Sanger sequencing and deconvolution of polyclonal infections: a quantitative approach to monitor drug-resistant Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance in Plasmodium falciparum is a major public health problem in malaria-endemic regions. Although various technical improvements in sequencing methods have been introduced to identify SNPs, the conventional approach with current tools does not discriminate mixed infections, and thus can be improved for more sensitive surveillance of anti-malarial resistance to better inform control strategies. METHODS: We developed a computational approach for deconvolution of chromatograms generated by standard Sanger sequencing of PCR amplicons in order to quantify molecular marker variants of anti-malarial drug resistance genes [Plasmodium falciparum dihydropteorate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr)]. We validated this computational approach using mixtures of V1/S and FCR3 at varying proportions between 0 and 100%, then applied it to field samples collected in Doneguebougou, Mali in 2018. We determined the mean fraction of resistance alleles in individual samples, as well as the prevalence of infections carrying resistant parasites. FINDINGS: We observed a highly significant correlation between the predicted and measured proportions of V1/S and FCR3 alleles in mixed laboratory samples (all p < 0.001). Among field samples, the mean fraction of resistant Pfdhps alleles was 4.7% 431V, 95.9% 436F/A, 49.9% 437G, 0.0% 540E, 1.2% 581G and 1.5% 613S/T; corresponding prevalences were 50.0%, 100%, 72.5%, 0.0%, 25.0%, and 12.5%, respectively. The mean fraction of resistant Pfdhfr alleles was 0.6% 16V, 11.1% 50R, 89.0% 51I, 98.3% 59R, 74.7% 108T/N, 8.6% 140L and 8.7% 164L; corresponding prevalences were 12.5%, 75.0%, 100%, 100%, 100%, 50.0%, and 28.6%, respectively. We identified two new point mutations on the Pfdhps gene at codons D484T and D545N. INTERPRETATION: Computational deconvolution of sequencing chromatograms can discriminate varying proportions of antimalarial drug-sensitive versus -resistant alleles. This cost-effective and quantitative variant-sequencing approach will be useful for population-based surveys that characterize mixed infections at the individual level to survey known and unknown mutations in P. falciparum drug-resistance genes. FUNDING: This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). HM was supported by the African Postdoctoral Training Initiative (APTI) Fellowship program jointly managed by the US NIH, The African Academy of Sciences (AAS) and Bill & Melinda Gates Foundation (BMGF); Grant Reference Number: APTI-18-01.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Prevalence of Plasmodium falciparum haplotypes associated with resistance to sulfadoxine-pyrimethamine and amodiaquine before and after upscaling of seasonal malaria chemoprevention in seven African countries: a genomic surveillance study.

BACKGROUND: Seasonal malaria chemoprevention is used in 13 countries in the Sahel region of Africa to prevent malaria in children younger than 5 years. Resistance of Plasmodium falciparum to seasonal malaria chemoprevention drugs across the region is a potential threat to this intervention. METHODS: Between December, 2015, and March, 2016, and between December, 2017, and March, 2018, immediately following the 2015 and 2017 malaria transmission seasons, community surveys were done among children younger than 5 years and individuals aged 10-30 years in districts implementing seasonal malaria chemoprevention with sulfadoxine-pyrimethamine and amodiaquine in Burkina Faso, Chad, Guinea, Mali, Nigeria, Niger and The Gambia. Dried blood samples were collected and tested for P falciparum DNA by PCR. Resistance-associated haplotypes of the P falciparum genes crt, mdr1, dhfr, and dhps were identified by quantitative PCR and sequencing of isolates from the collected samples, and survey-weighted prevalence and prevalence ratio between the first and second surveys were estimated for each variant. FINDINGS: 5130 (17·5%) of 29 274 samples from 2016 and 2176 (7·6%) of 28 546 samples from 2018 were positive for P falciparum on quantitative PCR. Among children younger than 5 years, parasite carriage decreased from 2844 of 14 345 samples (19·8% [95% CI 19·2-20·5]) in 2016 to 801 of 14 019 samples (5·7% [5·3-6·1]) in 2018 (prevalence ratio 0·27 [95% CI 0·24-0·31], p<0·0001). Genotyping found no consistent evidence of increasing prevalence of amodiaquine resistance-associated variants of crt and mdr1 between 2016 and 2018. The dhfr haplotype IRN (consisting of 51Ile-59Arg-108Asn) was common at both survey timepoints, but the dhps haplotype ISGEAA (431Ile-436Ser-437Gly-540Glu-581Ala-613Ala), crucial for resistance to sulfadoxine-pyrimethamine, was always rare. Parasites carrying amodiaquine resistance-associated variants of both crt and mdr1 together with dhfr IRN and dhps ISGEAA occurred in 0·05% of isolates. The emerging dhps haplotype VAGKGS (431Val-436Ala-437Gly-540Lys-581Gly-613Ser) was present in four countries. INTERPRETATION: In seven African countries, evidence of a significant reduction in parasite carriage among children receiving seasonal malaria chemoprevention was found 2 years after intervention scale-up. Combined resistance-associated haplotypes remained rare, and seasonal malaria chemoprevention with sulfadoxine-pyrimethamine and amodiaquine is expected to retain effectiveness. The threat of future erosion of effectiveness due to dhps variant haplotypes requires further monitoring. FUNDING: Unitaid.

