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1.  Plasmodium knowlesi: Reservoir Hosts and Tracking the Emergence in Humans and Macaques 
PLoS Pathogens  2011;7(4):e1002015.
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.
Author Summary
We recently described the first focus of human infections with P. knowlesi, a malaria parasite of monkeys, and subsequently reported that these infections can be fatal. Whether mosquito transmission of infection depended on the monkey reservoir or was maintained by the human population was unknown. In the area of highest human infection incidence (within the Kapit Division of Sarawak, Malaysian Borneo), we surveyed 108 wild monkeys and found most were infected with malaria parasites, including P. knowlesi. We observed that the number of P. knowlesi genotypes per infection was much higher in monkeys than humans, some genotypes were shared between the two hosts and no major types were associated exclusively with either host. Evolutionary analyses of sequence data indicate that P. knowlesi existed in monkeys prior to human settlement in Southeast Asia and underwent a recent population expansion. Thus, P. knowlesi is essentially zoonotic; humans being infected with these parasites from the original and reservoir monkey hosts probably since they first entered the forests of Southeast Asia. We consider that the current increase in the human population, coupled with ecological changes due to deforestation, could result in a switch to humans as the preferred host for this pathogenic Plasmodium species.
PMCID: PMC3072369  PMID: 21490952
2.  Genetic diversity, haplotypes and allele groups of Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia 
Parasites & Vectors  2014;7:161.
The monkey malaria parasite Plasmodium knowlesi is now recognized as the fifth species of Plasmodium that can cause human malaria. Like the region II of the Duffy binding protein of P. vivax (PvDBPII), the region II of the P. knowlesi Duffy binding protein (PkDBPαII) plays an essential role in the parasite’s invasion into the host’s erythrocyte. Numerous polymorphism studies have been carried out on PvDBPII, but none has been reported on PkDBPαII. In this study, the genetic diversity, haplotyes and allele groups of PkDBPαII of P. knowlesi clinical isolates from Peninsular Malaysia were investigated.
Blood samples from 20 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. These samples were collected between 2010 and 2012. The PkDBPαII region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and haplotypes of PkDBPαII were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes.
Fifty-three PkDBPαII sequences from human infections and 6 from monkeys were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence showed 52 synonymous and 76 nonsynonymous mutations. Analysis on the rate of these mutations indicated that PkDBPαII was under purifying (negative) selection. At the amino acid level, 36 different PkDBPαII haplotypes were identified. Twelve of the 20 human and 1 monkey blood samples had mixed haplotype infections. These haplotypes were clustered into 2 distinct allele groups. The majority of the haplotypes clustered into the large dominant group.
Our present study is the first to report the genetic diversity and natural selection of PkDBPαII. Hence, the haplotypes described in this report can be considered as novel. Although a high level of genetic diversity was observed, the PkDBPαII appeared to be under purifying selection. The distribution of the haplotypes was skewed, with one dominant major and one minor group. Future study should investigate PkDBPαII of P. knowlesi from Borneo, which hitherto has recorded the highest number of human knowlesi malaria.
PMCID: PMC4022242  PMID: 24693997
Plasmodium knowlesi; Duffy binding protein; Diversity; Selection; Haplotypes; Allele groups
3.  Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members 
Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥0.34, p = <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region.
Author Summary
Plasmodium knowlesi, a parasite of Southeast Asian macaques, has entered the human population. Approximately 10% of P. knowlesi infections are severe, 1–2% are fatal, in Sarawak, Malaysian Borneo. Increase in parasitaemia is associated with disease severity, but little is known about parasite virulence in this newly described human pathogen. Here we present the results of a study on P. knowlesi parasites collected from 147 patients. We use the isolates to produce DNA sequences from a polymorphic (genetically variable) region of two P. knowlesi genes, Pknbpxa and Pknbpxb, that are involved in parasite entry into host red blood cells. We addressed the question that some parasite genotypes may have an invasion advantage leading to severe disease in human infections. We analysed the DNA sequences with matched clinical and laboratory data from the patient cohort (n = 147). We found that specific DNA sequences (Pknbpxa and Pknbpxb alleles) clustered with high parasitaemia and markers of disease severity. Here, for the first time, we provide evidence that variant alleles of the Plasmodium Reticulocyte Binding-Like Protein invasion gene family can influence disease progression in patients with malaria. The biological characteristics of the variants will be studied to aid our understanding of malaria pathophysiology and to inform intervention strategies.
PMCID: PMC4133233  PMID: 25121807
4.  First case of detection of Plasmodium knowlesi in Spain by Real Time PCR in a traveller from Southeast Asia 
Malaria Journal  2010;9:219.
