A case of severe Plasmodium knowlesi in a splenectomized patient

Plasmodium knowlesi, a zoonotic malaria, is now considered the fifth species of Plasmodium causing malaria in humans. With its 24-hour erythrocytic stage of development, it has raised concern regarding its high potential in replicating and leading to severe illness. Spleen is an important site for removal of parasitized red blood cells and generating immunity. We reported a case of knowlesi malaria in a non-immune, splenectomized patient. We observed the delay in parasite clearance, high parasitic counts, and severe illness at presentation. A thorough search through literature revealed several case reports on falciparum and vivax malaria in splenectomized patients. However, literature available for knowlesi malaria in splenectomized patient is limited. Further studies need to be carried out to clarify the role of spleen in host defense against human malaria especially P. knowlesi.     

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 new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: 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. CONCLUSIONS: 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.     

Plasmodium knowlesi infecting humans in Southeast Asia: What’s next?

Plasmodium knowlesi, a simian malaria parasite, has been in the limelight since a large focus of human P. knowlesi infection was reported from Sarawak (Malaysian Borneo) in 2004. Although this infection is transmitted across Southeast Asia, the largest number of cases has been reported from Malaysia. The increasing number of knowlesi malaria cases has been attributed to the use of molecular tools for detection, but environmental changes including deforestation likely play a major role by increasing human exposure to vector mosquitoes, which coexist with the macaque host. In addition, with the reduction in human malaria transmission in Southeast Asia, it is possible that human populations are at a greater risk of P. knowlesi infection due to diminishing cross-species immunity. Furthermore, the possibility of increasing exposure of humans to other simian Plasmodium parasites such as Plasmodium cynomolgi and Plasmodium inui should not be ignored. We here review the current status of these parasites in humans, macaques, and mosquitoes to support necessary reorientation of malaria control and elimination in the affected areas.     

Knowlesi malaria: newly emergent and of public health importance?

Several questions on public health impact have arisen from the discovery of a large focus of the simian malaria parasite, Plasmodium knowlesi, in the human population. P. knowlesi malaria is not newly emergent and was overlooked until molecular tools to distinguish between P. knowlesi and the morphologically similar Plasmodium malariae became available. Knowlesi malaria is a zoonosis that is widely distributed in Southeast Asia and can be fatal. Information on knowlesi malaria should be included in medical and public health guidelines to encourage the accurate diagnosis and treatment of patients, and monitor the incidence and distribution of cases. A complete emergence of P. knowlesi into the human population could be overwhelming and, although challenging, the prevention of this situation deserves serious consideration.     

Deaths due to Plasmodium knowlesi malaria in Sabah, Malaysia: association with reporting as Plasmodium malariae and delayed parenteral artesunate

ABSTRACT: BACKGROUND: 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. METHODS: 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. RESULTS: 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. vivaxassociated severe illness did not receive parenteral treatment. In contrast six of seven patients with severe falciparum malaria received immediate parenteral treatment. CONCLUSION: 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.     

Rural health centres, communities and malaria case detection in Zambia using mobile telephones: a means to detect potential reservoirs of infection in unstable transmission conditions

Background

Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here.

Case presentation

A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent.

Conclusions

The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans.     

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

Plasmodium knowlesi as a Threat to Global Public Health

Malaria is a tropical disease caused by protozoans of the Plasmodium genus. Delayed diagnosis and misdiagnosis are strongly associated with higher mortality. In recent years, a greater importance is attributed to Plasmodium knowlesi, a species found mainly in Southeast Asia. Routine parasitological diagnostics are associated with certain limitations and difficulties in unambiguous determination of the parasite species based only on microscopic image. Recently, molecular techniques have been increasingly used for predictive diagnosis. The aim of the study is to draw attention to the risk of travelling to knowlesi malaria endemic areas and to raise awareness among personnel involved in the therapeutic process.     

New serological test for malaria antibodies.

In an enzyme-linked immunosorbent assay test for malaria antibodies, antibodies to Plasmodium vivax and P. falciparum in man are detected using a crude antigen prepared from the simian malaria parasite P. knowlesi. The test may be suitable for epidemiological studies.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Malaria parasites do not contain or synthesize sialic acids

The capacity of Plasmodia to synthesize sialic acids was investigated by adding radioactive acetate to short-term in vitro cultures of the intraerythrocytic asexual forms of three malaria parasites (the human malaria Plasmodium falciparum in Aotus trivirgatus erythrocytes; the simian malaria P. knowlesi in rhesus monkey erythrocytes; the rodent malaria P. berghei in mouse erythrocytes) and to cultures of extracellular zygotes of the avian malaria P. gallinaceum. Radioactive acetate was added to normal rhesus monkey erythrocytes and to cells of the murine myeloma NS-1 for comparison. Although [1-14C]-acetate labeled many proteins with each malaria parasite and the NS-1 cells, analysis of purified sialic acids revealed that only with the NS-1 cells was radioactivity incorporated into sialic acids. Furthermore, N-acetyl[6-3H]mannosamine was not incorporated into sialic acids or malarial glycoproteins when added to P. knowlesi cultures. All of the malaria parasites underwent growth or differentiation during these experiments as measured by [35S]methionine uptake into protein and by light microscopy. Extracellular parasites largely free of erythrocyte membranes were prepared to determine whether Plasmodia contain sialic acids that are not labeled by exogenous precursors. Purified merozoites of P. knowlesi and zygotes of P. gallinaceum did not contain detectable amounts of sialic acids on chemical analysis. Thus, although we could show that Plasmodia can incorporate radioactive sugars such as glucosamine, galactose and mannose into proteins, presumably glycoproteins, they do not synthesize sialic acids or sialo-glycoproteins, nor do they contain sialo-glycoconjugates of host origin.     

