keywords Malaria, Plasmodium falciparum, artemisinin-based combination therapy, drug resistance, Somalia

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1 Tropical Medicine and International Health doi: /tmi volume 20 no 4 pp april 2015 Treatment of uncomplicated malaria with artesunate plus sulfadoxine pyrimethamine is failing in Somalia: evidence from therapeutic efficacy studies and Pfdhfr and Pfdhps mutant alleles Marian Warsame 1, Abdillahi Mohamed Hassan 2, Amy Barrette 1, Ali Mohamed Jibril 3, Husein Haji Elmi 4, Abdulkadir Mohamed Arale 5, Hanan El Mohammady 6, Rania A. Nada 6, Jamal Ghilan Hefzullah Amran 7, Abdikarim Muse 8, Fahmi Essa Yusuf 8 and Abdiqani Sheikh Omar 5 1 Global Malaria Programme, World Health Organization, Geneva, Switzerland 2 World Health Organization, WHO Office, Mogadishu, Somalia 3 INTERSOS, Jowhar Hospital, Middle Shabele, Somalia 4 Muslim Aid UK, Jamame, Lower Juba Region, Somalia 5 Ministry of Health and Human Service, Mogadishu, Somalia 6 US Naval Medical Research Unit-3, Cairo, Egypt 7 World Health Organization, WHO Somalia, Warwick Centre UN Avenue, Nairobi, Kenya 8 World Health Organization, WHO Office, Hargeisa, Somalia Abstract objective Artesunate plus sulfadoxine pyrimethamine (AS + SP) has been Somalia s national treatment policy since Routine monitoring of first-line malaria treatment is needed to ensure appropriate national malaria treatment policy and early detection of drug resistance. For this purpose, we conducted therapeutic efficacy studies of AS + SP for the treatment of uncomplicated malaria in Somalia in methods Studies were conducted in three sentinel sites. Eligible patients were evaluated for clinical and parasitological outcomes according to the WHO standard protocol. Molecular surveillance was conducted on resistance conferring mutations in the P.falciparum dihydrofolate reductase (dfhr) and dihydropteroate synthase (dhps) genes. results The proportion of PCR-corrected treatment failures was high in Jamame (22%, 95% CI: %) and low (<5%) in Janale and Jowhar. All patients cleared parasites by day 3. Molecular markers associated with SP resistance were detected in all three sites. Treatment failure was associated with the presence of the double mutant dhps A437G/K540E (OR = 22.4, 95% CI: ), quadruple mutant dhfr N51I/S108N+dhps A437G/K540E (OR = 5.5, 95% CI: ), quintuple mutant dhfr N51I/C59R/S108N+dhps A437G/K540E (OR = 3.5, 95% CI: ) and younger age (OR=0.86, 95% CI: ). conclusions The high treatment failure rate observed in Jamame, together with the presence of molecular mutations associated with SP resistance, indicates P. falciparum resistance to SP. In Jowhar, high treatment failure rates were absent despite the presence of molecular mutations; signs of resistance in vivo may have been masked by the stronger immunity of the older study population. The study underscores the need to update Somalia s national malaria treatment policy. keywords Malaria, Plasmodium falciparum, artemisinin-based combination therapy, drug resistance, Somalia Introduction Malaria remains an important public health problem in Somalia [1]. Transmission is moderate along the Juba and Shabele rivers, while the remaining areas of the country are characterised by low transmission, with occasional epidemics [2, 3]. Artesunate plus sulfadoxine pyrimethamine (AS + SP) is the current national treatment policy and one of five artemisinin-based combination therapies (ACTs) recommended by WHO [4, 5]. Therapeutic efficacy studies John Wiley & Sons Ltd

2 of AS + SP were conducted in Somalia between 2003 and 2006; high treatment efficacy (95 100%) was observed in each of the country s three sentinel sites [3]. These studies led to the adoption and implementation of AS + SP as Somalia s first-line treatment for uncomplicated falciparum malaria in The ACT is the recommended firstline treatment for uncomplicated P.falciparum malaria in most endemic countries located in the Middle East and in Central Asia [4]. Sulfadoxine pyrimethamine (SP) is the recommended medicine for intermittent preventive treatment in pregnancy (IPTp) in areas of moderate to high malaria transmission in Sub-Saharan Africa [6] including Somalia. In Somalia, SP is available in private drug stores and is frequently used as a monotherapy (M. Warsame, personal communication). Routine monitoring of the therapeutic efficacy of ACTs is needed for the early detection of drug resistance, which