Genetic polymorphism and clustering of the Plasmodium cynomolgi Duffy binding protein 1 region II of recent macaque isolates from Peninsular Malaysia.

Plasmodium cynomolgi is a simian malaria parasite that has been increasingly infecting humans. It is naturally present in the long-tailed and pig-tailed macaques in Southeast Asia. The P. cynomolgi Duffy binding protein 1 region II [PcDBP1(II)] plays an essential role in the invasion of the parasite into host erythrocytes. This study investigated the genetic polymorphism, natural selection and haplotype clustering of PcDBP1(II) from wild macaque isolates in Peninsular Malaysia. The genomic DNA of 50 P. cynomolgi isolates was extracted from the macaque blood samples. Their PcDBP1(II) gene was amplified using a semi-nested PCR, cloned into a plasmid vector and subsequently sequenced. The polymorphism, natural selection and haplotypes of PcDBP1(II) were analysed using MEGA X and DnaSP ver.6.12.03 programmes. The analyses revealed high genetic polymorphism of PcDBP1(II) (π = 0.026 ± 0.004; Hd = 0.996 ± 0.001), and it was under purifying (negative) selection. A total of 106 haplotypes of PcDBP1(II) were identified. Phylogenetic and haplotype analyses revealed two groups of PcDBP1(II). Amino acid length polymorphism was observed between the groups, which may lead to possible phenotypic difference between them.

Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia.

Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo.

Plasmodium knowlesi Duffy binding protein alpha region II (PkDBPαII) in clinical isolates from Peninsular Malaysia and Malaysian Borneo exhibit different immune responses in animal models.

Plasmodium knowlesi utilizes the Duffy binding protein alpha (PkDBPα) to facilitate its invasion into human erythrocytes. PkDBPα region II (PkDBPαII) from Peninsular Malaysia and Malaysian Borneo has been shown to occur as distinct haplotypes, and the predominant haplotypes from these geographical areas demonstrated differences in binding activity to human erythrocytes in erythrocyte binding assays. This study aimed to determine the effects of genetic polymorphisms in PkDBPαII to immune responses in animal models. The recombinant PkDBPαII (~ 45 kDa) of Peninsular Malaysia (PkDBPαII-H) and Malaysian Borneo (PkDBPαII-S) were expressed in a bacterial expression system, purified, and used in mice and rabbit immunization. The profile of cytokines IL-1ra, IL-2, IL-6, IL-10, TNF-α, and IFN-γ in immunized mice spleen was determined via ELISA. The titer and IgG subtype distribution of raised antibodies was characterized. Immunized rabbit sera were purified and used to perform an in vitro merozoite invasion inhibition assay. The PkDBPαII-immunized mice sera of both groups showed high antibody titer and a similar IgG subtype distribution pattern: IgG2b > IgG1 > IgG2a > IgG3. The PkDBPαII-H group was shown to have higher IL-1ra (P = 0.141) and IL-6 (P = 0.049) concentrations, with IL-6 levels significantly higher than that of the PkDBPαII-S group (P ≤ 0.05). Merozoite invasion inhibition assay using purified anti-PkDBPαII antibodies showed a significantly higher inhibition rate in the PkDBPαII-H group than the PkDBPαII-S group (P ≤ 0.05). Besides, anti-PkDBPαII-H antibodies were able to exhibit inhibition activity at a lower concentration than anti-PkDBPαII-S antibodies. PkDBPαII was shown to be immunogenic, and the PkDBPαII haplotype from Peninsular Malaysia exhibited higher responses in cytokines IL-1ra and IL-6, antibody IgM level, and merozoite invasion inhibition assay than the Malaysian Borneo haplotype. This suggests that polymorphisms in the PkDBPαII affect the level of immune responses in the host.

Genetic Diversity and Clustering of the Rhoptry Associated Protein-1 of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo.

Human infection with simian malaria Plasmodium knowlesi is a cause for concern in Southeast Asian countries, especially in Malaysia. A previous study on Peninsular Malaysia P. knowlesi rhoptry associated protein-1 (PkRAP1) gene has discovered the existence of dimorphism. In this study, genetic analysis of PkRAP1 in a larger number of P. knowlesi samples from Malaysian Borneo was conducted. The PkRAP1 of these P. knowlesi isolates was PCR-amplified and sequenced. The newly obtained PkRAP1 gene sequences (n = 34) were combined with those from the previous study (n = 26) and analysed for polymorphism and natural selection. Sequence analysis revealed a higher genetic diversity of PkRAP1 compared to the previous study. Exon II of the gene had higher diversity (π = 0.0172) than exon I (π = 0.0128). The diversity of the total coding region (π = 0.0167) was much higher than those of RAP1 orthologues such as PfRAP-1 (π = 0.0041) and PvRAP1 (π = 0.00088). Z-test results indicated that the gene was under purifying selection. Phylogenetic tree and haplotype network showed distinct clustering of Peninsular Malaysia and Malaysian Borneo PkRAP1 haplotypes. This geographical-based clustering of PkRAP1 haplotypes provides further evidence of the dimorphism of the gene and possible existence of 2 distinct P. knowlesi lineages in Malaysia.

