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Author summary
Plasmodium knowlesi, a common malaria parasite of long-tailed and pig-tailed macaques, is now recognized as a significant cause of human malaria, accounting for up to 70% of malaria cases in certain areas in Southeast Asia including Malaysian Borneo. Rapid human population growth, deforestation and encroachment on wild macaque habita...
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During development within the host erythrocyte malaria parasites generate nascent membranous structures which serve as a pathway for parasite protein transport to modify the host cell. The molecular basis of such membranous structures is not well understood, particularly for malaria parasites other than Plasmodium falciparum. To characterize the st...
The five major Plasmodium spp. that cause human malaria appear similar under light microscopy, which raises the possibility that misdiagnosis could routinely occur in clinical settings. Assessing the extent of misdiagnosis is of particular importance for monitoring P. knowlesi , which cocirculates with the other Plasmodium spp. We performed a syste...
Background:
Plasmodium knowlesi is reported increasingly across Southeast Asia, and is the commonest cause of malaria in Malaysia. No randomized trials have assessed comparative efficacy of artemether-lumefantrine (AL) for knowlesi malaria.
Methods:
A randomized-controlled trial was conducted in 3 district hospitals in Sabah, Malaysia to compare...
Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which...
Citations
... fascicularis; Mn-Pk -M. nemestrina) 13,14 . Genomic dimorphism among the groups have been revealed 15 . ...
... The lowest nucleotide diversity occurred genome-wide in the Borneo clusters (Mf-Pk and Mn-Pk), with identical fragments found on chromosomes 5, 8, 10, 11 and 13 (Fig. 3). Chromosome 8 has been linked with chromosomal-segment exchange events between Borneo populations 14 . The joint nucleotide diversity for the Peninsular and Mn-associated clusters decreased slightly in regions on chromosomes 7, 12 and 13, potentially contributing to the mosaic pattern. ...
... Pen-Pk versus Mn-Pk 0734500 0734600, 0812000, 1402300 (GAP) 0714500 (UFD1), 0734500, 0734600, 1234000 0100500, 0215700, 0713100 (UBC9), 0713500, 0722800, 0727700, 0727800, 0728400, 0728700 (LRR8), 0731600, 0733600, 0733900 (VPS33), 0734200, 0811800, 1136300, 1234900, 1240100, 1249600, 1354000, 1434300 (14), SR140 (14), and SLARP (13). These loci are involved across different parasite life cycle stages. ...
The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax malaria has been the focus of control efforts. Understanding of the genetic diversity of P. knowlesi parasites can provide insights into its evolution, population structure, diagnostics, transmission dynamics, and the emergence of drug resistance. Previous work has revealed that P. knowlesi fall into three main sub-populations distinguished by a combination of geographical location and macaque host (Macaca fascicularis and M. nemestrina). It has been shown that Malaysian Borneo groups display profound heterogeneity with long regions of high or low divergence resulting in mosaic patterns between sub-populations, with some evidence of chromosomal-segment exchanges. However, the genetic structure of non-Borneo sub-populations is less clear. By gathering one of the largest collections of P. knowlesi whole-genome sequencing data, we studied structural genomic changes across sub-populations, with the analysis revealing differences in Borneo clusters linked to mosquito-related stages of the parasite cycle, in contrast to differences in host-related stages for the Peninsular group. Our work identifies new genetic exchange events, including introgressions between Malaysian Peninsular and M. nemestrina-associated clusters on various chromosomes, including in parasite invasion genes (DBPβ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}, NBPXα\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document} and NBPXβ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}), and important proteins expressed in the vertebrate parasite stages. Recombination events appear to have occurred between the Peninsular and M. fascicularis-associated groups, including in the DBPβ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document} and DBPγ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document} invasion associated genes. Overall, our work finds that genetic exchange events have occurred among the recognised contemporary groups of P. knowlesi parasites during their evolutionary history, leading to apparent mosaicism between these sub-populations. These findings generate new hypotheses relevant to parasite evolutionary biology and P. knowlesi epidemiology, which can inform malaria control approaches to containing the impact of zoonotic malaria on human communities.
... Control P. knowlesi DNA was extracted from the experimental line P. knowlesi A1-H.1 adapted to in vitro culture in human erythrocytes, the culture kindly donated by Robert Moon (Moon et al., 2013). In order to distinguish the genome data generated here for P. knowlesi A1-H.1 from those already existing, we use the unique abbreviation StAPkA1H1 (Diez Benavente et al., 2017;Benavente et al., 2018). ...
