients before day 3, even in patients presenting with high initial parasitemias. In contrast to the rapid parasite clearance after ACTs, 58 of 161 patients (36.0%) treated with ATPG (25 from the ATPG-1 group and 33 from the ATPG-2 group; PP populations) still presented with parasite-positive smears on day 3 (P <.001, compared to the ASAQ and AS-ATPG groups). Among these 58 patients, the geometric mean parasitemia on day 3 was 222 asexual parasites/µl (95% CI, asexual parasites/µl; range, asexual parasites/µl). By day 7, with the exception of 3 patients who were also febrile and required rescue treatment on day 3 for early treatment failure (Table 5), 55 of 58 patients had parasite-negative smears. Logistic regression analysis failed to identify a factor (ie, age, sex, weight, body temperature, or hematocrit) that may predict a delayed parasitological response on day 3. Pretreatment hematocrit values were available for all 338 enrolled patients. Day 14 hematocrit values were available for 323 patients (ITT population, 95.6%). In each treatment group, there was a statistically significant (P <.001) increase in hematocrit from day 0 to day 14. Among all treatment groups, the mean increase in hematocrit was between 13% and 20%. In Vitro and Molecular Assays The in vitro activity of atovaquone was determined in 65 isolates. The geometric mean IC 50 of 55 pretreatment clinical isolates with interpretable data was 1.32 nm (95% CI, nm;range, nm). The cytochrome b sequence was determined in isolates from all patients treated with ATPG or AS-ATPG. No mutation was found. DISCUSSION The present study confirmed our earlier findings that ASAQ is well tolerated and rapidly effective in clearing fever and parasitemia in young Cameroonian children. Young African children aged <5 years constitute the largest and most vulnerable population that bears most of the global malaria burden in terms of morbidity and mortality, primarily because of inadequate level of acquired immunity. Children with symptomatic malaria require rapid and accurate diagnosis, followed by rapidly acting, effective treatment to prevent the progression of the disease toward potentially fatal, severe, and complicated malaria. ASAQ and other ACTs fulfill this requirement for rapid clearance of fever and parasitemia. Recent clinical studies have suggested that the best available means to detect early signs of decline in artemisinin efficacy is the time to attain 50% of the pretreatment parasitemia [6]. The main disadvantage of this measure is the requirement for a close, 6 8-hour monitoring of parasitemia during the first 48 hours of treatment. For retrospective analysis of clinical data, the best available secondary outcome to evaluate the artemisinin efficacy is the proportion of patients who have parasite-positive smears on day 3 [35]. Our previous studies of the efficacy of ASAQ conducted between 2005 and 2007 in young children in Yaoundé have shown that a small minority of patients (2 of 206 [0.97%]) remain smear positive on day 3 [40]. Likewise, other ACTs evaluated in young children resulted in rapid fever and parasite clearance. By contrast, amodiaquine monotherapy and amodiaquine-sulfadoxine-pyrimethamine combination resulted in higher proportions of positive smears (8 of 62 patients Table 5. Evolution of Body Temperature and Parasitemia in 8 Patients Responding With Early Treatment Failure After Atovaquone- Proguanil Treatment Patient Code Age in mo; Sex Weight, kg Day 0 Hematocrit, % Rectal Temperature, C Parasitemia, Asexual Parasites/µL Day 0 Day 1 Day 2 Day 3 Day 0 Day 2 Day 3 NKO-43/1 11; M NKO-36/2 30; M NKO-58/2 7; M NKO-103/1 15; M NKO-60/1 59; F NKO-129/1 24; F NKO-112/1 48; F NKO-138/1 10; M Two subsets of patients fulfilled one of the 4 criteria of early treatment failure: (1) fever (rectal temperature, 38.0 C) and positive parasitemia on day 3 (patients NKO-43/1, NKO-36/2, and NKO-58/2) or (2) parasitemia on day 2 higher than the initial parasitemia on day 0, with or without fever on day 2 (patients NKO-103/1, NKO- 60/1, NKO-129/1, NKO-112/1, and NKO-138/1). Atovaquone-Proguanil Treatment for Malaria JID 7

