Treatment Failure in a UK Malaria Patient Harboring Genetically Variant Plasmodium falciparum From Uganda With Reduced In Vitro Susceptibility to Artemisinin and Lumefantrine.

BACKGROUND: Recent cases of clinical failure in malaria patients in the United Kingdom (UK) treated with artemether-lumefantrine have implications for malaria chemotherapy worldwide. METHODS: Parasites were isolated from an index case of confirmed Plasmodium falciparum treatment failure after standard treatment, and from comparable travel-acquired UK malaria cases. Drug susceptibility in vitro and genotypes at 6 resistance-associated loci were determined for all parasite isolates and compared with clinical outcomes for each parasite donor. RESULTS: A traveler, who returned to the UK from Uganda in 2022 with Plasmodium falciparum malaria, twice failed treatment with full courses of artemether-lumefantrine. Parasites from the patient exhibited significantly reduced susceptibility to artemisinin (ring-stage survival, 17.3% [95% confidence interval {CI}, 13.6%-21.1%]; P < .0001) and lumefantrine (effective concentration preventing 50% of growth = 259.4 nM [95% CI, 130.6-388.2 nM]; P = .001). Parasite genotyping identified an allele of pfk13 encoding both the A675V variant in the Pfk13 propeller domain and a novel L145V nonpropeller variant. In vitro susceptibility testing of 6 other P. falciparum lines of Ugandan origin identified reduced susceptibility to artemisinin and lumefantrine in 1 additional line, also from a 2022 treatment failure case. These parasites did not harbor a pfk13 propeller domain variant but rather the novel nonpropeller variant T349I. Variant alleles of pfubp1, pfap2mu, and pfcoronin were also identified among the 7 parasite lines. CONCLUSIONS: We confirm, in a documented case of artemether-lumefantrine treatment failure imported from Uganda, the presence of pfk13 mutations encoding L145V and A675V. Parasites with reduced susceptibility to both artemisinin and lumefantrine may be emerging in Uganda.

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty.

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.

Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines.

Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.

Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum.

BACKGROUND: Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes. RESULTS: Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates. CONCLUSIONS: Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.

Trisubstituted thiazoles as potent and selective inhibitors of Plasmodium falciparum protein kinase G (PfPKG).

A series of trisubstituted thiazoles have been identified as potent inhibitors of Plasmodium falciparum (Pf) cGMP-dependent protein kinase (PfPKG) through template hopping from known Eimeria PKG (EtPKG) inhibitors. The thiazole series has yielded compounds with improved potency, kinase selectivity and good in vitro ADME properties. These compounds could be useful tools in the development of new anti-malarial drugs in the fight against drug resistant malaria.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

BACKGROUND: Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. METHODS: Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. RESULTS: The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. CONCLUSIONS: Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.

Variation in Plasmodium falciparum erythrocyte invasion phenotypes and merozoite ligand gene expression across different populations in areas of malaria endemicity.

Plasmodium falciparum merozoites use diverse alternative erythrocyte receptors for invasion and variably express cognate ligands encoded by the erythrocyte binding antigen (eba) and reticulocyte binding-like homologue (Rh) gene families. Previous analyses conducted on parasites from single populations in areas of endemicity revealed a wide spectrum of invasion phenotypes and expression profiles, although comparisons across studies have been limited by the use of different protocols. For direct comparisons within and among populations, clinical isolates from three different West African sites of endemicity (in Ghana, Guinea, and Senegal) were cryopreserved and cultured ex vivo after thawing in a single laboratory to assay invasion of target erythrocytes pretreated with enzymes affecting receptor subsets. Complete invasion assay data from 67 isolates showed no differences among the populations in the broad range of phenotypes measured by neuraminidase treatment (overall mean, 40.6% inhibition) or trypsin treatment (overall mean, 83.3% inhibition). The effects of chymotrypsin treatment (overall mean, 79.2% inhibition) showed heterogeneity across populations (Kruskall-Wallis P = 0.023), although the full phenotypic range was seen in each. Schizont-stage transcript data for a panel of 8 invasion ligand genes (eba175, eba140, eba181, Rh1, Rh2a, Rh2b, Rh4, and Rh5) were obtained for 37 isolates, showing similar ranges of variation in each population except that eba175 levels tended to be higher in parasites from Ghana than in those from Senegal (whereas levels of eba181 and Rh2b were lower in parasites from Ghana). The broad diversity in invasion phenotypes and gene expression seen within each local population, with minimal differences among them, is consistent with a hypothesis of immune selection maintaining parasite variation.

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.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.

Genomic variation during culture adaptation of genetically complex Plasmodium falciparum clinical isolates.

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes.

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 96 clinical isolates from 4 populations with various levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator ap2-g. Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing mspdbl2 did not express ap2-g, consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. IMPORTANCE These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial ex vivo generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and mspdbl2 gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission. Single-cell transcriptome data confirm the absence of a direct correlation with the ap2-g master regulator of sexual development, indicating that the MSPDBL2-positive subpopulation has a separate function in asexual survival and replication under conditions that promote terminal sexual differentiation.

Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum.

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature. Therefore, to focus discovery efforts on genes that are polymorphic, we scanned all available shotgun genome sequence data from laboratory lines of P. falciparum and chose six loci with more than five single nucleotide polymorphisms per kilobase (including PF10_0348) for in-depth frequency-based analyses in a Kenyan population (allele sample sizes >50 for each locus) and comparison of Hudson-Kreitman-Aguade (HKA) ratios of population diversity (π) to interspecific divergence (K) from the chimpanzee parasite Plasmodium reichenowi. Three of these (the msp3/6-like genes PF10_0348 and PF10_0355 and the surf(4.1) gene PFD1160w) showed exceptionally high positive values of Tajima's D and Fu and Li's F indices and have the highest HKA ratios, indicating that they are under balancing selection and should be prioritized for studies of their protein products as candidate targets of immunity. Combined with earlier results, there is now strong evidence that high HKA ratio (as well as the frequency-independent ratio of Watterson's /K) is predictive of high values of Tajima's D. Thus, the former offers value for use in genome-wide screening when numbers of genome sequences within a species are low or in combination with Tajima's D as a 2D test on large population genomic samples.

Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level.

The central role that erythrocyte invasion plays in Plasmodium falciparum survival and reproduction makes this process an attractive target for therapeutic or vaccine development. However, multiple invasion-related genes with complementary and overlapping functions afford the parasite the plasticity to vary ligands used for invasion, leading to phenotypic variation and immune evasion. Overcoming the challenge posed by redundant ligands requires a deeper understanding of conditions that select for variant phenotypes and the molecular mediators. While host factors including receptor heterogeneity and acquired immune responses may drive parasite phenotypic variation, we have previously shown that host-independent changes in invasion phenotype can be achieved by continuous culturing of the W2mef and Dd2 P. falciparum strains in moving suspension as opposed to static conditions. Here, we have used a highly biologically replicated whole transcriptome sequencing approach to identify the molecular signatures of variation associated with the phenotype switch. The data show increased expression of particular invasion-related genes in switched parasites, as well as a large number of genes encoding proteins that are either exported or form part of the export machinery. The genes with most markedly increased expression included members of the erythrocyte binding antigens (EBA), reticulocyte binding homologues (RH), surface associated interspersed proteins (SURFIN), exported protein family 1 (EPF1) and Plasmodium Helical Interspersed Sub-Telomeric (PHIST) gene families. The data indicate changes in expression of a repertoire of genes not previously associated with erythrocyte invasion phenotypes, suggesting the possibility that moving suspension culture may also select for other traits.

Multi-locus genotyping reveals established endemicity of a geographically distinct Plasmodium vivax population in Mauritania, West Africa.

BACKGROUND: Plasmodium vivax has been recently discovered as a significant cause of malaria in Mauritania, although very rare elsewhere in West Africa. It has not been known if this is a recently introduced or locally remnant parasite population, nor whether the genetic structure reflects epidemic or endemic transmission. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the P. vivax population genetic structure in Mauritania and compare with populations previously analysed elsewhere, multi-locus genotyping was undertaken on 100 clinical isolates, using a genome-wide panel of 38 single nucleotide polymorphisms (SNPs), plus seven SNPs in drug resistance genes. The Mauritanian P. vivax population is shown to be genetically diverse and divergent from populations elsewhere, indicated consistently by genetic distance matrix analysis, principal components analyses, and fixation indices. Only one isolate had a genotype clearly indicating recent importation, from a southeast Asian source. There was no linkage disequilibrium in the local parasite population, and only a small number of infections appeared to be closely genetically related, indicating that there is ongoing genetic recombination consistent with endemic transmission. The P. vivax diversity in a remote mining town was similar to that in the capital Nouakchott, with no indication of local substructure or of epidemic population structure. Drug resistance alleles were virtually absent in Mauritania, in contrast with P. vivax in other areas of the world. CONCLUSIONS/SIGNIFICANCE: The molecular epidemiology indicates that there is long-standing endemic transmission that will be very challenging to eliminate. The virtual absence of drug resistance alleles suggests that most infections have been untreated, and that this endemic infection has been more neglected in comparison to P. vivax elsewhere.

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites.

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.

Gene copy number variation throughout the Plasmodium falciparum genome.

BACKGROUND: Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome. RESULTS: We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, approximately 40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified. CONCLUSION: CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.

Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria.

The pathogenesis of infectious diseases depends on the interaction of host and pathogen. In Plasmodium falciparum malaria, host and parasite processes can be assessed by dual RNA sequencing of blood from infected patients. We performed dual transcriptome analyses on samples from 46 malaria-infected Gambian children to reveal mechanisms driving the systemic pathophysiology of severe malaria. Integrating these transcriptomic data with estimates of parasite load and detailed clinical information allowed consideration of potentially confounding effects due to differing leukocyte proportions in blood, parasite developmental stage, and whole-body pathogen load. We report hundreds of human and parasite genes differentially expressed between severe and uncomplicated malaria, with distinct profiles associated with coma, hyperlactatemia, and thrombocytopenia. High expression of neutrophil granule-related genes was consistently associated with all severe malaria phenotypes. We observed severity-associated variation in the expression of parasite genes, which determine cytoadhesion to vascular endothelium, rigidity of infected erythrocytes, and parasite growth rate. Up to 99% of human differential gene expression in severe malaria was driven by differences in parasite load, whereas parasite gene expression showed little association with parasite load. Coexpression analyses revealed interactions between human and P. falciparum, with prominent co-regulation of translation genes in severe malaria between host and parasite. Multivariate analyses suggested that increased expression of granulopoiesis and interferon-γ-related genes, together with inadequate suppression of type 1 interferon signaling, best explained severity of infection. These findings provide a framework for understanding the contributions of host and parasite to the pathogenesis of severe malaria and identifying new treatments.

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential for parasite sexual development and transmission. We identified a major structural dimorphism with alternative 1.5 kb and 1.0 kb sequence deletions at different positions of the 3'-intergenic region, in tight linkage disequilibrium with the most highly differentiated single nucleotide polymorphism, one of the alleles being very frequent in Senegal and The Gambia but rare in the other locations. Long non-coding RNA transcripts were previously shown to include the entire antisense of the gdv1 coding sequence and the portion of the intergenic region with allelic deletions, suggesting adaptive regulation of parasite sexual development and transmission in response to local conditions.