can emerge to the artemisinin and the partner drug independently, due to their different modes of action. Resistance to artemisinin is suspected when 10% of patients fail to clear parasites by day 3 after treatment with an ACT [7]. WHO recommends a change in national malaria treatment policy if the total treatment failure rate is 10%, as assessed through in vivo monitoring of therapeutic efficacy [4, 5]. Resistance to sulfadoxine pyrimethamine is mediated through point mutations that accumulate at several sites in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of P. falciparum [8]. Detection of these mutations has been an effective tool for monitoring SP resistance [9]. Mutations associated with SP resistance have been identified in codons 16, 51, 59, 108, 140 and 164 in the dhfr gene, and codons 436, 437, 540, 581 and 613 in the dhps gene [10]. The occurrence of the dhfr triple mutant (N51I/C59R/S108N) in combination with the dhps double mutant (A437G/K540E), called a quintuple mutant, has been associated with SP treatment failure [8, 11 13]. Other findings have suggested that the dhfr triple mutant (N51I/C59R/S108N) alone is a useful predictor of treatment failure [14, 15]. This study investigated the therapeutic efficacy of AS + SP and the presence of SP resistance conferring molecular markers in three surveillance sites in Somalia in This study is the first to examine molecular markers for SP resistance (dhfr and dhps) in Somalia. The findings have important implications for Somalia s current national malaria treatment policy. Methods Study sites The studies were conducted during the peak malaria transmission period in Somalia s three sentinel sites. The sites are situated along the belt of Juba and Shabele rivers: Jamame in the Lower Juba region, Janale in the Lower Shabele region and Jowhar in the Middle Shabele region. Mobile clinics were set up for patients living in rural communities within 10 km of each site. Studies were conducted in 2011, between July and September in Jamame and Jowhar, and from September to October in Janale. Patients Patients were eligible for inclusion if they were 6 months of age or above, had fever or history of fever in the previous 24 hours ( 37.5 C) and P. falciparum mono-infection with parasite density of asexual parasites/ll. Patients were excluded if they had one or more signs of severe or complicated malaria, mixed infection or infection with another species, or a febrile illness other than malaria. Female minors and unmarried women were also excluded, due to social and cultural norms related to requesting a pregnancy test from this population. Study procedures The study was an open-label, one-arm prospective evaluation of the therapeutic efficacy of AS + SP for the treatment of uncomplicated malaria. Methods were based on the standardised WHO protocol for monitoring antimalarial drug efficacy [16]. Clinical examination, including axillary temperature, was performed at enrolment (day 0) and on days 1, 2, 3, 7, 14, 21 and 28. Blood smear for determining parasite counts was taken on the same days except for day 1. Patients were also asked to report to the clinic at any time if new or recurrent symptoms occurred. Blood was obtained through finger prick for blood smears and stained with 2.5% Giemsa for 30 min. Parasite densities were determined on Giemsa-stained thick films and recorded as the number of asexual parasites per 200 white blood cells (or per 500, if the count was <10 parasites/200 white blood cells), assuming a white blood cell count of 8000 ll. A smear was declared negative if no parasites were seen after 1000 white blood cells were counted. For Plasmodium species detection, thin blood smears were taken on day 0. For quality assurance, all blood smears were re-read by a second microscopist. Blood smears with non-concordant results (difference in species or in parasite density of >50%) were re-read by a third microscopist, and the average parasite density of the two most concordant counts was used. Prior to enrolment, individual informed consent was obtained for all study patients. Consent was requested 2015 John Wiley & Sons Ltd 511

