Population Genomic Structure and Recent Evolution of Plasmodium knowlesi, Peninsular Malaysia.

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

Efficient Surveillance of Plasmodium knowlesi Genetic Subpopulations, Malaysian Borneo, 2000-2018.

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

Macaca fascicularis and Macaca nemestrina infected with zoonotic malaria parasites are widely distributed in Sarawak, Malaysian Borneo

Human infections with Plasmodium knowlesi, a malaria parasite of Macaca fascicularis and Macaca nemestrina (long-tailed and pig-tailed macaques respectively), occur throughout Southeast Asia, especially Malaysian Borneo. Other naturally-acquired human infections with malaria parasites from macaques in Southeast Asia are P. cynomolgi, P. inui-like, P. coatneyi and P. simiovale. In Sarawak, Malaysian Borneo, M. fascicularis and M. nemestrina from only the Kapit Division have been examined previously for malaria parasites. In order to determine the distribution of P. knowlesi and other zoonotic malaria parasites, 73 macaque blood samples derived from 7 other administrative divisions in Sarawak were studied. Of 45 blood samples from M. fascicularis and 28 from M. nemestrina tested by nested PCR assays, 23 (51.1%) M. fascicularis and 15 (53.6%) M. nemestrina samples were positive for Plasmodium DNA. Thirty-two of these macaques from 7 divisions sampled, harboured either single (n = 12), double (n = 9), triple (n = 7) or quadruple (n = 4) infections of P. knowlesi, P. inui, P. cynomolgi and P. coatneyi, while the infecting species of Plasmodium could not be identified for 6 samples. P. knowlesi was detected in 15.5% (7/45) M. fascicularis and in 7.1% (2/28) M. nemestrina sampled. Despite the small number of samples analysed from each administrative division, the current study indicates that macaques infected with the zoonotic malaria parasites P. knowlesi, P. cynomolgi, P. inui and P. coatneyi are widely distributed throughout Sarawak, Malaysian Borneo. Travelers to forested areas in Sarawak should be made aware of the potential risk of acquiring zoonotic malaria

A comparison of the clinical, laboratory and epidemiological features of two divergent subpopulations of Plasmodium knowlesi

Plasmodium knowlesi, a simian malaria parasite responsible for all recent indigenous cases of malaria in Malaysia, infects humans throughout Southeast Asia. There are two genetically distinct subpopulations of Plasmodium knowlesi in Malaysian Borneo, one associated with long-tailed macaques (termed cluster 1) and the other with pig-tailed macaques (cluster 2). A prospective study was conducted to determine whether there were any between-subpopulation differences in clinical and laboratory features, as well as in epidemiological characteristics. Over 2 years, 420 adults admitted to Kapit Hospital, Malaysian Borneo with knowlesi malaria were studied. Infections with each subpopulation resulted in mostly uncomplicated malaria. Severe disease was observed in 35/298 (11.7%) of single cluster 1 and 8/115 (7.0%) of single cluster 2 infections (p = 0.208). There was no clinically significant difference in outcome between the two subpopulations. Cluster 1 infections were more likely to be associated with peri-domestic activities while cluster 2 were associated with interior forest activities consistent with the preferred habitats of the respective macaque hosts. Infections with both P. knowlesi subpopulations cause a wide spectrum of disease including potentially life-threatening complications, with no implications for differential patient management

New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Background Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak. Methods Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis. Results Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo. Conclusions Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak

Three Divergent Subpopulations of the Malaria Parasite Plasmodium knowlesi

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

Low levels of polymorphisms and no evidence for diversifying selection on the Plasmodium knowlesi Apical Membrane Antigen 1 gene.

International audienceInfection with Plasmodium knowlesi, a zoonotic primate malaria, is a growing human health problem in Southeast Asia. P. knowlesi is being used in malaria vaccine studies, and a number of proteins are being considered as candidate malaria vaccine antigens, including the Apical Membrane Antigen 1 (AMA1). In order to determine genetic diversity of the ama1 gene and to identify epitopes of AMA1 under strongest immune selection, the ama1 gene of 52 P. knowlesi isolates derived from human infections was sequenced. Sequence analysis of isolates from two geographically isolated regions in Sarawak showed that polymorphism in the protein is low compared to that of AMA1 of the major human malaria parasites, P. falciparum and P. vivax. Although the number of haplotypes was 27, the frequency of mutations at the majority of the polymorphic positions was low, and only six positions had a variance frequency higher than 10%. Only two positions had more than one alternative amino acid. Interestingly, three of the high-frequency polymorphic sites correspond to invariant sites in PfAMA1 or PvAMA1. Statistically significant differences in the quantity of three of the six high frequency mutations were observed between the two regions. These analyses suggest that the pkama1 gene is not under balancing selection, as observed for pfama1 and pvama1, and that the PkAMA1 protein is not a primary target for protective humoral immune responses in their reservoir macaque hosts, unlike PfAMA1 and PvAMA1 in humans. The low level of polymorphism justifies the development of a single allele PkAMA1-based vaccine

Three-dimensional distribution of polymorphic amino acid residues of PkAMA1, PfAMA1 and PvAMA1.

<p>Two views of a surface representation, rotated by 180° with respect to each other, are shown for the crystal structures of (A) PkAMA1 (PDB entry 4UV6), (B) PfAMA1 (PDB entry 2Z8V) and (C) PvAMA1 (PDB entry 1W8L), with each orthologue oriented at equivalent angles. PkAMA1 polymorphisms are from this study, PfAMA1 polymorphisms are from refs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124400#pone.0124400.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124400#pone.0124400.ref024" target="_blank">24</a>] and PvAMA1 polymorphisms are from ref [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124400#pone.0124400.ref025" target="_blank">25</a>]. Polymorphic residues are labeled and colored in red for high frequency polymorphisms (> 10%) and in blue for low frequency polymorphisms (< 10%). Domains 1 and 2 from crystal structures of all three orthologues are shown in white. Domain 3, shown in grey, was modeled for PkAMA1 and PfAMA1 from the crystal structure of PvAMA1 since the crystal structure of this domain has not been determined for these two orthologues. (PkAMA1 Domain 3 polymorphic residues 411 and 481 are not shown as these are disordered and thus not visible in the PvAMA1 structure).</p

Polymorphic residues in the PkAMA1 protein.

<p>High frequency polymorphic sites in red, bold and highlighted in white; low frequency polymorphic sites in black.</p