Overall and Gender-Specific Effects of Intermittent Preventive Treatment of Malaria with Artemisinin-Based Combination Therapies among Schoolchildren in Mali: A Three-Group Open Label Randomized Controlled Trial.

Intermittent preventive treatment of malaria among schoolchildren (IPTsc) reduces clinical malaria, asymptomatic parasitemia, and anemia. The effects of IPTsc by gender have not been studied longitudinally. We investigated overall IPTsc efficacy and conducted a secondary analysis to explore gender-specific differences. We enrolled schoolchildren aged 6-13 years in an open-label, rolling-cohort randomized controlled trial between September 2007 and February 2013 in Kolle, Mali. Annually, schoolchildren received two full-treatment courses of sulfadoxine-pyrimethamine (SP) plus artesunate, or amodiaquine (AQ) plus artesunate, or no malaria treatment as control. We used mixed-effects generalized linear models to estimate differences in treatment outcomes across groups with interaction terms to explore gender-specific differences associated with Plasmodium falciparum infection, hemoglobin, and grade point averages (GPA) based on standardized testing. Overall, 305 students contributed 4,564 observations. Compared with the control, SP plus artesunate and AQ plus artesunate reduced the odds of P. falciparum infection (odds ratio [OR]: 0.33, 95% CI: 0.26-0.43; OR: 0.46, 95% CI: 0.36-0.59). We found strong evidence of increased mean hemoglobin concentrations (g/dL) in the SP plus artesunate group versus control (difference +0.37, 95% CI: 0.13-0.58). Collectively, schoolchildren given AQ plus artesunate had higher mean GPA (difference +0.36, 95% CI: 0.02-0.69) relative to control. Schoolgirls, compared with schoolboys, given SP plus artesunate had greater improvement in GPA (+0.50, 95% CI: -0.02 to 1.02 versus -0.27, 95% CI: -0.71 to 0.16); interaction P = 0.048, respectively. The IPTsc decreases P. falciparum infections in schoolchildren. Treatment regimens that include longer-acting drugs may be more effective at decreasing malaria-related anemia and improving educational outcomes as observed among girls in this setting.

Selection of pfcrt K76 and pfmdr1 N86 Coding Alleles after Uncomplicated Malaria Treatment by Artemether-Lumefantrine in Mali.

BACKGROUND: Artemether-lumefantrine is a highly effective artemisinin-based combination therapy that was adopted in Mali as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study was designed to measure the efficacy of artemether-lumefantrine and to assess the selection of the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genotypes that have been associated with drug resistance. METHODS: A 28-day follow-up efficacy trial of artemether-lumefantrine was conducted in patients aged 6 months and older suffering from uncomplicated falciparum malaria in four different Malian areas during the 2009 malaria transmission season. The polymorphic genetic markers MSP2, MSP1, and Ca1 were used to distinguish between recrudescence and reinfection. Reinfection and recrudescence were then grouped as recurrent infections and analyzed together by PCR-restriction fragment length polymorphism (RFLP) to identify candidate markers for artemether-lumefantrine tolerance in the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multi-drug resistance 1 (pfmdr1) gene. RESULTS: Clinical outcomes in 326 patients (96.7%) were analyzed and the 28-day uncorrected adequate clinical and parasitological response (ACPR) rate was 73.9%. The total PCR-corrected 28-day ACPR was 97.2%. The pfcrt 76T and pfmdr1 86Y population prevalence decreased from 49.3% and 11.0% at baseline (n = 337) to 38.8% and 0% in patients with recurrent infection (n = 85); p = 0.001), respectively. CONCLUSION: Parasite populations exposed to artemether-lumefantrine in this study were selected toward chloroquine-sensitivity and showed a promising trend that may warrant future targeted reintroduction of chloroquine or/and amodiaquine.