Seroprevalence of sarcocystosis in the local communities of Pangkor and Tioman Islands using recombinant surface antigens 3 (rSAG3) of Sarcocystis falcatula.

OBJECTIVE: To investigate the seroprevalence of Sarcocystosis in the local communities of Pangkor and Tioman islands, Malaysia, by using antigenic recombinant surface antigens 2 and 3 from Sarcocystis falcatula (rSfSAG2 and rSfSAG3) as the target proteins via Western blot and ELISA assays. METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis. RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3. CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.

The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

BACKGROUND: The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. METHODS: The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. RESULTS: Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. CONCLUSION: This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia.

Infections of humans with the zoonotic simian malaria parasite Plasmodium knowlesi occur throughout Southeast Asia, although most cases have occurred in Malaysia, where P. knowlesi is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 P. knowlesi genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct P. knowlesi types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia.

Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

Genetic diversity and natural selection in the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia.

BACKGROUND: The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite's invasion of red blood cells. Although there is some evidence that the protein is recognized by the host's immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953-1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1. METHODS: Blood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs. RESULTS: Thirty PkRAP-1 sequences were obtained. The nucleotide diversity (π) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima's D test and Fu and Li's D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes. CONCLUSIONS: The present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated purifying (negative) selection of the gene. The separation of PkRAP-1haplotypes into two groups provides further evidence to the postulation of two distinct P. knowlesi types or lineages.

Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo.

BACKGROUND: The zoonotic malaria parasite Plasmodium knowlesi has become an emerging threat to South East Asian countries particular in Malaysia. A recent study from Sarawak (Malaysian Borneo) discovered two distinct normocyte binding protein xa (Pknbpxa) types of P. knowlesi. In the present study, the Pknbpxa of clinical isolates from Peninsular Malaysia and Sabah (Malaysian Borneo) were investigated for the presence of Pknbpxa types and natural selection force acting on the gene. METHOD: Blood samples were collected from 47 clinical samples from Peninsular Malaysia (n = 35) and Sabah (Malaysian Borneo, n = 12) were used in the study. The Pknbpxa gene was successfully amplified and directly sequenced from 38 of the samples (n = 31, Peninsular Malaysia and n = 7, Sabah, Malaysian Borneo). The Pknbpxa sequences of P. knowlesi isolates from Sarawak (Malaysian Borneo) were retrieved from GenBank and included in the analysis. Polymorphism, genetic diversity and natural selection of Pknbpxa sequences were analysed using DNAsp v 5.10, MEGA5. Phylogentics of Pknbpxa sequences was analysed using MrBayes v3.2 and Splits Tree v4.13.1. The pairwise F ST indices were used to determine the genetic differentiation between the Pknbpxa types and was calculated using Arlequin 3.5.1.3. RESULTS: Analyses of the sequences revealed Pknbpxa dimorphism throughout Malaysia indicating co-existence of the two types (Type-1 and Type-2) of Pknbpxa. More importantly, a third type (Type 3) closely related to Type 2 Pknbpxa was also detected. This third type was found only in the isolates originating from Peninsular Malaysia. Negative natural selection was observed, suggesting functional constrains within the Pknbpxa types. CONCLUSIONS: This study revealed the existence of three Pknbpxa types in Malaysia. Types 1 and 2 were found not only in Malaysian Borneo (Sarawak and Sabah) but also in Peninsular Malaysia. A third type which was specific only to samples originating from Peninsular Malaysia was discovered. Further genetic studies with a larger sample size will be necessary to determine whether natural selection is driving this genetic differentiation and geographical separation.

Field evaluation of a PfHRP-2/pLDH rapid diagnostic test and light microscopy for diagnosis and screening of falciparum malaria during the peak seasonal transmission in an endemic area in Yemen.

