Therapeutic efficacy of artemether-lumefantrine and artesunate-amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Mali, 2015-2016.

BACKGROUND: The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali. METHODS: Children between 6 and 59 months of age with uncomplicated Plasmodium falciparum infection and 2000-200,000 asexual parasites/µL of blood were enrolled, randomly assigned to either AL or ASAQ, and followed up for 42 days. Uncorrected and PCR-corrected efficacy results at days 28 and 42. were calculated. Known markers of resistance in the Pfk13, Pfmdr1, and Pfcrt genes were assessed using Sanger sequencing. RESULTS: A total of 449 patients were enrolled: 225 in the AL group and 224 in the ASAQ group. Uncorrected efficacy at day 28 was 83.4% (95% CI 78.5-88.4%) in the AL arm and 93.1% (95% CI 89.7-96.5%) in the ASAQ arm. The per protocol PCR-corrected efficacy at day 28 was 91.0% (86.0-95.9%) in the AL arm and 97.1% (93.6-100%) in the ASAQ arm. ASAQ was significantly (p < 0.05) better than AL for each of the aforementioned efficacy outcomes. No mutations associated with artemisinin resistance were identified in the Pfk13 gene. Overall, for Pfmdr1, the N86 allele and the NFD haplotype were the most common. The NFD haplotype was significantly more prevalent in the post-treatment than in the pre-treatment isolates in the AL arm (p < 0.01) but not in the ASAQ arm. For Pfcrt, the CVIET haplotype was the most common. CONCLUSIONS: The findings indicate that both AL and ASAQ remain effective for the treatment of uncomplicated malaria in Sélingué, Mali.

Mosquito microevolution drives Plasmodium falciparum dynamics.

Malaria, a major cause of child mortality in Africa, is engendered by Plasmodium parasites that are transmitted by anopheline mosquitoes. Fitness of Plasmodium parasites is closely linked to the ecology and evolution of its anopheline vector. However, whether the genetic structure of vector populations impacts malaria transmission remains unknown. Here, we describe a partitioning of the African malaria vectors into generalists and specialists that evolve along ecological boundaries. We next identify the contribution of mosquito species to Plasmodium abundance using Granger causality tests for time-series data collected over two rainy seasons in Mali. We find that mosquito microevolution, defined by changes in the genetic structure of a population over short ecological timescales, drives Plasmodium dynamics in nature, whereas vector abundance, infection prevalence, temperature and rain have low predictive values. Our study demonstrates the power of time-series approaches in vector biology and highlights the importance of focusing local vector control strategies on mosquito species that drive malaria dynamics.

Measuring the impact of seasonal malaria chemoprevention as part of routine malaria control in Kita, Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a new strategy recommended by WHO in areas of highly seasonal transmission in March 2012. Although randomized controlled trials (RCTs) have shown SMC to be highly effective, evidence and experience from routine implementation of SMC are limited. METHODS: A non-randomized pragmatic trial with pre-post design was used, with one intervention district (Kita), where four rounds of SMC with sulfadoxine + amodiaquine (SP + AQ) took place in August-November 2014, and one comparison district (Bafoulabe). The primary aims were to evaluate SMC coverage and reductions in prevalence of malaria and anaemia when SMC is delivered through routine programmes using existing community health workers. Children aged 3-59 months from 15 selected localities per district, sampled with probability proportional to size, were surveyed and blood samples collected for malaria blood smears, haemoglobin (Hb) measurement, and molecular markers of drug resistance in two cross-sectional surveys, one before SMC (July 2014) and one after SMC (December 2014). Difference-in-differences regression models were used to assess and compare changes in malaria and anaemia in the intervention and comparison districts. Adherence and tolerability of SMC were assessed by cross-sectional surveys 4-7 days after each SMC round. Coverage of SMC was assessed in the post-SMC survey. RESULTS: During round 1, 84% of targeted children received at least the first SMC dose, but coverage declined to 67% by round 4. Across the four treatment rounds, 54% of children received four complete SMC courses. Prevalence of parasitaemia was similar in intervention and comparison districts prior to SMC (23.4 vs 29.5%, p = 0.34) as was the prevalence of malaria illness (2.4 vs 1.9%, p = 0.75). After SMC, parasitaemia prevalence fell to 18% in the intervention district and increased to 46% in the comparison district [difference-in-differences (DD) OR = 0.35; 95% CI 0.20-0.60]. Prevalence of malaria illness fell to a greater degree in the intervention district versus the comparison district (DD OR = 0.20; 95% CI 0.04-0.94) and the same for moderate anaemia (Hb < 8 g/dL) (DD OR = 0.26, 95% CI 0.11-0.65). The frequency of the quintuple mutation (dhfr N51I, C59R and S108N + dhps A437G and K540E) remained low (5%) before and after intervention in both districts. CONCLUSIONS: Routine implementation of SMC in Mali substantially reduced malaria and anaemia, with reductions of similar magnitude to those seen in previous RCTs. Improving coverage could further strengthen SMC impact. Trial registration clinical trial registration number NCT02894294.

