Sarcocystis nesbitti is an intracellular protozoan parasite found as sarcocysts within muscle fibers of intermediate hosts (monkey and baboon). The definitive host is suspected to be the snake. We report two cases from a larger cohort of 89 patients who had fever, headache, and generalized myalgia after a trip to Pangkor Island, Malaysia. Sarcocysts were detected in skeletal muscle biopsy specimens by light and electron microscopy from these two patients. DNA sequencing based on the 18S ribosomal DNA region identified the Sarcocystis species as S. nesbitti. We also identified S. nesbitti sequences in the stools of a snake (Naja naja). Phylogenetic analysis showed that these sequences form a cluster with most of the other known Sarcocystis species for which the snake is a definitive host. We believe these two patients were likely to have symptomatic acute muscular sarcocystosis after S. nesbitti infection that may have originated from snakes.
From the 17th to 19th January 2012, a group of 92 college students and teachers attended a retreat in a hotel located on Pangkor Island, off the west coast of Peninsular Malaysia. Following the onset of symptoms in many participants who presented to our institute, an investigation was undertaken which ultimately identified Sarcocystis nesbitti as the cause of this outbreak.
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.
Entamoeba histolytica infection is the third-greatest parasitic disease responsible for death in the world. Wild rats harbouring E. histolytica can be the possible reservoir hosts for human amoebiasis. There were numerous studies on prevalence of intestinal parasites among wild rats in Malaysia but none has reported E. histolytica. Rats were captured from Sentul and Chow Kit areas, Kuala Lumpur, Malaysia. The preserved stool samples were used for microscopy examination and molecular analysis. Out of 137 samples collected, 12 were positive for E. histolytica / E. dispar / E. moshkovskii microscopically. Two E. histolytica (1.4%), 1 E. dispar (0.7%) and 6 mixed infections of E. histolytica and E. dispar (4.3%) were detected using PCR. This is the first report of molecular detection of E. histolytica/dispar infection among wild rats in Malaysia. This study provides useful information about the potential risks of zoonotic agents and the importance of developing control measures to prevent zoonotic transmission.
Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
Malaria causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium sp. and its infection is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119) appears as a potential candidate for malaria blood stage vaccine as it could induce protective immunity. In this study, codon optimized P. knowlesi MSP-119 (pkMSP-119) was expressed and purified in yeast Pichia pastoris expression system. The purified recombinant protein was further evaluated using Western blot assay using knowlesi malaria, non-knowlesi human malaria, non-malarial parasitic infections and healthy serum samples (n = 50). The sensitivity of purified pkMSP-119 towards detection of knowlesi infection was as 28.6% (2/7). pkMSP-119 did not react with all non-malarial parasitic infections and healthy donor sera, yet reacted with some non-knowlesi human malaria sera, therefore lead to a specificity of 86.0% (37/43).
Toxoplasma gondii, an obligate intracellular protozoan parasite, causes a disease called toxoplasmosis which can sometimes be acquired congenitally by a newborn from an infected mother. This study aimed to determine the seroprevalence of Toxoplasma infection and its associated risks among 219 and 215 pregnant women from Malaysia and Myanmar, respectively.
Malaria is caused by parasitic protozoans of the genus Plasmodium and is one of the most prevalent infectious diseases in tropical and subtropical regions. For this reason, effective and practical diagnostic methods are urgently needed to control the spread of malaria. The aim of the current study was to identify a panel of new malarial markers, which could be used to diagnose patients infected with various Plasmodium species, including P. knowlesi, P. vivax and P. falciparum. Sera from malaria-infected patients were pooled and compared to control sera obtained from healthy individuals using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. Mass spectrometry was used to identify serum proteins and quantify their relative abundance. We found that the levels of several proteins were increased in pooled serum from infected patients, including cell adhesion molecule-4 and C-reactive protein. In contrast, the serum concentration of haptoglobin was reduced in malaria-infected individuals, which we verified by western blot assay. Therefore, these proteins might represent infectious markers of malaria, which could be used to develop novel diagnostic tools for detecting P. knowlesi, P. vivax and P. falciparum. However, these potential malarial markers will need to be validated in a larger population of infected individuals.