Previously, Plasmodium knowlesi was not considered as a species of Plasmodium that could cause malaria in human beings, as it is parasite of long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques found in Southeast Asia. A case of infection by P. knowlesi is described in a Spanish traveller, who came back to Spain with daily fever after his last overseas travel, which was a six-month holiday in forested areas of Southeast Asia between 2008 and 2009. His P. knowlesi infection was detected by multiplex Real time quantitative PCR and confirmed by sequencing the amplified fragment. Using nested multiplex malaria PCR (reference method in Spain) and a rapid diagnostic test, the P. knowlesi infection was negative. This patient was discharged and asymptomatic when the positive result to P. knowlesi was reported. Prior to this case, there have been two more reports of European travellers with malaria caused by P. knowlesi, a Finnish man who travelled to Peninsular Malaysia during four weeks in March 2007, and a Swedish man who did a short visit to Malaysian Borneo in October 2006. Taken together with this report of P. knowlesi infection in a Spanish traveller returning from Southeast Asia, this is the third case of P. knowlesi infection in Europe, indicating that this simian parasite can infect visitors to endemic areas in Southeast Asia. This last European case is quite surprising, given that it is an untreated-symptomatic P. knowlesi in human, in contrast to what is currently known about P. knowlesi infection. Most previous reports of human P. knowlesi malaria infections were in adults, often with symptoms and relatively high parasite densities, up to the recent report in Ninh Thuan province, located in the southern part of central Vietnam, inhabited mainly by the Ra-glai ethnic minority, in which all P. knowlesi infections were asymptomatic, co-infected with P. malariae, with low parasite densities and two of the three identified cases were very young children under five years old.
PMCID: PMC2921078  PMID: 20663184
5.  A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi 
PLoS ONE  2012;7(2):e31848.
Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.
Methodology and Significant Findings
We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.
The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.
PMCID: PMC3282782  PMID: 22363751
6.  Transfection of the Primate Malaria Parasite Plasmodium knowlesi Using Entirely Heterologous Constructs 
The Journal of Experimental Medicine  1997;185(8):1499-1504.
The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite–host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.
PMCID: PMC2196274  PMID: 9126931
7.  A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi 
Malaria Journal  2010;9:344.
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.
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.
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.
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.
PMCID: PMC3009662  PMID: 21114872
8.  Genome Comparison of Human and Non-Human Malaria Parasites Reveals Species Subset-Specific Genes Potentially Linked to Human Disease 
PLoS Computational Biology  2011;7(12):e1002320.
Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.
Author Summary
With more than 250 million infections and over a million deaths each year, malaria remains one of the most devastating infectious diseases worldwide. With the availability of complete genome sequences of both human and non-human Plasmodium parasites, the causative agents of malaria, it is now possible to use comparative genomics as a tool to look for genes that are present in some but not all Plasmodium species. Such species subset-specific genes possibly underlie important phenotypic differences between malaria parasites and could provide important clues for the development of new strategies to prevent and treat malaria in humans. In this study, we performed a comprehensive computational comparison of the published genomes of six Plasmodium species, including two human (P. falciparum and P. vivax), one monkey (P. knowlesi), and three rodent malaria parasites (P. berghei, P. yoelii, and P. chabaudi). This comparison revealed many species subset-specific genes that are potentially linked to human pathogenicity, human-to-human transmissibility, and human virulence. These genes can now be examined further by targeted experimental analyses to test predicted phenotypic associations and to elucidate gene function.
PMCID: PMC3245289  PMID: 22215999
9.  High proportion of knowlesi malaria in recent malaria cases in Malaysia 
Malaria Journal  2014;13:168.
Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.
A total of 457 microscopically confirmed, malaria-positive blood samples were collected from 22 state and main district hospitals in Malaysia between September 2012 and December 2013. Nested PCR assay targeting the 18S rRNA gene was used to determine the infecting Plasmodium species.
A total of 453 samples were positive for Plasmodium species by using nested PCR assay. Plasmodium knowlesi was identified in 256 (56.5%) samples, followed by 133 (29.4%) cases of Plasmodium vivax, 49 (10.8%) cases of Plasmodium falciparum, two (0.4%) cases of Plasmodium ovale and one (0.2%) case of Plasmodium malariae. Twelve mixed infections were detected, including P. knowlesi/P. vivax (n = 10), P. knowlesi/P. falciparum (n = 1), and P. falciparum/P. vivax (n = 1). Notably, P. knowlesi (Included mixed infections involving P. knowlesi (P. knowlesi/P. vivax and P. knowlesi /P. falciparum)) showed the highest proportion in Sabah (84/115 cases, prevalence of 73.0%), Sarawak (83/120, 69.2%), Kelantan (42/56, 75.0%), Pahang (24/25, 96.0%), Johor (7/9, 77.8%), and Terengganu (4/5, 80.0%,). In contrast, the rates of P. knowlesi infection in Selangor and Negeri Sembilan were found to be 16.2% (18/111 cases) and 50.0% (5/10 cases), respectively. Sample of P. knowlesi was not obtained from Kuala Lumpur, Melaka, Perak, Pulau Pinang, and Perlis during the study period, while a microscopically-positive sample from Kedah was negative by PCR.