Plasmodium knowlesi, a malaria parasite of Old World macaque monkeys, is used extensively to model Plasmodium biology. Recently, P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes uncomplicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such, P. knowlesi presents a unique opportunity to develop experimental translational model systems for malaria pathophysiology informed by clinical data from same-species human infections. Experimental lines of P. knowlesi represent well-characterized genetically stable parasites, and to maximize their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence and de novo assemble P. knowlesi genomes from frozen clinical samples. The sequencing platform and assembly pipelines were designed to facilitate capturing data and describing, for the first time, P. knowlesi schizont-infected cell agglutination (SICA) var and Knowlesi-Interspersed Repeats (kir) multiple gene families in parasites acquired from nature. The SICAvar gene family members code for antigenically variant proteins analogous to the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) multiple var gene family. Evidence presented here suggests that the SICAvar family members have arisen through a process of gene duplication, selection pressure, and variation. Highly evolving genes including PfEMP1family members tend to be restricted to relatively unstable sub-telomeric regions that drive change with core genes protected in genetically stable intrachromosomal locations. The comparable SICAvar and kir gene family members are counter-intuitively located across chromosomes. Here, we demonstrate that, in contrast to conserved core genes, SICAvar and kir genes occupy otherwise gene-sparse chromosomal locations that accommodate rapid evolution and change. The novel methods presented here offer the malaria research community not only new tools to generate comprehensive genome sequence data from small clinical samples but also new insight into the complexity of clinically important real-world parasites.
... Microsatellite genotyping that targets multiple loci across the genome allows comprehensive screening of the whole genome at high resolution and identify loci that are under selection [161]. Studies based on whole-genome analysis revealed the existence of genomic mosaicism among the P. knowlesi subpopulations, indicative of chromosomalsegment exchanges events between two distinct Malaysian Borneo subpopulations associated with either M. fascicularis or M. nemestrina [160,164]. On the other hand, another study reported that several fragments of genotype in the peninsular Malaysia subpopulation were similar to the M. nemestrina-associated Malaysian Borneo subpopulation [162]. Genetic studies enable large volume of complex information to be generated rapidly. ...
Plasmodium knowlesi is a zoonotic malaria parasite that has gained increasing medical interest over the past two decades. This zoonotic parasitic infection is prevalent in Southeast Asia and causes many cases with fulminant pathology. Despite several biogeographical restrictions that limit its distribution, knowlesi malaria cases have been reported in different parts of the world due to travelling and tourism activities. Here, breakthroughs and key information generated from recent (over the past five years, but not limited to) studies conducted on P. knowlesi were reviewed, and the knowledge gap in various research aspects that need to be filled was discussed. Besides, challenges and strategies required to control and eradicate human malaria with this emerging and potentially fatal zoonosis were described.
... The first two clusters are thought to have separated due to sympatric divergence between the two species of macaques and the ecological areas they inhabit, with pigtailed macaques living mostly in remote forest areas and long-tailed macaques being found in both forested and urban areas, whilst the third is thought to have occurred due to allopatric separation as a result of the ocean barrier between Peninsular Malaysia and Malaysian Borneo. Although the separation of the clusters is clear, there has been some evidence of mixing between clusters 1 and 2, which is thought to have been caused by deforestation and the associated changes in vector populations [85]. Furthermore, parasites from cluster 1 have been more commonly found in human infections compared to those from cluster 2 [86]. ...
Background
The zoonotic malaria parasite Plasmodium knowlesi has emerged across Southeast Asia and is now the main cause of malaria in humans in Malaysia. A critical priority for P. knowlesi surveillance and control is understanding whether transmission is entirely zoonotic or is also occurring through human-mosquito-human transmission.
Methods
A systematic literature review was performed to evaluate existing evidence which refutes or supports the occurrence of sustained human-mosquito-human transmission of P. knowlesi. Possible evidence categories and study types which would support or refute non-zoonotic transmission were identified and ranked. A literature search was conducted on Medline, EMBASE and Web of Science using a broad search strategy to identify any possible published literature. Results were synthesized using the Synthesis Without Meta-analysis (SWiM) framework, using vote counting to combine the evidence within specific categories.
Results
Of an initial 7,299 studies screened, 131 studies were included within this review: 87 studies of P. knowlesi prevalence in humans, 14 studies in non-human primates, 13 studies in mosquitoes, and 29 studies with direct evidence refuting or supporting non-zoonotic transmission. Overall, the evidence showed that human-mosquito-human transmission is biologically possible, but there is limited evidence of widespread occurrence in endemic areas. Specific areas of research were identified that require further attention, notably quantitative analyses of potential transmission dynamics, epidemiological and entomological surveys, and ecological studies into the sylvatic cycle of the disease.
