Evidence of Submicroscopic Plasmodium knowlesi Mono-Infection in Remote Indigenous Communities in Kelantan, Peninsular Malaysia.

Malaysia has maintained zero cases of indigenous human malaria since 2018. However, zoonotic malaria is still prevalent in underdeveloped areas and hard-to-reach populations. This study aimed to determine the prevalence of malaria among remote indigenous communities in Peninsular Malaysia. A cross-sectional survey was conducted in six settlements in Kelantan state, from June to October 2019. Blood samples were tested for malaria using microscopy and nested polymerase chain reaction (nPCR) targeting the Plasmodium cytochrome c oxidase subunit III (cox3) gene. Of the 1,954 individuals who appeared healthy, no malaria parasites were found using microscopy. However, nPCR revealed seven cases of Plasmodium knowlesi mono-infection (0.4%), and six out of seven infections were in the group of 19 to 40 years old (P = 0.026). No human malaria species were detected by nPCR. Analysis of the DNA sequences also showed high similarity that reflects common ancestry to other P. knowlesi isolates. These findings indicate low submicroscopic P. knowlesi infections among indigenous communities in Malaysia, requiring PCR-based surveillance to support malaria control activities in the country.

Naturally acquired antibody response to Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Peninsular Malaysia.

Malaria remains a public health problem in many parts of the world. In Malaysia, the significant progress towards the national elimination programme and effective disease notification on malaria has resulted in zero indigenous human malaria cases since 2018. However, the country still needs to determine the extent of malaria exposure and transmission patterns, particularly in high-risk populations. In this study, a serological method was used to measure transmission levels of Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Kelantan, Peninsular Malaysia. A community-based cross-sectional survey was conducted in three Orang Asli communities (i.e., Pos Bihai, Pos Gob, and Pos Kuala Betis) in Kelantan from June to July 2019. Antibody responses to malaria were assessed by enzyme-linked immunosorbent assay (ELISA) using two P. falciparum (PfAMA-1 and PfMSP-119) and two P. vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCRs). Multiple logistic regression was used to investigate factors associated with malaria exposure. The overall malaria seroprevalence was 38.8% for PfAMA-1, 36.4% for PfMSP-119, 2.2% for PvAMA-1, and 9.3% for PvMSP-119. Between study areas, the proportion of seropositivity for any P. falciparum and P. vivax antigens was significantly highest in Pos Kuala Betis with 34.7% (p < 0.001) and 13.6% (p < 0.001), respectively. For all parasite antigens except for PvAMA-1, the proportion of seropositive individuals significantly increased with age (all p < 0.001). Based on the SCR, there was a higher level of P. falciparum transmission than P. vivax in the study area. Multivariate regression analyses showed that living in Pos Kuala Betis was associated with both P. falciparum (adjusted odds ratio [aOR] 5.6, p < 0.001) and P. vivax (aOR 2.1, p < 0.001) seropositivities. Significant associations were also found between age and seropositivity to P. falciparum and P. vivax antigens. Analysis of community-based serological data helps describe the level of transmission, heterogeneity, and factors associated with malaria exposure among indigenous communities in Peninsular Malaysia. This approach could be an important adjunct tool for malaria monitoring and surveillance in low malaria transmission settings in the country.

A survey of simian Plasmodium infections in humans in West Kalimantan, Indonesia.

The simian parasite Plasmodium knowlesi is the predominant species causing human malaria infection, including hospitalisations for severe disease and death, in Malaysian Borneo. By contrast, there have been only a few case reports of knowlesi malaria from Indonesian Borneo. This situation seems paradoxical since both regions share the same natural macaque hosts and Anopheles mosquito vectors, and therefore have a similar epidemiologically estimated risk of infection. To determine whether there is a true cross-border disparity in P. knowlesi prevalence, we conducted a community-based malaria screening study using PCR in Kapuas Hulu District, West Kalimantan. Blood samples were taken between April and September 2019 from 1000 people aged 6 months to 85 years attending health care facilities at 27 study sites within or close to jungle areas. There were 16 Plasmodium positive samples by PCR, five human malarias (two Plasmodium vivax, two Plasmodium ovale and one Plasmodium malariae) and 11 in which no species could be definitively identified. These data suggest that, if present, simian malarias including P. knowlesi are rare in the Kapuas Hulu District of West Kalimantan, Indonesian Borneo compared to geographically adjacent areas of Malaysian Borneo. The reason for this discrepancy, if confirmed in other epidemiologically similar regions of Indonesian Borneo, warrants further studies targeting possible cross-border differences in human activities in forested areas, together with more detailed surveys to complement the limited data relating to monkey hosts and Anopheles mosquito vectors in Indonesian Borneo.