In addition to Sabah and Sarawak, which have been known for high prevalence of P. knowlesi infection, the findings from this study highlight the widespread distribution of P. knowlesi in many Peninsular Malaysia states.
PMCID: PMC4016780  PMID: 24886266
Malaria distribution; Plasmodium knowlesi; Malaysia Borneo; Peninsular Malaysia; Nested PCR; Microscopy; SSU rRNA gene
10.  Evaluation of three rapid diagnostic tests for the detection of human infections with Plasmodium knowlesi 
Malaria Journal  2014;13:60.
Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, infects humans and can cause fatal malaria. It is difficult to diagnose by microscopy because of morphological similarity to Plasmodium malariae. Nested PCR assay is the most accurate method to distinguish P. knowlesi from other Plasmodium species but is not cost effective in resource-poor settings. Rapid diagnostic tests (RDTs) are recommended for settings where malaria is prevalent. In this study, the effectiveness of three RDTs in detecting P. knowlesi from fresh and frozen patient blood samples was evaluated.
Forty malaria patients (28 P. knowlesi, ten P. vivax and two P. falciparum) diagnosed by microscopy were recruited in Sarawak, Malaysian Borneo during a 16-month period. Patient blood samples were used to determine parasitaemia by microscopy, confirm the Plasmodium species present by PCR and evaluate three RDTs: OptiMAL-IT, BinaxNOW® Malaria and Paramax-3. The RDTs were also evaluated using frozen blood samples from 41 knowlesi malaria patients.
OptiMAL-IT was the most sensitive RDT, with a sensitivity of 71% (20/28; 95% CI = 54-88%) for fresh and 73% (30/41; 95% CI = 59-87%) for frozen knowlesi samples. However, it yielded predominantly falciparum-positive results due to cross-reactivity of the P. falciparum test reagent with P. knowlesi. BinaxNOW® Malaria correctly detected non-P. falciparum malaria in P. knowlesi samples but was the least sensitive, detecting only 29% (8/28; 95% CI = 12-46%) of fresh and 24% (10/41; 95% CI = 11-37%) of frozen samples. The Paramax-3 RDT tested positive for P. vivax with PCR-confirmed P. knowlesi samples with sensitivities of 40% (10/25; 95% CI = 21-59%) with fresh and 32% (13/41; 95% CI = 17-46%) with frozen samples. All RDTs correctly identified P. falciparum- and P. vivax-positive controls with parasitaemias above 2,000 parasites/μl blood.
The RDTs detected Plasmodium in P. knowlesi-infected blood samples with poor sensitivity and specificity. Patients with P. knowlesi could be misdiagnosed as P. falciparum with OptiMAL-IT, P. vivax with Paramax-3 and more correctly as non-P. vivax/non-P. falciparum with BinaxNOW® Malaria. There is a need for a sensitive and specific RDT for malaria diagnosis in settings where P. knowlesi infections predominate.
PMCID: PMC3931291  PMID: 24548805
Plasmodium knowlesi; Malaria diagnostics; Rapid diagnostic tests
11.  Zoonotic Malaria – Global Overview and Research and Policy Needs 
The four main Plasmodium species that cause human malaria, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, are transmitted between humans by mosquito vectors belonging to the genus Anopheles. It has recently become evident that Plasmodium knowlesi, a parasite that typically infects forest macaque monkeys, can be transmitted by anophelines to cause malaria in humans in Southeast Asia. Plasmodium knowlesi infections are frequently misdiagnosed microscopically as P. malariae. Direct human to human transmission of P. knowlesi by anophelines has not yet been established to occur in nature. Knowlesi malaria must therefore be presently considered a zoonotic disease. Polymerase chain reaction is now the definitive method for differentiating P. knowlesi from P. malariae and other human malaria parasites. The origin of P. falciparum and P. vivax in African apes are examples of ancient zoonoses that may be continuing at the present time with at least P. vivax, and possibly P. malariae and P. ovale. Other non-human primate malaria species, e.g., Plasmodium cynomolgi in Southeast Asia and Plasmodium brasilianum and Plasmodium simium in South America, can be transmitted to humans by mosquito vectors further emphasizing the potential for continuing zoonoses. The potential for zoonosis is influenced by human habitation and behavior as well as the adaptive capabilities of parasites and vectors. There is insufficient knowledge of the bionomics of Anopheles vector populations relevant to the cross-species transfer of malaria parasites and the real extent of malaria zoonoses. Appropriate strategies, based on more research, need to be developed for the prevention, diagnosis, and treatment of zoonotic malaria.