Preventive malaria treatment among school-aged children in sub-Saharan Africa: a systematic review and meta-analyses.

BACKGROUND: The burden of malaria infection in sub-Saharan Africa among school-aged children aged 5-15 years is underappreciated and represents an important source of human-to-mosquito transmission of Plasmodium falciparum. Additional interventions are needed to control and eliminate malaria. We aimed to assess whether preventive treatment of malaria might be an effective means of reducing P falciparum infection and anaemia in school-aged children and lowering parasite transmission. METHODS: In this systematic review and two meta-analyses, we searched the online databases PubMed, Embase, Cochrane CENTRAL, and Clinicaltrials.gov for intervention studies published between Jan 1, 1990, and Dec 14, 2018. We included randomised studies that assessed the effect of antimalarial treatment among asymptomatic school-aged children aged 5-15 years in sub-Saharan Africa on prevalence of P falciparum infection and anaemia, clinical malaria, and cognitive function. We first extracted data for a study-level meta-analysis, then contacted research groups to request data for an individual participant data meta-analysis. Outcomes of interest included prevalence of P falciparum infection detected by microscopy, anaemia (study defined values or haemoglobin less than age-adjusted and sex-adjusted values), clinical malaria (infection and symptoms on the basis of study-specific definitions) during follow-up, and code transmission test scores. We assessed effects by treatment type and duration of time protected, and explored effect modification by transmission setting. For study-level meta-analysis, we calculated risk ratios for binary outcomes and standardised mean differences for continuous outcomes and pooled outcomes using fixed-effect and random-effects models. We used a hierarchical generalised linear model for meta-analysis of individual participant data. This study is registered with PROSPERO, CRD42016030197. FINDINGS: Of 628 studies identified, 13 were eligible for the study-level meta-analysis (n=16 309). Researchers from 11 studies contributed data on at least one outcome (n=15 658) for an individual participant data meta-analysis. Interventions and study designs were highly heterogeneous; overall risk of bias was low. In the study-level meta-analysis, treatment was associated with reductions in P falciparum prevalence (risk ratio [RR] 0·27, 95% CI 0·17-0·44), anaemia (0·77, 0·65-0·91), and clinical malaria (0·40, 0·28-0·56); results for cognitive outcomes are not presented because data were only available for three trials. In our individual participant data meta-analysis, we found treatment significantly decreased P falciparum prevalence (adjusted RR [ARR] 0·46, 95% CI 0·40-0·53; p<0·0001; 15 648 individuals; 11 studies), anaemia (ARR 0·85, 0·77-0·92; p<0·0001; 15 026 individuals; 11 studies), and subsequent clinical malaria (ARR 0·50, 0·39-0·60; p<0·0001; 1815 individuals; four studies) across transmission settings. We detected a marginal effect on cognitive function in children older than 10 years (adjusted mean difference in standardised test scores 0·36, 0·01-0·71; p=0·044; 3962 individuals; five studies) although we found no significant effect when combined across all ages. INTERPRETATION: Preventive treatment of malaria among school-aged children significantly decreases P falciparum prevalence, anaemia, and risk of subsequent clinical malaria across transmission settings. Policy makers and programme managers should consider preventive treatment of malaria to protect this age group and advance the goal of malaria elimination, while weighing these benefits against potential risks of chemoprevention. FUNDING: US National Institutes of Health and Burroughs Wellcome Fund/ASTMH Fellowship.

Two-Year Scale-Up of Seasonal Malaria Chemoprevention Reduced Malaria Morbidity among Children in the Health District of Koutiala, Mali.