Molecular incidence and clearance of Plasmodium falciparum infection.

BACKGROUND: Although the epidemiology of malaria has been based primarily on microscopy and rapid diagnostic tests, molecular methods are necessary to understand the complexity of natural infection in regions where transmission is intense and simultaneous infection with multiple parasite genotypes is common such as sub-Saharan Africa. METHODS: To compare microscopic and molecular estimates of the incidence and clearance of Plasmodium falciparum infection, we followed 80 children monthly for 1 year in the village of Bancoumana in Mali. RESULTS AND DISCUSSION: Similar seasonal patterns were observed with both methods (rainy season peak, dry season nadir), although molecular methods detected more infections than microscopy (571 vs 331 in 906 specimens), more new infections (311 vs 104 during 829 person-months) and spontaneous clearance events (317 vs 116) and found higher incidence (0.38 vs 0.13 new genotypes/person/month, p < 0.001) and spontaneous clearance rates (0.38 vs 0.14 genotypes cleared/person/month, p < 0.001). These differences were greatest for persistently-infected subjects in whom neither new infections nor the clearance of old infections could be detected by microscopy (0.71 new infections and 0.73 cleared infections per month using molecular methods vs 0.000 by microscopy, p < 0.001). CONCLUSIONS: Molecular methods provide information about genetic diversity, the intensity of transmission and spontaneous clearance in the absence of drug treatment that cannot be obtained by microscopy. They will be necessary to evaluate the efficacy of vaccines, drugs and other control strategies for diseases such as malaria in which simultaneous infection with more than one organism (genotype) is common.

[Study of the impact of breeding sites on the infectivity of P. falciparum gametocytes to An. gambiae s.l in malaria-endemic area of Nanguilabougou.-Mali]. / Etude de l'impact des gites larvaires sur l'infectivite des gametocytes de Plasmodium Falciparum chez Anopheles Gambiae s.l en zone d'endemie palustre de Nanguilabougou - Mali.

Numerous studies have shown the influence of some bacteria colonizing the breeding sites on the development of the parasite in Anopheles malaria vectors. The aim of this study was to evaluate the impact of the breeding sites of Plasmodium falciparum on the gametocytes infectivity of Anopheles gambiae sl using the membrane feeding method. We carried out an experimental infestation study from September 2010 to January 2011 in a village located in the southern savanna of Mali. Cross sectional surveys were conducted to collect larvae and to select gametocyte carriers. Female offspring of L3 and L4 stage larvae were used for the experimental infestation. The gametocyte carriers were children aged within 4 to 12 years. Bacteria identified in breeding water of larvae and adult mosquitoes were: Escherichia coli, Salmonella spp, Klebsiella oxytoca, Pseudomonas spp, Staphylococcus spp, Shigella sp. The oocyst load of infected mosquitoes did not significantly vary according to the samples (F = 1.517, P = 0.230). Low infection rates (5.4% and 2.8%) were observed in the larval bedding and in mosquitoes containing K. oxytoca and Shigella. The blood feeding rates (62.4%; 60.9%; 67.7%) and mortality (66.4%; 64.9%; 61.9%) of An. gambiae did not significantly vary depending on the breeding sites [(P = 0.15); (P = 0.22)].

