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    Patients’ dispositions.

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    A, Incidence of vomiting per treatment arm. B, Plasmodium falciparum infection clearance per treatment arm. AS + SP cleared parasite faster than AS + AQ or AS (P < 0.05). C, Fever clearance per treatment arm. AS + AQ cleared fever faster than AS (P < 0.001); differences between all other pairs did not reach statistical significance. D, Evolution of gametocyte carriage over 28 days of follow-up.

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Efficacy, Safety, and Selection of Molecular Markers of Drug Resistance by Two ACTs in Mali

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  • 1 Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Bamako, Mali

We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine–pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 100% efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > 0.05). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < 0.001), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < 0.001). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.

INTRODUCTION

The spread of malaria parasites resistant to safe and affordable drugs is greatly hampering the control of malaria in Africa. Resistance to the cheap monotherapies chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) is established in East, Southern, and Central Africa.13 Although rates of antimalarial drug resistance were lower in West Africa, an increase in chloroquine resistance was directly linked to increased malaria mortality and morbidity in Senegal.4 In Mali, a steady increase of Plasmodium falciparum resistance to chloroquine was reported.5 All sub-Saharan African countries have either changed their antimalarial treatment guidelines or are in the process of doing so.68

As with other infectious diseases, combination therapies rather than monotherapies have been shown to be better suitable for the treatment of malaria.9,10 Artemisinin and its derivatives are the most potent current antimalarial drugs.11 They produce a sharp and rapid decrease in the parasite biomass followed by a resolution of malaria symptoms. However, their short half-lives make patients treated with these drugs prone to reappearance of parasitemia in a few days,12 hence the necessity to combine artemisinin and its derivatives with another antimalarial drug with a longer half-life. These artemisinin-based combination therapies (ACTs) were first tested in South-East Asia where they demonstrated better cure rates, potentials to deter the spread of antimalarial drug resistance and to decrease malaria transmission.13,14

The World Health Organization (WHO) now recommends the use of ACTs for the treatment of uncomplicated malaria in nearly all malarious areas. The leading ACTs used in most of sub-Saharan Africa and contemplated for introduction into Mali were artesunate + amodiaquine (AS + AQ), artemether–lumefantrine (AR-L, Coartem; Novartis, Basel, Switzerland), and artesunate + sulfadoxine–pyrimethamine (AS + SP). Several studies aimed at evaluating the efficacy and safety of ACTs have been conducted.1419 Most of these studies compared the ACTs to monotherapies with SP or amodiaquine (AQ). However, no study comparing these ACTs to AS monotherapy in the same epidemiologic setting has been reported. Besides our recent report on the efficacy and safety of AR-L versus artesunate + sulfamethoxypyrazine–pyrimethamine in Mali, no study of the efficacy and safety of the remaining leading ACTs in the country has yet been published.20 Furthermore, very little is known on the effect of ACTs on the selection of molecular markers associated with the partner drug of the artemisinin derivative. Here we report the efficacy, safety, and impact on molecular markers of antimalarial drug resistance of a 3-day treatment with AS + AQ or AS + SP compared with 5 days of AS monotherapy (the reference treatment of uncomplicated malaria at the initiation of this study).

PATIENTS AND METHODS

The study was conducted from December 2002 to October 2004 in Bougoula-Hameau, a village of 5,000 people in Southern Mali. P. falciparum is hyperendemic with seasonal peaks.21 Day 14 prevalences of CQ and SP resistance were 25% and 7%, respectively (our unpublished data).

We conducted a randomized single-blinded in vivo efficacy study. We tested the hypothesis that AS + AQ and AS + SP given over 3 days were non-inferior to 5 days of AS monotherapy. Patients weighing more than 5 kg and presenting with uncomplicated malaria caused by any of the 4 human malaria parasites (P. falciparum, Plasmodium malaria, Plasmodium ovalae, and Plasmodium vivax) were eligible for inclusion. After ensuring that inclusion criteria were met, written informed consent or assent was obtained from the patients or guardians. The following criteria needed to be met for entry into the study: adults or children aged 6 months or older and weighing more than 5 kg; residents of Bougoula-Hameau during the entire follow-up period; oral treatment possible; axillary temperature ≥ 37.5°C; infection with Plasmodium species with parasite density between 2,000 and 200,000 trophozoites/mm3; informed consent or assent obtained from the patient or parent/guardian. Patients were excluded if they had symptoms of severe malaria as defined by WHO; allergy to one of the study drugs; pregnancy (disclosed or clinically patent pregnancy); or documented consumption of 1 of the study drugs during the preceding 7 days.