Conclusion
There are key questions about P. knowlesi that remain within the areas of research that require more attention. These questions have significant implications for malaria elimination and eradication programs. This paper considers limited but varied research and provides a methodological framework for assessing the likelihood of different transmission patterns for emerging zoonotic diseases.
... The parasite shows highly differentiated subgroups, broadly associated with their natural vertebrate host species: long tailed (Macaca fascicularis) and pig tailed (Macaca nemestrina) macaques [84]. Both are found in human infections in Bornean Malaysia [85][86][87][88]. A third divergent population is documented in both humans and macaques on Peninsular Malaysia [89][90][91]. ...
... Further substructure is seen within this Peninsular Malaysia population [90]. Moreover, whole genome sequencing has shown evidence of introgression and a mosaic structure of the P. knowlesi genome, indicating a breakdown of genetic isolation between divergent groups [85][86][87][88]. This complex population structure should be studied further as it may provide important insights into transmission and pathogenesis in this zoonotic parasite. ...
Genomics has revolutionised the study of the biology of parasitic diseases. The first Eukaryotic parasite to have its genome sequenced was the malaria parasite Plasmodium falciparum. Since then, Plasmodium genomics has continued to lead the way in the study of the genome biology of parasites, both in breadth—the number of Plasmodium species’ genomes sequenced—and in depth—massive-scale genome re-sequencing of several key species. Here, we review some of the insights into the biology, evolution and population genetics of Plasmodium gained from genome sequencing, and look at potential new avenues in the future genome-scale study of its biology.
... Particularly, the initial study of Cluster 1 in Malaysian Borneo indicated recent selection on several genomic loci, the strongest signature being identified on chromosome 8 . This signature of positive selection on chromosome 8 was confirmed by a secondary analysis of the sequence data performed after mapping to an alternatively-generated reference genome sequence (Diez Benavente et al., 2017). In contrast, analysis of the Cluster 3 subpopulation in Peninsular Malaysia did not indicate such a strong signature of selection on chromosome 8, but indicated that there are loci on four other chromosomes (numbers 1, 7, 9 and 12) that have been under recent positive directional selection (Hocking et al., 2020) (Fig. 10). ...
Plasmodium knowlesi, a simian malaria parasite of great public health concern has been reported from most countries in Southeast Asia and exported to various countries around the world. Currently P. knowlesi is the predominant species infecting humans in Malaysia. Besides this species, other simian malaria parasites such as P. cynomolgi and P. inui are also infecting humans in the region. The vectors of P. knowlesi and other Asian simian malarias belong to the Leucosphyrus Group of Anopheles mosquitoes which are generally forest dwelling species. Continual deforestation has resulted in these species moving into forest fringes, farms, plantations and human settlements along with their macaque hosts. Limited studies have shown that mosquito vectors are attracted to both humans and macaque hosts, preferring to bite outdoors and in the early part of the night. We here review the current status of simian malaria vectors and their parasites, knowledge of vector competence from experimental infections and discuss possible vector control measures. The challenges encountered in simian malaria elimination are also discussed. We highlight key knowledge gaps on vector distribution and ecology that may impede effective control strategies.
... knowlesi subgroups and Mn-P. knowlesi subpopulations [50]. These exchanges potentially relate to genes associated with P. knowlesi's interaction with mosquitoes during its lifecycle [50]. ...
... knowlesi subpopulations [50]. These exchanges potentially relate to genes associated with P. knowlesi's interaction with mosquitoes during its lifecycle [50]. ...
... Specifically, on chromosome 8, Benavente et al. [50], described that the Mf associated P. knowlesi genotype from Borneo Malaysia had two distinct geographical sub-groups that correspond with Kapit and Betong regions. Exemplifying significant chromosomal variation between geographical locations which has led to the further subdivision of cluster one [50]. ...
Zoonotic malaria, Plasmodium knowlesi, threatens the global progression of malaria elimination. Southeast Asian regions are fronting increased zoonotic malaria rates despite the control measures currently implemented—conventional measures to control human-malaria neglect P. knowlesi’s residual transmission between the natural macaque host and vector. Initiatives to control P. knowlesi should adopt themes of the One Health approach, which details that the management of an infectious disease agent should be scrutinized at the human-animal-ecosystem interface. This review describes factors that have conceivably permitted the emergence and increased transmission rates of P. knowlesi to humans, from the understanding of genetic exchange events between subpopulations of P. knowlesi to the downstream effects of environmental disruption and simian and vector behavioral adaptations. These factors are considered to advise an integrative control strategy that aligns with the One Health approach. It is proposed that surveillance systems address the geographical distribution and transmission clusters of P. knowlesi and enforce ecological regulations that limit forest conversion and promote ecosystem regeneration. Furthermore, combining individual protective measures, mosquito-based feeding trapping tools and biocontrol strategies in synergy with current control methods may reduce mosquito population density or transmission capacity.
