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Abstract Background The World Health Organization 2022 malaria chemoprevention guidelines recommend providing a full course of antimalarial treatment at pre-defined intervals, regardless of malaria status to prevent illness among children resident in moderate to high perennial malaria transmission settings as perennial malaria chemoprevention (PMC) with sulfadoxine-pyrimethamine (SP). The dhps I431V mutation circulating in West Africa has unknown effect on SP protective efficacy. Methods This protocol is for a three-arm, parallel, double-blinded, placebo-controlled, randomised trial in Cameroon among children randomly assigned to one of three directly-observed treatment groups: (i) Group 1 (n = 450) receives daily artesunate (AS) placebo on days − 7 to -1, then active SP plus placebo amodiaquine (AQ) on day 0, and placebo AQ on days 1 and 2; (ii) Group 2 (n = 250) receives placebo AS on days − 7 to -1, then active SP and AQ on day 0, and active AQ on days 1 and 2; and (iii) Group 3 (n = 200) receives active AS on days − 7 to -1, then placebo SP on day 0 and placebo AQ on days 0 to 2. On days 0, 2, 5, 7, and thereafter weekly until day 28, children provide blood for thick smear slides. Dried blood spots are collected on the same days and weekly from day 28 to day 63 for quantitative polymerase chain reaction (qPCR) and genotype analyses. Discussion Our aim is to quantify the chemopreventive efficacy of SP, and SP plus AQ, and measure the effect of the parasite genotypes associated with SP resistance on parasite clearance and protection from infection when exposed to SP chemoprevention. We will report unblinded results including: (i) time-to-parasite clearance among SP and SP plus AQ recipients who were positive on day 0 by qPCR and followed to day 63; (ii) mean duration of SP and SP plus AQ protection against infection, and (iii) mean duration of symptom-free status among SP and SP plus AQ recipients who were parasite free on day 0 by qPCR. Our study is designed to compare the 28-day follow-up of the new WHO malaria chemoprevention efficacy study protocol with extended follow-up to day 63. Trial registration ClinicalTrials.gov NCT06173206; 15/12/2023.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Objectives: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). Methods: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. Results: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation (‘prozone-like effect’), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. Conclusions: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

A tool that analyzes the genome of parasites found in the blood of malaria patients can help inform policy decisions on how best to tackle the rise in drug-resistant infections.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Drug resistance is a recurrent problem in the fight against malaria. Genetic and epidemiological surveillance of antimalarial resistant parasite alleles is crucial to guide drug therapies and clinical management. New antimalarial compounds are currently at various stages of clinical trials and regulatory evaluation. Using ∼2000 Plasmodium falciparum genome sequences, we investigated the genetic diversity of eleven gene-targets of promising antimalarial compounds and assessed their potential efficiency across malaria endemic regions. We determined if the loci are under selection prior to the introduction of new drugs and established a baseline of genetic variance, including potential resistant alleles, for future surveillance programmes. Keywords: Antimalarial drugs, Drug-resistant mutations, Gene targets, Genetic diversity

