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Background Carriers of persistent asymptomatic Plasmodium falciparum infections constitute an infectious reservoir that maintains malaria transmission. Understanding the extent of carriage and characteristics of carriers specific to endemic areas could guide use of interventions to reduce infectious reservoir. Methods In eastern Gambia, an all-age cohort from four villages was followed up from 2012 to 2016. Each year, cross-sectional surveys were conducted at the start and end of the malaria transmission season to determine asymptomatic P. falciparum carriage by microscopy while passive case detection was conducted during the transmission to determine incidence of clinical malaria. Association between carriage at the end of a season and at the start of the next and risk factors for this were assessed. Effect of carriage at the start of the season on risk of clinical malaria during the season was also examined. Results A total of 1403 individuals − 1154 from a semi-urban village and 249 from three rural villages were enrolled; median age was 12 years (IQR 6, 30) and 12 years (IQR 7, 27) respectively. In adjusted analysis, asymptomatic P. falciparum carriage at the end of a transmission season and at the start of the next were strongly associated (OR = 19.99; 95% CI 12.57–31.77, P P. falciparum carriage at the end of a transmission season strongly predicts carriage at the start the next season. Odds of carriage across seasons was higher in villages with relatively higher transmission and in older children. Interventions that reduce persistent asymptomatic infections can be applied in a targeted manner and may supress the initiation of yearly seasonal malaria transmission.

Background: Polymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown.Methods: Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygote (carriers of the minor allele, C/C), heterozygote (T/C) and wildtype (T/T) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank.Results: Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ±159 and 4261±283] , for homozygotes, heterozygotes and wildtypes respectively, p½ ± SD = 4.7±0.5, 1.8±0.3 and 1.9±0.4 min, pPlasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (pvar-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50=122uM CI: 110-134) confirming its sensitivity to calcium channel blockade.Conclusion: The data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimising all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.

Background Polymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown. Methods Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygotes (carriers of the minor alleles, T/T (variant), heterozygote T/C and wildtype C/C (ancestral)) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank. Results Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ± 159 and 4261 ± 283], for homozygotes, heterozygotes and wildtypes respectively, p ½ ± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50 = 122uM CI: 110–134) confirming its sensitivity to calcium channel blockade. Conclusion The data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.

Background: Recent genome-wide association and cross-sectional studies have identified novel human genetic polymorphisms associated with alterations in severe malaria risk. Some of the strongest associations have been identified in the ATP2B4 gene. ATP2B4 codes for PMCA4b, the primary regulator of erythrocyte calcium concentration. Methods: To investigate the cellular mechanisms by which polymorphisms in ATP2B4 alter malaria pathogenesis, we performed in vitro assays that model malaria pathogenesis on red blood cells (RBCs) collected from individuals (n=96) with and without copies of ATP2B4 variant haplotype. Findings: We found reduced rates of PMCA4b expression (p

Artemisinin combination therapies are the current frontline therapy for falciparum malaria. Artemisinin is activated by heme iron, and the consequent production of reactive oxygen species and carbon-centered radicals results in rapid parasite clearance. Red blood cells (RBCs) from anemic iron-deficient individuals have decreased levels of heme, and such deficiencies are highly prevalent among children and pregnant women in malaria-endemic countries. We, therefore, investigated the possibility that host anemia could impair artemisinin activity and alter the drug sensitivity of artemisinin-resistant strains of Plasmodium falciparum. We collected RBCs from anemic (n = 35) and nonanemic (n = 11) Gambian children between the ages of 2 and 24 months. Parasites grown in RBCs from both groups were assessed in vitro using the ring-stage survival assay with artemisinin-resistant and artemisinin-sensitive strains of P. falciparum. No differences were found in artemisinin sensitivity (P > 0.05), and there was no correlation between artemisinin activity and host hemoglobin levels. Standard antimalarial drug activity assays for representatives of the major classes of antimalarial drugs found no differences in the IC(50) values against P. falciparum between anemic and nonanemic RBCs. We conclude that host anemia does not influence artemisinin activity.

The FPN Q248H mutation protects children from anemia, hemolysis, and iron deficiency, but not malaria or bacterial infection., Iron acquisition is critical for life. Ferroportin (FPN) exports iron from mature erythrocytes, and deletion of the Fpn gene results in hemolytic anemia and increased fatality in malaria-infected mice. The FPN Q248H mutation (glutamine to histidine at position 248) renders FPN partially resistant to hepcidin-induced degradation and was associated with protection from malaria in human studies of limited size. Using data from cohorts including over 18,000 African children, we show that the Q248H mutation is associated with modest protection against anemia, hemolysis, and iron deficiency, but we found little evidence of protection against severe malaria or bacteremia. We additionally observed no excess Plasmodium growth in Q248H erythrocytes ex vivo, nor evidence of selection driven by malaria exposure, suggesting that the Q248H mutation does not protect from malaria and is unlikely to deprive malaria parasites of iron essential for their growth.