BACKGROUND: Previous controlled studies demonstrated seasonal malaria chemoprevention (SMC) reduces malaria morbidity by >80% in children aged 3-59 months. Here, we assessed malaria morbidity after large-scale SMC implementation during a pilot campaign in the health district of Koutiala, Mali. METHODS: Starting in August 2012, children received three rounds of SMC with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ). From July 2013 onward, children received four rounds of SMC. Prevalence of malaria infection, clinical malaria and anemia were assessed during two cross-sectional surveys conducted in August 2012 and June 2014. Investigations involved 20 randomly selected clusters in 2012 against 10 clusters in 2014. RESULTS: Overall, 662 children were included in 2012, and 670 in 2014. Children in 2014 versus those surveyed in 2012 showed reduced proportions of malaria infection (12.4% in 2014 versus 28.7% in 2012 (p = 0.001)), clinical malaria (0.3% versus 4.2%, respectively (p < 0.001)), and anemia (50.1% versus 67.4%, respectively (p = 0.001)). A propensity score approach that accounts for environmental differences showed that SMC conveyed a significant protective effect against malaria infection (IR = 0.01, 95% CI (0.0001; 0.09), clinical malaria (OR = 0.25, 95% CI (0.06; 0.85)), and hemoglobin concentration (ß = 1.3, 95% CI (0.69; 1.96)) in 2012 and 2014, respectively. CONCLUSION: SMC significantly reduced frequency of malaria infection, clinical malaria and anemia two years after SMC scale-up in Koutiala.

Impact of Three-Year Intermittent Preventive Treatment Using Artemisinin-Based Combination Therapies on Malaria Morbidity in Malian Schoolchildren.

Previous studies have shown that a single season of intermittent preventive treatment in schoolchildren (IPTsc) targeting the transmission season has reduced the rates of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. Efficacy over the course of multiple years of IPTsc has been scantly investigated. METHODS: An open, randomized-controlled trial among schoolchildren aged 6-13 years was conducted from September 2007 to January 2010 in Kolle, Mali. Students were included in three arms: sulphadoxine-pyrimethamine+artesunate (SP+AS), amodiaquine+artesunate (AQ+AS), and control (C). All students received two full doses, given 2 months apart, and were compared with respect to the incidence of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. RESULTS: A total of 296 students were randomized. All-cause clinic visits were in the SP+AS versus control (29 (20.1%) vs. 68 (47.2%); 20 (21.7%) vs. 41 (44.6%); and 14 (21.2%) vs. 30 (44.6%); p < 0.02) in 2007, 2008, and 2009, respectively. The prevalence of asymptomatic parasitemia was lower in the SP+AS compared to control (38 (7.5%) vs. 143 (28.7%); and 47 (12.7%) vs. 75 (21.2%); p < 0.002) in 2007 and 2008, respectively. Hemoglobin concentration was significantly higher in children receiving SP+AS (11.96, 12.06, and 12.62 g/dL) than in control children (11.60, 11.64, and 12.15 g/dL; p < 0.001) in 2007, 2008, and 2009, respectively. No impact on clinical malaria was observed. CONCLUSION: IPTsc with SP+AS reduced the rates of all-cause clinic visits and anemia during a three-year implementation.

Reduced ex vivo susceptibility of Plasmodium falciparum after oral artemether-lumefantrine treatment in Mali.

BACKGROUND: Artemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether-lumefantrine (AL) treatment. METHODS: Study samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing. RESULTS: Overall 61 samples were successfully analysed in ex vivo studies. Mean IC50s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%. CONCLUSION: Malian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

Seasonal Malaria Chemoprevention with Sulphadoxine-Pyrimethamine and Amodiaquine Selects Pfdhfr-dhps Quintuple Mutant Genotype in Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ) is being scaled up in Sahelian countries of West Africa. However, the potential development of Plasmodium falciparum resistance to the respective component drugs is a major concern. METHODS: Two cross-sectional surveys were conducted before (August 2012) and after (June 2014) a pilot implementation of SMC in Koutiala, Mali. Children aged 3-59 months received 7 rounds of curative doses of SP plus AQ over two malaria seasons. Genotypes of P. falciparum Pfdhfr codons 51, 59 and 108; Pfdhps codons 437 and 540, Pfcrt codon 76 and Pfmdr1codon 86 were analyzed by PCR on DNA from samples collected before and after SMC, and in non-SMC patient population as controls (November 2014). RESULTS: In the SMC population 191/662 (28.9%) and 85/670 (12.7%) of children were P. falciparum positive by microscopy and were included in the molecular analysis before (2012) and after SMC implementation (2014), respectively. In the non-SMC patient population 220/310 (71%) were successfully PCR analyzed. In the SMC children, the prevalence of all molecular markers of SP resistance increased significantly after SMC including the Pfdhfr-dhps quintuple mutant genotype, which was 1.6% before but 7.1% after SMC (p = 0.02). The prevalence of Pfmdr1-86Y significantly decreased from 26.7% to 15.3% (p = 0.04) while no significant change was seen for Pfcrt 76T. In 2014, prevalence of all molecular markers of SP resistance were significantly higher among SMC children compared to the non-SMC population patient (p < 0.01). No Pfdhfr-164 mutation was found neither at baseline nor post SMC. CONCLUSION: SMC increased the prevalence of molecular markers of P. falciparum resistance to SP in the treated children. However, there was no significant increase of these markers of resistance in the general parasite population after 2 years and 7 rounds of SMC.