The presence of Plasmodium malariae and Plasmodium knowlesi in near malaria elimination setting in western Indonesia.

BACKGROUND: Indonesia is progressing towards malaria elimination. To achieve this goal, intervention measures must be addressed to cover all Plasmodium species. Comprehensive control measures and surveillance programmes must be intensified. This study aims to determine the prevalence of microscopic and submicroscopic malaria in Langkat district, North Sumatera Province, Indonesia. METHODS: A cross-sectional survey was conducted in six villages in Langkat district, North Sumatera Province in June 2019. Data were recorded using a standardized questionnaire. Finger pricked blood samples were obtained for malaria examination using rapid diagnostic test, thick and thin blood smears, and polymerase chain reaction. RESULTS: A total of 342 individuals were included in the study. Of them, one (0.3%) had a microscopic Plasmodium malariae infection, no positive RDT examination, and three (0.9%) were positive for P. malariae (n = 1) and Plasmodium knowlesi (n = 2). The distribution of bed net ownership was owned by 40% of the study participants. The participants had a house within a radius of 100-500 m from the forest (86.3%) and had the housing material of cement floor (56.1%), a tin roof (82.2%), wooden wall (35.7%), bamboo wall (28.1%), and brick wall (21.6%). CONCLUSION: Malaria incidence has substantially decreased in Langkat, North Sumatera, Indonesia. However, submicroscopic infection remains in the population and may contribute to further transmission. Surveillance should include the detection of microscopic undetected parasites, to enable the achievement of malaria elimination.

Seroepidemiological surveillance, community perceptions and associated risk factors of malaria exposure among forest-goers in Northeastern Thailand.

Malaria remains a major public health challenge in Thailand. Continuous assessment and understanding of the behavior and perceptions related to malaria exposure in the high-risk group are necessary to achieve the elimination goal. This study aimed to investigate the parasite prevalence, seroprevalence rate, knowledge, attitudes, and practices (KAP), and malaria risk factors in rural communities living close to a forested area in the northeastern part of Thailand. A community-based cross-sectional survey was conducted in three forest-goer communities (i.e., Ban Khok, Ban Koh, and Dong Yang) located in Khamcha-i district, Mukdahan Province, Thailand, from July to August 2019. Demographic, socioeconomic information and KAP data were collected using a structured questionnaire. Parasite prevalence was determined by microscopy. Seroprevalence was determined via ELISA using two Plasmodium falciparum (PfAMA-1 and PfMSP-119) and two Plasmodium vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analyzed using a reversible catalytic model to calculate seroconversion rate (SCR). Malaria parasite was not detected in any of the 345 participants. The overall malaria seroprevalence was 72.2% for PfAMA-1, 18.8% for PfMSP-119, 32.5% for PvAMA-1, and 4.4% for PvMSP-119. The proportion of seroprevalence for P. falciparum and P. vivax antigens was significantly highest in Ban Koh (35.1%, P < 0.001) and Don Yang (18.8%, P < 0.001), respectively. For all parasite antigens except PvMSP-119, the proportion of seropositive individuals significantly increased with age (P < 0.001). Based on the SCRs, there was a higher level of P. falciparum transmission than P. vivax. Regarding KAP, almost all respondents showed adequate knowledge and awareness about malaria. Nevertheless, significant effort is needed to improve positive attitudes and practices concerning malaria prevention measures. Multivariate regression analyses showed that living in Ban Koh was associated with both P. falciparum (adjusted odds ratio [aOR] 12.87, P < 0.001) and P. vivax (aOR 9.78, P < 0.001) seropositivities. We also found significant associations between age and seropositivity against P. falciparum and P. vivax antigens. The data suggest that seroepidemiological surveillance using AMA-1 and MSP-119 antigens may provide further evidence to reconstruct malaria exposure history. The absence of weak evidence of recent malaria transmission in Mukdahan Province is promising in the context of the disease elimination program.

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.

Molecular epidemiology and population genomics of Plasmodium knowlesi.

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.

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.