Antimalarial interventions have yielded a significant decline in malaria prevalence in The Gambia, where artemether-lumefantrine (AL) has been used as a first-line antimalarial for a decade. Clinical Plasmodium falciparum isolates collected from 2012 to 2015 were analyzed ex vivo for antimalarial susceptibility and genotyped for drug resistance markers ( pfcrt K76T, pfmdr1 codons 86, 184, and 1246, and pfk13 ) and microsatellite variation. Additionally, allele frequencies of single nucleotide polymorphisms (SNPs) from other drug resistance-associated genes were compared from genomic sequence data sets from 2008 ( n = 79) and 2014 ( n = 168). No artemisinin resistance-associated pfk13 mutation was found, and only 4% of the isolates tested in 2015 showed significant growth after exposure to dihydroartemisinin. Conversely, the 50% inhibitory concentrations (IC 50 s) of amodiaquine and lumefantrine increased within this period. pfcrt 76T and pfmdr1 184F mutants remained at a prevalence above 80%. pfcrt 76T was positively associated with higher IC 50 s to chloroquine. pfmdr1 NYD increased in frequency between 2012 and 2015 due to lumefantrine selection. The TNYD ( pfcrt 76T and pfmdr1 NYD wild-type haplotype) also increased in frequency following AL implementation in 2008. These results suggest selection for pfcrt and pfmdr1 genotypes that enable tolerance to lumefantrine. Increased tolerance to lumefantrine calls for sustained chemotherapeutic monitoring in The Gambia to minimize complete artemisinin combination therapy (ACT) failure in the future.

In 2006, artemether-lumefantrine (AL) became the first-line treatment of uncomplicated malaria in Senegal, Mali, and the Gambia. To monitor its efficacy, between August 2011 and November 2014, children with uncomplicated Plasmodium falciparum malaria were treated with AL and followed up for 42 days. A total of 463 subjects were enrolled in three sites (246 in Senegal, 97 in Mali, and 120 in Gambia). No early treatment failure was observed and malaria infection cleared in all patients by day 3. Polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR) was 100% in Mali, and the Gambia, and 98.8% in Senegal. However, without PCR adjustment, ACPR was 89.4% overall; 91.5% in Mali, 98.8% in Senegal, and 64.3% in the Gambia (the lower value in the Gambia attributed to poor compliance of the full antimalarial course). However, pfmdr1 mutations were prevalent in Senegal and a decrease in parasite sensitivity to artesunate and lumefantrine (as measured by ex vivo drug assay) was observed at all sites. Recrudescent parasites did not show Kelch 13 (K13) mutations and AL remains highly efficacious in these west African sites.

BackgroundChlorproguanil-dapsone (Lapdap), developed as a low-cost antimalarial, was withdrawn in 2008 after concerns about safety in G6PD deficient patients. This trial was conducted in 2004 to evaluate the safety and effectiveness of CD and comparison with artemether-lumefantrine (AL) under conditions of routine use in G6PD normal and G6PD deficient patients with uncomplicated malaria in The Gambia. We also examined the effects of a common genetic variant that affects chlorproguanil metabolism on risk of treatment failure.Methods1238 children aged 6 months to 10 years with uncomplicated malaria were randomized to receive CD or artemether-lumefantrine (AL) and followed for 28 days. The first dose was supervised, subsequent doses given unsupervised at home. G6PD genotype was determined to assess the interaction between treatment and G6PD status in their effects on anaemia. The main endpoints were clinical treatment failure by day 28, incidence of severe anaemia (HbFindingsOne third of patients treated with AL, and 6% of patients treated with CD, did not complete their course of medication. 18% (109/595) of children treated with CD and 6.1% (36/587) with AL required rescue medication within 4 weeks, risk difference 12% (95%CI 8.9%-16%). 23 children developed severe anaemia (17 (2.9%) treated with CD and 6 (1.0%) with AL, risk difference 1.8%, 95%CI 0.3%-3.4%, P = 0.02). Haemoglobin concentration on day 3 was lower among children treated with CD than AL (difference 0.43 g/dL, 95% CI 0.24 to 0.62), and within the CD group was lower among those children who had higher parasite density at enrollment. Only 17 out of 1069 children who were typed were G6PD A- deficient, of these 2/9 treated with CD and 1/8 treated with AL developed severe anaemia. 5/9 treated with CD had a fall of 2 g/dL or more in haemoglobin concentration by day 3.InterpretationAL was well tolerated and highly effective and when given under operational conditions despite poor adherence to the six-dose regimen. There were more cases of severe malaria and anaemia after CD treatment although G6PD deficiency was uncommon.Trial registrationClinicaltrials.gov NCT00118794.