BackgroundSeasonal malaria chemoprevention (SMC) is recommended in the Sahel region of Africa for children under 5 years of age, for up to 4 months of the year. It may be appropriate to include older children, and to provide protection for more than 4 months. We evaluated the effectiveness of SMC using sulfadoxine-pyrimethamine plus amodiaquine given over 5 months to children under 10 years of age in Saraya district in south-east Senegal in 2011.Methods and findingsTwenty-four villages, including 2,301 children aged 3-59 months and 2,245 aged 5-9 years, were randomised to receive SMC with community case management (CCM) (SMC villages) or CCM alone (control villages). In all villages, community health workers (CHWs) were trained to treat malaria cases with artemisinin combination therapy after testing with a rapid diagnostic test (RDT). In SMC villages, CHWs administered SMC to children aged 3 months to 9 years once a month for 5 months. The study was conducted from 27 July to 31 December 2011. The primary outcome was malaria (fever or history of fever with a positive RDT). The prevalence of anaemia and parasitaemia was measured in a survey at the end of the transmission season. Molecular markers associated with resistance to SMC drugs were analysed in samples from incident malaria cases and from children with parasitaemia in the survey. SMC was well tolerated with no serious adverse reactions. There were 1,472 RDT-confirmed malaria cases in the control villages and 270 in the SMC villages. Among children under 5 years of age, the rate difference was 110.8/1,000/month (95% CI 64.7, 156.8; p < 0.001) and among children 5-9 years of age, 101.3/1,000/month (95% CI 66.7, 136.0; p < 0.001). The mean haemoglobin concentration at the end of the transmission season was higher in SMC than control villages, by 6.5 g/l (95% CI 2.0, 11; p = 0.007) among children under 5 years of age, and by 5.2 g/l (95% CI 0.4, 9.9; p = 0.035) among children 5-9 years of age. The prevalence of parasitaemia was 18% in children under 5 years of age and 25% in children 5-9 years of age in the control villages, and 5.7% and 5.8%, respectively, in these 2 age groups in the SMC villages, with prevalence differences of 12.5% (95% CI 6.8%, 18.2%; p < 0.001) in children under 5 years of age and 19.3% (95% CI 8.3%, 30.2%; p < 0.001) in children 5-9 years of age. The pfdhps-540E mutation associated with clinical resistance to sulfadoxine-pyrimethamine was found in 0.8% of samples from malaria cases but not in the final survey. Twelve children died in the control group and 14 in the SMC group, a rate difference of 0.096/1,000 child-months (95% CI 0.99, 1.18; p = 0.895). Limitations of this study include that we were not able to obtain blood smears for microscopy for all suspected malaria cases, such that we had to rely on RDTs for confirmation, which may have included false positives.ConclusionsIn this study SMC for children under 10 years of age given over 5 months was feasible, well tolerated, and effective in preventing malaria episodes, and reduced the prevalence of parasitaemia and anaemia. SMC with CCM achieved high coverage and ensured children with malaria were promptly treated with artemether-lumefantrine.Trial registrationwww.clinicaltrials.gov NCT01449045.

BackgroundSeasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ), given each month during the transmission season, is recommended for children living in areas of the Sahel where malaria transmission is highly seasonal. The recommendation for SMC is currently limited to children under five years of age, but, in many areas of seasonal transmission, the burden in older children may justify extending this age limit. This study was done to determine the effectiveness of SMC in Senegalese children up to ten years of age.Methods and findingsSMC was introduced into three districts over three years in central Senegal using a stepped-wedge cluster-randomised design. A census of the population was undertaken and a surveillance system was established to record all deaths and to record all cases of malaria seen at health facilities. A pharmacovigilance system was put in place to detect adverse drug reactions. Fifty-four health posts were randomised. Nine started implementation of SMC in 2008, 18 in 2009, and a further 18 in 2010, with 9 remaining as controls. In the first year of implementation, SMC was delivered to children aged 3-59 months; the age range was then extended for the latter two years of the study to include children up to 10 years of age. Cluster sample surveys at the end of each transmission season were done to measure coverage of SMC and the prevalence of parasitaemia and anaemia, to monitor molecular markers of drug resistance, and to measure insecticide-treated net (ITN) use. Entomological monitoring and assessment of costs of delivery in each health post and of community attitudes to SMC were also undertaken. About 780,000 treatments were administered over three years. Coverage exceeded 80% each month. Mortality, the primary endpoint, was similar in SMC and control areas (4.6 and 4.5 per 1000 respectively in children under 5 years and 1.3 and 1.2 per 1000 in children 5-9 years of age; the overall mortality rate ratio [SMC: no SMC] was 0.90, 95% CI 0.68-1.2, p = 0.496). A reduction of 60% (95% CI 54%-64%, p < 0.001) in the incidence of malaria cases confirmed by a rapid diagnostic test (RDT) and a reduction of 69% (95% CI 65%-72%, p < 0.001) in the number of treatments for malaria (confirmed and unconfirmed) was observed in children. In areas where SMC was implemented, incidence of confirmed malaria in adults and in children too old to receive SMC was reduced by 26% (95% CI 18%-33%, p < 0.001) and the total number of treatments for malaria (confirmed and unconfirmed) in these older age groups was reduced by 29% (95% CI 21%-35%, p < 0.001). One hundred and twenty-three children were admitted to hospital with a diagnosis of severe malaria, with 64 in control areas and 59 in SMC areas, showing a reduction in the incidence rate of severe disease of 45% (95% CI 5%-68%, p = 0.031). Estimates of the reduction in the prevalence of parasitaemia at the end of the transmission season in SMC areas were 68% (95% CI 35%-85%) p = 0.002 in 2008, 84% (95% CI 58%-94%, p < 0.001) in 2009, and 30% (95% CI -130%-79%, p = 0.56) in 2010. SMC was well tolerated with no serious adverse reactions attributable to SMC drugs. Vomiting was the most commonly reported mild adverse event but was reported in less than 1% of treatments. The average cost of delivery was US$0.50 per child per month, but varied widely depending on the size of the health post. Limitations included the low rate of mortality, which limited our ability to detect an effect on this endpoint.ConclusionsSMC substantially reduced the incidence of outpatient cases of malaria and of severe malaria in children, but no difference in all-cause mortality was observed. Introduction of SMC was associated with an overall reduction in malaria incidence in untreated age groups. In many areas of Africa with seasonal malaria, there is a substantial burden in older children that could be prevented by SMC. SMC in older children is well tolerated and effective and can contribute to reducing malaria transmission.Trial registrationClinicalTrials.gov NCT00712374.