De nombreuses études ont montré l'influence de certaines bactéries vivant dans les gites larvaires sur le développement du parasite chez les anophèles vecteurs du paludisme. Le but du présent travail consistait à étudier l'impact des gites larvaires sur l'infectivité des gamétocytes de Plasmodium falciparum chez Anopheles gambiae s.l par la technique d'infestation sur membrane. Pour réaliser ce travail, une étude d'infestation expérimentale a été conduite de septembre 2010 à janvier 2011 dans un village de savane sud soudanienne du Mali. Des passages transversaux ont été effectués pour la collecte des larves et la sélection des porteurs de gamétocytes. Les femelles issues des larves de stades L3 et L4 ont été utilisées pour les séances d'infestation expérimentale. Les enfants porteurs de gamétocytes étaient âgés de 4 à 12 ans. Les bactéries identifiées dans l'eau des gites chez les larves et les adultes de moustiques étaient : Escherichia coli, Salmonella s.p., Klebsiella oxytoca, Pseudomonas s.p., Staphylococcus s.p., Shigella sp. La charge oocystique des moustiques infectés n'avait pas varié de façon significative en fonction des gites (F=1,517 ; P=0,230). De faibles taux d'infection (5,4% et 2,8%) ont été observés dans les gites et chez les moustiques contenant K. oxytoca et Shigella. Le taux d'engorgement (62,4% ; 60,9% ; 67,7%) et le taux de mortalité (66,4% ; 64,9% ; 61,9%) d'An. gambiae n'ont pas varié significativement en fonction des gites larvaires,[(P= 0,15) ; (P= 0,22)].

Co-infection of long-term carriers of Plasmodium falciparum with Schistosoma haematobium enhances protection from febrile malaria: a prospective cohort study in Mali.

BACKGROUND: Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear. METHODS: We evaluated the effect of baseline S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥ 37.5 °C) and parasitemia (≥ 2500 asexual parasites/µl). Secondary definitions of malaria using different parasite densities were also explored. RESULTS: After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression (hazard ratios 0.71 and 0.44; P = 0.01 and 0.02; reference group: uninfected at baseline). Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode. CONCLUSIONS: Co-infection with S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.

Inter-observer agreement according to malaria parasite density.

BACKGROUND: Recent developments in diagnostic techniques for malaria, particularly DNA probes and sero-immunology, have raised questions as to how these techniques might be used to facilitate malaria diagnosis at the most peripheral levels of the primary health care system. At present, malaria diagnosis is based on the standard microscopic examination of blood films in most field epidemiologic studies and is likely to remain so in the immediate future in Africa. The objective of this study was to assess inter-observer agreement for the examination of Giemsa-stained slides for Plasmodium falciparum parasites. METHODS: Children aged 0 to 10 years were enrolled yearly in Bancoumana village (West Africa), mainly during the transmission season (June to October). The blood smears obtained from the persistently negative children in June 1996, August 1996, October 1996 and March 1997 were systematically re-examined. A stratified random sample (10%) proportional to the following parasite density classes 1-100, 101-5000, and 5001 and over was taken from the slides collected. The kappa statistics and the intra-class correlation were used as measures of agreement the first and the second slide examinations. RESULTS: The weighted kappa statistic, widely used as a chance-corrected measure for nominal agreement, showed excellent inter-observer agreement (κ(w)=0.7926; 95% CI [0.7588, 0.8263]; p=0.01). The intra-class correlation co-efficient had the same value of 0.7926 confirming the appropriateness of the weighted kappa statistic. Inter-observer agreement for slides read as negative by one observer, or as containing more than 100 parasites per µl, was excellent: 97% (493/506) and 92% (145/158), respectively. In contrast, the inter-observer agreement for slides read by one observer as containing 1-100 parasites/µl was poor, 36% (96/268). CONCLUSIONS: In field conditions in Mali, there was a high reproducibility for slides reported as negative or as having more than 100 parasites per µl. However, smears with readings of 1-100 parasites per µl were less reproducible and should be re-examined carefully.

Sickle cell trait protects against Plasmodium falciparum infection.

Although sickle cell trait protects against severe disease due to Plasmodium falciparum, it has not been clear whether sickle trait also protects against asymptomatic infection (parasitemia). To address this question, the authors identified 171 persistently smear-negative children and 450 asymptomatic persistently smear-positive children in Bancoumana, Mali (June 1996 to June 1998). They then followed both groups for 2 years using a cohort-based strategy. Among the 171 children with persistently negative smears, the median time for conversion to smear-positive was longer for children with sickle trait than for children without (274 vs. 108 days, P < 0.001; Cox hazard ratio = 0.56, 95% confidence interval: 0.33, 0.96; P = 0.036). Similar differences were found in the median times to reinfection after spontaneous clearance without treatment (365 days vs. 184 days; P = 0.01). Alternatively, among the 450 asymptomatic children with persistently positive smears, the median time for conversion to smear-negative (spontaneous clearance) was shorter for children with sickle trait than for children without (190 vs. 365 days; P = 0.02). These protective effects of sickle trait against asymptomatic P. falciparum infection under conditions of natural transmission were demonstrable using a cohort-based approach but not when the same data were examined using a cross-sectional approach.

Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium falciparum gametocyte carriers.

INTRODUCTION: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. METHODS: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. RESULTS: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70). CONCLUSIONS: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene.

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.

Use of a pLDH-based dipstick in the diagnostic and therapeutic follow-up of malaria patients in Mali.

BACKGROUND: Malaria is a major public health problem in Mali and diagnosis is typically based on microscopy. Microscopy requires a well trained technician, a reliable power source, a functioning microscope and adequate supplies. The scarcity of resources of community health centres (CHC) does not allow for such a significant investment in only one aspect of malaria control. In this context, Rapid Diagnostic Tests (RDTs) may improve case management particularly in remote areas. METHODS: This multicentre study included 725 patients simultaneously screened with OptiMal-IT test and thick smears for malaria parasite detection. While evaluating the therapeutic efficacy of choroquine in 2 study sites, we compared the diagnostic values of thick smear microscopy to OptiMal-IT test applying the WHO 14 days follow-up scheme using samples collected from 344 patients. RESULTS: The sensitivity and the specificity of OptiMal-IT compared to thick smear was 97.2% and 95.4%, whereas the positive and negative predictive values were 96.7 and 96.1%, respectively. The percent agreement between the two diagnostic tests was 0.93. The two tests were comparable in detecting malaria at day 0, day 3 and day 14. The only difference was observed at day 7 due to high gametocytemia. Subjectively, health care providers found OptiMal-IT easier to use and store under field conditions. CONCLUSION: OptiMal-IT test revealed similar results when compared to microscopy which is considered the gold standard for malaria diagnostics. The test was found to have a short processing time and was easier to use. These advantages may improve malaria case management by providing a diagnostic and drug efficacy follow-up tool to peripheral health centres with limited resources.

Superiority of 3 over 2 doses of intermittent preventive treatment with sulfadoxine-pyrimethamine for the prevention of malaria during pregnancy in mali: a randomized controlled trial.

BACKGROUND: In 2003, Mali introduced intermittent preventive therapy in pregnancy (ITPp) with sulfadoxine-pyrimethamine (SP) for the control of malaria in pregnancy, consisting of 2 doses of SP given in the 2nd and 3rd trimester. This widely used regimen, although very effective, leaves many women unprotected from malaria during the last 4-to-8 weeks of gestation, which is a pivotal period for fetal weight gain. The aim of the study was to compare the efficacy and safety of 3-dose versus 2-dose IPTp-SP for the prevention of placental malaria and associated low birth weight (LBW). METHODS: We conducted a parallel-group, open-label, individually randomized controlled superiority trial involving 814 women of all gravidity, enrolled from April 2006 through March 2008. All women were seen at least 3 times and received either 2 (n = 401) or 3 (n = 413) doses of IPTp-SP. The primary endpoint measured was placental malaria, LBW, preterm births, and maternal anemia were secondary endpoints, and severe maternal skin reactions and neonatal jaundice were safety endpoints. RESULTS: Among the 96% of study subjects who were followed up until delivery, the prevalence of placental malaria was 2-fold lower in the 3-dose group (8.0%) than in the 2-dose group (16.7%); the adjusted prevalence ratio (APR) was 0.48 (95% confidence interval [CI], 0.32-0.71). LBW and preterm births were also reduced; the prevalence of LBW was 6.6% in the 3-dose group versus 13.3% in the 2-dose group (APR, 0.50; 95% CI, 0.32-0.79), and the prevalence of preterm births was 3.2% versus 8.9% (APR, 0.37; 95% CI, 0.19-0.71). No significant reductions in maternal anemia or differences in safety endpoints were observed. CONCLUSIONS: Adding a third dose of ITPp-SP halved the risk of placental malaria, LBW, and preterm births in all gravidae, compared with the standard 2-dose regimen, in this area of highly seasonal transmission with low levels of SP resistance. CLINICAL TRIALS REGISTRATION: ISRCTN 74189211.

Efficacy of artesunate + sulfamethoxypyrazine/pyrimethamine versus praziquantel in the treatment of Schistosoma haematobium in children.

