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  1. Molecular epidemiology and population genomics of Plasmodium knowlesi
    Divis PCS, Singh B, Conway DJ
    Adv Parasitol, 2021;113:191-223.
    PMID: 34620383 DOI: 10.1016/bs.apar.2021.08.003
    Molecular epidemiology has been central to uncovering P. knowlesi as an important cause of human malaria in Southeast Asia, and to understanding the complex nature of this zoonosis. Species-specific parasite detection and characterization of sequences were vital to show that P. knowlesi was distinct from the human parasite species that had been presumed to cause all malaria. With established sensitive and specific molecular detection tools, surveys subsequently indicated the distribution of P. knowlesi infections in humans, wild primate reservoir host species, and mosquito vector species. The importance of studying P. knowlesi genetic polymorphism was indicated initially by analysing a few nuclear gene loci as well as the mitochondrial genome, and subsequently by multi-locus microsatellite analyses and whole-genome sequencing. Different human infections generally have unrelated P. knowlesi genotypes, acquired from the diverse local parasite reservoirs in macaques. However, individual human infections are usually less genetically complex than those of wild macaques which experience more frequent superinfection with different P. knowlesi genotypes. Multi-locus analyses have revealed deep population subdivisions within P. knowlesi, which are structured both geographically and in relation to different macaque reservoir host species. Simplified genotypic discrimination assays now enable efficient large-scale surveillance of the sympatric P. knowlesi subpopulations within Malaysian Borneo. The whole-genome sequence analyses have also identified loci under recent positive natural selection in the P. knowlesi genome, with evidence that different loci are affected in different populations. These provide a foundation to understand recent adaptation of the zoonotic parasite populations, and to track and interpret future changes as they emerge.
  2. Fulltext Genome-wide mosaicism in divergence between zoonotic malaria parasite subpopulations with separate sympatric transmission cycles
    Divis PCS, Duffy CW, Kadir KA, Singh B, Conway DJ
    Mol Ecol, 2018 02;27(4):860-870.
    PMID: 29292549 DOI: 10.1111/mec.14477
    Plasmodium knowlesi is a significant cause of human malaria transmitted as a zoonosis from macaque reservoir hosts in South-East Asia. Microsatellite genotyping has indicated that human infections in Malaysian Borneo are an admixture of two highly divergent sympatric parasite subpopulations that are, respectively, associated with long-tailed macaques (Cluster 1) and pig-tailed macaques (Cluster 2). Whole-genome sequences of clinical isolates subsequently confirmed the separate clusters, although fewer of the less common Cluster 2 type were sequenced. Here, to analyse population structure and genomic divergence in subpopulation samples of comparable depth, genome sequences were generated from 21 new clinical infections identified as Cluster 2 by microsatellite analysis, yielding a cumulative sample size for this subpopulation similar to that for Cluster 1. Profound heterogeneity in the level of intercluster divergence was distributed across the genome, with long contiguous chromosomal blocks having high or low divergence. Different mitochondrial genome clades were associated with the two major subpopulations, but limited exchange of haplotypes from one to the other was evident, as was also the case for the maternally inherited apicoplast genome. These findings indicate deep divergence of the two sympatric P. knowlesi subpopulations, with introgression likely to have occurred recently. There is no evidence yet of specific adaptation at any introgressed locus, but the recombinant mosaic types offer enhanced diversity on which selection may operate in a currently changing landscape and human environment. Loci responsible for maintaining genetic isolation of the sympatric subpopulations need to be identified in the chromosomal regions showing fixed differences.
  3. Extreme geographical fixation of variation in the Plasmodium falciparum gamete surface protein gene Pfs48/45 compared with microsatellite loci
    Conway DJ, Machado RL, Singh B, Dessert P, Mikes ZS, Povoa MM, et al.
    Mol Biochem Parasitol, 2001 Jul;115(2):145-56.
    PMID: 11420101
    Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.