8 [12.9%] and 9 of 65 patients [13.8%], respectively; PP analysis). Based on this criterion of day-3 parasite clearance, ATPG performed poorly (58 of 161 patients [36.0%] with positive smears; PP analysis), as compared with ACTs and even amodiaquine monotherapy and amodiaquine-sulfadoxine-pyrimethamine. The slow action of ATPG in clearing parasitemia is one of its major disadvantages that may also explain some of the cases of early treatment failure observed with this drug. In an earlier study conducted in Gabon [26], early treatment failure due to a rebound parasitemia resulting in a parasite count on day 2 that was higher than that on day 0 was observed in one pediatric patient after treatment with ATPG, but the blood smear of this patient became negative for parasites on day 3 without any additional treatment. In the present study, 8 cases of early treatment failure were observed among ATPG-treated patients. Three patients had fever and parasitemia on day 3 and required rescue treatment on day 3. Five patients were classified as experiencing early treatment failure, based on the parasitological criterion of parasitemia on day 2 higher than that on day 0, with or without fever, and were censored on day 2. However, because of the delay in determining parasitemia by independent microscopists, these 5 patients, who did not manifest any sign of clinical aggravation and were afebrile on days 1, 2, and 3, were followed beyond day 2 without rescue treatment. Two of these patients were aparasitemic on days 7, 14, and 21 and afebrile from day 1 to day 21 but on day 28 had recurrent P. falciparum parasitemia and fever. Alternative treatment was administered on day 28. Genotyping demonstrated that, in both cases, reappearance of parasitemia on day 28 was due to reinfection. Three additional patients classified as experiencing early treatment failure were afebrile from day 1 onward and had parasite-negative smears from day 7 to day 28. These patients were followed until day 28 and did not require any alternative treatment. These latter cases illustrate that the criterion of early treatment failure (ie, parasitemia on day 2, with or without fever, that was greater than parasitemia on day 0) may not always predict clinical failure. Although this criterion may overestimate early treatment failure for slowacting drugs, it is necessary to ensure patients safety and welfare in a standardized protocol for drug studies. There were 16 late treatment failures occurring on or after day 7: 8 were in the ASAQ group, 8 were in the ATPG-1 and ATPG-2 combined groups, and none were in the AS-ATPG group. Only 2 of 8 failures occurring in the ASAQ group were due to recrudescence; the other 6 failures were due to reinfection. In the combined ATPG-1 and ATPG-2 groups, 7 of 8 failures were due to reinfection, while 1 failure was attributable to recrudescence, which was not associated with any mutation in cytochrome b. Therefore, most late failures were reclassified as new infections and censored in the ASAQ, ATPG-1, and ATPG-2 arms. The clinical observation was in agreement with the high in vitro activity of atovaquone and the absence of mutations, particularly in codon 268 of the cytochrome b gene. Moreover, mutant codon 268 is rarely found in Africa [49, 50]. In the present study, comparison of the tolerability profile was not planned since it was assessed in several earlier studies [20 27]. Most individual adverse events reported by the patients parents or legal guardians, such as headache, fever, myalgia, arthralgia, abdominal pain, gastrointestinal disorders, asthenia, loss of appetite, nausea, dizziness, vomiting, and cough, were present at enrollment and persisted during the first 2 3 days of follow-up. Formal links of these signs and symptoms with drug intake, rather than being the consequences of malarial disease, are difficult to establish, particularly because a large majority of parents reported disappearance of these signs and symptoms on or before day 7. No serious adverse effect was observed with any of the 3 drugs evaluated in the present study. In summary, ATPG was characterized by a slow blood schizontocidal action resulting in parasite-positive smears on day 3 and early treatment failure in some patients. ASAQ and ATPG were generally effective but were associated with reinfections, mostly occurring between day 14 and day 28. One of the limiting factors of artesunate-atovaquone-proguanil is the unavailability of a coformulated product, which may compromise patient compliance unless treatment is supervised. However, artesunate-atovaquone-proguanil was a highly effective alternative treatment that was not associated with any recrudescence or reinfection during the 28-day follow-up period. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online ( 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. 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