Enrolled patients were randomly assigned to treatment groups. They underwent clinical and/or biologic follow-up on days 1, 2, 3, 4, 5, 7, 14, 21, and 28 or on days of recurrent illness. The randomization list was concealed to the clinicians. Drug administration and follow-up were done exactly alike among the 3 study arms. Cases of treatment failures were treated with quinine.

Thick blood films and blood on filter paper samples were made on days 0, 1, 2, 3, 7, 14, 21, and 28 and on any days of recurrent illness. Parasitemia and rate of gametocyte carriage were measured as described.20 On Day 0 prior to treatment and on Days 7, 14, and 28, 2–5 mL of blood was collected for glucose, hemoglobin measurements (HemoCue, ngelholm, Ä Sweden), complete blood count (CBC) with differential count, liver enzyme (ALT, alanine aminotransferase), and serum creatinine. If clinically significant abnormal laboratory values were found on days 7 or 14, a repeat measurement was made on Day 28.

All drug doses were administered under supervision of the study personnel. Patients were observed for an hour after administration of the treatment. Full drug doses were re-administered if the patients vomited study drug within 0.5 hour. Vomiting of re-administered study drugs resulted in withdrawal of the patient.

Toxicity ranges were computed according to standard laboratory ranges, and any values above or below those ranges were considered as abnormal. However, only clinically significant abnormal laboratory values found on days 7 or 14 led to repeat measurements on Day 28. In addition, toxicity was also evaluated according to the Division of Microbiology and Infectious Diseases (DMID)-defined grades of toxicity in the respective age categories. (DMID Adult Toxicity Table, May 2001, and DMID Pediatric Toxicity Tables, May 2001).

Study arm 1 was Arsucam (AS 50 mg/AQ 153 mg; Sanofi-Aventis, Paris, France), AS 4 mg/kg/day + AQ 10 mg/kg/day, 3 days of treatment. Arm 2 was Arsumax (AS 50 mg; Sanofi-Aventis) + sulfadoxine–pyrimethamine (S = 500 mg/P = 25 mg, Roche), AS 4 mg/kg/day for 3 days + SP 1 tablet/20 kg, single dose administered on Day 0. Arm 3 was Arsumax (AS 50 mg; Sanofi-Aventis), monotherapy, 4 mg/kg on Day 0 followed with 2 mg/kg/day on Days 1–4. 14 Adults received a maximum of 200 mg of artesunate, 4 tablets of amodiaquine (153 mg base), and 3 tablets of SP. Doses for children were rounded to the nearest quarter tablet.

The primary endpoints were adequate clinical and parasitological response (ACPR) by Day 14 and tolerance and safety, i.e., incidence of clinical adverse events and modification of biologic parameters. Adverse events were defined as previously reported.20 The first 53 patients were included using the WHO 1996 protocols.22 The remainder of the study used the current (2003) WHO guidelines.23 Briefly, the presence of parasitemia without fever on days 14–28 was considered as a cure in the WHO 1996 protocol while the same case was counted as a late parasitological failure according to the WHO 2003 protocol. Additional endpoints included parasite and fever clearance times, evolution of anemia and gametocytemia, and clinical and parasitogical cure at Day 28.

Filter-paper samples were used for molecular analyses of msp1 and msp2 polymorphisms to distinguish recrudescence from new infections.20,24,25 Cases of post-treatment mixed infection that contained parasites from Day 0 were counted as recrudescent. Change of profile in any or both loci was counted as re-infection. Molecular corrected ACPRs (cACPR) were recalculated by limiting the cases of failure to true recrudescent parasites.

With AS + AQ or AS monotherapy, P. falciparum chloroquine-resistance transporter (PfCRT) K76T and P. falciparum multidrug resistance 1 (PfMDR1) N86Y mutations were genotyped in randomly selected pretreatment samples and in parasitemias occurring between days 7 and 28.26 Post-treatment infections were included in the analysis regardless of their status of new infections or recrudescences. DHFR S108N, C59R, and N51I and DHPS A437G and K540E were detected in randomly selected baseline samples and in post AS + SP or AS treatment samples.27 With all polymorphisms, cases of mixed infections were considered as mutants for data-analysis purposes.