Effect of artemether-lumefantrine (Coartem) on cytomegalovirus urine viral load during and following treatment for malaria in children.

BACKGROUND: Artemisinins, commonly used to treat malaria, have shown activity against cytomegalovirus (CMV) in vitro, in an animal model, and in case reports; however, the in vivo anti-CMV activity has not been well investigated. OBJECTIVES: To evaluate whether artemisinins affect CMV shedding among subjects co-infected with CMV and malaria. STUDY DESIGN: A prospective observational study of children in Mali (6 month-10 year) presenting with fever. Urine samples were collected at day 0, 3, and 14 from children treated with artemether-lumefantrine (Coartem(®)) for malaria and those who had other illnesses not treated with Coartem. CMV DNA was quantified using a real-time PCR. Resulting urine viral loads were compared between the groups at three time points. RESULTS: 164 malaria cases and 143 non-malaria comparisons were enrolled. Eighty-one (49%) cases and 88 (62%) comparisons shed CMV at day 0. Day 0 and day 3 viral loads were similar, but at day 14 the median viral load of cases was lower than that of comparisons (360 vs 720 copies/mL or 2.56 vs 2.86 log10), p=0.059. A stratified analysis of day 0 high viral shedders (defined as >1000 copies/mL) showed significantly lower median viral load at day 14 among cases (490 copies/mL, 2.69 log10) vs comparisons (1200 copies/mL, 3.08 log10), p=0.045. CONCLUSION: A high rate of urinary CMV shedding was found in a malaria-endemic area. Among high virus shedders artemether-lumefantrine decreased urine viral load, but the effect was not observed when analysis of both high and low shedders was performed. These results support additional studies of artemisinin dosing and duration in CMV infection.

Efficacy of sulphadoxine-pyrimethamine + artesunate, sulphadoxine-pyrimethamine + amodiaquine, and sulphadoxine-pyrimethamine alone in uncomplicated falciparum malaria in Mali.

BACKGROUND: Plasmodium falciparum resistance to artemisinin has been reported in South-East Asia. Long half-life drugs are increasingly being used for malaria prevention. The potential spread of parasite resistance to these regimens is real and makes regular efficacy surveillance a priority. METHODS: From August to December 2004 and July to December 2005, a randomized open label trial of sulphadoxine-pyrimethamine (SP) + artesunate (AS) versus SP + amodiaquine (AQ), and SP alone, was conducted in two villages of Mali. PCR was used to distinguish new infections from recrudescent P. falciparum infections. Patients were followed for 28 days to assess treatment efficacy. RESULTS: Overall 912 children aged between six to 59 months, with uncomplicated P. falciparum malaria were recruited. Baseline characteristics were similar in the three treatment arms. Crude ACPRs were 94.9%; 98.6% and 93.5% for SP + AS; SP + AQ and SP alone arms respectively (SP + AS versus SP + AQ, p = 0.01; SP + AS versus SP, p = 0.5; SP + AQ versus SP, p = 0.001). After PCR adjustment, cACPRs were 99%; 100% and 97.2% for SP + AS; SP + AQ and SP alone arms, respectively (SP + AS versus SP + AQ, p = 0.25; SP + AS versus SP, p = 0.12; SP + AQ versus SP, p = 0.007). CONCLUSION: Sulphadoxine-pyrimethamine + amodiaquine therapy was as efficacious as sulphadoxine-pyrimethamine + artesunate, but more efficacious than sulphadoxine-pyrimethamine alone in the treatment of uncomplicated P. falciparum malaria in Mali.

Quinine treatment selects the pfnhe-1 ms4760-1 polymorphism in Malian patients with Falciparum malaria.