Perceptions and prevention practices on malaria among the indigenous Orang Asli community in Kelantan, Peninsular Malaysia.

BACKGROUND: Malaysia is on track towards malaria elimination. However, several cases of malaria still occur in the country. Contributing factors and communal aspects have noteworthy effects on any malaria elimination activities. Thus, assessing the community's knowledge, attitudes and practices (KAP) towards malaria is essential. This study was performed to evaluate KAP regarding malaria among the indigenous people (i.e. Orang Asli) in Peninsular Malaysia. METHODS: A household-based cross-sectional study was conducted in five remote villages (clusters) of Orang Asli located in the State of Kelantan, a central region of the country. Community members aged six years and above were interviewed. Demographic, socio-economic and KAP data on malaria were collected using a structured questionnaire and analysed using descriptive statistics. RESULTS: Overall, 536 individuals from 208 households were interviewed. Household indoor residual spraying (IRS) coverage and bed net ownership were 100% and 89.2%, respectively. A majority of respondents used mosquito bed nets every night (95.1%), but only 50.2% were aware that bed nets were used to prevent malaria. Nevertheless, almost all of the respondents (97.9%) were aware that malaria is transmitted by mosquitoes. Regarding practice for managing malaria, the most common practice adopted by the respondents was seeking treatment at the health facilities (70.9%), followed by self-purchase of medication from a local shop (12.7%), seeking treatment from a traditional healer (10.5%) and self-healing (5.9%). Concerning potential zoonotic malaria, about half of the respondents (47.2%) reported seeing monkeys from their houses and 20.1% reported entering nearby forests within the last 6 months. CONCLUSION: This study found that most populations living in the villages have an acceptable level of knowledge and awareness about malaria. However, positive attitudes and practices concerning managing malaria require marked improvement.

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.

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.

Genome-wide mosaicism in divergence between zoonotic malaria parasite subpopulations with separate sympatric transmission cycles.

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.

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.

Contribution of Plasmodium knowlesi to Multispecies Human Malaria Infections in North Sumatera, Indonesia.

Background: As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province. Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi. Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%). Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi.

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.

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.

Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques.

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.

A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi.

BACKGROUND: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples. METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples. RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/µL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/µL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA. CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

Clinical and parasitological response to oral chloroquine and primaquine in uncomplicated human Plasmodium knowlesi infections.

BACKGROUND: Plasmodium knowlesi is a cause of symptomatic and potentially fatal infections in humans. There are no studies assessing the detailed parasitological response to treatment of knowlesi malaria infections in man and whether antimalarial resistance occurs. METHODS: A prospective observational study of oral chloroquine and primaquine therapy was conducted in consecutive patients admitted to Kapit Hospital, Sarawak, Malaysian Borneo with PCR-confirmed single P. knowlesi infections. These patients were given oral chloroquine for three days, and at 24 hours oral primaquine was administered for two consecutive days, primarily as a gametocidal agent. Clinical and parasitological responses were recorded at 6-hourly intervals during the first 24 hours, daily until discharge and then weekly to day 28. Vivax malaria patients were studied as a comparator group. RESULTS: Of 96 knowlesi malaria patients who met the study criteria, 73 were recruited to an assessment of the acute response to treatment and 60 completed follow-up over 28 days. On admission, the mean parasite stage distributions were 49.5%, 41.5%, 4.0% and 5.6% for early trophozoites, late trophozoites, schizonts and gametocytes respectively. The median fever clearance time was 26.5 [inter-quartile range 16-34] hours. The mean times to 50% (PCT50) and 90% (PCT90) parasite clearance were 3.1 (95% confidence intervals [CI] 2.8-3.4) hours and 10.3 (9.4-11.4) hours. These were more rapid than in a group of 23 patients with vivax malaria 6.3 (5.3-7.8) hours and 20.9 (17.6-25.9) hours; P = 0.02). It was difficult to assess the effect of primaquine on P. knowlesi parasites, due to the rapid anti-malarial properties of chloroquine and since primaquine was administered 24 hours after chloroquine. No P. knowlesi recrudescences or re-infections were detected by PCR. CONCLUSIONS: Chloroquine plus primaqine is an inexpensive and highly effective treatment for uncomplicated knowlesi malaria infections in humans and there is no evidence of drug resistance. Further studies using alternative anti-malarial drugs, including artemisinin derivatives, would be desirable to define optimal management strategies for P. knowlesi.