Monkey Malaria Research Articles

Genetic diversity, haplotypes and allele groups of Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia

BackgroundThe 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.MethodsBlood 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.ResultsFifty-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.ConclusionsOur 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.

Genomic insights into the other malaria

Plasmodium vivax has received less attention and study than Plasmodium falciparum, due in part to difficulties in culturing this pathogen. Whole-genome sequencing of both P. vivax and Plasmodium cynomolgi and characterization of genetic variation in these species provide a genetic toolbox for tertian malaria and new insights into the monkey malaria clade.

Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade

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.

Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes

BackgroundPlasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb).MethodsOverlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis.ResultsAn N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant.ConclusionsThe data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

Ketotifen is an antimalarial prodrug of norketotifen with blood schizonticidal and liver-stage efficacy

Ketotifen is known to exhibit antimalarial activity in mouse and monkey malaria models. However, the low plasma levels and short half life of the drug do not adequately explain its in vivo efficacy. We synthesized most of the known metabolites of ketotifen and evaluated their antimalarial activity and pharmacokinetics in mice. Norketotifen, the de-methylated metabolite of ketotifen, was a more potent antimalarial in vitro as compared to ketotifen, and exhibited equivalent activity in vivo against asexual blood and developing liver-stage parasites. After ketotifen dosing, norketotifen levels were much higher than ketotifen relative to the IC50s of the compounds against Plasmodium falciparum in vitro. The data support the notion that the antimalarial activity of ketotifen in mice is mediated through norketotifen.

Plasmodium knowlesi: A Malaria Parasite of Monkeys and Humans

Plasmodium knowlesi is a malaria parasite of monkeys of Southeast Asia that is transmitted by mosquitoes of the Anopheles leucosphyrus group. Humans are frequently infected with this parasite and misdiagnosed as being infected with Plasmodium malariae. The parasite was a major monkey animal model for developing antimalarial vaccines and investigations of the biology of parasite invasion. P. knowlesi is the first monkey malaria parasite genome to be sequenced and annotated.

The past and present of malaria diagnosis

Within 30 years of Laveran’s discovery of human malaria parasites in 1880, four species, Plasmodium falciparum, P. vivax, P. malar-iae and P. ovale, had been described and their morphological distinguishing features clarified. Although other diagnostic techniques have been developed, 130 years later the gold standard is still examination of thick and thin blood films.Recently a fifth species, the monkey malaria Plasmodium knowlesi, has been found to infect humans in parts of Southeast Asia. With human activities continuing to impinge on the habitats of other primates, it is possible that other malaria species may cross over to humans as well.

Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil

In some states of the Brazilian extra-Amazonian region, such as the Atlantic Forest area, autochthonous human cases of malaria were related to simian malarias and vice versa. To verify the presence of Plasmodium, 50 blood samples of howler monkeys (Alouatta guariba clamitans) rescued from the Metropolitan Region of Saõ Paulo city, where the Atlantic Forest is present, were analyzed. The samples were submitted to microscopy (thin and thick blood smears), enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescent assay (IFA), and polymerase chain reaction (PCR). Only one smear showed forms reminiscent of Plasmodium vivax. In ELISA, the frequencies of antibodies against synthetic peptides corresponding to circumsporozoite protein of P. vivax VK210 'classic' (Pvc), P. vivax VK247, human P. vivax-like (Pvk and Pvl), P. malariae/P. brasilianum (Pm), and P. falciparum (Pf) were 24.0% (12/50) for Pvc, 8.0% (04/50) for Pvk, 6.0% (03/50) for Pvl, 24.0% (12/50) for Pm, and 28.0% (14/50) for Pf, while the frequency of antibodies against PvMSP119 recombinant proteins was 42.0% (21/50). No serum reacted against PfMSP1-19. In IFA,the seropositivity of antibodies against asexual forms of P. malariae was 31.3% (15/48). We utilized three PCR protocols to develop a molecular consensus (positive results in, at least, two protocols). The frequency of Plasmodium infections detected by PCR was 18.0% (09/50) for P. vivax, 4.0% (02/50) for P. malariae, and 76.0% (38/50) of samples were negative. The molecular consensus was not seen in 4.0% (02/50) of samples. These results suggest that a possible interaction between human and simian malaria coming from a zoonotic cycle cannot be discarded because simians that live in the areas of the Atlantic Forest could play a role as a reservoir for Plasmodium.