BACKGROUND: The mechanism of Plasmodium falciparum resistance to quinine is not known. In vitro quantitative trait loci mapping suggests involvement of a predicted P. falciparum sodium-hydrogen exchanger (pfnhe-1) on chromosome 13. METHODS: We conducted prospective quinine efficacy studies in 2 villages, Kollé and Faladié, Mali. Cases of clinical malaria requiring intravenous therapy were treated with standard doses of quinine and followed for 28 days. Treatment outcomes were classified using modified World Health Organization protocols. Molecular markers of parasite polymorphisms were used to distinguish recrudescent parasites from new infections. The prevalence of pfnhe-1 ms4760-1 among parasites before versus after quinine treatment was determined by direct sequencing. RESULTS: Overall, 163 patients were enrolled and successfully followed. Without molecular correction, the mean adequate clinical and parasitological response (ACPR) was 50.3% (n = 163). After polymerase chain reaction correction to account for new infections, the corrected ACPR was 100%. The prevalence of ms4760-1 increased significantly, from 26.2% (n = 107) before quinine treatment to 46.3% (n = 54) after therapy (P = .01). In a control sulfadoxine-pyrimethamine study, the prevalence of ms4760-1 was similar before and after treatment. CONCLUSIONS: This study supports a role for pfnhe-1 in decreased susceptibility of P. falciparum to quinine in the field.

Similar efficacy and safety of artemether-lumefantrine (Coartem®) in African infants and children with uncomplicated falciparum malaria across different body weight ranges.

BACKGROUND: Artemisinin-based combination therapy, including artemether-lumefantrine (AL), is currently recommended for the treatment of uncomplicated Plasmodium falciparum malaria. The objectives of the current analysis were to compare the efficacy and safety of AL across different body weight ranges in African children, and to examine the age and body weight relationship in this population. METHODS: Efficacy, safety and pharmacokinetic data from a randomized, investigator-blinded, multicentre trial of AL for treatment of acute uncomplicated P. falciparum malaria in infants and children in Africa were analysed according to body weight group. RESULTS: The trial included 899 patients (intent-to-treat population 886). The modified intent-to-treat (ITT) population (n = 812) comprised 143 children 5 to < 10 kg, 334 children 10 to < 15 kg, 277 children 15 to < 25 kg, and 58 children 25 to < 35 kg. The 28-day PCR cure rate, the primary endpoint, was comparable across all four body weight groups (97.2%, 98.9%, 97.8% and 98.3%, respectively). There were no clinically relevant differences in safety or tolerability between body weight groups. In the three AL body weight dosing groups (5 to < 15 kg, 15 to < 25 kg and 25 to < 35 kg), 80% of patients were aged 10-50 months, 46-100 months and 90-147 months, respectively. CONCLUSION: Efficacy of AL in uncomplicated falciparum malaria is similar across body weight dosing groups as currently recommended in the label with no clinically relevant differences in safety or tolerability. AL dosing based on body weight remains advisable.

Effects of amodiaquine and artesunate on sulphadoxine-pyrimethamine pharmacokinetic parameters in children under five in Mali.

BACKGROUND: Sulphadoxine-pyrimethamine, in combination with artesunate or amodiaquine, is recommended for the treatment of uncomplicated malaria and is being evaluated for intermittent preventive treatment. Yet, limited data is available on pharmacokinetic interactions between these drugs. METHODS: In a randomized controlled trial, children aged 6-59 months with uncomplicated falciparum malaria, received either one dose of sulphadoxine-pyrimethamine alone (SP), one dose of SP plus three daily doses of amodiaquine (SP+AQ) or one dose of SP plus 3 daily doses of artesunate (SP+AS). Exactly 100 µl of capillary blood was collected onto filter paper before drug administration at day 0 and at days 1, 3, 7, 14, 21 and 28 after drug administration for analysis of sulphadoxine and pyrimethamine pharmacokinetic parameters. RESULTS: Fourty, 38 and 31 patients in the SP, SP+AQ and SP+AS arms, respectively were included in this study. The concentrations on day 7 (that are associated with therapeutic efficacy) were similar between the SP, SP+AQ and SP+AS treatment arms for sulphadoxine (median [IQR] 35.25 [27.38-41.70], 34.95 [28.60-40.85] and 33.40 [24.63-44.05] µg/mL) and for pyrimethamine (56.75 [46.40-92.95], 58.75 [43.60-98.60] and 59.60 [42.45-86.63] ng/mL). There were statistically significant differences between the pyrimethamine volumes of distribution (4.65 [3.93-6.40], 4.00 [3.03-5.43] and 5.60 [4.40-7.20] L/kg; p = 0.001) and thus elimination half-life (3.26 [2.74 -3.82], 2.78 [2.24-3.65] and 4.02 [3.05-4.85] days; p < 0.001). This study confirmed the lower SP concentrations previously reported for young children when compared with adult malaria patients. CONCLUSION: Despite slight differences in pyrimethamine volumes of distribution and elimination half-life, these data show similar exposure to SP over the critical initial seven days of treatment and support the current use of SP in combination with either AQ or AS for uncomplicated falciparum malaria treatment in young Malian children.