The required sample size was determined using the assumption of non-inferiority of the 2 ACTs to AS monotherapy and referred to the published 95% efficacy of AS + AQ at Day 14.15 The maximum accepted difference in efficacy between the 2 regimens was set at 5% (5.3% relative acceptability limit), α at 0.05, and β at 0.2. A total of 251 patients per treatment arm (753 patients total, including 10% lost to follow-up) were needed. Data entry and analysis were performed as reported.20 Frequencies of molecular markers in pre- and post-treatment were compared using Pearson’s χ2 test.

The study protocol and all amendments were approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Bamako, Mali. Informed consent was obtained from all adult participants and from parents or legal guardians of minors.

Role of the study sponsor.

The study sponsor was involved with protocol development and reporting of severe adverse events (SAEs) and provided the external monitoring of the study. The sponsor was not involved with any data collection, data analysis, or data interpretation.

RESULTS

Descriptive results.

Overall, 753 patients were included into the study. The mean age was 4.4 years, and 47% of the included patients were males. At baseline, the treatment arms were comparable with regard to median age, sex, prevalence of anemia, mean asexual parasitemia, and prevalence of gametocyte carriage (Table 1). Overall, 17 (2.2%) patients were excluded from the analysis for the reasons shown in Figure 1.

Treatment outcome in P. falciparum mono-infections.

Table 2 shows that noncorrected Day 14 ACPR was > 99% for both AS + AQ and AS + SP but was 92.9% for AS (P < 0.001). After identification of recrudescences by genotyping (molecular correction), the differences in efficacy between each of the ACTs and the reference were no longer statistically significant (Table 2). Day 28 analysis showed that AS + SP was more efficacious than both AS + AQ and AS and that AS + AQ was more efficacious than AS (P < 0.001 for each comparison; Table 2). However, after molecular correction all 3 treatments regimens achieved > 96% efficacy, and the differences were no longer statistically significant (Table 2).

Treatment outcome in nonfalciparum malaria or in malaria with mixed species.

We found no case of P. vivax and < 5% of P. malaria and P. ovalae in association with P. falciparum at Day 0. In all cases of mixed infections, the nonfalciparum parasites were completely cured from Day 3 onward. We found 5 cases of P. malaria mono-infection (2, 1, and 2 in the AS + AQ, AS + SP, and AS arms, respectively). All but the 2 infections in the AS arm were cleared by Day 28.

Impact of treatment on vomiting, parasite, and fever clearance and gametocyte carriage.

There was no difference in the incidence of vomiting during the first 5 days of treatment (Figure 2A). The Day 1 parasite clearance was significantly faster with AS + SP, followed by AS monotherapy and AS + AQ (P < 0.05; Figure 2B). Figure 2C shows that fever clearance by Day 1 post-treatment was significantly better with AS + AQ than AS + SP and AS monotherapy (P < 0.001). All treatments sharply decreased the prevalence of anemia in the population, and at each time point the prevalence of anemia was comparable among the 3 study arms (data not shown).

All treatment arms induced a transient, nonstatistically significant rise in gametocyte carriage at Day 3 post-treatment followed by a steady decrease. At no time during follow-up did we observe a statistically significant difference in the rate of gametocyte carriage between the 3 arms (Figure 2D).

Safety evaluation.

No clinically significant abnormal laboratory values were found. All treatment decreased the prevalence and incidence of abnormal values of creatinin, leukocytes, and platelets. The baseline and Day 7 post-treatment distributions of ALT toxicity (mostly DMID grade 1) were comparable between groups (details not shown). However, at Day 14 post-treatment, the prevalence of grade 1 ALT toxicity was 9.7%, (N = 247), 2.5% (N = 249), and 4.8% (N = 249) in the AS + AQ, AS + SP, and AS, respectively (P = 0.006). Similarly, the incidence of abnormal ALT during follow-up was 10.6% (N = 226), 3.6% (N = 225), and 3.1% (N = 226) for the treatments listed above (P = 0.001).