The neglected tropical diseases, echinococcosis, schistosomiasis and toxoplasmosis are all globally widespread zoonotic diseases with potentially harmful consequences. There is very limited data available on the prevalence of these infections, except for schistosmiasis, in underdeveloped countries. This study aimed to determine the seroprevalence of Echinococcus multilocularis, Schistosoma mansoni, and Toxoplasma gondii antibodies in populations from the Monduli and Babati districts in Tanzania.
Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.
Toxoplasma gondii infects all warm-blooded animals, including humans. Early diagnosis and determining the infective stage are critical for effectively treating immunosuppressed individuals and pregnant women with toxoplasmosis. Among the rhoptry proteins of the parasite, Rhoptry protein 8 (ROP8), is known to be expressed during the early stages of T. gondii infection and is involved in parasitophorous vacuole formation. In this study, we have investigated the diagnostic efficacy of recombinant ROP8 (rROP8).
Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.).
Toxoplasma gondii, is one of the infectious agents of congenital TORCH infections, causes severe clinical outcomes in fetus and newborns. Nevertheless this life-threatening parasitic disease is preventable by simple preventive measures related to lifestyle during pregnancy. We aim to study on the knowledge about toxoplasmosis and practices that prevents this infection among the pregnant women. Total of 2598 pregnant women from Malaysia, Philippines, and Thailand were randomly surveyed to determine the knowledge and their practices on Toxoplasma infection. The questionnaire covered respondents' general information and knowledge on plausible risks factors, symptoms, timing of infection, prevention knowledge, and preventive behavior regarding Toxoplasma infection. Majority of these pregnant women were in their age group of 20-29 years (50.9%), completed secondary level of education (51.7%), in their second trimester of pregnancies (38.1%), non-parous (36.6%), and had no history of abortion (90.4%). Based on this survey, only 11% of these pregnant women had read, heard, or seen information regarding toxoplasmosis and 3.5% of them were aware of being tested for the infection. A small percentage of these pregnant women knew that T. gondii were shed in the feces of infected cats (19.4%) and sometimes found in the raw or undercooked meat (11.0%). There was 16.1% of responding women knew that toxoplasmosis is caused by an infection. Demographic profiles such as age group, level of education, pregnancy term, and number of children of the pregnant women showed significant association with their responses toward prevention knowledge and preventive behavior related questions (P < 0.05). Thus, it is suggested that health education on toxoplasmosis and primary behavioral practices should be consistently offered to reproductive age women in general and pregnant women in particular. This information could help to reduce vertical transmission of Toxoplasma infection during pregnancy.
Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene.
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.
Toxoplasmosis, being one of the TORCH's infections in pregnant women, is caused by Toxoplasma gondii, an obligate intracellular protozoan parasite. This parasitic infection in pregnancy congenitally causes severe outcomes to their fetus and newborn. This study aimed to determine the seroprevalence and stages of Toxoplasma infection in pregnant women and its associated risks exposures.
Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines.
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.
The Duffy blood group is of major interest in clinical medicine as it plays an important role in Plasmodium knowlesi and Plasmodium vivax infection. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Peninsular Malaysia were determined. The blood group of 60 healthy blood donors and 51 P. knowlesi malaria patients were genotyped using allele specific polymerase chain reaction (ASP-PCR). The data was analyzed using Fisher's exact test in order to assess the significance of the variables. Our results show a high proportion of the FY*A/FY*A genotype (>85% for both groups) and a high frequency of the FY*A allele (>90% for both groups). The FY*A/FY*A genotype was the most predominant genotype in both infected and healthy blood samples. The genotype frequency did not differ significantly between the donor blood and the malaria patient groups. Also, there was no significant correlation between susceptibility to P. knowlesi infection with any Duffy blood genotype.
In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).