A molecular map of chloroquine resistance in Mali.

Plasmodium falciparum chloroquine resistance (CQR) transporter point mutation (PfCRT 76T) is known to be the key determinant of CQR. Molecular detection of PfCRT 76T in field samples may be used for the surveillance of CQR in malaria-endemic countries. The genotype-resistance index (GRI), which is obtained as the ratio of the prevalence of PfCRT 76T to the incidence of CQR in a clinical trial, was proposed as a simple and practical molecular-based addition to the tools currently available for monitoring CQR in the field. In order to validate the GRI model across populations, time, and resistance patterns, we compiled data from the literature and generated new data from 12 sites across Mali. We found a mean PfCRT 76T mutation prevalence of 84.5% (range 60.9-95.1%) across all sites. CQR rates predicted from the GRI model were extrapolated onto a map of Mali to show the patterns of resistance throughout the participating regions. We present a comprehensive map of CQR in Mali, which strongly supports recent changes in drug policy away from chloroquine.

Intermittent preventive treatment using artemisinin-based combination therapy reduces malaria morbidity among school-aged children in Mali.

OBJECTIVE: To assess the efficacy of intermittent preventive treatment (IPT) against malaria in school-aged children. METHODS: This was an open randomized controlled trial of seasonal IPT among school children (IPTsc) aged 6-13 years in Kollé, Mali. The study began in September 2007 and completed follow-up in May 2008. Students were randomized to one of three study arms: Sulfadoxine-pyrimethamine plus artesunate (SP/AS), amodiaquine plus artesunate (AQ/AS) or vitamin C. All students received two full treatment doses, given 2 months apart during the season of high transmission from September to December. Groups were compared with respect to incidence of clinical malaria, asymptomatic parasitemia and haemoglobin concentration. RESULTS: A total of 296 students were randomized, and retention in the study was 99.3%. Clinical malaria incidence in the SP/AS and AQ/AS arms was reduced by 66.6% and 46.5%, respectively, vs. vitamin C (P < 0.001). There were fewer clinic visits for any cause among the children receiving SP/AS or AQ/AS (P = 0.024). The prevalence of asymptomatic parasitemia was fivefold higher in the vitamin C arm than either SP/AS or AQ/AS at each post-treatment evaluation (P < 0.001). At the end of the transmission period, children treated with IPT had lower rates of anaemia (SP/AS, 17.7%; AQ/AS, 16.0%; vitamin C, 29.6%; P = 0.039). CONCLUSION: IPT among school children reduced the rates of clinical malaria, all-cause acute clinic visits, asymptomatic parasitemia and anaemia among school-aged children.

Efficacy and safety of a fixed dose artesunate-sulphamethoxypyrazine-pyrimethamine compared to artemether-lumefantrine for the treatment of uncomplicated falciparum malaria across Africa: a randomized multi-centre trial.

BACKGROUND: The efficacy of artemisinin-based combination therapy has already been demonstrated in a number of studies all over the world, and some of them can be regarded as comparably effective. Ease of administration of anti-malarial treatments with shorter courses and fewer tablets may be key determinant of compliance. METHODS: Patients with uncomplicated falciparum malaria and over six months of age were recruited in Cameroon, Mali, Rwanda and Sudan. 1,384 patients were randomly assigned to receive artesunate-sulphamethoxypyrazine-pyrimethamine (AS-SMP) three-day (once daily for 3 days) regimen (N = 476) or AS-SMP 24-hour (0 h, 12 h, 24 h) regimen (N = 458) or artemether-lumefantrine (AL), the regular 6 doses regimen (N = 450). The primary objective was to demonstrate non-inferiority (using a margin of -6%) of AS-SMP 24 hours or AS-SMP three days versus AL on the PCR-corrected 28-day cure rate. RESULTS: The PCR corrected 28-day cure rate on the intention to treat (ITT) analysis population were: 96.0%(457/476) in the AS-SMP three-day group, 93.7%(429/458) in the AS-SMP 24-hour group and 92.0%(414/450) in the AL group. Likewise, the cure rates on the PP analysis population were high: 99.3%(432/437) in the AS-SMP three-day group, 99.5%(416/419) in the AS-SMP 24-hour group and 99.7(391/394)% in the AL group. Most common drug-related adverse events were gastrointestinal symptoms (such as vomiting and diarrhea) which were slightly higher in the AS-SMP 24-hour group. CONCLUSION: AS-SMP three days or AS-SMP 24 hours are safe, are as efficacious as AL, and are well tolerated. TRIAL REGISTRATION: NCT00484900 http://www.clinicaltrials.gov.

Efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria: revisiting molecular markers in an area of emerging AQ and SP resistance in Mali.

BACKGROUND: To update the National Malaria Control Programme of Mali on the efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria. METHODS: During the malaria transmission seasons of 2002 and 2003, 455 children--between six and 59 months of age, with uncomplicated malaria in Kolle, Mali, were randomly assigned to one of three treatment arms. In vivo outcomes were assessed using WHO standard protocols. Genotyping of msp1, msp2 and CA1 polymorphisms were used to distinguish reinfection from recrudescent parasites (molecular correction). RESULTS: Day 28 adequate clinical and parasitological responses (ACPR) were 14.1%, 62.3% and 88.9% in 2002 and 18.2%, 60% and 85.2% in 2003 for chloroquine, amodiaquine and sulphadoxine-pyrimethamine, respectively. After molecular correction, ACPRs (cACPR) were 63.2%, 88.5% and 98.0% in 2002 and 75.5%, 85.2% and 96.6% in 2003 for CQ, AQ and SP, respectively. Amodiaquine was the most effective on fever. Amodiaquine therapy selected molecular markers for chloroquine resistance, while in the sulphadoxine-pyrimethamine arm the level of dhfr triple mutant and dhfr/dhps quadruple mutant increased from 31.5% and 3.8% in 2002 to 42.9% and 8.9% in 2003, respectively. No infection with dhps 540E was found. CONCLUSION: In this study, treatment with sulphadoxine-pyrimethamine emerged as the most efficacious on uncomplicated falciparum malaria followed by amodiaquine. The study demonstrated that sulphadoxine-pyrimethamine and amodiaquine were appropriate partner drugs that could be associated with artemisinin derivatives in an artemisinin-based combination therapy.

Artemisinin-based combinations versus amodiaquine plus sulphadoxine-pyrimethamine for the treatment of uncomplicated malaria in Faladje, Mali.

BACKGROUND: Because of the emergence of chloroquine resistance in Mali, artemether-lumefantrine (AL) or artesunate-amodiaquine (AS+AQ) are recommended as first-line therapy for uncomplicated malaria, but have not been available in Mali until recently because of high costs. METHODS: From July 2005 to January 2006, a randomized open-label trial of three oral antimalarial combinations, namely AS+AQ, artesunate plus sulphadoxine-pyrimethamine (AS+SP), and amodiaquine plus sulphadoxine-pyrimethamine (AQ+SP), was conducted in Faladje, Mali. Parasite genotyping by polymerase chain reaction (PCR) was used to distinguish new from recrudescent Plasmodium falciparum infections. RESULTS: 397 children 6 to 59 months of age with uncomplicated Plasmodium falciparum malaria were enrolled, and followed for 28 days to assess treatment efficacy. Baseline characteristics were similar in all three treatment groups. The uncorrected rates of adequate clinical and parasitologic response (ACPR) were 55.7%, 90.8%, and 97.7% in AS+AQ, AS+SP, and AQ+SP respectively (p < 0.001); after PCR correction ACPR rates were similar among treatment groups: 95.4%, 96.9%, and 99.2% respectively (p = 0.17). Mean haemoglobin concentration increased across all treatment groups from Day 0 (9.82 +/- 1.68 g/dL) to Day 28 (10.78 +/- 1.49 g/dL) (p < 0.001), with the greatest improvement occurring in children treated with AQ+SP. On Day 2, the prevalence of parasitaemia was significantly greater among children treated with AQ+SP (50.8%) than in children treated with AS+AQ (10.5%) or AS+SP (10.8%) (p < 0.001). No significant difference in gametocyte carriage was found between groups during the follow-up period. CONCLUSION: The combination of AQ+SP provides a potentially low cost alternative for treatment of uncomplicated P. falciparum infection in Mali and appears to have the added value of longer protective effect against new infection.