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Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima’s D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb region within and flanking the gdv1 gene that regulates the early stages of gametocyte development, which is likely related to the extreme differences in mosquito vector abundance and seasonality that determine the transmission opportunities for the sexual stage of the parasite.

Background Malaria parasite population genetic structure varies among areas of differing endemicity, but this has not been systematically studied across Plasmodium falciparum populations in Africa where most infections occur. Methods Ten polymorphic P. falciparum microsatellite loci were genotyped in 268 infections from eight locations in four West African countries (Republic of Guinea, Guinea Bissau, The Gambia and Senegal), spanning a highly endemic forested region in the south to a low endemic Sahelian region in the north. Analysis was performed on proportions of mixed genotype infections, genotypic diversity among isolates, multilocus standardized index of association, and inter-population differentiation. Results Each location had similar levels of pairwise genotypic diversity among isolates, although there were many more mixed parasite genotype infections in the south. Apart from a few isolates that were virtually identical, the multilocus index of association was not significant in any population. Genetic differentiation between populations was low (most pairwise F ST values < 0.03), and an overall test for isolation by distance was not significant. Conclusions Although proportions of mixed genotype infections varied with endemicity as expected, population genetic structure was similar across the diverse sites. Very substantial reduction in transmission would be needed to cause fragmented or epidemic sub-structure in this region.

A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors. We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007-2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area. Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.

Targeting parasite's protein kinaseMalaria elimination goals are constantly eroded by the challenge of emerging drug and insecticide resistance. Alam et al. have taken established drug targets—CLK protein kinases involved in regulation of RNA splicing—and investigated how inhibition of the parasite's enzymes blocks completion of its complex life cycle. They identified an inhibitor of the parasite's CLK protein kinase that was 100-fold less active against the most closely related human protein kinase and effective at clearing rodent malaria parasites. Not only does this compound halt the development of sexual stages but it also limits transmission to the mosquito vector of the parasite, a key requirement for malaria drugs.Science, this issue p. eaau1682INTRODUCTIONDespite the positive effects of intervention strategies that include insecticide-impregnated bed nets and artemisinin-based drug therapies, malaria still kills nearly 500,000 people per year and infects more than 200 million individuals globally. This, together with the emerging resistance of the parasite to frontline antimalarials, means that there is an urgent need for novel treatments that not only offer a cure for malaria but also prevent transmission. We show that by inhibiting an essential protein kinase that is a key regulator of RNA processing, we are able to kill the parasite in the blood and liver stages as well as prevent the development of the sexual-stage gametocytes, thereby blocking transmission to the mosquito.RATIONALEOur group has previously published a list of 36 protein kinases that are essential for blood-stage survival of the most virulent form of the human malaria parasite, Plasmodium falciparum. Here, we focused on one of these protein kinases from the P. falciparum CLK (cyclin-dependent–like kinase) family, PfCLK3, and reasoned that inhibition of this protein kinase by a small drug-like molecule would be effective at killing blood-stage parasites. We further hypothesized that because PfCLK3 plays a key role in RNA splicing, inhibition of this kinase would be effective at killing the parasite at all stages of the life cycle where RNA splicing is required. This would include blood, liver, and sexual stages.RESULTSBy screening a focused library of nearly 30,000 compounds, we identified a probe molecule that selectively inhibited PfCLK3 and killed blood-stage P. falciparum. Using a combination of evolved resistance and chemogenetics, we established that our probe molecule had parasiticidal activity by inhibition of PfCLK3. We further showed that inhibition of PfCLK3 in parasites resulted in a reduction in more than 400 gene transcripts known to be essential for parasite survival. The finding that the vast majority of the genes down-regulated by PfCLK3 inhibition contained introns supported the notion that inhibition of PfCLK3 killed the malaria parasite by preventing the splicing of essential parasite genes. Because there is a high degree of homology between orthologs of CLK3 in other Plasmodium species, it might be expected that our probe molecule would both inhibit CLK3 contained in other malaria parasite species and have effective parasiticidal activity in these parasites. This was indeed found to be the case, with our molecule showing potent inhibition of CLK3 from P. vivax and P. berghei as well as killing the blood stages of P. berghei and P. knowlesi. Furthermore, we demonstrated that CLK3 inhibition also kills liver-stage P. berghei parasites and prevents P. berghei infection in mice. Finally, we showed that inhibition of PfCLK3 prevents the development of P. falciparum gametocytes, thereby blocking the infection of mosquitoes.CONCLUSIONWe found that inhibition of the essential malaria protein kinase CLK3 can kill multiple species of malaria parasites at the blood stage as well as killing liver-stage parasites and blocking transmission of the parasite to mosquitoes by preventing gametocyte development. In this way, we validate Plasmodium spp. CLK3 as a target that can offer prophylactic, curative, and transmission-blocking potential.The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi. Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure—to be prophylactic and transmission blocking in malaria.

The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T was high (allele freq. = 0.96-1), but did not vary by resting location. The overall sporozoite positivity rate was 1.3% (95% CI: 0.5-2%) in mosquito populations. Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis fed on animal blood. In this study, high levels of resistance mutations were observed that could be influencing the mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour demonstrated in the mosquito populations suggest that larval source management could be an intervention to complement vector control in this setting.