PMCID: PMC4135302  PMID: 25184118
African apes; Anopheles vectors; human malaria; malaria control; malaria transmission; non-human primate malaria; Plasmodium; zoonosis
12.  Increasing Incidence of Plasmodium knowlesi Malaria following Control of P. falciparum and P. vivax Malaria in Sabah, Malaysia 
The simian parasite Plasmodium knowlesi is a common cause of human malaria in Malaysian Borneo and threatens the prospect of malaria elimination. However, little is known about the emergence of P. knowlesi, particularly in Sabah. We reviewed Sabah Department of Health records to investigate the trend of each malaria species over time.
Reporting of microscopy-diagnosed malaria cases in Sabah is mandatory. We reviewed all available Department of Health malaria notification records from 1992–2011. Notifications of P. malariae and P. knowlesi were considered as a single group due to microscopic near-identity.
From 1992–2011 total malaria notifications decreased dramatically, with P. falciparum peaking at 33,153 in 1994 and decreasing 55-fold to 605 in 2011, and P. vivax peaking at 15,857 in 1995 and decreasing 25-fold to 628 in 2011. Notifications of P. malariae/P. knowlesi also demonstrated a peak in the mid-1990s (614 in 1994) before decreasing to ≈100/year in the late 1990s/early 2000s. However, P. malariae/P. knowlesi notifications increased >10-fold between 2004 (n = 59) and 2011 (n = 703). In 1992 P. falciparum, P. vivax and P. malariae/P. knowlesi monoinfections accounted for 70%, 24% and 1% respectively of malaria notifications, compared to 30%, 31% and 35% in 2011. The increase in P. malariae/P. knowlesi notifications occurred state-wide, appearing to have begun in the southwest and progressed north-easterly.
A significant recent increase has occurred in P. knowlesi notifications following reduced transmission of the human Plasmodium species, and this trend threatens malaria elimination. Determination of transmission dynamics and risk factors for knowlesi malaria is required to guide measures to control this rising incidence.
Author Summary
The simian parasite Plasmodium knowlesi is a common cause of malaria in Malaysian Borneo; however, little is known about its emergence over time, particularly in Sabah. We reviewed all available Sabah Department of health malaria notification records from 1992–2011, and considered notifications of P. malariae and P. knowlesi as a single group due to their microscopic similarity. We found that malaria notifications in Sabah have decreased dramatically, with P. falciparum and P. vivax notifications peaking at 33,153 and 15,877 respectively during 1994–1995, and falling to 605 and 628 respectively in 2011. Notifications of P. malariae/P. knowlesi fell from a peak of 614 in 1994 to ≈100/year in the late 1990s/early 2000s, however increased >10-fold between 2004 (n = 59) and 2011 (n = 703). In 1992 P. falciparum, P. vivax and P. malariae/P. knowlesi monoinfections accounted for 70%, 24% and 1% respectively of malaria notifications, compared to 30%, 31% and 35% in 2011. The increase in P. malariae/P. knowlesi notifications occurred state-wide, appearing to have begun in the southwest and progressed north-easterly. This significant recent increase in P. knowlesi notifications following reduced transmission of the human Plasmodium species threatens malaria elimination; further research is required to determine transmission dynamics and risk factors for knowlesi malaria.
PMCID: PMC3554533  PMID: 23359830
13.  First case of a naturally acquired human infection with Plasmodium cynomolgi 
Malaria Journal  2014;13:68.
Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.
The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.
Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.
This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.
Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.
The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.
PMCID: PMC3937822  PMID: 24564912
Plasmodium vivax; Plasmodium cynomolgi; Molecular methods; Malaysia; Simian malaria
In the studies of complement fixation described in this paper, the antigens were prepared from (a) normal monkey red cells, (b) parasitized red cells of monkeys dying with Plasmodium knowlesi infection, (c) the spleens of monkeys dying with Plasmodium knowlesi infection; the sera came from (a) normal human beings, (b) patients with syphilis, (c) patients with paresis who were receiving malaria therapy with Plasmodium knowlesi, Plasmodium vivax, or Plasmodium falciparum, and (d) patients with malaria alone. The malarial antigens gave negative complement fixation reactions with 70 to 80 per cent of the luetic and normal sera and weak or doubtful reactions with the remaining 20 to 30 per cent. With the exception of one antigen prepared from spleen, there was no evidence that the malarial antigens were more reactive with Wassermann-positive than with Wassermann-negative sera. Some human sera give weak complement fixation with antigens prepared from normal monkey erythrocytes, and the percentage of these positive reactions is slightly higher with malarial sera than with normal or luetic sera. The most sensitive and specific malarial antigen was prepared from dried parasitized red cells by extraction with saline, freezing, and thawing. This P. knowlesi antigen gives strong complement fixation with malarial sera from human beings infected with P. knowlesi, P. vivax, or P. falciparum. The titer of complement-fixing antibodies reaches a maximum about 1 month after the beginning of the acute infection. At this time all of the P. knowlesi sera tested were positive. After 4 months the reaction diminishes rapidly in titer but may remain positive for 12 months or longer. With P. knowlesi infections in man, the complement fixation reaction remains positive for some time after the infection has apparently disappeared as judged by daily smears and inoculation of monkeys with the blood. The complement fixation reaction in malaria is group-specific rather than species-specific. Sera from patients infected with P. vivax or P. falciparum react in the same way with the P. knowlesi antigen as the homologous sera. Absorption of malarial human sera with normal monkey erythrocytes does not remove the immune bodies which fix complement with malarial antigens.