BACKGROUND: This study was conducted to determine the efficacy of the antimalarial artemisinin-based combination therapy (ACT) artesunate +sulfamethoxypyrazine/pyrimethamine (As+SMP), administered in doses used for malaria, to treat Schistosoma haematobium in school aged children. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in Djalakorodji, a peri-urban area of Bamako, Mali, using a double blind setup in which As+SMP was compared with praziquantel (PZQ). Urine samples were examined for Schistosoma haematobium on days -1, 0, 28 and 29. Detection of haematuria, and haematological and biochemical exams were conducted on day 0 and day 28. Clinical exams were performed on days 0, 1, 2, and 28. A total of 800 children were included in the trial. The cure rate obtained without viability testing was 43.9% in the As+SMP group versus 53% in the PZQ group (Chi(2) = 6.44, p = 0.011). Egg reduction rates were 95.6% with PZQ in comparison with 92.8% with As+SMP, p = 0.096. The proportion of participants who experienced adverse events related to the medication was 0.5% (2/400) in As+SMP treated children compared to 2.3% (9/399) in the PZQ group (p = 0.033). Abdominal pain and vomiting were the most frequent adverse events in both treatment arms. All adverse events were categorized as mild. CONCLUSIONS/SIGNIFICANCE: The study demonstrates that PZQ was more effective than As+SMP for treating Schistosoma haematobium. However, the safety and tolerability profile of As+SMP was similar to that seen with PZQ. Our findings suggest that further investigations seem justifiable to determine the dose/efficacy/safety pattern of As+SMP in the treatment of Schistosoma infections. TRIAL REGISTRATION: ClinicalTrials.gov NCT00510159.

Randomized, multicentre assessment of the efficacy and safety of ASAQ--a fixed-dose artesunate-amodiaquine combination therapy in the treatment of uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The use of artemisinin derivative-based combination therapy (ACT) such as artesunate plus amodiaquine is currently recommended for the treatment of uncomplicated Plasmodium falciparum malaria. Fixed-dose combinations are more adapted to patients than regimens involving multiple tablets and improve treatment compliance. A fixed-dose combination of artesunate + amodiaquine (ASAQ) was recently developed. To assess the efficacy and safety of this new combination and to define its optimum dosage regimen (once or twice daily) in the treatment of uncomplicated P. falciparum malaria, a multicentre clinical study was conducted. METHODS: A multicentre, randomized, controlled, investigator-blinded, parallel-group study was conducted in five African centers in Cameroon, Madagascar, Mali and Senegal from March to December 2006. Efficacy and safety of ASAQ were assessed compared to those of artemether + lumefantrine (AL). The WHO protocol with a 28-day follow-up for assessing the drug therapeutic efficacy was used. Patients suffering from uncomplicated P. falciparum malaria were randomized to receive ASAQ orally once daily (ASAQ1), ASAQ twice daily (ASAQ2) or AL twice daily (AL) for three days. The primary outcome was PCR-corrected parasitological cure rate and clinical response. RESULTS: Of 941 patients initially randomized and stratified into two age groups (<5 years, and >or=5 years), 936 (99.5%) were retained for the intent to treat (ITT) analysis, and 859 (91.3%) patients for the per protocol (PP) analysis. Among ITT population, up to D28, PCR-corrected adequate parasitological and clinical response rates were 95.2% in the ASAQ1 group, 94.9% in the ASAQ2 group and 95.5% in the AL group. Moreover, the cure rate evaluated among PP population was >or=98.5% in both ASAQ therapeutic arms. Therapeutic response rates did not display any significant differences between age groups or between one geographical site and another. Altogether, this demonstrates the non-inferiority of ASAQ1 regimen compared to both ASAQ2 and AL regimens. During follow-up mild and moderate adverse events including gastrointestinal and/or nervous disorders were reported in 29.3% of patients, with no difference between groups in the nature, frequency or intensity of adverse events. CONCLUSION: The non-inferiority of ASAQ compared with AL was demonstrated. The fixed-dose combination artesunate + amodiaquine (ASAQ) is safe and efficacious even in young children under 5 years of age. Whilst administration on a twice-a-day basis does not improve the efficacy of ASAQ significantly, a once-a-day intake of this new combination clearly appears as an effective and safe therapy in the treatment of uncomplicated P. falciparum malaria both in adults and children. Implications of such findings are of primary importance in terms of public health especially in African countries. As most national policies plan to strengthen malaria control to reach the elimination of this disease, anti-malarial drugs such as the artesunate + amodiaquine fixed-dose ACT will play a pivotal role in this process. TRIAL REGISTRATION: The protocol was registered with the www.clinicaltrials.gov open clinical trial registry under the identifier number NCT00316329.

