Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species.

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

Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species

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 longtailed 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

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

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

Changing epidemiology of malaria in Sabah, Malaysia : increasing incidence of Plasmodium knowlesi

Background: While Malaysia has had great success in controlling Plasmodium falciparum and Plasmodium vivax, notifications of Plasmodium malariae and the microscopically near-identical Plasmodium knowlesi increased substantially over the past decade. However, whether this represents microscopic misdiagnosis or increased recognition of P. knowlesi has remained uncertain. Methods: To describe the changing epidemiology of malaria in Sabah, in particular the increasing incidence of P. knowlesi, a retrospective descriptive study was undertaken involving a review of Department of Health malaria notification data from 2012–2013, extending a previous review of these data from 1992–2011. In addition, malaria PCR and microscopy data from the State Public Health Laboratory were reviewed to estimate the accuracy of the microscopy-based notification data. Results: Notifications of P. malariae/P. knowlesi increased from 703 in 2011 to 815 in 2012 and 996 in 2013. Notifications of P. vivax and P. falciparum decreased from 605 and 628, respectively, in 2011, to 297 and 263 in 2013. In 2013, P. malariae/P. knowlesi accounted for 62% of all malaria notifications compared to 35% in 2011. Among 1,082 P. malariae/P. knowlesi blood slides referred for PCR testing during 2011–2013, there were 924 (85%) P. knowlesi mono-infections, 30 (2.8%) P. falciparum, 43 (4.0%) P. vivax, seven (0.6%) P. malariae, six (0.6%) mixed infections, 31 (2.9%) positive only for Plasmodium genus, and 41 (3.8%) Plasmodium-negative. Plasmodium knowlesi mono-infection accounted for 32/156 (21%) and 33/87 (38%) blood slides diagnosed by microscopy as P. falciparum and P. vivax, respectively. Twenty-six malaria deaths were reported during 2010–2013, including 12 with ‘P. malariae/P. knowlesi’ (all adults), 12 with P. falciparum (seven adults), and two adults with P. vivax. Conclusions: Notifications of P. malariae/P. knowlesi in Sabah are increasing, with this trend likely reflecting a true increase in incidence of P. knowlesi and presenting a major threat to malaria control and elimination in Malaysia. With the decline of P. falciparum and P. vivax, control programmes need to incorporate measures to protect against P. knowlesi, with further research required to determine effective interventions.Published versio

Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries - Fig 3

<p><b>Sub-region neighbour joining tree analyses of the 1.6kb partial <i>gag-pol</i> genes sequenced in two clusters of (A) subtype B'/C and (B) B'/G recombinants characterised in the population.</b> Based on the informative sites analyses, recombination breakpoints were estimated for each strain and the partial <i>gag-pol</i> sequences (HXB2:1753–3440) were then sub-divided into different regions for phylogenetic reconstruction. Putative HIV-1 parental reference genotypes used in bootscan were 90THCM235 (CRF01_AE), CNRL42 (subtype B' of Thai origin), 95IN21068 (subtype C) and 01NGPL0674 (subtype G). Incident or prevalent HIV-1 infections for each strain, as determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) were identified by orange triangles or red circles, respectively. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p

Correlation between degree of cluster admixture and multi-locus linkage disequilibrium (standardised index of association).

<p>The degree of <i>P</i>. <i>knowlesi</i> cluster admixture was estimated as the local cluster mixedness (<i>p1</i>*<i>p2</i>) based on the proportions of infections designated as Cluster 1 (<i>p1</i>) and Cluster 2 (<i>p2</i>) at each of 10 sampling sites for human infections across Malaysia (Spearman’s Rho = 0.678, P = 0.015). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.s012" target="_blank">S5 Table</a> gives details of each of these values for each population.</p

Phylogenetic reconstruction of 136 partial <i>gag-pol</i> gene sequences of 1.6kb amplified among the blood donors in Kuala Lumpur, Malaysia between 2013 and 2014.

<p>HIV-1 incidence was estimated using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) to identify recent (incident) and long-standing (prevalent) infections as indicated where available. Neighbour-joining tree was constructed in MEGA 5.05 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161853#pone.0161853.ref022" target="_blank">22</a>] using Kimura 2-parameter method of nucleotide substitutions and the reliability of the branching nodes were assessed by bootstrap analysis of 1000 replicates. Eleven partial <i>gag</i>-PR (834bp) and two RT gene sequences (966bp) were genotyped separately using similar methods and their prevalence was reported in this study (figures not shown for clarity). Relevant HIV-1 reference genotypes in Southeast Asia include subtype B, CRF01_AE, CRF33_01B, CRF34_01B, CRF48_01B, CRF52_01B, CRF53_01B, CRF54_01B, CRF58_01B and CRF74_01B. Reference sequences of other genotypes prevalent in China (CRF07_BC, CRF08_BC and other recently-described B'/C CRFs) and Africa (subtype G, CRF02_AG and CRF45_cpx) were also included in the analysis. The reference sequences were labelled in the following order: genotype, country of origin, isolate name and GenBank accession number. A well-supported cluster of Malaysian subtype G strains was also highlighted as G_MY within the subtype G clade of African reference strains. All 12 unique recombinant forms were denoted by closed diamonds and labelled according to incident or prevalent infection status. Clusters of novel B'/C recombinants (strains 13MYNBB108, 14MYNBB084, 14MYNBB090 and 14MYNBB164) and B'/G recombinants (13MYNBB064 and 13MYNBB065) were highlighted in the tree. Simian immunodeficiency virus (SIVcpz) reference strains were included as outgroup. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p

HIV-1 genotype distribution of incident and prevalent infections among 127 blood donors in Kuala Lumpur.

<p>A limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) was used to distinguish incident from prevalent HIV-1 infections. Out of 179 samples available for incidence assay testing, 70.9% (n = 127) were successfully genotyped and comprised of 29 (22.8%) incident HIV-1 infections.</p