  4. Fragmented population structure of plasmodium falciparum in a region of declining endemicity
    Anthony TG, Conway DJ, Cox-Singh J, Matusop A, Ratnam S, Shamsul S, et al.
    J Infect Dis, 2005 May 1;191(9):1558-64.
    PMID: 15809916
    The population genetic structure of Plasmodium falciparum differs between endemic regions, but the characteristics of a population recently fragmented by effective malaria control have been unknown.
  5. Fulltext Population Genomic Structure and Recent Evolution of Plasmodium knowlesi, Peninsular Malaysia
    Hocking SE, Divis PCS, Kadir KA, Singh B, Conway DJ
    Emerg Infect Dis, 2020 08;26(8):1749-1758.
    PMID: 32687018 DOI: 10.3201/eid2608.190864
    Most malaria in Malaysia is caused by Plasmodium knowlesi parasites through zoonotic infection from macaque reservoir hosts. We obtained genome sequences from 28 clinical infections in Peninsular Malaysia to clarify the emerging parasite population structure and test for evidence of recent adaptation. The parasites all belonged to a major genetic population of P. knowlesi (cluster 3) with high genomewide divergence from populations occurring in Borneo (clusters 1 and 2). We also observed unexpected local genetic subdivision; most parasites belonged to 2 subpopulations sharing a high level of diversity except at particular genomic regions, the largest being a region of chromosome 12, which showed evidence of recent directional selection. Surprisingly, we observed a third subpopulation comprising P. knowlesi infections that were almost identical to each other throughout much of the genome, indicating separately maintained transmission and recent genetic isolation. Each subpopulation could evolve and present a broader health challenge in Asia.
  6. Fulltext Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques
    Lee KS, Divis PC, Zakaria SK, Matusop A, Julin RA, Conway DJ, et al.
    PLoS Pathog, 2011 Apr;7(4):e1002015.
    PMID: 21490952 DOI: 10.1371/journal.ppat.1002015
    Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000-40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.
  7. Fulltext Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening
    Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, et al.
    Clin Infect Dis, 2008 Jan 15;46(2):165-71.
    PMID: 18171245 DOI: 10.1086/524888
    BACKGROUND: Until recently, Plasmodium knowlesi malaria in humans was misdiagnosed as Plasmodium malariae malaria. The objectives of the present study were to determine the geographic distribution of P. knowlesi malaria in the human population in Malaysia and to investigate 4 suspected fatal cases.

    METHODS: Sensitive and specific nested polymerase chain reaction was used to identify all Plasmodium species present in (1) blood samples obtained from 960 patients with malaria who were hospitalized in Sarawak, Malaysian Borneo, during 2001-2006; (2) 54 P. malariae archival blood films from 15 districts in Sabah, Malaysian Borneo (during 2003-2005), and 4 districts in Pahang, Peninsular Malaysia (during 2004-2005); and (3) 4 patients whose suspected cause of death was P. knowlesi malaria. For the 4 latter cases, available clinical and laboratory data were reviewed.

    RESULTS: P. knowlesi DNA was detected in 266 (27.7%) of 960 of the samples from Sarawak hospitals, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. Only P. knowlesi DNA was detected in archival blood films from the 4 patients who died. All were hyperparasitemic and developed marked hepatorenal dysfunction.

    CONCLUSIONS: Human infection with P. knowlesi, commonly misidentified as the more benign P. malariae, are widely distributed across Malaysian Borneo and extend to Peninsular Malaysia. Because P. knowlesi replicates every 24 h, rapid diagnosis and prompt effective treatment are essential. In the absence of a specific routine diagnostic test for P. knowlesi malaria, we recommend that patients who reside in or have traveled to Southeast Asia and who have received a "P. malariae" hyperparasitemia diagnosis by microscopy receive intensive management as appropriate for severe falciparum malaria.

  8. Fulltext Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi
    Sutton PL, Luo Z, Divis PCS, Friedrich VK, Conway DJ, Singh B, et al.