BACKGROUND: Malaria is a public health threat in Yemen, with 149,451 cases being reported in 2013. Of these, Plasmodium falciparum represents 99%. Prompt diagnosis by light microscopy (LM) and rapid diagnostic tests (RTDs) is a key element in the national strategy of malaria control. The heterogeneous epidemiology of malaria in the country necessitates the field evaluation of the current diagnostic strategies, especially RDTs. Thus, the present study aimed to evaluate LM and an RDT, combining both P. falciparum histidine-rich protein-2 (PfHRP-2) and Plasmodium lactate dehydrogenase (pLDH), for falciparum malaria diagnosis and survey in a malaria-endemic area during the transmission season against nested polymerase chain reaction (PCR) as the reference method. METHODS: A household-based, cross-sectional malaria survey was conducted in Mawza District, a malaria-endemic area in Taiz governorate. A total of 488 participants were screened using LM and PfHRP-2/pLDH RDT. Positive samples (160) and randomly selected negative samples (52) by both RDT and LM were further analysed using 18S rRNA-based nested PCR. RESULTS: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RDT were 96.0% (95% confidence interval (CI): 90.9-98.3), 56.0% (95% CI: 44.7-66.8), 76.3% (95% CI: 69.0-82.3), and 90.4% (95% CI: 78.8-96.8), respectively. On the other hand, LM showed sensitivity of 37.6% (95% CI: 29.6-46.3), specificity of 97.6% (95% CI: 91.7-99.7), PPV of 95.9% (95% CI: 86.3-98.9), and NPV of 51.3% (95% CI: 43.2-59.2). The sensitivity of LM dropped to 8.5% for detecting asymptomatic malaria. Malaria prevalence was 32.8% (32.1 and 37.5% for ≥10 and <10 years, respectively) with the RDT compared with 10.7% (10.8 and 9.4% for age groups of ≥10 and <10 years, respectively) with LM. Among asymptomatic malaria individuals, LM and RDT-based prevalence rates were 1.6 and 25.6%, respectively. However, rates of 88.2 and 94.1% of infection with P. falciparum were found among patients who reported fever in the 48 h prior to the survey by LM and PfHRP-2/pLDH RDT, respectively. CONCLUSIONS: The PfHRP-2/pLDH RDT shows high sensitivity for the survey of falciparum malaria even for asymptomatic malaria cases. Although the RDT had high sensitivity, its high false-positivity rate limits its utility as a single diagnostic tool for clinical diagnosis of malaria. On the other hand, low sensitivity of LM indicates that a high proportion of malaria cases is missed, underestimating the true prevalence of malaria in the community. Higher NPV of PfHRP-2/pLDH RDT than LM can give a straightforward exclusion of malaria among febrile patients, helping to avoid unnecessary presumptive treatments.

Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia.

BACKGROUND: Plasmodium knowlesi is one of the monkey malaria parasites that can cause human malaria. The Duffy binding protein of P. knowlesi (PkDBPαII) is essential for the parasite's invasion into human and monkey erythrocytes. A previous study on P. knowlesi clinical isolates from Peninsular Malaysia reported high level of genetic diversity in the PkDBPαII. Furthermore, 36 amino acid haplotypes were identified and these haplotypes could be separated into allele group I and allele group II. In the present study, the PkDBPαII of clinical isolates from the Malaysian states of Sarawak and Sabah in North Borneo was investigated, and compared with the PkDBPαII of Peninsular Malaysia isolates. METHODS: Blood samples from 28 knowlesi malaria patients were used. These samples were collected between 2011 and 2013 from hospitals in North Borneo. The PkDBPαII region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and phylogenetics of PkDBPαII haplotypes were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes. RESULTS: Forty-nine PkDBPαII sequences were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence revealed 58 synonymous and 102 non-synonymous mutations. Analysis on these mutations showed that PkDBPαII was under purifying (negative) selection. At the amino acid level, 38 different PkDBPαII haplotypes were identified. Twelve of the 28 blood samples had mixed haplotype infections. Phylogenetic analysis revealed that all the haplotypes were in allele group I, but they formed a sub-group that was distinct from those of Peninsular Malaysia. Wright's FST fixation index indicated high genetic differentiation between the North Borneo and Peninsular Malaysia haplotypes. CONCLUSIONS: This study is the first to report the genetic diversity and natural selection of PkDBPαII of P. knowlesi from Borneo Island. The PkDBPαII haplotypes found in this study were distinct from those from Peninsular Malaysia. This difference may not be attributed to geographical separation because other genetic markers studied thus far such as the P. knowlesi circumsporozoite protein gene and small subunit ribosomal RNA do not display such differentiation. Immune evasion may possibly be the reason for the differentiation.