The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr).Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72-76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene.Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Y and 184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/μl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and presence of mutations in the Pfcrt, Pfmdr1 or Pfdhfr genes (P>0.05).Markers of resistance to chloroquine and pyrimethamine were high, whereas cycloguanil-resistance marker was not present in the studied population. The low level of mutations in the Pfmdr1gene indicates likely efficacy of amodiaquine against malaria in pregnancy.

Five human cases of infection with the simian malaria parasite Plasmodium knowlesi from Palawan, the Philippines, were confirmed by nested PCR. This study suggests that this zoonotic infection is found across a relatively wide area in Palawan and documents autochthonous cases in the country.

Circumsporozoite protein (CS) is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates) and Malawi (235 isolates), we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

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.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.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

In the Sahel, most malaria deaths occur among children 1-4 years old during a short transmission season. A trial of seasonal intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine (SP) and a single dose of artesunate (AS) showed an 86% reduction in the incidence of malaria in Senegal but this may not be the optimum regimen. We compared this regimen with three alternatives.2102 children aged 6-59 months received either one dose of SP plus one dose of AS (SP+1AS) (the previous regimen), one dose of SP plus 3 daily doses of AS (SP+3AS), one dose of SP plus three daily doses of amodiaquine (AQ) (SP+3AQ) or 3 daily doses of AQ and AS (3AQ+3AS). Treatments were given once a month on three occasions during the malaria transmission season. The primary end point was incidence of clinical malaria. Secondary end-points were incidence of adverse events, mean haemoglobin concentration and prevalence of parasites carrying markers of resistance to SP.The incidence of malaria, and the prevalence of parasitaemia at the end of the transmission season, were lowest in the group that received SP+3AQ: 10% of children in the group that received SP+1AS had malaria, compared to 9% in the SP+3AS group (hazard ratio HR 0.90, 95%CI 0.60, 1.36); 11% in the 3AQ+3AS group, HR 1.1 (0.76-1.7); and 5% in the SP+3AQ group, HR 0.50 (0.30-0.81). Mutations associated with resistance to SP were present in almost all parasites detected at the end of the transmission season, but the prevalence of Plasmodium falciparum was very low in the SP+3AQ group.Monthly treatment with SP+3AQ is a highly effective regimen for seasonal IPT. Choice of this regimen would minimise the spread of drug resistance and allow artemisinins to be reserved for the treatment of acute clinical malaria.