Worldwide sequence conservation of transmission-blocking vaccine candidate Pvs230 in Plasmodium vivax

Pfs230, surface protein of gametocyte/gamete of the human malaria parasite, Plasmodium falciparum, is a prime candidate of malaria transmission-blocking vaccine. Plasmodium vivax has an ortholog of Pfs230 (Pvs230), however, there has been no study in any aspects on Pvs230 to date. To investigate whether Pvs230 can be a vivax malaria transmission-blocking vaccine, we performed evolutionary and population genetic analysis of the Pvs230 gene (pvs230: PVX_003905). Our analysis of Pvs230 and its orthologs in eight Plasmodium species revealed two distinctive parts: an interspecies variable part (IVP) containing species-specific oligopeptide repeats at the N-terminus and a 7.5kb interspecies conserved part (ICP) containing 14 cysteine-rich domains. Pvs230 was closely related to its orthologs, Pks230 and Pcys230, in monkey malaria parasites. Analysis of 113 pvs230 sequences obtained from worldwide, showed that nucleotide diversity is remarkably low in the non-repeat 8-kb region of pvs230 (θπ=0.00118) with 77 polymorphic nucleotide sites, 40 of which results in amino acid replacements. A signature of purifying selection but not of balancing selection was seen on pvs230. Functional and/or structural constraints may limit the level of polymorphism in pvs230. The observed limited polymorphism in pvs230 should ground for utilization of Pvs230 as an effective transmission-blocking vaccine.

Insights from monkey malaria that can change thinking about human infections

Despite prolonged and intense effort to understand and control the health impact of malaria morbidity and mortality, malaria remains third in the global ranking for severe and fatal infections. P. falciparum is responsible for most malaria morbidity and mortality [1]. The more widespread Plasmodium vivax is considered less pathogenic but now appears to cause severe acute disease with some fatal cases [2]. Nevertheless among the human malarias virulent acute P. falciparum malaria attracts most research attention. Falciparum malaria has several characteristics notfound in P. vivax or the other two human adapted species, Plasmodium ovale and Plasmodium malariae. Differences include parasitemias >100 000/uL and the ability to sequester from the peripheral blood in deep post-capillary venules. Both are strong contenders for increased pathophysiology in this species. Unfortunately because of few similarities between the four human-host malarias it is difficult distinguish virulence factors from interspecies differentiation. The monkey malaria, Plasmodium knowlesi may change this and our views on malaria pathophysiology. The discovery of severe and fatal cases of knowlesi malaria in the human population was unexpected and for the first time offers a comparator disease for falciparum malaria. In Sarawak, Malaysian Borneo 10% of P. knowlesi cases are complicated, with at least 1% of all human cases becoming fatal [3]. Complicated knowlesi malaria presents as organ dysfunction, acute and delayed acute respiratory distress syndrome, but not coma or severe malarial anaemia that are often associated with falciparum malaria. Preliminary studies show a direct correlation between P. knowlesi parasitemia and complications, parasitemias >100 000/uL are common. Blood vessels in the brain from a fatal knowlesi case were congested with sequestered heavily pigmented mature stage parasites [4]. The patient did not present with cerebral malaria, a complication often associated with P. falciparum sequestration in post capillaries of the brain. Descriptions of naturally acquired P. knowlesi malaria in humans are enhanced by the wealth of baseline molecular information available for P. knowlesi gathered from experimental primate infections and the knowlesi genome project [5]. P. knowlesi is permissive in various primates and some infections are representative of human knowlesi malaria. Pathophysiology of naturally acquired P. knowlesi malaria in humans and P. falciparum compare well and there are important differences. Comparisons will lead to the identification of testable candidate virulence factors. The ability to specifically test virulence in representative primate models may well offer much needed means to understand malaria pathophysiology and to help distinguish P. falciparum virulence from inconsequential species-specific characteristics.

Interview with the Expert: William E. Collins, Ph.D.

William Collins, Ph.D., received his B.S. and M.Sc. degrees in entomology from Michigan State University. He completed his Ph.D. at Rutgers University in two years, just before being inducted into the Army to serve in the Korean War. He was assigned to Fort Detrick at the Biological Warfare Research Laboratories and after three years returned to Rutgers as an extension entomologist. He accepted a position in 1959 with the U.S. Public Health Service, with which he has worked for the last 50 years. In 1963, the Public Health Service laboratory moved to Atlanta and Dr. Collins' group began working with non-human primates following the discovery that monkey malarias were transmissible to humans. Parasites from monkeys or apes isolated in Asia, South America, and Africa were sent to the laboratory in Chamblee, Georgia, where they were adapted and transmitted to laboratory-maintained primates and their life cycles described and characterized. Transmissions to human volunteers were also attempted. In 1973, the laboratory operation was transferred to the CDC, and the emphasis changed from the study of monkey malaria in monkeys to that of human malaria in monkeys. During the last 25 to 30 years, different isolates of human malaria parasites have been adapted to New World monkeys to characterize the isolates for the development and testing of drugs and vaccines. Dr. Collins' task has been to identify and choose the best combination of vector-parasite-host combinations for testing each vaccine candidate. He has co-authored more than 450 manuscripts and has been awarded the U.S. Public Health Service Superior Service Award, The Joseph A. LaPrince Medal for Malariology from the American Society of Tropical Medicine and Hygiene, The Distinguished Service Award of the Department of Health and Human Services, the William Watson Medal of Excellence from the Centers for Disease Control and Prevention, and the Hoogstraal Medal from the American Committee of Medical Entomology.

Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam

The feasibility of identifying parasite DNA and specific mRNAs from wild-caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi DNA by conventional PCR and, furthermore, detected P. falciparum gametocyte-specific genes, pfg377 and pfs16 mRNA, P. knowlesi circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2) mRNA by reverse transcription-PCR. Using this technique, we were able to confirm the presence of P. vivax, P. falciparum and P. knowlesi in one particular wild-caught mosquito. These results indicate that P. knowlesi may be transmitted by the primary human malaria vector in forested areas in Vietnam. This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria.

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-γ-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-γ-producing CD8 + T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.

Monkey Malaria in Humans: A Diagnostic Dilemma with Conflicting Laboratory Data

Plasmodium knowlesi has recently been recognized as the fifth Plasmodium species causing malaria in humans. Diagnosis is difficult morphologically, and currently, available rapid tests have not been comprehensively evaluated with this pathogen. We report a case of P. knowlesi malaria that was confirmed after the initial clue of discordant microscopy and immunochromatographic results, highlighting the importance of molecular diagnostics in cases with the relevant clinical and epidemiologic history.

Use of Malaria Rapid Diagnostic Test to IdentifyPlasmodium knowlesiInfection

Reports of human infection with Plasmodium knowlesi, a monkey malaria, suggest that it and other nonhuman malaria species may be an emerging health problem. We report the use of a rapid test to supplement microscopic analysis in distinguishing the 5 malaria species that infect humans.

Use of Malaria Rapid Diagnostic Test to Identify Plasmodium knowlesi Infection

Reports of human infection with Plasmodium knowlesi, a monkey malaria, suggest that it and other nonhuman malaria species may be an emerging health problem. We report the use of a rapid test to supplement microscopic analysis in distinguishing the 5 malaria species that infect humans.

Monkey Malaria in a European Traveler Returning from Malaysia

In 2007, a Finnish traveler was infected in Peninsular Malaysia with Plasmodium knowlesi, a parasite that usually causes malaria in monkeys. P. knowlesi has established itself as the fifth Plasmodium species that can cause human malaria. The disease is potentially life-threatening in humans; clinicians and laboratory personnel should become more aware of this pathogen in travelers.

Is a Monkey Malaria from Borneo an Emerging Human Disease?

In 2004, a team of researchers from Malaysia surprised the medical community by reporting that a significant number of people on the island of Borneo were becoming infected with a malaria parasite of monkeys, Plasmodium knowlesi [1]. The world was still unaware of the extent of the problem until 2008 when the same research group gave us a clear view of the gravity of the situation [2]. They found that P. knowlesi infections accounted for 71% of the cases of malaria in one regional hospital. Of even more concern, the team has shown that this form of malaria is potentially life threatening. The medical community is now asking whether P. knowlesi is a ‘fifth human malaria’ with the potential to spread to other parts of Southeast Asia and beyond. Many species of malaria parasite that naturally infect monkeys will also develop in humans. As early as the 1930s, human infections by monkey malarias were studied under controlled conditions in a number of laboratories, including extensive studies carried out in the USA and Romania. These studies showed that although the infections were not initially life threatening, they became increasingly virulent when cycled through humans in the absence of an intermediate passage through mosquitoes [William C, Pers. Comm] [3]. The virulence associated with the Malaysian infections raises the possibility that a new and more virulent strain of P. knowlesi is involved as well as the possibility that, in the cases of more virulent infection, some means of parasite transmission other than that initiated by the bite of an infected mosquito is playing a role. First reported in the Lancet in 2004, Singh and collaborators described their investigation of 208 cases of malaria from the Kapit division of Malaysian Borneo [1]. Their interest had arisen because of the severity of the disease in patients that had been diagnosed as being infected with Plasmodium malariae, which causes a relatively benign form of human malaria. They found that over half of these cases were the result of infection with a monkey malaria, P. knowlesi. In 2008, the group published the results of a more extensive study on the distribution of human infection by P. knowlesi in Sarawak and Sabah in Borneo and Pahang peninsular Malaysia. They demonstrated that these infections are occurring in significant numbers in many of the district hospitals. Furthermore, their investigation of four deaths in the region indicated high levels of infection with P. knowlesi and hepatorenal dysfunction, which is often associated with high parasitemias. This suggested that infection with P. knowlesi is potentially life threatening. Singh, Cox-Singh and Conway now proceed with their ground-breaking research on several fronts, including epidemiological studies to unravel the complicated story of how this emerging disease is being transmitted and further investigation of fatalities in the region to determine the extent to which the threat presents to the people of their region.

Plasmodium knowlesi: The Fifth Human Malaria Parasite

In 1932, when Knowles and Das Gupta [ 1 ] succeeded in transmitting to humans the monkey malaria they had discovered, it appeared that a new agent for malaria therapy had been discovered. Since the Nobel Prize-winning research of Julius WagnerJauregg, malaria therapy had become widely used for the treatment of general paralysis of the insane (neurosyphilis), one of the main reasons for admission to psychiatric institutions. But it soon became apparent that this infection could rapidly become uncontrollable, and after several fatalities, its use was largely discontinued in favor of the less virulent human parasite Plasmodium vivax. Malaria parasites are generally rather choosy, both about their mammalian, avian, or reptilian hosts and their respective mosquito vectors. Transmission of Plasmodium knowlesiy for malaria therapy, from human to human was by blood passage. So initially, it was uncertain whether natural infection could take place and, thus, whether this could be a zoonosis. In 1960, Eyles et al. [2] demonstrated the first experimental mosquito transmission of a

Recent independent evolution of msp1 polymorphism in Plasmodium vivax and related simian malaria parasites

The Plasmodium MSP-1 is a promising malaria vaccine candidate. However, the highly polymorphic nature of the MSP-1 gene ( msp1) presents a potential obstacle for effective vaccine development. To investigate the evolutionary history of msp1 polymorphism in P. vivax, we construct phylogenetic trees of msp1 from P. vivax and related monkey malaria parasite species. All P. vivax msp1 alleles cluster in the P. vivax lineage and are not distributed among other species. Similarly, all P. cynomolgi msp1 alleles cluster in the P. cynomolgi lineage. This suggests that, in contrast to presumed ancient origin of P. falciparum msp1 polymorphism, the origin of P. vivax msp1 polymorphism is relatively recent. We observed positive selection in the P. vivax lineage but not in P. cynomolgi. Also, positive selection acts on different regions of msp1 in P. vivax and P. falciparum. This study shows that the evolutionary history of msp1 differs greatly among parasite lineages.