Detection of human malaria using recombinant Plasmodium knowlesi merozoire surface protein-1 (MSP-119) expressed in Escherichia coli.

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Human infection with Plasmodium knowlesi is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119), which plays an important role in protective immunity against asexual blood stage malaria parasites, appears as a leading immunogenic antigen of Plasmodium sp. We evaluated the sensitivity and specificity of recombinant P. knowlesi MSP-119 (rMSP-119) for detection of malarial infection. rMSP-119 was expressed in Escherichia coli expression system and the purified rMSP-119 was evaluated with malaria, non-malaria and healthy human serum samples (n = 215) in immunoblots. The sensitivity of rMSP-119 for detection of P. knowlesi, Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale infection was 95.5%, 75.0%, 85.7% and 100%, respectively. rMSP-119 did not react with all the non-malaria and healthy donor sera, which represents 100% specificity. The rMSP-119 could be used as a potential antigen in serodiagnosis of malarial infection in humans.

HETEROLOGOUS EXPRESSION OF TOXOPLASMA GONDII DENSE GRANULE PROTEIN 2 AND 5.

Toxoplasmosis is a foodborne disease caused by Toxoplasma gondii, an obligate intracellular parasite. The parasite remains protected within a parasitophorous vacuole (PV), a specialized compartment formed within the infected host cell during and after invasion. Dense granules (GRA) are T. gondii specialized secretory organelles involved in PV development. GRA2 contributes to the formation of intravacuolar network in the PV, allowing nutrients transportation to nourish the parasites. GRA5 helps to inhibit apoptosis of the infected cells thereby protecting the parasites. As such, these two essential antigens have been selected as the target subjects. Heterologous expression in E. coli BL21 pLysS (DE3) of GRA2 and GRA5 fragment was achieved by transfecting with recombinant expression GRA2- and GRA5-pRSET B plasmid, respectively. His-tagged recombinant proteins were affinity purified using a Nickel-nitrilotriacetic acid column. The identities of recombinant rGRA2 (30 kDa) and 5 (20 kDa) proteins were confirmed by western blotting using immune serum from a patient with toxoplasmosis and by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified T. gondii antigens provide candidates for future development of diagnostic kits of human infection as well as vaccines.

High proportion of knowlesi malaria in recent malaria cases in Malaysia.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The prevalence of simian malaria in wild long-tailed macaques throughout Peninsular Malaysia.

The parasite Plasmodium knowlesi has been the sole cause of malaria in Malaysia from 2018 to 2022. The persistence of this zoonotic species has hampered Malaysia's progress towards achieving the malaria-free status awarded by the World Health Organisation (WHO). Due to the zoonotic nature of P. knowlesi infections, it is important to study the prevalence of the parasite in the macaque host, the long-tailed macaque (Macaca fascicularis). Apart from P. knowlesi, the long-tailed macaque is also able to harbour Plasmodium cynomolgi, Plasmodium inui, Plasmodium caotneyi and Plasmodium fieldi. Here we report the prevalence of the 5 simian malaria parasites in the wild long-tailed macaque population in 12 out of the 13 states in Peninsular Malaysia using a nested PCR approach targeting the 18s ribosomal RNA (18s rRNA) gene. It was found that all five Plasmodium species were widely distributed throughout Peninsular Malaysia except for states with major cities such as Kuala Lumpur and Putrajaya. Of note, Pahang reported a malaria prevalence of 100% in the long-tailed macaque population, identifying it as a potential hotspot for zoonotic transmission. Overall, this study shows the distribution of the 5 simian malaria parasite species throughout Peninsular Malaysia, the data of which could be used to guide future malaria control interventions to target zoonotic malaria.

Cloning, expression, and immunocharacterization of surface protein containing an altered thrombospondin repeat domain (SPATR) from Plasmodium knowlesi.

