Alfred Amambua-Ngwa

Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

K13-Propeller Polymorphisms in Plasmodium falciparum Parasites From Sub-Saharan Africa

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Population Genomic Scan for Candidate Signatures of Balancing Selection to Guide Antigen Characterization in Malaria Parasites

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

Genomic epidemiology of artemisinin resistant malaria.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in West Africa

BackgroundMalaria 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.MethodsTen 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.ResultsEach 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 FST values < 0.03), and an overall test for isolation by distance was not significant.ConclusionsAlthough 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.

Erythrocyte Invasion and Merozoite Ligand Gene Expression in Severe and Mild Plasmodium falciparum Malaria

Erythrocyte invasion is central to malaria parasite replication and virulence. Plasmodium falciparum parasites use different alternative erythrocyte receptors and vary in expression of erythrocyte-binding antigenic (EBA) proteins and reticulocyte-binding protein homologues (Rh). Parasite invasion phenotypes and schizont-stage transcript expression profiles of the 8 eba and Rh protein-coding genes without internal stop codons were determined for 163 clinical isolates cultured ex vivo in The Gambia. There was extensive diversity in ability to invade erythrocytes treated with neuraminidase, trypsin, or chymotrypsin, and severe malaria isolates were less restricted by trypsin treatment than were mild malaria isolates (P = .015). Expression profiles of the eba and Rh genes showed distinct clusters indicating coordinated alternative transcription. The most divergent of 5 major clusters was dominated by Rh2b, with virtually no expression of eba175 or eba140 genes (which were dominant in the other 4 clusters). Particular transcripts were significantly correlated with parasitemia (Rh5 was positively correlated and eba140 negatively correlated; P < .01 for both) and age of patients (eba181 was positively correlated and eba175 negatively correlated; P < .001 for both) but not with invasion phenotypes or severity of malaria. Severe and mild malaria isolates were also evenly represented across the different expression clusters.

Prospective Identification of Malaria Parasite Genes under Balancing Selection

Background Endemic human pathogens are subject to strong immune selection, and interrogation of pathogen genome variation for signatures of balancing selection can identify important target antigens. Several major antigen genes in the malaria parasite Plasmodium falciparum have shown such signatures in polymorphism-versus-divergence indices (comparing with the chimpanzee parasite P. reichenowi), and in allele frequency based indices. Methodology/Principal Findings To compare methods for prospective identification of genes under balancing selection, 26 additional genes known or predicted to encode surface-exposed proteins of the invasive blood stage merozoite were first sequenced from a panel of 14 independent P. falciparum cultured lines and P. reichenowi. Six genes at the positive extremes of one or both of the Hudson-Kreitman-Aguade (HKA) and McDonald-Kreitman (MK) indices were identified. Allele frequency based analysis was then performed on a Gambian P. falciparum population sample for these six genes and three others as controls. Tajimas D (TjD) index was most highly positive for the msp3/6-like PF10_0348 (TjD = 1.96) as well as the positive control ama1 antigen gene (TjD = 1.22). Across the genes there was a strong correlation between population TjD values and the relative HKA indices (whether derived from the population or the panel of cultured laboratory isolates), but no correlation with the MK indices. Conclusions/Significance Although few individual parasite genes show significant evidence of balancing selection, analysis of population genomic and comparative sequence data with the HKA and TjD indices should discriminate those that do, and thereby identify likely targets of immunity.

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.

Statistical estimation of cell-cycle progression and lineage commitment in Plasmodium falciparum reveals a homogeneous pattern of transcription in ex vivo culture.

We have cultured Plasmodium falciparum directly from the blood of infected individuals to examine patterns of mature-stage gene expression in patient isolates. Analysis of the transcriptome of P. falciparum is complicated by the highly periodic nature of gene expression because small variations in the stage of parasite development between samples can lead to an apparent difference in gene expression values. To address this issue, we have developed statistical likelihood-based methods to estimate cell cycle progression and commitment to asexual or sexual development lineages in our samples based on microscopy and gene expression patterns. In cases subsequently matched for temporal development, we find that transcriptional patterns in ex vivo culture display little variation across patients with diverse clinical profiles and closely resemble transcriptional profiles that occur in vitro. These statistical methods, available to the research community, assist in the design and interpretation of P. falciparum expression profiling experiments where it is difficult to separate true differential expression from cell-cycle dependent expression. We reanalyze an existing dataset of in vivo patient expression profiles and conclude that previously observed discrete variation is consistent with the commitment of a varying proportion of the parasite population to the sexual development lineage.