Conserved group-specific epitopes of the circumsporozoite proteins revealed by antibodies to synthetic peptides

The immunogenic properties of sporozoites are associated mainly with the circumsporozoite (CS) protein that covers the surface of mature sporozoites. This stage-specific protein has an immunodominant region with repetitive epitopes. Rabbits that are repeatedly immunized with sporozoites of Plasmodium knowlesi, a monkey malaria parasite, also recognize two synthetic peptides (N2 and C2) representing other polar domains of the CS protein. We show in this report that antibodies to the N2 and C2 synthetic peptides react not only with P. knowlesi but also with conserved regions of the surface membrane of other human, monkey, and rodent (but not avian) malaria sporozoites. Moreover, antibodies to N2 partially neutralize the infectivity of sporozoites of P. berghei, a rodent malaria parasite. In contrast, antibodies to synthetic peptides representing the repetitive epitope of P. knowlesi were strictly species specific.     

Evolutionary relatedness of some primate models of Plasmodium

Primate--and, specifically, monkey--malaria infections are commonly used for understanding the pathology of and immune response to the human disease because they are thought to resemble most closely the host-parasite relationship found in humans. Plasmodium cynomolgi is used extensively as a model for the human parasite, P. vivax, and P. knowlesi is used primarily as a model for the development of erythrocytic-stage vaccines. Both of these simian parasites can naturally infect man, resulting in mildly symptomatic episodes of the disease. The phylogenetic relationship between these two simian parasites and previously characterized Plasmodium species, including P. vivax, was examined by comparison of the asexually expressed small- subunit ribosomal RNA genes. Our analysis confirmed that P. vivax is most closely related to P. cynomolgi and that it remains an appropriate model of the human pathogen. Furthermore, with P. knowlesi and P. fragile, these two species form a group of closely related species, distant from other Plasmodium species. What is considered to be the most ancient of the human malaria pathogens, P. malariae, was also included in the analysis and does not group at all with other simian or human parasites.      

Plasmodium knowlesi in the marmoset (Callithrix jacchus).

Common marmosets were shown to be susceptible to Plasmodium knowlesi malaria. The morphology of the parasite was indistinguishable from the observed in the natural host (Macaca fascicularis) and the common laboratory model (Macaca mulatta). A differential susceptibility to P. knowlesi was observed in the 8 marmosets studied. Multiplication rates of parasites were variable over 24 h periods. Five animals died of a fulminating infection within 12--17 days after challenge. Three animals recovered spontaneously from infection and were subsequently resistant to challenge with homologous and heterologous variants and strains of P. knowlesi. This resistance was maintained for intervals up to 100 days between challenge infections.     

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.     

Focus: Zoonotic Disease: An Ecologically Framed Comparison of The Potential for Zoonotic Transmission of Non-Human and Human-Infecting Species of Malaria Parasite

The threats, both real and perceived, surrounding the development of new and emerging infectious diseases of humans are of critical concern to public health and well-being. Among these risks is the potential for zoonotic transmission to humans of species of the malaria parasite, Plasmodium, that have been considered historically to infect exclusively non-human hosts. Recently observed shifts in the mode, transmission, and presentation of malaria among several species studied are evidenced by shared vectors, atypical symptoms, and novel host-seeking behavior. Collectively, these changes indicate the presence of environmental and ecological pressures that are likely to influence the dynamics of these parasite life cycles and physiological make-up. These may be further affected and amplified by such factors as increased urban development and accelerated rate of climate change. In particular, the extended host-seeking behavior of what were once considered non-human malaria species indicates the specialist niche of human malaria parasites is not a limiting factor that drives the success of blood-borne parasites. While zoonotic transmission of non-human malaria parasites is generally considered to not be possible for the vast majority of Plasmodium species, failure to consider the feasibility of its occurrence may lead to the emergence of a potentially life-threatening blood-borne disease of humans. Here, we argue that recent trends in behavior among what were hitherto considered to be non-human malaria parasites to infect humans call for a cross-disciplinary, ecologically-focused approach to understanding the complexities of the vertebrate host/mosquito vector/malaria parasite triangular relationship. This highlights a pressing need to conduct a multi-species investigation for which we recommend the construction of a database to determine ecological differences among all known Plasmodium species, vectors, and hosts. Closing this knowledge gap may help to inform alternative means of malaria prevention and control.     

Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites

Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8(+) T cells that kill parasites developing in the liver. We were curious to know if CD8(+) T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8(+) T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8(+) T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8(+) T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8(+) T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.