3 from parent/guardians of children aged less than 17 years. Permission to conduct the studies was obtained from the Ministry of Health of the Transitional Government of Somalia. The study team also sought permission from the community leaders and held public meetings in each study community to inform about the study objectives and procedures. The study information was read to each parent/guardian or patient at the time of individual patient recruitment by the study clinician, in the presence of the accompanying relative/friend. In cases where there was no accompanying relative/friend, a community representative was present. The required sample size was estimated based on an expected treatment failure rate of 5%. With a confidence level of 95% and precision of 5%, the target sample size was 73 patients per site. An additional 20% was added to ensure the sample size would be achieved after exclusion of patients due to loss to follow up and withdrawal. Treatment regimen All patients received the standard therapeutic dose of artesunate (4 mg/kg on days 0, 1 and 2), co-administered with SP (single dose of 25 mg sulfadoxine/1.25 mg pyrimethamine per kg with first artesunate dose). Study medicine was provided by WHO. All tablets were divided into quarters and given under direct supervision. If a patient vomited within 30 min of treatment, a full dose was readministered. All patients who failed treatment were treated with artemether plus lumefantrine. Drug tolerability and safety were assessed clinically. Adverse events and severe adverse events were monitored at enrolment and at each follow-up visit. An adverse event was defined as any untoward medical occurrence irrespective of its suspected relationship to the study medications. Severe adverse events were defined as any untoward medical occurrence requiring hospitalisation or resulting in death. Treatment outcomes Treatment outcomes were based on parasitological and clinical outcomes according to the methods recommended by WHO [16]. Therapeutic response was classified as either: early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The proportion of cases positive on day 3 was also recorded. Polymerase chain reaction (PCR) analysis was conducted to distinguish between recrudescence and reinfection. Parasite DNA was extracted from blood spots on day 0 and on the day of reappearance of asexual parasitemia. Spots were tested using nested PCR targeting polymorphic variant genes msp1, msp2 (merozoite surface proteins) and glurp (glutamate-rich protein) according to WHO recommended methods [17]. Molecular markers of sulfadoxine pyrimethamine resistance Mutations conferring resistance to sulphadoxine pyrimethamine were detected by analysing extracted DNA from all day 0 samples, to determine the mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes. Briefly, both dhfr and dhps genes were amplified using nested PCR, as described elsewhere [18]. Subsequently, positive PCR products were subjected to DNA sequencing using BigDye terminator chemistry (Applied Biosystems, Foster City, CA) on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). DNA sequences were assembled and mutations were verified by inspection of both forward and reverse strands using BioEdit version [19]. DNA sequencing analysis of the dhfr fragment was carried out to detect mutations at codons 16, 51, 59, 108 and 164, whereas dhps fragment was evaluated at codons 436, 437, 540, 581 and 613. Statistical analysis Data were double-entered and validated using a programme developed by WHO ( malaria/publications/atoz/ /en/). Statistical analysis was performed using Stata/IC 11.0 (Stata Corporation, College Station, TX, USA). Patient characteristics at baseline were compared across the three sites. Patients who were subsequently lost to follow up, had reinfections, or unknown PCR, were excluded from the per-protocol analysis of treatment outcomes, but included in the Kaplan Meier analysis until the day of withdrawal from the study. Differences in categorical data were compared using the chi-square test or the Fisher s exact test where cell counts were 5. Comparisons of mean age between groups were computed using the t-test. Exact confidence intervals were calculated for proportions. Logistic regression was used to estimate the association between treatment failure and the presence of dhfr/dhps mutations and patient age. Results A total of 283 patients were enrolled in the three sites, of which 89 were in Jamame, 92 in Janale and 102 in Jowhar. Patient characteristics on admission are summarised for each site in Table 1. Patient age ranged from John Wiley & Sons Ltd