... Compared to microsatellites, SNP markers are more suitable for comparisons of both strongly and weakly diverged populations, and in revealing ancestral patterns of genetic structuring [24]. By leveraging off whole genome sequencing for population characterisation [14,15,25,26], a number of SNP-based barcodes have been derived for P. falciparum. A barcode based on 24 SNPs in the nuclear genome ("24-SNP barcode") has been used to identify and track isolates from an endemic population in Senegal [27]. ...
Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.
... As a result of these filters, 103 isolates, including 20 of the 26 isolates undergoing SWGA, were carried forward for further analysis (see Supplementary Table 1). A neighbour-joining tree was constructed using the SNP data ( Fig. 2) and revealed 3 predominant clusters, consistent with recent findings 23,25 . In particular, these clusters relate to the specific geographic Peninsular-Malaysia subpopulation (purple, π = 3.4 × 10 −9 ), and Borneo macaque Mn-Pk (green, π = 2.23 × 10 −9 ) and Mf-Pk (blue, π = 3.29 × 10 −9 ) associated subpopulations. ...
... It has been shown that the subpopulations of P. knowlesi in Malaysian Borneo, although presenting a strong genetic differentiation, are not genetically isolated. In particular, we have identified genetic exchanges predominantly between the Mf-Pk and Mn-Pk clusters 23 . We sought to investigate whether these events are also found in the clinical isolates from Peninsular Malaysia, by estimating SNP nucleotide diversity (SNP π) across the genome in sliding 50 kbp windows. ...
... Previous population genetics studies on P. knowlesi subpopulations among human and macaque isolates from Malaysian Borneo provided evidence that chromosomal-segment exchanges between subpopulations have occurred recently 23 . This observation could be indicative of subpopulations that diverged in isolation and have www.nature.com/scientificreports ...
The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPβ and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.
... Given the geographical separation of the archipelagic part of Southeast Asia, it is hypothesized that different locations will host different genetic variants of the P. knowlesi parasite [58,59]. Most human infections of P. knowlesi are dominated by single genotypes from one of three main genomic clusters that show evidence of recent strong selection [60,61], two clusters from each of the macaque hosts in Borneo (Macaca fascicularis and Macaca nemestrina Borneo) and a third cluster from Peninsular Malaysia found in humans [60,[62][63][64][65]. Geographical separation of the island of Borneo from the peninsular by sea-level rise at the end of the last ice age would have prevented macaque movement and been responsible for any allopatric divergence [66]. ...
... However, recent genomic analysis has uncovered chromosomal-segment exchanges between these subpopulations [61]. Benavente and colleagues [61] investigated the P. knowlesi genotype from Cluster 1 (Mf-Pk Borneo) and found two geographic subgroups corresponding to different geographical regions: the Kapit and Betong regions in Sarawak. ...
... However, recent genomic analysis has uncovered chromosomal-segment exchanges between these subpopulations [61]. Benavente and colleagues [61] investigated the P. knowlesi genotype from Cluster 1 (Mf-Pk Borneo) and found two geographic subgroups corresponding to different geographical regions: the Kapit and Betong regions in Sarawak. Chromosome 8 of the genome presented an anomaly where distinct genetic differences were noted for this chromosome between the two geographical locations. ...
Abstract Plasmodium knowlesi is a zoonotic malaria parasite normally residing in long-tailed and pig-tailed macaques (Macaca fascicularis and Macaca nemestrina, respectively) found throughout Southeast Asia. Recently, knowlesi malaria has become the predominant malaria affecting humans in Malaysian Borneo, being responsible for approximately 70% of reported cases. Largely as a result of anthropogenic land use changes in Borneo, vectors which transmit the parasite, along with macaque hosts, are both now frequently found in disturbed forest habitats, or at the forest fringes, thus having more frequent contact with humans. Having access to human hosts provides the parasite with the opportunity to further its adaption to the human immune system. The ecological drivers of the transmission and spread of P. knowlesi are operating over many different spatial (and, therefore, temporal) scales, from the molecular to the continental. Strategies to prevent and manage zoonoses, such as P. knowlesi malaria require interdisciplinary research exploring the impact of land use change and biodiversity loss on the evolving relationship between parasite, reservoir hosts, vectors, and humans over multiple spatial scales.