No death occurred in this study. Two SAEs (1 in the AS arm and 1 in the AS + AQ arm) were seen, including 1 case of severe anemia and 1 case of respiratory distress and severe anemia. Both patients were referred to the Regional Hospital of Sikasso and fully recovered.

The majority of malaria related symptoms (headaches, body aches, joint aches, asthenia, malaise, stomach aches) resolved by Day 3 following each of the treatments. However, the prevalence of diarrhea on Day 3 was 2% (N = 250), 3.1% (N = 249), and 8% (N = 251) in the AS + AQ, AS + SP, and AS arms, respectively (P < 0.01). Adverse event (AE) distribution was unremarkable. AEs with the highest incidence were vomiting followed by stomach aches and headaches.

ACT treatment and molecular markers of resistance to the partner drug.

Thirty-six pre-treatment and all but 1 post-treatment infections of the AS + SP arm were genotyped for the above-mentioned DHFR and DHPS mutations. The rates of the genotypes associated with SP resistance, i.e., the triple-mutant genotype (DHFR 108N + 59R + 51I) and quadruple-mutant genotype (triple DHFR mutant and DHPS 437G) were significantly higher in post-treatment infections compared with baseline infections (P < 0.001 with both genotypes) (Table 3). No DHPS 540E and hence no quintuple-mutation genotype was found in this study.

Overall, 91 randomly selected pre-treatment samples yielded complete genotypes for PfCRT and PfMDR1 polymorphisms in the AS + AQ arm. Thirty-two randomly selected post-AS + AQ infections were genotyped. We found that AS + AQ treatment significantly selected PfCRT 76T and PfMDR1 86Y mutations (P < 0.001 for each genotype; Table 3).

Thirty-four samples were genotyped before and after AS treatment in monotherapy. The prevalence of DHFR, DHPS, PfCRT, and PfMDR1 mutations were comparable in the baseline infections and in the post-treatment ones (Table 3).

DISCUSSION

We have shown that 3 days of artesunate in combination with 3 days of amodiaquine (AS + AQ) or a single dose of sulfadoxine–pyrimethamine (AS + SP) are as efficacious as 5 days of artesunate (AS) monotherapy in the hyperendemic malaria setting of Mali. After molecular correction, near-complete efficacy was achieved with both ACTs at days 14 and 28. At the initiation of this study, 5 days of AS was considered to be the optimum treatment of uncomplicated malaria.

This high efficacy of combination regimens is similar to previous reports in other African countries.8,14,17 However, the tested ACTs showed better efficacy than in Kenya, where rates of drug resistance are known to be higher than in Mali.28,29 AS + SP was less efficacious than AS + AQ in the Democratic Republic of Congo, which may be explained by high SP resistance in that study setting.18

Twenty-four hours after initiation of therapy, AS + SP showed faster parasite clearance while fever clearance was fastest with AS + AQ. During the first 7 days of follow-up, all 3 groups showed comparable efficacy against anemia, vomiting, headaches, body aches, joint aches, asthenia, and malaise. There were significantly more cases of diarrhea in the AS arm on Day 3.

In addition to P. falciparum infections, we show that the ACTs were efficacious against P. malariae and P. ovale. Most studies of uncomplicated malaria in Africa limit inclusion to patients carrying P. falciparum monospecific infections.15 We included all other human malaria parasites to test the efficacy of the ACTs on these other species. Indeed, a cyclic appearance of different malaria species has been described.30 Two mono-infections with P. malariae failed to clear after treatment with AS monotherapy. No molecular correction was performed on these infections, and the significance of these observations is not known.

The discrepancies between the raw efficacy and the molecular corrected efficacy can be easily explained by the intensity of malaria transmission in this village21 as well as by the high efficacy of SP in Mali31 and its long elimination half-life.32 We note that our molecular distinction of recrudescent versus re-infections was based solely on size polymorphism in 2 markers (MSP1 and MSP2).33 Restriction digestion of the amplified products34 or the use of neutral markers such as microsatellites35 could have yielded a higher degree of polymorphism and increased the sensitivity of the distinction. No in vitro drug efficacy assay was performed. Therefore, the few infections classified as recrudescent in the AS arm at Day 14 or 28 may not be considered as true AS-resistant parasites.