4 6 months to 60 years. The study population in Jamame was younger than the other two sites, with a greater proportion of patients under 5 years of age (n = 32, 36%, 95% CI: ) than in Janale (n = 16, 17.4%, 95% CI: %, v 2 = 8.0, P = 0.005) and Jowhar (n = 12, 11.8%, 95% CI: %, v 2 = 15.7, P < 0.001). Jamame also had fewer older patients than the other two sites, with only 1 patient over 15 years of age (1.1%, 95% CI: 0 6.1%), compared to 17 patients in Janale (18.5%, 95% CI: %, P < 0.001) and 27 patients in Jowhar (26.5%, 95% CI: %, P < 0.001). No other differences in admission characteristics were observed between the three sites. Of the 283 patients, 14 were censored from the analysis of PCR-corrected treatment outcomes: 5 patients were lost to follow up (1 in Jamame on day 14 and 4 in Jowhar on day 7), 6 patients were excluded due to reinfections (2 on day 21 and 3 on day 28 in Jamame, 1 in Jowhar on day 21), and 3 were excluded due to unknown PCR results (2 in Jamame and 1 in Janale). As shown in Table 2, all patients cleared parasitaemia before day 3. The percentage of PCR-corrected treatment failures was higher in Jamame (n = 18, 22.2%, 95% CI: ) than in Janale (n = 4, 4.4%, 95% CI: %, P < 0.001) and Jowhar (n = 1, 1%, 95% CI: 0 5.6%, P < 0.001). Overall, patients with treatment Table 1 Patient characteristics at baseline Jamame (n = 89) Janale (n = 92) Jowhar (n = 102) Number of 55/34 46/46 68/34 male/female patients Age at baseline n (%) (95% CI) n (%) (95% CI) n (%) (95% CI) <5 years 32 (36.0) ( )* 16 (17.4) ( ) 12 (11.8) ( ) 5 15 years 56 (62.9) ( ) 59 (64.1) ( ) 63 (61.8) ( ) >15 years 1 (1.1) (0 6.1)* 17 (18.5) ( ) 27 (26.5) ( ) Temperature C mean (SD) a mean (SD) a mean (SD) a 38.2 (0.7) 38.1 (0.4) 37.7 (0.8) Parasitemia ll mean b (95% CI) mean b (95% CI) mean b (95% CI) ( ) ( ) ( ) a standard deviation, b geometric mean. *Statistically significant difference between Jamame and both of the other two sites (P < 0.05). Table 2 Clinical and parasitological outcomes Jamame (n = 89) Janale (n = 92) Jowhar (n = 102) Outcomes n (%) 95% CI n (%) 95% CI n (%) 95% CI Parasitemia, D3 0 (0) (0 4.0) 0 (0) (0 4.0) 0 (0) (0 4.0) PCR-uncorrected, D28 Lost to follow up/excluded 1 (1.1) (0 6.1) 0 (0) (0 0.4) 4 (3.9) ( ) ACPR 63 (71.6) ( )* 87 (94.6) ( ) 96 (98.0) ( ) Treatment failure 25 (28.4) ( )* 5 (5.4) ( ) 2 (2.0) ( ) LCF 7 (8.0) ( ) 5 (5.4) ( ) 2 (2.0) ( ) LPF 18 (20.5) ( )* 0 (0) (0 0.4) 0 (0) ( ) PCR-corrected, D28 Lost to follow up/excluded 8 (9.0) ( ) 1 (1.1) ( ) 5 (4.9) ( ) ACPR 63 (77.8) ( )* 87 (95.6) ( ) 96 (99) ( ) Treatment failure 18 (22.2) ( )* 4 (4.4) ( ) 1 (1.0) (0 5.6) LCF 4 (4.9) ( ) 4 (4.4) ( ) 1 (1.0) (0 5.6) LPF 14 (17.3) ( )* 0 (0) (0 4.0) 0 (0) (0 3.7) Kaplan Meier 18 (20.7) ( )* 4 (4.4) ( ) 1 (1.0) ( ) LCF, late clinical failure; LPF, late parasitological failure; ACPR, adequate clinical and parasitological response. *Statistically significant difference between Jamame and the other two sites (P < 0.05) John Wiley & Sons Ltd 513

5 Percentage of patients S108N dhfr N51I/S108N Jamame C59R/S108N Janale N51I/C59R/S108N wild type Jowhar dhps S436A A437G A437G/K540E Figure 1 Distribution of the prevalence of dfhr and dhps mutations, at sentinel sites at enrolment. failure were younger in age (n = 23, mean = 5.7 years, 95% CI: %) than those with treatment success (n = 246, mean = 11.1 years, 95% CI: ), (t = 2.47, P = 0.01). All therapeutic regimens of the study drug were well-tolerated and safe. No adverse events or deaths occurred. The prevalence of dhfr and dhps resistance conferring mutations is shown for each site in Figure 1. The presence of molecular markers could not be determined for 14 of the 283 patients who had missing values for both dhfr and dhps (1 from Jamame and 13 from Janale), and 2 additional patients who had missing values for dhps only (both from Jamame). All study patients carried the dhfr S108N mutation; however, only 5 patients, all from Janale, carried this as a single mutation. Double dhfr mutations (N51I/S108N or C59R/S108N) were observed among 60 (68.2%) patients in Jamame, 52 (65.8%) patients in Janale, and 52 (51%) patients in Jowhar. Between the two possible double mutations, the dhfr N51I/S108N combination pre-dominated in each site. The triple dhfr mutation (N51I/C59R/S108N) was observed among 28 (31.8%) patients in Jamame, 22 (27.9%) patients in Janale and 50 (49%) patients in Jowhar. No patients were observed to carry mutations at dhfr codons 16 or 164. Overall, dhps mutations were less prevalent (Figure 1). The dhps wild type was found among 33 (38.4%) patients in Jamame, 70 (88.6%) patients in Janale and 58 (56.9%) patients in Jowhar. The proportion of dhps sensitive patients in Janale was significantly higher than the other two sites (v 2 = 37.6, P < 0.001). Single mutations at S436A and A437G were found among 7 patients. The double dhps A437G/K540E mutation was found among 51 (59.3%) patients in Jamame, 6 (7.6%) patients in Janale and 42 (41.2%) patients in Jowhar. All 99 patients with the dhps double mutant A437G/K540E also had either a quadruple or quintuple mutation (discussed below). No mutations were observed at dhps codons 581 or 613. The distribution of dhps and dhfr mutations occurring in combination is shown for each site in Figure 2. The majority of patients in Jamame (n = 51, 59.3%) carried a quadruple or quintuple mutation, significantly more than the 6 patients in Janale (7.6%, v 2 = 48.7, P < 0.001), or the 42 patients in Jowhar (41.2%, v 2 = 6.1, P < 0.05). Only 5 of the 28 patients in Jamame who carried the dhfr N51I/C59R/S108N triple mutant were found to carry the dhps wild type. In Janale, most patients carried either the dhfr N51I/S108N or C59R/S108N double mutant and the dhps wild type, observed in 47 (59.5%) patients, or the dhfr N51I/C59R/S108N triple mutant and dhps wild type, observed in 18 (22.8%) patients. Jowhar had a more even distribution of mutations, with the dhfr double mutant (N51I/S108N) and dhps wild type found among 23 (22.5%) patients, the dhfr triple mutant (N51I/C59R/S108N) and dhps wild type among 34 (33.3%) patients, the quadruple mutant (dhfr N51I, S108N + dhps A437G, K540E) among 23 (22.6%) patients, and the quintuple mutant (dhfr N51I/C59R/ S108N + dhps A437G/K540E) among 16 (15.7%) patients. Further analysis revealed an association between treatment failure, multiple mutations and age. The odds of treatment failure were increased among patients who Percentage of patients Jamame (n = 86) Janale (n = 79) Quintuple Quadruple Triple Double Single Other Treatment failure 10% treatment failure Jowhar threshold for policy (n = 102) change Figure 2 Frequency distribution of patients with dfhr/dhps mutations at sentinel sites at enrolment and treatment failure rates at day 28. quintuple: dhfr N51I/C59R/S108N + dhps A437G/K540E; quadruple: dhfr N51I/S108N + dhps A437G/ K540E, or dhfr C59R/S108N + dhps A437G/K540E; triple: dhfr N51I/C59R/S108N + dhps wild type; double: dhfr N51I/ S108N + dhps wild type, or dhfr C59R/S108N + dhps wild type; single: dhfr S108N + dhps wild type; other: dhfr N51I/ S108N + dhps S436A, or dhfr C59R/S108N + dhps S436A, or dhfr C59R/S108N + dhps A437G John Wiley & Sons Ltd

6 carried the double mutant dhps A437G/K540E (n = 21, OR = 22.4, 95% CI: ), the quadruple mutant dhfr N51I/S108N + dhps A437G/K540E (n = 11, OR = 5.5, 95% CI: ), and the quintuple mutant (n = 8, OR = 3.5 (95% CI: ). Patients who carried either the quadruple or quintuple mutation and experienced treatment failure (n = 21) also had a lower mean age (5.6 years, 95% CI: ) than the patients with quadruple or quintuple mutations who recovered following treatment (n = 74, 9.9 years, 95% CI: years), t = 2.7, P < Overall, treatment failure was associated with younger age (OR = 0.86, 95% CI: ). Discussion In this study, the PCR-corrected treatment failure rate in one of the three study sites, Jamame (22.2%), surpassed the threshold of 10% which is recommended by WHO for reviewing and updating treatment policy [4, 5]. The high proportion of treatment failures and the high prevalence of molecular mutations associated with SP resistance provide clear evidence of the presence of resistance to the partner drug in Jamame. Artemisinins tend to kill the mass of parasites, after which the partner drug eliminates the remaining parasites. Given that all patients in this study cleared parasites by microscopy on day 3, the artemisinin component of the combination is still effective in all three sites [4]. It is worth noting that patients who had recrudescence likely had submicroscopic levels of parasitemia on day 3. Therefore, SP failed to clear the residual parasites. The odds of treatment failure were higher among younger patients and patients with quadruple and quintuple mutations. The high treatment failure rate in Jamame was expected, given the high prevalence of multiple mutations. Studies previously conducted in Kenya, Malawi and Uganda found an association between quintuple mutations