BACKGROUND The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS We used polymerase chain reaction–based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P = 0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P = 0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.)

BackgroundSingle nucleotide polymorphism (SNP) genotyping provides the means to develop a practical, rapid, inexpensive assay that will uniquely identify any Plasmodium falciparum parasite using a small amount of DNA. Such an assay could be used to distinguish recrudescence from re-infection in drug trials, to monitor the frequency and distribution of specific parasites in a patient population undergoing drug treatment or vaccine challenge, or for tracking samples and determining purity of isolates in the laboratory during culture adaptation and sub-cloning, as well as routine passage.MethodsA panel of twenty-four SNP markers has been identified that exhibit a high minor allele frequency (average MAF > 35%), for which robust TaqMan genotyping assays were constructed. All SNPs were identified through whole genome sequencing and MAF was estimated through Affymetrix array-based genotyping of a worldwide collection of parasites. These assays create a "molecular barcode" to uniquely identify a parasite genome.ResultsUsing 24 such markers no two parasites known to be of independent origin have yet been found to have the same allele signature. The TaqMan genotyping assays can be performed on a variety of samples including cultured parasites, frozen whole blood, or whole blood spotted onto filter paper with a success rate > 99%. Less than 5 ng of parasite DNA is needed to complete a panel of 24 markers. The ability of this SNP panel to detect and identify parasites was compared to the standard molecular methods, MSP-1 and MSP-2 typing.ConclusionThis work provides a facile field-deployable genotyping tool that can be used without special skills with standard lab equipment, and at reasonable cost that will unambiguously identify and track P. falciparum parasites both from patient samples and in the laboratory.

To study the effects of malaria-control interventions on parasite population genomics, we examined a set of 1,007 samples of the malaria parasite Plasmodium falciparum collected in Thiès, Senegal between 2006 and 2013. The parasite samples were genotyped using a molecular barcode of 24 SNPs. About 35% of the samples grouped into subsets with identical barcodes, varying in size by year and sometimes persisting across years. The barcodes also formed networks of related groups. Analysis of 164 completely sequenced parasites revealed extensive sharing of genomic regions. In at least two cases we found first-generation recombinant offspring of parents whose genomes are similar or identical to genomes also present in the sample. An epidemiological model that tracks parasite genotypes can reproduce the observed pattern of barcode subsets. Quantification of likelihoods in the model strongly suggests a reduction of transmission from 2006-2010 with a significant rebound in 2012-2013. The reduced transmission and rebound were confirmed directly by incidence data from Thiès. These findings imply that intensive intervention to control malaria results in rapid and dramatic changes in parasite population genomics. The results also suggest that genomics combined with epidemiological modeling may afford prompt, continuous, and cost-effective tracking of progress toward malaria elimination. malaria | genomics | epidemiology I ntensive intervention to reduce the burden of malaria has proven successful in a number of countries in Africa (1). In certain regions of Senegal, implementation of a redesigned National Malaria Control Program (NMCP) in 2006 that included rapid diagnostic tests, artemisinin combination therapies, enhanced insecticide-treated bed nets, and indoor residual spraying resulted in a more than 95% decrease in the number of confirmed cases by 2009 (2). We had been collecting parasite samples in one of these regions annually since 2006. These samples afford a unique opportunity to determine the extent to which intensive intervention is manifested in genetic changes in the parasite population. Genetic changes would be expected to include bottlenecks in the parasite population size, increased random genetic drift, reduced genetic variation, greater self-fertilization during transmission, and increased allele sharing and identity by descent.A key question for tracking malaria elimination is whether such genomic changes would be large enough to be detected in a cost-effective manner in samples of reasonable size. If changes in parasite population genomics took place rapidly enough after intervention, and if they were large enough to be detected, then parasite genomics could play an important role in malaria elimination. Given sufficiently rapid onset and detectability of changes in parasite genomics, an epidemiological model that incorporates parasite genotypes could in principle be used to estimate the epidemiological parameters that most closely match the genomic observations. Estimates of epidemiological parameters s...

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified ≈500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.

SignificanceThe spread of Plasmodium falciparum with reduced susceptibility to artemisinin (ART) in Southeast Asia threatens global malaria control. Most failures of ART treatment are attributed to mutations in the pfkelch13 locus acting through an unclear mechanism. The role of pfkelch13 in reducing ART susceptibility was first identified in an in vitro selection experiment. We carried out a similar in vitro selection and discovered mutations in a different gene that reduce susceptibility to ART. The gene encodes PfCoronin, a conserved protein that in other organisms binds with actin to enhance cytoskeletal plasticity or is involved in vesicular transport. PfCoronin and PfKelch13 share structural similarities, and this finding may yield insights into the molecular mechanisms of ART resistance.

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.   Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

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