Characterizing the impact of sustained sulfadoxine/pyrimethamine use upon the Plasmodium falciparum population in Malawi.

BACKGROUND: Malawi experienced prolonged use of sulfadoxine/pyrimethamine (SP) as the front-line anti-malarial drug, with early replacement of chloroquine and delayed introduction of artemisinin-based combination therapy. Extended use of SP, and its continued application in pregnancy is impacting the genomic variation of the Plasmodium falciparum population. METHODS: Whole genome sequence data of P. falciparum isolates covering 2 years of transmission within Malawi, alongside global datasets, were used. More than 745,000 SNPs were identified, and differences in allele frequencies between countries assessed, as well as genetic regions under positive selection determined. RESULTS: Positive selection signals were identified within dhps, dhfr and gch1, all components of the parasite folate pathway associated with SP resistance. Sitting predominantly on a dhfr triple mutation background, a novel copy number increase of ~twofold was identified in the gch1 promoter. This copy number was almost fixed (96.8% frequency) in Malawi samples, but found at less than 45% frequency in other African populations, and distinct from a whole gene duplication previously reported in Southeast Asian parasites. CONCLUSIONS: SP resistance selection pressures have been retained in the Malawian population, with known resistance dhfr mutations at fixation, complemented by a novel gch1 promoter duplication. The effects of the duplication on the fitness costs of SP variants and resistance need to be elucidated.

Imputation-based population genetics analysis of Plasmodium falciparum malaria parasites.

Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86 k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima's D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and association analyses, and supporting global surveillance for drug resistance markers and candidate vaccine antigens.

A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains.

Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide.

Whole-genome scans provide evidence of adaptive evolution in Malawian Plasmodium falciparum isolates.

BACKGROUND: Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. METHODS: We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure and identify loci under selection. RESULTS: High genetic diversity (π = 2.4 × 10(-4)), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency-based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparison of the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries (Kenya, Burkina Faso, Mali, Cambodia, and Thailand) detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. CONCLUSIONS: The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs and may be useful in formulating local malaria treatment guidelines.

estMOI: estimating multiplicity of infection using parasite deep sequencing data.

Individuals living in endemic areas generally harbour multiple parasite strains. Multiplicity of infection (MOI) can be an indicator of immune status and transmission intensity. It has a potentially confounding effect on a number of population genetic analyses, which often assume isolates are clonal. Polymerase chain reaction-based approaches to estimate MOI can lack sensitivity. For example, in the human malaria parasite Plasmodium falciparum, genotyping of the merozoite surface protein (MSP1/2) genes is a standard method for assessing MOI, despite the apparent problem of underestimation. The availability of deep coverage data from massively parallizable sequencing technologies means that MOI can be detected genome wide by considering the abundance of heterozygous genotypes. Here, we present a method to estimate MOI, which considers unique combinations of polymorphisms from sequence reads. The method is implemented within the estMOI software. When applied to clinical P.falciparum isolates from three continents, we find that multiple infections are common, especially in regions with high transmission.

PlasmoView: a web-based resource to visualise global Plasmodium falciparum genomic variation.

Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (eg, biological function) in a geographic setting. The first release contains over 600,000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania).

Mechanistic Studies of the Negative Epistatic Malaria-protective Interaction Between Sickle Cell Trait and α+thalassemia.

BACKGROUND: Individually, the red blood cell (RBC) polymorphisms sickle cell trait (HbAS) and α+thalassemia protect against severe Plasmodium falciparum malaria. It has been shown through epidemiological studies that the co-inheritance of both conditions results in a loss of the protection afforded by each, but the biological mechanisms remain unknown. METHODS: We used RBCs from >300 donors of various HbAS and α+thalassemia genotype combinations to study the individual and combinatorial effects of these polymorphisms on a range of putative P. falciparum virulence phenotypes in-vitro, using four well-characterised P. falciparum laboratory strains. We studied cytoadhesion of parasitized RBCs (pRBCs) to the endothelial receptors CD36 and ICAM1, rosetting of pRBCs with uninfected RBCs, and pRBC surface expression of the parasite-derived adhesion molecule P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). FINDINGS: We confirmed previous reports that HbAS pRBCs show reduced cytoadhesion, rosetting and PfEMP1 expression levels compared to normal pRBC controls. Furthermore, we found that co-inheritance of HbAS with α+thalassemia consistently reversed these effects, such that pRBCs of mixed genotype showed levels of cytoadhesion, rosetting and PfEMP1 expression indistinguishable from those seen in normal pRBCs. However, pRBCs with α+thalassemia alone showed parasite strain-specific effects on adhesion, and no consistent reduction in PfEMP1 expression. INTERPRETATION: Our data support the hypothesis that the negative epistasis between HbAS and α+thalassemia observed in epidemiological studies might be explained by host genotype-specific changes in the pRBC-adhesion properties that contribute to parasite sequestration and disease pathogenesis in vivo. The mechanism by which α+thalassemia on its own protects against severe malaria remains unresolved.

Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome.

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases.