Patrick L. Sutton

The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

The complexities of malaria disease manifestations with a focus on asymptomatic malaria

Malaria is a serious parasitic disease in the developing world, causing high morbidity and mortality. The pathogenesis of malaria is complex, and the clinical presentation of disease ranges from severe and complicated, to mild and uncomplicated, to asymptomatic malaria. Despite a wealth of studies on the clinical severity of disease, asymptomatic malaria infections are still poorly understood. Asymptomatic malaria remains a challenge for malaria control programs as it significantly influences transmission dynamics. A thorough understanding of the interaction between hosts and parasites in the development of different clinical outcomes is required. In this review, the problems and obstacles to the study and control of asymptomatic malaria are discussed. The human and parasite factors associated with differential clinical outcomes are described and the management and treatment strategies for the control of the disease are outlined. Further, the crucial gaps in the knowledge of asymptomatic malaria that should be the focus of future research towards development of more effective malaria control strategies are highlighted.

Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Malaria in India: The Center for the Study of Complex Malaria in India

Malaria is a major public health problem in India and one which contributes significantly to the overall malaria burden in Southeast Asia. The National Vector Borne Disease Control Program of India reported ∼1.6 million cases and ∼1100 malaria deaths in 2009. Some experts argue that this is a serious underestimation and that the actual number of malaria cases per year is likely between 9 and 50 times greater, with an approximate 13-fold underestimation of malaria-related mortality. The difficulty in making these estimations is further exacerbated by (i) highly variable malaria eco-epidemiological profiles, (ii) the transmission and overlap of multiple Plasmodium species and Anopheles vectors, (iii) increasing antimalarial drug resistance and insecticide resistance, and (iv) the impact of climate change on each of these variables. Simply stated, the burden of malaria in India is complex. Here we describe plans for a Center for the Study of Complex Malaria in India (CSCMi), one of ten International Centers of Excellence in Malaria Research (ICEMRs) located in malarious regions of the world recently funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The CSCMi is a close partnership between Indian and United States scientists, and aims to address major gaps in our understanding of the complexity of malaria in India, including changing patterns of epidemiology, vector biology and control, drug resistance, and parasite genomics. We hope that such a multidisciplinary approach that integrates clinical and field studies with laboratory, molecular, and genomic methods will provide a powerful combination for malaria control and prevention in India.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Plasmodium falciparum and Plasmodium vivax infections in the Peruvian Amazon: propagation of complex, multiple allele-type infections without super-infection.

Outcrossing potential between Plasmodium parasites is defined by the population-level diversity (PLD) and complexity of infection (COI). There have been few studies of PLD and COI in low transmission regions. Since the 1995-1998 Peruvian Amazon epidemic, there has been sustained transmission with < 0.5 P. falciparum and < 1.6 P. vivax infections/person/year. Using weekly active case detection, we described PLD by heterozygosity (H(e)) and COI using P. falciparum Pfmsp1-B2 and P. vivax Pvmsp3alpha. Not being homologous genes, we limited comparisons to within species. P. falciparum (N = 293) had low (H(e) = 0.581) and P. vivax (N = 186) had high (H(e) = 0.845) PLD. A total of 9.5% P. falciparum infections and 26.3% P. vivax infections had COI > 1. Certain allele types were in more mixed infections than expected by chance. The few appearances of new alleles could be explained by stochastic polymerase chain reaction detection or synchronization/sequestration. The results suggest propagation of mixed infections by multiple inocula, not super-infection, implying decade-long opportunity for outcrossing in these mixed infections.

Plasmodium falciparum Genetic Diversity Maintained and Amplified Over 5 Years of a Low Transmission Endemic in the Peruvian Amazon

Plasmodium falciparum entered into the Peruvian Amazon in 1994, sparking an epidemic between 1995 and 1998. Since 2000, there has been sustained low P. falciparum transmission. The Malaria Immunology and Genetics in the Amazon project has longitudinally followed members of the community of Zungarococha (N = 1,945, 4 villages) with active household and health center-based visits each year since 2003. We examined parasite population structure and traced the parasite genetic diversity temporally and spatially. We genotyped infections over 5 years (2003–2007) using 14 microsatellite (MS) markers scattered across ten different chromosomes. Despite low transmission, there was considerable genetic diversity, which we compared with other geographic regions. We detected 182 different haplotypes from 302 parasites in 217 infections. Structure v2.2 identified five clusters (subpopulations) of phylogenetically related clones. To consider genetic diversity on a more detailed level, we defined haplotype families (hapfams) by grouping haplotypes with three or less loci differences. We identified 34 different hapfams identified. The Fst statistic and heterozygosity analysis showed the five clusters were maintained in each village throughout this time. A minimum spanning network (MSN), stratified by the year of detection, showed that haplotypes within hapfams had allele differences and haplotypes within a cluster definition were more separated in the later years (2006–2007). We modeled hapfam detection and loss, accounting for sample size and stochastic fluctuations in frequencies overtime. Principle component analysis of genetic variation revealed patterns of genetic structure with time rather than village. The population structure, genetic diversity, appearance/disappearance of the different haplotypes from 2003 to 2007 provides a genome-wide “real-time” perspective of P. falciparum parasites in a low transmission region.

A call to arms: on refining Plasmodium vivax microsatellite marker panels for comparing global diversity

BackgroundMicrosatellite (MS) markers have become an important tool for studying the population diversity, evolutionary history and multiplicity of infection (MOI) of malaria parasite infections. MS are typically selected on the basis of being highly polymorphic. However, it is known that the polymorphic potential (mutability) of each marker can vary as much as two orders of magnitude, which radically changes how diversity is represented in the genome from one marker to the next. Over the past decade, approximately 240 Plasmodium vivax MS have been published, comprising nine major panels of markers. Inconsistent usage of each panel has resulted in a surfeit of descriptive genetic diversity data that are largely incomparable between populations. The objective of this study was to statistically evaluate the quality of individual MS markers in order to validate a refined panel of markers that will provide a balanced picture of P. vivax population diversity.MethodsAll previously published data, including genetic diversity indices, MS parameters, and population parameters, were assembled from 18 different global studies into a flat file to facilitate statistical analysis and modelling using JMP® Genomics 6.0 (SAS Institute Inc, Cary, NC, USA). Statistical modeling was employed to down-select markers with extreme variation among the mean number of alleles, expected heterozygosity, maximum repeat length and/or chromosomal location of the repeat. Individual MS were analysed by step-down whole model linear regression and standard least squares fit models, both stratified by annual parasite incidence to identify MS markers with values significantly different from the mean.ResultsOf the 42 MS under evaluation in this study, 18 (nine high priority) were identified as ideal candidates for measuring population diversity between global regions, while five (two high priority) additional markers were identified as candidates for MOI studies.ConclusionsMS diversity was found to be a function of endemicity and motif structure. Evaluation of individual MS permitted the assembly of a refined panel of markers that can be reliably utilized in the field to compare population structures between global regions.

Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India.

BackgroundAssessing the Plasmodium vivax burden in India is complicated by the potential threat of an emerging chloroquine (CQ) resistant parasite population from neighbouring countries in Southeast Asia. Chennai, the capital of Tamil Nadu and an urban setting for P. vivax in southern India, was selected as a sentinel site for investigating CQ efficacy and sensitivity in vivax malaria.MethodsCQ efficacy was evaluated with a 28-day in vivo therapeutic study, while CQ sensitivity was measured with an in vitro drug susceptibility assay. In both studies, isolates also underwent molecular genotyping to investigate correlations between parasite diversity and drug susceptibility to CQ. Molecular genotyping included sequencing a 604 base pair (bp) fragment of the P. vivax multidrug resistant gene-1 (Pvmdr1) for single nucleotide polymorphisms (SNPs) and also the amplification of eight microsatellite (MS) loci located across the genome on eight different chromosomes.ResultsIn the 28-day in vivo study (N=125), all subjects were aparasitaemic by Day 14. Passive case surveillance continuing beyond Day 28 in 22 subjects exposed 17 recurrent infections, which ranged from 44 to 148 days post-enrollment. Pvmdr1 sequencing of these recurrent infections revealed that 93.3% had identical mutant haplotypes (958M/Y976/1076L) to their baseline Day 0 infection. MS genotyping further revealed that nine infection pairs were related with ≥75% haplotype similarity (same allele at six or more loci). To test the impact of this mutation on CQ efficacy, an in vitro drug assay (N=68) was performed. No correlation between IC50 values and the percentage of ring-stage parasites prior to culture was observed (rsadj: -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values.ConclusionsPlasmodium vivax was found to be susceptible to CQ drug treatment in both the in vivo therapeutic drug study and the in vitro drug assay. Though the mutant 1076L of Pvmdr1 was found in a majority of isolates tested, this single mutation did not associate with CQ resistance. MS haplotypes revealed strong heterogeneity in this population, indicating a low probability of reinfection with highly related haplotypes.

The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen.

BackgroundPlasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (< 0.5 infections/person/year). Purportedly non-immune individuals with discrete (non-overlapping) P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this populations antibody response to Pf MSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for Pf MSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of Pf MSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to Pf MSP1-19KD are determined.MethodsThe 42KD region in Pf MSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2) was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether Pf MSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of Pf MSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types.ResultsDespite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4%) were the Q-KSNG-F haplotype and 1 (0.6%) was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of Pf MSP1-19KD.ConclusionsA non-allele specific antibody response in Pf MSP1-19KD may explain why other allelic forms have not been maintained or evolved in this population. This has important implications for the use of Pf MSP1-19KD as a vaccine candidate. It is possible that Peruvians have increased antibody responses to the shared sites of Pf MSP1-19KD, either due to exposure/parasite characteristics or due to a human-genetic predisposition. Alternatively, these allelic polymorphisms are not immune-specific even in other geographic regions, implying these polymorphisms may be less important in immune evasion that previous studies suggest.