Our data confirm previous reports showing a transient increase of gametocyte carriage at Day 3 after AS + AQ treatment.36 This could be due to the slower pace of Day 1 parasite clearance seen with this combination, which may leave more time for asexual parasites to differentiate into gametocytes. As with previous studies, we found a steady reduction of gametocyte carriage following treatment by all artemisinin-containing regimens.14

No significant adverse event attributable to any of the study drugs was evident from this study. Therefore, the combination regimens were comparable to AS with regard to safety and tolerability. Furthermore, each treatment regimen decreased the prevalence of abnormal values of markers of blood and kidney toxicity. In the AQ + AS group, the prevalence and incidence of abnormal titers of liver enzymes were noted, but these were not clinically significant and may be related to the known liver toxicity of amodiaquine.37 The incidence of diarrhea was significantly higher in the AS arm than in the ACTs.

We show a highly significant selection of PfCRT 76T and PfMDR1 86Y after AS + AQ treatment. These mutations are known to be selected by AQ monotherapy.38 Conversely, AS + SP treatment exerted a significant selection on DHFR and DHPS mutations that are associated with SP resistance. The data are consistent with the recent report of selection of PfCRT, PfMDR1, DHFR, and DHPS mutations by AQ + SP.39 A recent report found a selection of PfMDR1 86Y but not PfCRT 76T in post AS + AQ infections. As rightfully pointed by the authors, this may be explained by the near fixation of that mutant in that setting.40 We found that AS did not exert any selection upon PfCRT, PfMDR1, or DHFR and DHPS mutations, which are associated with resistance to AQ and SP, respectively. To our knowledge, this is the first report of the effect of AS monotherapy on these markers in Africa. The main rationale for combining artemisinin derivatives with another drug is the need for protection against the selection of resistant parasite to both drug partners.41 Our data indicate that the current combination scheme may protect artemisinin but not the other drug.

This study demonstrates that 3 days of artesunate combined either with amodiaquine or sulfadoxine– pyrimethamine can safely cure uncomplicated malaria in Mali. However, the selection of molecular markers associated with resistance to the respective partner drugs raises concerns over the lifespan of these combinations in Africa. Further studies are needed to measure the long-term impact of these treatments on malaria morbidity, transmission, and drug resistance.

Table 1

Descriptive results, the 3 treatment arms were comparable with regard to median age, sex, baseline parasitemia, prevalence of anemia, and prevalence of gametocyte carriage*

DrugMedian age (years)Sex (% male)Anemia† (%)Parasitemia (geometric mean)Gametocytemia (%)
* P > 0.05 in all comparisons.
† Hemoglobin < 10 g/dL.
AS + AQ (N = 252)3.9 (0.6–38)44.841.814,371 (12,585–16,409)6.8
AS + SP (N = 250)3.0 (0.6–24)42.245.816,897 (14,860–19,213)5.6
AS (N = 251)3.0 (0.6–24)54.24315,760 (13,887–17,887)6
Table 2

Molecular noncorrected and corrected Day 14 and Day 28 efficacies of the 3 treatment regimens

ETF*LCFLPFACPRcACPR
AS + AQ % (N)AS + SP % (N)AS % (N)AS + AQ % (N)AS + SP % (N)AS % (N)AS + AQ % (N)AS + SP % (N)AS % (N)AS + AQ % (N)AS + SP % (N )AS % (N )AS + AQ % (N )AS + SP % (N)AS % (N )
* See text for abbreviations.
† AS + AQ vs. AS + SP, P = 0.002; AS + AQ vs. AS, P < 0.001; AS + SP vs. AS, P < 0.001.
‡ AS + AQ vs. AS + SP, P < 0.001; AS + AQ vs. AS, P < 0.001; AS + SP vs. AS, P < 0.001.
§ AS + AQ vs. AS + SP, P = not significant; AS + AQ vs. AS, P < 0.001; AS + SP vs. AS, P < 0.001.
¶ AS + AQ vs. AS + SP, P < 0.001; AS + AQ vs. AS, P < 0.001; AS + SP vs. AS, P < 0.001.
|| AS + AQ vs. AS + SP, P = not significant; AS + AQ vs. AS, P = not significant; and AS + SP vs. AS, P = not significant.
Day 140 (237)0 (236)0 (237)0 (237)0 (236)0.8 (237)0 (237)0.8 (236)6.3 (237)100.0 (237)99.2 (236)92.9§ (237)100.0 (232)100 (234)98.3|| (234)
Day 280 (235)0 (232)0 (234)5.9 (235)0.9 (232)17.1† (234)12.8 (235)3.4 (232)25.2‡ (234)81.3 (235)95.7 (232)57.7¶ (234)99.1 (229)100 (229)96.5|| (231)
Table 3