    Infect Genet Evol, 2016 06;40:243-252.
    PMID: 26980604 DOI: 10.1016/j.meegid.2016.03.009
    Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes.
  9. Fulltext Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species
    Divis PC, Singh B, Anderios F, Hisam S, Matusop A, Kocken CH, et al.
    PLoS Pathog, 2015 May;11(5):e1004888.
    PMID: 26020959 DOI: 10.1371/journal.ppat.1004888
    Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.
  10. A large focus of naturally acquired Plasmodium knowlesi infections in human beings
    Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, et al.
    Lancet, 2004 Mar 27;363(9414):1017-24.
    PMID: 15051281
    About a fifth of malaria cases in 1999 for the Kapit division of Malaysian Borneo had routinely been identified by microscopy as Plasmodium malariae, although these infections appeared atypical and a nested PCR assay failed to identify P malariae DNA. We aimed to investigate whether such infections could be attributable to a variant form of P malariae or a newly emergent Plasmodium species.
  11. Fulltext Three Divergent Subpopulations of the Malaria Parasite Plasmodium knowlesi
    Divis PC, Lin LC, Rovie-Ryan JJ, Kadir KA, Anderios F, Hisam S, et al.
    Emerg Infect Dis, 2017 04;23(4):616-624.
    PMID: 28322705 DOI: 10.3201/eid2304.161738
    Multilocus microsatellite genotyping of Plasmodium knowlesi isolates previously indicated 2 divergent parasite subpopulations in humans on the island of Borneo, each associated with a different macaque reservoir host species. Geographic divergence was also apparent, and independent sequence data have indicated particularly deep divergence between parasites from mainland Southeast Asia and Borneo. To resolve the overall population structure, multilocus microsatellite genotyping was conducted on a new sample of 182 P. knowlesi infections (obtained from 134 humans and 48 wild macaques) from diverse areas of Malaysia, first analyzed separately and then in combination with previous data. All analyses confirmed 2 divergent clusters of human cases in Malaysian Borneo, associated with long-tailed macaques and pig-tailed macaques, and a third cluster in humans and most macaques in peninsular Malaysia. High levels of pairwise divergence between each of these sympatric and allopatric subpopulations have implications for the epidemiology and control of this zoonotic species.
  12. Fulltext Efficient Surveillance of Plasmodium knowlesi Genetic Subpopulations, Malaysian Borneo, 2000-2018
    Divis PCS, Hu TH, Kadir KA, Mohammad DSA, Hii KC, Daneshvar C, et al.
    Emerg Infect Dis, 2020 07;26(7):1392-1398.
    PMID: 32568035 DOI: 10.3201/eid2607.190924
    Population genetic analysis revealed that Plasmodium knowlesi infections in Malaysian Borneo are caused by 2 divergent parasites associated with long-tailed (cluster 1) and pig-tailed (cluster 2) macaques. Because the transmission ecology is likely to differ for each macaque species, we developed a simple genotyping PCR to efficiently distinguish between and survey the 2 parasite subpopulations. This assay confirmed differences in the relative proportions in areas of Kapit division of Sarawak state, consistent with multilocus microsatellite analyses. Analyses of 1,204 human infections at Kapit Hospital showed that cluster 1 caused approximately two thirds of cases with no significant temporal changes from 2000 to 2018. We observed an apparent increase in overall numbers in the most recent 2 years studied, driven mainly by increased cluster 1 parasite infections. Continued monitoring of the frequency of different parasite subpopulations and correlation with environmental alterations are necessary to determine whether the epidemiology will change substantially.
  13. Fulltext Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi
    Assefa S, Lim C, Preston MD, Duffy CW, Nair MB, Adroub SA, et al.
    Proc Natl Acad Sci U S A, 2015 Oct 20;112(42):13027-32.
    PMID: 26438871 DOI: 10.1073/pnas.1509534112
    Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.
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