Evaluation and optimization of membrane feeding compared to direct feeding as an assay for infectivity.

BACKGROUND: Malaria parasite infectivity to mosquitoes has been measured in a variety of ways and setting, includind direct feeds of and/or membrane feeding blood collected from randomly selected or gametocytemic volunteers. Anopheles gambiae s.l is the main vector responsible of Plasmodium falciparum transmission in Bancoumana and represents about 90% of the laboratory findings, whereas Plasmodium malariae and Plasmodium ovale together represent only 10%. MATERIALS AND METHODS: Between August 1996 and December 1998, direct and membrane feeding methods were compared for the infectivity of children and adolescent gametocyte carriers to anopheline mosquitoes in the village of Bancoumana in Mali. Gametocyte carriers were recruited twice a month through a screening of members of 30 families using Giemsa-stained thick blood smears. F1 generation mosquitoes issued from individual female wild mosquitoes from Bancoumana were reared in a controlled insectary conditions and fed 5% sugar solution in the laboratory in Bamako, until the feeding day when they are starved 12 hours before the feeding experiment. These F1 generation mosquitoes were divided in two groups, one group fed directly on gametocyte carriers and the other fed using membrane feeding method. RESULTS: Results from 372 Plasmodium falciparum gametocyte carriers showed that children aged 4-9 years were more infectious than adolescents (p = 0.039), especially during the rainy season. Data from 35 carriers showed that mosquitoes which were used for direct feeding were about 1.5 times more likely to feed (p < 0.001) and two times more likely to become infected, if they fed (p < 0.001), than were those which were used for membrane feeding. Overall, infectivity was about three-times higher for direct feeding than for membrane feeding (p < 0.001). CONCLUSION: Although intensity of infectivity was lower for membrane feeding, it could be a surrogate to direct feeding for evaluating transmission-blocking activity of candidate malaria vaccines. An optimization of the method for future trials would involve using about three-times more mosquitoes than would be used for direct feeding.

Space-time clustering of childhood malaria at the household level: a dynamic cohort in a Mali village.

BACKGROUND: Spatial and temporal heterogeneities in the risk of malaria have led the WHO to recommend fine-scale stratification of the epidemiological situation, making it possible to set up actions and clinical or basic researches targeting high-risk zones. Before initiating such studies it is necessary to define local patterns of malaria transmission and infection (in time and in space) in order to facilitate selection of the appropriate study population and the intervention allocation. The aim of this study was to identify, spatially and temporally, high-risk zones of malaria, at the household level (resolution of 1 to 3 m). METHODS: This study took place in a Malian village with hyperendemic seasonal transmission as part of Mali-Tulane Tropical Medicine Research Center (NIAID/NIH). The study design was a dynamic cohort (22 surveys, from June 1996 to June 2001) on about 1300 children (<12 years) distributed between 173 households localized by GPS. We used the computed parasitological data to analyzed levels of Plasmodium falciparum, P. malariae and P. ovale infection and P. falciparum gametocyte carriage by means of time series and Kulldorff's scan statistic for space-time cluster detection. RESULTS: The time series analysis determined that malaria parasitemia (primarily P. falciparum) was persistently present throughout the population with the expected seasonal variability pattern and a downward temporal trend. We identified six high-risk clusters of P. falciparum infection, some of which persisted despite an overall tendency towards a decrease in risk. The first high-risk cluster of P. falciparum infection (rate ratio = 14.161) was detected from September 1996 to October 1996, in the north of the village. CONCLUSION: This study showed that, although infection proportions tended to decrease, high-risk zones persisted in the village particularly near temporal backwaters. Analysis of this heterogeneity at the household scale by GIS methods lead to target preventive actions more accurately on the high-risk zones identified. This mapping of malaria risk makes it possible to orient control programs, treating the high-risk zones identified as a matter of priority, and to improve the planning of intervention trials or research studies on malaria.

Efficacy of chloroquine and sulfadoxine/pyrimethamine for the treatment of uncomplicated falciparum malaria in Koumantou, Mali.