PMCID: PMC2133745  PMID: 19870853
15.  Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species 
PLoS Pathogens  2015;11(5):e1004888.
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.
Author Summary
Extraordinary phases of pathogen evolution may occur during an emerging zoonosis, potentially involving adaptation to human hosts, with changes in patterns of virulence and transmission. In a large population genetic survey, we show that the malaria parasite Plasmodium knowlesi in humans is an admixture of two highly divergent parasite populations, each associated with different forest-dwelling macaque reservoir host species. Most of the transmission and sexual reproduction occurs separately in each of the two parasite populations. In addition to the reservoir host-associated parasite population structure, there was also significant genetic differentiation that correlated with geographical distance. Although both P. knowlesi types co-exist in the same areas, the divergence between them is similar to or greater than that seen between sub-species in other sexually reproducing eukaryotes. This may offer particular opportunities for evolution of virulence and host-specificity, not seen with other malaria parasites, so studies of ongoing adaptation and interventions to reduce transmission are urgent priorities.
PMCID: PMC4447398  PMID: 26020959
16.  Characterization and tissue-specific expression patterns of the Plasmodium chabaudi cir multigene family 
Malaria Journal  2011;10:272.
Variant antigens expressed on the surface of parasitized red blood cells (pRBCs) are important virulence factors of malaria parasites. Whereas Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) are responsible for sequestration of mature parasites, little is known about putative ligands mediating cytoadherence to host receptors in other Plasmodium species. Candidates include members of the pir superfamily found in the human parasite Plasmodium vivax (vir), in the simian pathogen Plasmodium knowlesi (kir) and in the rodent malarias Plasmodium yoelii (yir), Plasmodium berghei (bir) and Plasmodium chabaudi (cir). The aim of this study was to reveal a potential involvement of cir genes in P. chabaudi sequestration.
Subfamilies of cir genes were identified by bioinformatic analyses of annotated sequence data in the Plasmodium Genome Database. In order to examine tissue-specific differences in the expression of cir mRNAs, RT-PCR with subfamily-specific primers was used. In total, 432 cDNA clones derived from six different tissues were sequenced to characterize the transcribed cir gene repertoire. To confirm differences in transcription profiles of cir genes, restriction fragment length polymorphism (RFLP) analyses were performed to compare different host tissues and to identify changes during the course of P. chabaudi infections in immunocompetent mice.
The phylogenetic analysis of annotated P. chabaudi putative CIR proteins identified two major subfamilies. Comparison of transcribed cir genes from six different tissues revealed significant differences in the frequency clones belonging to individual cir gene subgroups were obtained from different tissues. Further hints of difference in the transcription of cir genes in individual tissues were obtained by RFLP. Whereas only minimal changes in the transcription pattern of cir genes could be detected during the developmental cycle of the parasites, switching to expression of other cir genes during the course of an infection was observed around or after peak parasitemia.
The tissue-specific expression of cir mRNAs found in this study indicates correlation between expression of CIR antigens and distribution of parasites in inner organs. Together with comparable results for other members of the pir superfamily this suggests a role of cir and other pir genes in antigenic variation and sequestration of malaria parasites.
PMCID: PMC3189184  PMID: 21929749
17.  Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade 
Nature genetics  2012;44(9):1051-1055.
Plasmodium cynomolgi, a malaria parasite of Asian Old World monkeys, is the sister taxon of Plasmodium vivax, the most prevalent human malaria species outside Africa. Since P. cynomolgi shares many phenotypic, biologic and genetic characteristics of P. vivax, we generated draft genome sequences of three P. cynomolgi strains and performed comparative genomic analysis between them and P. vivax, as well as a third previously sequenced simian parasite, Plasmodium knowlesi. Here we show that genomes of the monkey malaria clade can be characterized by CNVs in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites, and CNVs in the P. cynomolgi genome, providing a map of genetic variation for mapping parasite traits and studying parasite populations. The P. cynomolgi genome is a critical step in developing a model system for P. vivax research, and to counteract the neglect of P. vivax.
PMCID: PMC3759362  PMID: 22863735
18.  Major Burden of Severe Anemia from Non-Falciparum Malaria Species in Southern Papua: A Hospital-Based Surveillance Study 
PLoS Medicine  2013;10(12):e1001575.
Ric Price and colleagues use hospital-based surveillance data to estimate the risk of severe anemia and mortality associated with endemic Plasmodium species in southern Papua, Indonesia.
Please see later in the article for the Editors' Summary
The burden of anemia attributable to non-falciparum malarias in regions with Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia.