Analysis of molecular markers of AQ and SP resistance, before and after treatment*

ASAS + SPAS + AQ
FR51 % (N )FR59 % (N)FR108 % (N)PS437 % (N )PS540 % (N)Triple % (N )Quad† % (N )% (N)MDR % (N)FR51 % (N)FR59 % (N)FR108 % (N)PS437 % (N )PS540 % (N)Triple % (N )Quad % (N)CRT % (N)MDR % (N )
* Definitions: Pre-treatment = before drug treatment; Post-treatment = after drug treatment; FR51 = DHFR 51I; FR59 = DHFR 59R; FR108 = DHFR 108N; PS437 = DHPS 437G; PS540 = DHPS 540E; Triple = (DHFR 108N + 59R + 51I); Quad = (DHFR 108N + 59R + 51I and DHPS 437G); CRT = PfCRT 76T; MDR = PfMDR1 86Y; NS = not significant.
Pre-treatment40.0 (35)37.1 (35)51.4 (35)40 (35)0.0 (35)25.7 (35)17.1 (35)81 (79)51.9 (79)36.1 (36)19.4 (36)30.6 (36)47.2 (36)0 (36)8.3 (36)5.6 (36)70.3 (91)62.6 (91)
Post-treatment35.7 (28)42.8 (28)50 (28)46.4 (28)0 (28)32.1 (28)17.9 (28)86 (57)40.4 (57)77.8 (9)88.9 (9)88.9 (9)88.9 (9)0 (9)77.8 (9)77.8 (9)100.0 (32)84.4 (32)
PNSNSNSNSNSNSNSNS0.06< 0.0010.0050.06< 0.001< 0.001< 0.001< 0.001
Figure 1.
Figure 1.

Patients’ dispositions.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.455

Figure 2.
Figure 2.

A, Incidence of vomiting per treatment arm. B, Plasmodium falciparum infection clearance per treatment arm. AS + SP cleared parasite faster than AS + AQ or AS (P < 0.05). C, Fever clearance per treatment arm. AS + AQ cleared fever faster than AS (P < 0.001); differences between all other pairs did not reach statistical significance. D, Evolution of gametocyte carriage over 28 days of follow-up.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.455

*

Address correspondence to Abdoulaye A. Djimdé, Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Bamako, PO Box 1805, Point G, Bamako, Mali (West Africa). E-mail: adjimde@mrtcbko.org

Authors’ addresses: Abdoulaye A. Djimdé, Bakary Fofana, Issaka Sagara, Bakary Sidibe, Sekou Toure, Demba Dembele, Souleymane Dama, Dinkorma Ouologuem, Alassane Dicko, and Ogobara K. Doumbo, Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Odonto-Stomatology (DEAP/FMPOS), University of Bamako, PO Box 1805, Point G, Bamako, Mali (West Africa), Telephone/Fax: + 223-222-81-09, E-mail: adjimde@mrtcbko.org.

Acknowledgments: The authors thank the local guides of Bougoula-Hameau (Chiaka Traore, Bourama Diarra, and Karim Traore), the Village council, the entire population of Bougoula-Hameau, and Health and Administrative authorities of Sikasso for their help and support during the study. The authors thank Mr. Ousmane Toure for help with graphics. We are grateful to Dr. Valerie Lameyre for her help and support for this study. This work was supported by Access to Medicines, Sanofi-Aventis, and by the International Atomic Energy Agency (grant RAF/6025). A.A.D. is supported by European and Developing Countries Clinical Trial Partnership Senior Fellowship (grant 2004.2.C.f1) and Howard Hughes Medical Institution International Scholarship (grant 55005502).

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Author Notes

Reprint requests: Abdoulaye A. Djimdé, MRTC/FMPOS, Point G, Bamako, Mali (West Africa), Telephone: + 223-222-8109, E-mail: adjimde@mrtcbko.org.
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