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 (F ST ) = 0.21, with 9,293 SNPs having fixed differences of F ST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean F ST 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.population genomics | Plasmodium diversity | reproductive isolation | zoonosis | adaptation

Background: Plasmodium knowlesi and Plasmodium cynomolgi are two malaria parasites naturally transmissible between humans and wild macaque through mosquito vectors, while Plasmodium inui can be experimentally transmitted from macaques to humans. One of their major natural hosts, the long-tailed macaque (Macaca fascicularis), is host to two other species of Plasmodium (Plasmodium fieldi and Plasmodium coatneyi) and is widely distributed in Southeast Asia. This study aims to determine the distribution of wild macaques infected with malarial parasites by examining samples derived from seven populations in five countries across Southeast Asia.Methods: Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in longtailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations. Results: Out of 276 samples tested, 177 were Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites (P. knowlesi, P. coatneyi, P. cynomolgi) with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively. Conclusions: The prevalence of malaria parasites, including those that are transmissible to humans, varied among all sampled regional populations of long-tailed macaques in Southeast Asia. The new discovery of P. knowlesi infection in Laos, and the high prevalence of P. cynomolgi infections in wild macaques in general, indicate the strong need of public advocacy in related countries.

P lasmodium spp. were identified in the late 1800s, and >30 species have been described in primates, including humans, apes, and monkeys (1,2). Of these, humans are natural hosts to 4 species: P. falciparum, P. malariae, P. vivax, and P. ovale. Human infections with simian malaria parasites were thought to be extremely rare until P. knowlesi was identified as a major cause of malaria in humans in Kapit, Malaysian Borneo (3). Subsequent human cases of infection with P. knowlesi have been reported across Southeast Asia (4-9). Most cases are reported in the Malaysian Borneo states of Sarawak and Sabah (4,10,11). The zoonotic capability of this parasite was confirmed with the aid of molecular techniques because P. knowlesi is morphologically

Z oonotic malaria caused by Plasmodium knowlesi, commonly found in long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina), is now a major emerging disease, particularly in Malaysia (1,2). Two other simian malaria parasites, P. cynomolgi (2-4) and P. inui (2), have also been shown to have the potential of zoonotic transmission to humans through the bites of infected mosquitoes under natural and experimental conditions. The risk of acquiring zoonotic malaria is highest for persons living at the forest fringe and working or venturing into the forest because of their proximity with the monkey reservoir hosts and the mosquito vectors (5,6). With the aid of molecular methods, we aimed to investigate whether human infections with simian malaria parasites were present among indigenous communities in Malaysia whose villages are situated in the forest or at the forest fringe. The StudyWe examined 645 archived blood samples that we had collected during 2011-2014 among indigenous populations of various subtribes from 14 villages in 7 states in Malaysia (Appendix Table 1, https://wwwnc. cdc.gov/EID/article/27/8/20-4502-App1.pdf). We fi rst screened the extracted DNA samples at Universiti Malaya (UM) for the presence of Plasmodium with the aid of genus-specifi c primers (rPLU1 and rPLU5; rPLU3 and rPLU4) (Appendix). Of the 645 indigenous community samples, 102 (15.8%) were positive for Plasmodium. Using species-specifi c nested PCR assays (Appendix), we identifi ed these infections as monoinfections with P. knowlesi (n = 40), P. vivax (n = 21), P. cynomolgi (n = 9), P. falciparum (n = 6), P. coatneyi (n = 3), P. inui (n = 3), P. malariae (n = 2), and P. ovale curtisi (n = 1) (Table 1). In 17 samples, the species could not be identifi ed despite repeated attempts. Our species-specifi c primer pairs were designed on the basis of either the asexually (A) or sexually (S) transcribed forms of Plasmodium small subunit (SSU) rRNA genes (7); the genus-specifi c primer pairs anneal to both asexual and sexual forms of the SSU rRNA genes, and therefore the genus-specifi c assay is more sensitive.We further characterized the 55 samples that tested positive for simian malaria parasites by amplifying a longer fragment of the SSU rRNA gene (914 bp-950 bp) for direct sequencing. Phylogenetic analysis using the neighbor-joining method (Figure 1) revealed the presence of P. 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.