and treatment failure [11, 20, 21]. Apart from the higher prevalence of drug resistance associated mutations, the lower age of the patients in Jamame may have contributed to the higher failure rate. Younger patients have less background immunity and children under five may have lower pyrimethamine and sulfadoxine levels than older children and adults [22]. However, in this study, we did not measure drug blood levels. In Jowhar, the treatment failure rate remained low (1%) despite a substantial number of patients carrying quadruple or quintuple mutations (41%). This inconsistency may be explained by the fact that patients in Jowhar were older than those in Jamame. Study patients in Jowhar are likely to have acquired a higher degree of immunity, which may have been responsible for clearing SP resistant parasites during treatment, effectively masking the presence of resistance. Similar findings have been reported elsewhere [21, 23, 24]. It is likely that in all sites, the prevalence of mutations will increase with sustained drug pressure. Unofficial use of SP as a monotherapy in Somalia is likely to further accelerate the proliferation of dhfr and dhps mutations [25]. Dhfr triple mutations (N51I/C59R/S108N) were observed in all three sites, indicating the presence of resistance to pyrimethamine. Previous studies have shown that the emergence of the triple dhfr (N51I/C59R/S108N) mutation precedes that of double dhps (A437G/K540E) mutant [8, 26, 27]. It is interesting to note that in this study, all patients who carried the dhps double mutant (A437G/K540E) also carried either the quadruple or quintuple mutation. The high prevalence of molecular markers associated with SP resistance detected in this study raises concerns about the life span of SP for IPTp. One study from Tanzania has shown that IPTp with SP fails to provide benefit in areas where SP resistance is widespread [28]. However, other evidence suggests that in sub-saharan Africa, in spite of the increased frequency of quintuple mutant dhfr/dhps haplotypes, IPTp-SP remains beneficial during pregnancy in preventing peripheral parasitemia, maternal anaemia, clinical malaria during pregnancy and is associated with reduced neonatal mortality [29 33]. Vigilant monitoring of the efficacy of SP for the prevention of malaria in pregnancy is encouraged. The high prevalence of treatment failures in Jamame, and the presence of dhfr and dhps mutations associated with SP resistance in all three sites, indicates the need to revise the current treatment policy of Somalia from AS + SP to a more effective ACT. With the failure of the partner drug SP, continued use of AS + SP as the first-line treatment in Somalia endangers patients lives. Furthermore, treatment effectively becomes a 3-day course of artesunate alone, which is insufficient to kill all parasites. Consequently, this will put the parasites under artesunate drug pressure, which facilitates the development of artemisinin resistance. Sustained use of an ACT with a failing partner drug will eventually compromise the artemisinin component. Routine monitoring of ACTs should be conducted in all malaria endemic countries for the early detection of P. falciparum resistance. Acknowledgements We would like to express our gratitude to all of the study patients, and to the parents and guardians of the children who participated. We thank the staff of the sentinel sites, 2015 John Wiley & Sons Ltd 515

7 especially Kaltumo Abdullahi Ali, Mohamed Mohamoud Jimale, Hussein Hirey Kulane, Mulki Nor Warsame and Aden Isak Ali in Janale; Ahmed Hassan Gomey, Ali Haji Mohamed, Liban Omar Afrah and Ibrahim Muse Mahaad in Jowhar; and Abdi Omar Abdi, Omar Ali Sharif and Ahmed Abdulkadir Mohamed and Ali Abdi Isak in Jamame. We are also grateful to Dr Pascal Ringwald for his collaboration. We thank the technical staff, especially Irene Kamal, Ismail Raafat and Mireille Kamel for their hard work. Financial support for the studies was provided by the Bill and Melinda Gates Foundation. References 1. WHO. World Malaria Report World Health Organization: Geneva, Noor AM, Clements AC, Gething PW et al. Spatial prediction of Plasmodium falciparum prevalence in Somalia. Malaria J 2008: 7: Warsame M, Atta H, Klena JD et al. Efficacy of monotherapies and artesunate-based combination therapies in children with uncomplicated malaria in Somalia. Acta Trop 2009: 109: WHO. 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