Methods and Findings
Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes (837,989 as outpatients and 84,131 as inpatients), a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. malariae infection had the lowest hemoglobin concentration (n = 1,608, mean = 8.93 [95% CI 8.81–9.06]), followed by those with mixed species infections (n = 8,645, mean = 9.22 [95% CI 9.16–9.28]), P. falciparum (n = 37,554, mean = 9.47 [95% CI 9.44–9.50]), and P. vivax (n = 19,858, mean = 9.53 [95% CI 9.49–9.57]); p-value for all comparisons <0.001. Severe anemia (hemoglobin <5 g/dl) was present in 8,151 (3.7%) patients. Compared to patients without malaria, those with mixed Plasmodium infection were at greatest risk of severe anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99–3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00–2.23), 1.87 (95% CI 1.74–2.01), and 2.18 (95% CI 1.76–2.67), respectively, p<0.001. Overall, 12.2% (95% CI 11.2%–13.3%) of severe anemia was attributable to non-falciparum infections compared with 15.1% (95% CI 13.9%–16.3%) for P. falciparum monoinfections. Patients with severe anemia had an increased risk of death (AOR = 5.80 [95% CI 5.17–6.50]; p<0.001). Not all patients had a hemoglobin measurement, thus limitations of the study include the potential for selection bias, and possible residual confounding in multivariable analyses.
In Papua P. vivax is the dominant cause of severe anemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria control strategies in areas of Plasmodium co-endemicity.
Please see later in the article for the Editors' Summary
Editors' Summary
Malaria—a mosquito-borne parasitic disease—is a global public health problem. Five parasites cause malaria—Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Of these, P. vivax is the commonest and most widely distributed, whereas P. falciparum causes the most deaths—about a million every year. All these parasites enter their human host when an infected mosquito takes a blood meal. The parasites migrate to the liver where they replicate and mature into a parasitic form known as merozoites. After 8–9 days, the merozoites are released from the liver cells and invade red blood cells where they replicate rapidly before bursting out and infecting more red blood cells. Malaria's recurring flu-like symptoms are caused by this cyclical increase in parasites in the blood. Malaria needs to be treated promptly with antimalarial drugs to prevent the development of potentially fatal complications. Infections with P. falciparum in particular can cause anemia (a reduction in red blood cell numbers) and can damage the brain and other vital organs by blocking the capillaries that supply these organs with blood.
Why Was This Study Done?
It is unclear what proportion of anemia is attributable to non-falciparum malarias in regions of the world where several species of malaria parasite are always present (Plasmodium co-endemicity). Public health officials in such regions need to know whether non-falciparum malarias are a major cause of anemia when designing malaria control strategies. If P. vivax, for example, is a major cause of anemia in an area where P. vivax and P. falciparum co-exist, then any malaria control strategies that are implemented need to take into account the biological differences between the parasites. In this hospital-based cohort study, the researchers investigate the burden of severe anemia from the endemic Plasmodium species in southern Papua, Indonesia.
What Did the Researchers Do and Find?
The researchers used hospital record numbers to link clinical and laboratory data for patients presenting to a referral hospital in southern Papua over an 8-year period. The hemoglobin level (an indicator of anemia) was measured in about a quarter of hospital presentations (some patients attended the hospital several times). A third of the presentations who had their hemoglobin level determined (67,696 presentations) had clinical malaria. Patients with P. malariae infection had the lowest average hemoglobin concentration. Patients with mixed species, P. falciparum, and P. vivax infections had slightly higher average hemoglobin levels but all these levels were below the normal range for people living in Papua. Among the patients who had their hemoglobin status assessed, 3.7% had severe anemia. After allowing for other factors that alter the risk of anemia (“confounding” factors such as age), patients with mixed Plasmodium infection were more than three times as likely to have severe anemia as patients without malaria. Patients with P. falciparum, P. vivax, or P. malariae infections were about twice as likely to have severe anemia as patients without malaria. About 12.2% of severe anemia was attributable to non-falciparum infections, 15.1% was attributable to P. falciparum monoinfections, and P. vivax was the dominant cause of severe anemia in infancy. Finally, compared to patients without anemia, patients with severe anemia had nearly a 6-fold higher risk of death.
What Do These Findings Mean?
These findings provide a comparative assessment of the pattern of anemia associated with non-falciparum malarias in Papua and an estimate of the public health importance of these malarias. Although the accuracy of these findings may be affected by residual confounding (for example, the researchers did not consider nutritional status when calculating how much malaria infection increases the risk of anemia) and other limitations of the study design, non-falciparum malarias clearly make a major contribution to the burden of anemia in southern Papua. In particular, these findings reveal the large contribution that P. vivax makes to severe anemia in infancy, show that the hematological (blood-related) impact of P. malariae is most apparent in adulthood, and suggest, in contrast to some previous reports, that mixed P. vivax/P. falciparum infection is associated with a higher risk of severe anemia than monoinfection with either species. These findings, which need to be confirmed in other settings, highlight the public health importance of implementing integrated malaria control strategies that aim to control all Plasmodium species rather than a single species in regions of Plasmodium co-endemicity.