BackgroundCurrent methods for detecting malaria parasites are invasive and associated with poor compliance when repeated sampling is required. New methods to detect and quantify parasites in a less-invasive manner would greatly enhance the potential for longitudinal surveillance in clinical trials MethodsSaliva, urine, and blood samples from 386 Gambian outpatients with suspected malaria infections were analyzed by nested polymerase chain reaction (nPCR) to detect infection and to evaluate diagnostic accuracy in comparison to expert microscopy. The amount of parasite DNA in malaria-positive samples was estimated using real-time quantitative PCR (qPCR) ResultsBlood parasite density as estimated by qPCR correlated well with parasite counts established by microscopy (ρ=0.94; P<.001). qPCR results for saliva had a significant correlation with microscopy counts (ρ=0.58; P<.001), whereas qPCR results for urine had a positive but poor correlation with microscopy counts (ρ=0.20; P=.117). The mean amounts of parasite DNA quantified in blood were greater than the mean amounts quantified in saliva and urine samples obtained concurrently from the same individual, by ∼600-fold and ∼2500-fold, respectively. When nPCR results were compared with microscopy results, nPCR of saliva had a sensitivity of 73% and a specificity of 97%; its sensitivity increased to 82% in samples with a parasite density of ⩾1000 parasites/μL. nPCR of urine had a sensitivity of 32% and a specificity of 98% Conclusion Saliva sampling is a promising less-invasive approach for detecting malaria infection

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 end of the malaria transmission season (January) and just before the start of the next one (June) to determine asymptomatic P. falciparum carriage. Passive case detection was conducted during each transmission season (August to January) to determine incidence of clinical malaria. Association between carriage at the end of the season and at start of the next one and the risk factors for this were assessed. Effect of carriage before 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 (interquartile range [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 carriage just before start of the next one were strongly associated (adjusted odds ratio [aOR] = 19.99; 95% CI 12.57–31.77, p < 0.001). The odds of persistent carriage (i.e. infected both in January and in June) were higher in rural villages (aOR = 13.0; 95% CI 6.33–26.88, p < 0.001) and in children aged 5–15 years (aOR = 5.03; 95% CI 2.47–10.23, p =  < 0.001). In the rural villages, carriage before start of the season was associated with a lower risk of clinical malaria during the season (incidence risk ratio [IRR] 0.48, 95% CI 0.27–0.81, p = 0.007). Conclusions Asymptomatic P. falciparum carriage at the end of a transmission season strongly predicted carriage just before start of the next one. Interventions that clear persistent asymptomatic infections when targeted at the subpopulation with high risk of carriage may reduce the infectious reservoir responsible for launching seasonal transmission.

Background Malaria elimination in Senegal requires accurate diagnosis of all Plasmodium species. Plasmodium falciparum is the most prevalent species in Senegal, although Plasmodium malariae , Plasmodium ovale, and recently Plasmodium vivax have also been reported. Nonetheless, most malaria control tools, such as Histidine Rich Protein 2 rapid diagnosis test (PfHRP2-RDT,) can only diagnose P. falciparum . Thus, PfHRP2-RDT misses non-falciparum species and P. falciparum infections that fall below the limit of detection. These limitations can be addressed using highly sensitive Next Generation Sequencing (NGS). This study assesses the burden of the four different Plasmodium species in western and eastern regions of Senegal using targeted PCR amplicon sequencing. Methods Three thousand samples from symptomatic and asymptomatic individuals in 2021 from three sites in Senegal (Sessene, Diourbel region; Parcelles Assainies, Kaolack region; Gabou, Tambacounda region) were collected. All samples were tested using PfHRP2-RDT and photoinduced electron transfer polymerase chain reaction (PET-PCR), which detects all Plasmodium species. Targeted sequencing of the nuclear 18S rRNA and the mitochondrial cytochrome B genes was performed on PET-PCR positive samples. Results Malaria prevalence by PfHRP2-RDT showed 9.4% (94/1000) and 0.2% (2/1000) in Diourbel (DBL) and Kaolack (KL), respectively. In Tambacounda (TAM) patients who had malaria symptoms and had a negative PfHRP2-RDT were enrolled. The PET-PCR had a positivity rate of 23.5% (295/1255) overall. The PET-PCR positivity rate was 37.6%, 12.3%, and 22.8% in Diourbel, Kaolack, and Tambacounda, respectively. Successful sequencing of 121/295 positive samples detected P. falciparum ( 93%), P. vivax ( 2.6%), P. malariae ( 4.4%), and P. ovale wallikeri (0.9%). Plasmodium vivax was co-identified with P. falciparum in thirteen samples. Sequencing also detected two PfHRP2-RDT-negative mono-infections of P. vivax in Tambacounda and Kaolack. Conclusion The findings demonstrate the circulation of P. vivax in western and eastern Senegal, highlighting the need for improved malaria control strategies and accurate diagnostic tools to better understand the prevalence of non-falciparum species countrywide.

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed.

Background As indicators of burden of malaria have substantially decreased in The Gambia, reaching a pre-elimination status may be attainable. Achieving this goal requires in-depth understanding of the current burden of Plasmodium falciparum infection. Methods A nationwide cross-sectional survey was conducted in 2012 to determine the prevalence of P. falciparum infection, and to describe its heterogeneity and associated risk factors. Finger-prick blood samples were collected for microscopy, species-specific PCR and haemoglobin measurement. Results A total of 9,094 participants were included and median age was 11.9 years (IQR 5, 28). Overall prevalence of P. falciparum was 16.01 % with marked heterogeneity between sites (4.32–36.75 %) and within villages in each site (1.63–49.13 %). Across all sites, 51.17 % (745/1,456) of infections were asymptomatic and 35.61 % (448/1,258) were sub-microscopic. The odds of P. falciparum infection were higher in older children; 5–15 years (OR = 1.90; 95 % CI 1.60–2.26), adults (OR = 1.48; 95 % CI 1.24–1.78) and participants with moderate anaemia (OR = 1.62; 95 % CI 1.32–1.99). Conclusions The current malaria control interventions are not sufficient to interrupt transmission in The Gambia as malaria prevalence is still relatively high in the eastern part of the country. New interventions aiming at interrupting transmission are needed and should be urgently evaluated.