BACKGROUND: Plasmodium knowlesi is the fifth species identified to cause malaria in humans and is often misdiagnosed as Plasmodium malariae due to morphological similarities. The development of an inexpensive, serological detection method utilizing antibodies specific to P. knowlesi would be a valuable tool for diagnosis. However, the identification of specific antigens for these parasites remains a major challenge for generating such assays. In this study, surface protein containing an altered thrombospondin repeat domain (SPATR) was selected as a potentially specific antigen from P. knowlesi. Its multistage expression by sporozoites, asexual erythrocytic forms and gametocytes, along with its possible role in liver cell invasion, suggests that SPATR could be used as a biomarker for diagnosis of P. knowlesi. METHODS: The spatr gene from P. knowlesi was codon optimized and cloned (pkhspatr). Recombinant pkHSPATR protein was expressed, purified, and evaluated for its sensitivity and specificity in immunoblot and ELISA-based assays for detecting P. knowlesi infection. RESULTS: The recombinant pkHSPATR protein allows sensitive detection of human P. knowlesi infection in serum samples by immunoblot and ELISA. CONCLUSIONS: With further research, recombinant pkHSPATR protein could be exploited as a marker for detection of P. knowlesi infection in humans. Therefore, this finding should contribute to the development of immunodiagnostic assays for the species-specific detection of malaria.

Immunogenicity of bacterial-expressed recombinant Plasmodium knowlesi merozoite surface protein-142 (MSP-142).

BACKGROUND: Plasmodium knowlesi is the fifth Plasmodium species that can infect humans. The Plasmodium merozoite surface protein-1(42) (MSP-1(42)) is a potential candidate for malaria vaccine. However, limited studies have focused on P. knowlesi MSP-1(42). METHODS: A ~42 kDa recombinant P. knowlesi MSP-1(42) (pkMSP-1(42)) was expressed using an Escherichia coli system. The purified pkMSP-1(42) was evaluated with malaria and non-malaria human patient sera (n = 189) using Western blots and ELISA. The immunogenicity of pkMSP-1(42) was evaluated in mouse model. RESULTS: The purified pkMSP-1(42) had a sensitivity of 91.0% for detection of human malaria in both assays. Specificity was 97.5 and 92.6% in Western blots and ELISA, respectively. Levels of cytokine interferon-gamma, interleukin-2, interleukin-4, and interleukin-10 significantly increased in pkMSP-1(42)-immunized mice as compared to the negative control mice. pkMSP-1(42)-raised antibody had high endpoint titres, and the IgG isotype distribution was IgG1 > IgG2b > IgG3 > IgG2a. CONCLUSIONS: pkMSP-1(42) was highly immunogenic and able to detect human malaria. Hence, pkMSP-1(42) would be a useful candidate for malaria vaccine development and seroprevalence studies.

Hyperparasitaemic human Plasmodium knowlesi infection with atypical morphology in peninsular Malaysia.

Plasmodium knowlesi is a potentially life-threatening zoonotic malaria parasite due to its relatively short erythrocytic cycle. Microscopic identification of P. knowlesi is difficult, with "compacted parasite cytoplasm" being one of the important identifying keys. This report is about a case of hyperparasitaemic human P. knowlesi infection (27% parasitaemia) with atypical amoeboid morphology. A peninsular Malaysian was admitted to the hospital with malaria. He suffered anaemia and acute kidney function impairment. Microscopic examination, assisted by nested PCR and sequencing confirmed as P. knowlesi infection. With anti-malarial treatment and several medical interventions, patient survived and recovered. One-month medical follow-up was performed after recovery and no recrudescence was noted. This case report highlights the extreme hyperparasitaemic setting, the atypical morphology of P. knowlesi in the patient's erythrocytes, as well as the medical interventions involved in this successfully treated case.

Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome.

BACKGROUND: Plasmodium ovale is one of the causative agents of human malaria. Plasmodium ovale infection has long been thought to be non-fatal. Due to its lower morbidity, P. ovale receives little attention in malaria research. METHODS: Two Malaysians went to Nigeria for two weeks. After returning to Malaysia, they fell sick and were admitted to different hospitals. Plasmodium ovale parasites were identified from blood smears of these patients. The species identification was further confirmed with nested PCR. One of them was successfully treated with no incident of relapse within 12-month medical follow-up. The other patient came down with malaria-induced respiratory complication during the course of treatment. Although parasites were cleared off the circulation, the patient's condition worsened. He succumbed to multiple complications including acute respiratory distress syndrome and acute renal failure. RESULTS: Sequencing of the malaria parasite DNA from both cases, followed by multiple sequence alignment and phylogenetic tree construction suggested that the causative agent for both malaria cases was P. ovale curtisi. DISCUSSION: In this report, the differences between both cases were discussed, and the potential capability of P. ovale in causing severe complications and death as seen in this case report was highlighted. CONCLUSION: Plasmodium ovale is potentially capable of causing severe complications, if not death. Complete travel and clinical history of malaria patient are vital for successful diagnoses and treatment. Monitoring of respiratory and renal function of malaria patients, regardless of the species of malaria parasites involved is crucial during the course of hospital admission.