Additional Information
Please access these websites via the online version of this summary at
This study is further discussed in a PLOS Medicine Perspective by Gosling and Hsiang
Information is available from the World Health Organization on malaria (in several languages); the 2012 World Malaria Report provides details of the current global malaria situation
The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish), including information on different Plasmodium species and a selection of personal stories about malaria
The Malaria Vaccine Initiative has fact sheets on Plasmodium falciparum malaria and on Plasmodium vivax malaria
MedlinePlus provides links to additional information on malaria and on anemia (in English and Spanish)
Information is available from the WorldWide Antimalarial Resistance Network on antimalarial drug resistance for P. falciparum and P. vivax
PMCID: PMC3866090  PMID: 24358031
19.  Deaths due to Plasmodium knowlesi malaria in Sabah, Malaysia: association with reporting as Plasmodium malariae and delayed parenteral artesunate 
Malaria Journal  2012;11:284.
The simian parasite Plasmodium knowlesi is recognized as a common cause of severe and fatal human malaria in Sabah, Malaysia, but is morphologically indistinguishable from and still commonly reported as Plasmodium malariae, despite the paucity of this species in Sabah. Since December 2008 Sabah Department of Health has recommended intravenous artesunate and referral to a general hospital for all severe malaria cases of any species. This paper reviews all malaria deaths in Sabah subsequent to the introduction of these measures. Reporting of malaria deaths in Malaysia is mandatory.
Details of reported malaria deaths during 2010-2011 were reviewed to determine the proportion of each Plasmodium species. Demographics, clinical presentations and management of severe malaria caused by each species were compared.
Fourteen malaria deaths were reported, comprising seven Plasmodium falciparum, six P. knowlesi and one Plasmodium vivax (all PCR-confirmed). Of the six P. knowlesi deaths, five were attributable to knowlesi malaria and one was attributable to P. knowlesi-associated enterobacter sepsis. Patients with directly attributable P. knowlesi deaths (N = 5) were older than those with P. falciparum (median age 51 [IQR 50-65] vs 22 [IQR 9-55] years, p = 0.06). Complications in fatal P. knowlesi included respiratory distress (N = 5, 100%), hypotension (N = 4, 80%), and renal failure (N = 4, 80%). All patients with P. knowlesi were reported as P. malariae by microscopy. Only two of five patients with severe knowlesi malaria on presentation received immediate parenteral anti-malarial treatment. The patient with P. vivax-associated severe illness did not receive parenteral treatment. In contrast six of seven patients with severe falciparum malaria received immediate parenteral treatment.
Plasmodium knowlesi was responsible, either directly or through gram-negative bacteraemia, for almost half of malaria deaths in Sabah. Patients with severe non-falciparum malaria were less likely to receive immediate parenteral therapy. This highlights the need in Sabah for microscopically diagnosed P. malariae to be reported as P. knowlesi to improve recognition and management of this potentially fatal species. Clinicians need to be better informed of the potential for severe and fatal malaria from non-falciparum species, and the need to treat all severe malaria with immediate intravenous artesunate.
PMCID: PMC3472242  PMID: 22905799
Malaria; Plasmodium knowlesi
20.  De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes 
Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion.
Author Summary
Plasmodium vivax is responsible for most malaria cases outside Africa, but is poorly understood, as the parasite is difficult to study in vitro. Genome sequencing studies offer a novel and exciting opportunity to better understand this parasite but, so far, have directly mapped reads onto the reference genome sequence generated from a single P. vivax strain. Here, we use sequence data generated from a field isolate to reconstruct long DNA sequences without relying on the reference genome. Our analyses reveal many P. vivax DNA sequences that are absent from the reference genome and contain 792 predicted genes. One of these novel genes encodes a predicted protein similar to known Plasmodium proteins involved in red blood cell invasion. This new gene is present in all P. vivax strains sequenced so far, except for the strain used to generate the reference genome, and is transcribed in blood-stage parasites. Overall, our analyses show that the catalogue of P. vivax genes was incomplete and that potentially important genes have been missed. We notably identified one putative invasion gene that seems functional and could dramatically change our understanding of the mechanisms determining red blood cell invasion by this important malaria parasite.
PMCID: PMC3854868  PMID: 24340114
21.  Plasmodium knowlesi – an emerging pathogen 
Isbt Science Series  2015;10(Suppl 1):134-140.