We report the results of an in vivo antimalarial efficacy study with chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) conducted between 2003 and 2004 in Koumantou, southern Mali. A total of 244 children were included in the study; 210 children were followed-up for 28 days according to WHO recommendations, with PCR genotyping to distinguish late recrudescence from re-infection. Global failure proportions at Day 14, without taking into account re-infections, were 44.2% (95% CI 34.9-53.5%) in the CQ group and 2.0% (95% CI 0.0-4.8%) in the SP group. PCR-adjusted failure proportions at Day 28 were even higher in the CQ group (90.5% (95/105), 95% CI 84.8-96.2%) and relatively low in the SP group (7.0% (7/100), 95% CI 1.9-12.1%). These results show that CQ is no longer efficacious in Koumantou. The use of SP in monotherapy is likely to compromise its efficacy. We recommend the use of artemisinin-based combination therapy as first-line treatment for uncomplicated Plasmodium falciparum malaria in Koumantou.

Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.

We surveyed an Anopheles gambiae population in a West African malaria transmission zone for naturally occurring genetic loci that control mosquito infection with the human malaria parasite, Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen resistance mechanisms in plants and mammals.

Season, fever prevalence and pyrogenic threshold for malaria disease definition in an endemic area of Mali.

BACKGROUND: Modelling malaria parasitaemia as function of fever has been proposed as best alternative to estimate the attributable fraction of malaria fever and the sensitivity and specificity of different case definitions of malaria disease. OBJECTIVES: To determine the prevalence of fever and its relation to malaria parasitaemia and to establish a pyrogenic threshold for malaria disease in the area. METHODS: We conducted two cross-sectional surveys in children of 6 months to 9 years of age (2434 during the rainy season of 1993 and 2353 during the dry season of 1994) randomly selected from 21 areas of Bandiagara district, Mali. RESULTS: The relationship between fever and Plasmodium falciparum parasitaemia depends strongly on the season, thus affecting the malaria-attributable fraction of fever cases and the sensitivity and specificity of malaria case definitions. The overall proportion of fever attributable to malaria parasitaemia was 33.6% during the rainy season and 23.3% during the dry season, with the highest proportion occurring among the youngest children. The cut-off value, where the sensitivity curve crosses the specificity curve, was around 3200 pf/microl for all age categories during the rainy season and 200 pf/microl during the dry season. CONCLUSIONS: Malaria remains a main cause of fever in this area of Mali. The pyrogenic threshold of parasitaemia depends strongly on the season, and different cut-off levels of parasitaemia should be used during the two seasons to define malaria cases in this area.

Risk factors for malaria infection and anemia for pregnant women in the Sahel area of Bandiagara, Mali.

Malaria infection and anemia during pregnancy are the primary causes of maternal and fetal morbidity and mortality. The aims of this study were to identify risk factors for malaria infection and to assess the relationship between malaria infection and anemia in pregnant women. Two cross-sectional surveys were conducted in September 1993 and then again in May 1994 (the end of the rainy and dry seasons respectively). A total of 235 pregnant women were randomly selected from both the rural and urban areas of Bandiagara, Mali. According to results from multivariate analysis, the risk of malaria infection was significantly higher during the rainy season (OR= 4.85, 95% CI 2.42-9.75) the first trimester of gestation (OR= 2.21, 95% CI 1.00-4.87, in younger women (OR= 2.48, 95% CI 1.19-5.16), and in women living in the rural area (2.49, 95% CI 0.99-6.27). The risk of anemia was also higher during the rainy season (OR= 1.93, 95% CI 1.10-3.39, in the rural area (OR= 3.55, 95% CI 1.46-8.62). The risk of anemia was lower during the first trimester of gestational age (OR= 0.45, 95% CI 0.22-0.92). The relationship between malaria infection and anemia also varied with season. During the rainy season, the risk for anemia was similar among malaria-infected and non-infected pregnant women. In contrast, the risk was higher among infected pregnant women during the dry season (OR= 3.43, 95% CI 1.09-10.07). In conclusion, the data suggest, that earlier gestation age, living in the rural area, and young age rather than parity are important risk factors for malaria infection in pregnant women. Further, malaria infection is strongly associated with anemia in pregnant women particularly during the dry season and is most likely the cause of anemia. Thus, control measures against malaria infection should target younger rural women in their first trimester of pregnancy.