Ten years have passed since the publication of a large focus of Plasmodium knowlesi infections in the human population. The discovery was made during a molecular investigation of atypical P. malariae cases in the Kapit Health Division, Sarawak, Malaysian Borneo. Patients were more symptomatic with higher parasite counts than expected in P. malariae infections. The investigation found only P. knowlesi DNA present in patient blood samples. Morphological similarity had allowed P. knowlesi to masquerade as P. malariae during routine diagnostic microscopy for malaria. P. knowlesi, a malaria parasite of macaque monkeys, had entered the human population. The subsequent development of P. knowlesi species-specific PCR assays soon demonstrated that the entry was not confined to the Kapit Division but extended across island and mainland Southeast Asia. Relevant clinical descriptions and guidelines for the treatment and management of patents with P. knowlesi malaria were not available. Nor was it clear whether P. knowlesi had undergone a host switch event into the human population or if infections were zoonotic. The outputs of studies on P. knowlesi malaria during the past 10 years will be summarized, highlighting major findings within the context of pathophysiology, virulence, host switch events, treatment, control and importantly malaria elimination.
PMCID: PMC4440384  PMID: 26029250
Plasmodium; knowlesi; malaria; pathophysiology; virulence; zoonosis
22.  Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain  
Infection and Immunity  2005;73(9):5402-5409.
Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.
PMCID: PMC1231135  PMID: 16113256
23.  Genome sequences reveal divergence times of malaria parasite lineages 
Parasitology  2010;138(13):1737-1749.
The evolutionary history of human malaria parasites (genus Plasmodium) has long been a subject of speculation and controversy. The complete genome sequences of the two most widespread human malaria parasites, P. falciparum and P. vivax, and of the monkey parasite P. knowlesi are now available, together with the draft genomes of the chimpanzee parasite P. reichenowi, three rodent parasites, P. yoelii yoelli, P. berghei and P. chabaudi chabaudi, and one avian parasite, P. gallinaceum.
We present here an analysis of 45 orthologous gene sequences across the eight species that resolves the relationships of major Plasmodium lineages, and provides the first comprehensive dating of the age of those groups.
Our analyses support the hypothesis that the last common ancestor of P. falciparum and the chimpanzee parasite P. reichenowi occurred around the time of the human-chimpanzee divergence. P. falciparum infections of African apes are most likely derived from humans and not the other way around. On the other hand, P. vivax, split from the monkey parasite P. knowlesi in the much more distant past, during the time that encompasses the separation of the Great Apes and Old World Monkeys.
The results support an ancient association between malaria parasites and their primate hosts, including humans.
PMCID: PMC3081533  PMID: 21118608
24.  Human Plasmodium knowlesi infection in Ranong province, southwestern border of Thailand 
Malaria Journal  2012;11:36.
Plasmodium knowlesi, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar.
Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for P. knowlesi using nested PCR assays. Positive samples were also investigated by PCR for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and were confirmed by sequencing the gene encoding the circumsporozoite protein (csp).
Two samples, one obtained from a Thai and the other a Myanmese, were positive for P. knowlesi only. Nucleotide sequences of the csp gene derived from these two patients were identical and phylogenetically indistinguishable from other P. knowlesi sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand.
This study indicates that transmission of P. knowlesi is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.
PMCID: PMC3293766  PMID: 22313518
Plasmodium knowlesi; Thailand; Myanmar; Circumsporozoite protein
25.  Plasmodium knowlesi in humans, macaques and mosquitoes in peninsular Malaysia 
Parasites & Vectors  2008;1:26.
Since a large focus of human infection with Plasmodium knowlesi, a simian malaria parasite naturally found in long-tailed and pig tailed macaques, was reported in Sarawak, Malaysian Borneo, it was pertinent to study the situation in peninsular Malaysia. A study was thus initiated to screen human cases of Plasmodium malariae using molecular techniques, to determine the presence of P. knowlesi in non- human primates and to elucidate its vectors.
Nested polymerase chain reaction (PCR) was used to identify all Plasmodium species present in the human blood samples sent to the Parasitology laboratory of Institute for Medical Research. At the same time, non-human primates were also screened for malaria parasites and nested PCR was carried out to determine the presence of P. knowlesi. Mosquitoes were collected from Pahang by human landing collection and monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites and nested PCR was carried out on positive glands. Sequencing of the csp genes were carried on P. knowlesi samples from humans, monkeys and mosquitoes, positive by PCR.
Results and Discussion
Plasmodium knowlesi was detected in 77 (69.37%) of the 111 human samples, 10 (6.90%) of the 145 monkey blood and in 2 (1.7%) Anopheles cracens. Sequence of the csp gene clustered with other P. knowlesi isolates.
Human infection with Plasmodium knowlesi is occurring in most states of peninsular Malaysia. An. cracens is the main vector. Economic exploitation of the forest is perhaps bringing monkeys, mosquitoes and humans into increased contact. A single bite from a mosquito infected with P. knowlesi is sufficient to introduce the parasite to humans. Thus, this zoonotic transmission has to be considered in the future planning of malaria control.
PMCID: PMC2531168  PMID: 18710577

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