The ultrastructure of the human skeletal muscle sarcocyst found in Malaysia is reported. Sarcocyst-positive, formalin-fixed tongue tissues were postfixed in osmium tetroxide. The primary cyst wall consisted of a thin membrane supported by osmiophilic material that was interrupted regularly by vesicle-like invaginations. Although there were no cytophaneres, stubby protrusions of the primary wall were observed. These protrusions were accentuated by dense, curvilinear material externally. The primary wall was wavy over about half the cross section of the cyst. The granular ground substance underlying the primary wall occasionally contained hitherto undescribed coiled microtubular structures. Branching septa extended from the ground substance into the cyst, separating mature merozoites into compartments. A few peripheral metrocytes and many laminated myelin figure-like structures, probably degenerating merozoites, were found. Although the human muscular sarcocyst has the same basic ultrastructure as those found in other animals, the stubby protrusions and coiled microtubular structures in the ground substance have not been described previously in nonhuman animals.
The ultrastructure of sarcocysts of macro- and microscopic species of Sarcocystis was compared from naturally infected water buffalo from India. Grossly visible sarcocysts had walls consisting of cauliflower-like villar protrusions, typical of S. fusiformis. The sarcocyst wall of the microscopic species of Sarcocystis was 6.4 microns thick and consisted of tightly packed conical villar protrusions that were 9.6 microns long and 3.7 microns wide at the base. At approximately 3 microns above the base, the distal two-thirds of the villar protrusion became conical shaped and was bent laterally at an angle of 45 degrees to the sarcocyst surface. The granular layer beneath the villar protrusions was 0.9 microns thick. In S. levinei the granular layer was 1.9 microns thick, the villar protrusions were narrow and it had a highly undulating primary cyst wall. Whether the microscopic S. levinei-like sarcocysts of Indian and Malaysian water buffalo are distinct species of Sarcocystis will require further investigation.
The ultrastructure of the cyst wall and zoites of a species of Sarcocystis from the skeletal muscles of a naturally-infected Malaysian long-tailed monkey, Macaca fascicularis, is described in detail. The wavy, electron-dense primary cyst wall is thin (55 nm) and invaginated. Cytophaneres are absent. The ground substance contains electron-dense granules and bundles of parallel, fibrillar elements in some areas. Thin trabeculae are present. The zoites measure 1.2 X 4.7 microns and have an interior conoid, 22 subpellicular microtubules, 50-60 micronemes, 4-6 rhoptries, and a posteriorly situated nucleus. Some ultrastructural aspects of the cyst wall and the zoites of this parasite resemble those of Sarcocystis species of the moonrat, rhesus monkey, tamarin, and baboon. The light microscopic appearance of this species from M. fascicularis also bears some resemblance to that of parasites from the four cases of human Sarcocystis reported in Malaysia. The cyst in all these human cases were thin-walled, with no cytophaners. Although the final hosts of these species of Sarcocystis are not known, it is quite possible that man, monkeys, and perhaps the moonrat (an insectivore) may serve as common intermediate hosts for one or several species of Sarcocystis.
The two species of Sarcocystis--S. levinei and S. fusiformis from the water buffalo, Bubalus bubalis, show some ultrastructural similarities in their cyst wall and zoites. The zoites of both species are of about the same size, banana-shaped and have 22 subpellicular microtubules, numerous micronemes, eight rhoptries, a micropore in the region of the micronemes, an elongated mitochondrion, and a nucleus. S. levinei has 200--300 micronemes and S. fusiformis has about 400. The sarcocysts of both species are trabeculated and their cyst walls have cytophaneres containing annulated fibrils and coarse, electron dense granules. The cytophaneres of S. levinei are sloping, with irregular, wavy outlines, whereas S. fusiformis has the cauliflower-type of cytophaneres. This difference in the appearance of the cytophaneres, together with the difference in size of the sarcocysts and their definitive hosts, further confirms that S. levinei and S. fusiformis are two distinct species in the water buffalo.
The second case of Sarcocystis infection in man is reported from an aborigine girl. It was an incidental finding at autopsy and two cysts were seen in the muscle of the oropharyngeal region. The cysts and zoites were similar to those in the previous case reported from Malaysia.
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.
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.
Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 x 24.5 µm and the thickness of the wall up to 2.5 µm. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.
Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.
Ultrastructural studies of sarcocysts obtained from Philippine water buffaloes revealed the presence of the commonly reported macroscopic species, Sarcocystis fusiformis, and the microscopic species Sarcocystis levinei (Dissanaike A, Kan S. Studies on Sarcocystis in Malaysia. I: Sarcocystis levinei n.sp. from the water buffalo Bubalus bubalis. Z Parasitenkd 1978;55:127-38), (Huong L, Dubey J, Uggla A. Redescription of Sarcocystis levinei Dissanaike and Kan, 1978 (Protozoa: Sarcocystidae) of the water buffalo (Bubalus bubalis). J Parasitol 1997;83:1148-52). The globular to oval microscopic cysts commonly observed in the muscles of the diaphragm and neck exhibit compartmentalized arrangement of zoites with septal partitions and measure 13-48 microns in diameter. The parasitophorous vacuolar membrane of sarcocyst bears minute and hair-like villar protrusions measuring 2.3-2.75 microns long emanating at certain distances from the primary cyst wall and lack microfilaments. Villar protrusions have expanded to dome-shaped base measuring 0.33-1.6 microns long by 0.22-1.0 micron wide, and intermediate and tapering distal segments bent approximately 90 degrees and run parallel to the cyst surface. The distal segments at some areas join to form conical tufts. The primary cyst wall bears numerous prominent undulations that are arranged in small clusters. The ground substance is 0.42-0.57 micron thick. This paper documents the first report of S. levinei in Philippine water buffaloes possessing the type 7 cyst wall.
Food-borne parasitic zoonoses have emerged as a major public health problem in many countries and are posing a medical challenge. They are not only important from the economic point of view but also because of their severe sequelae. In Malaysia, these parasitoses are a tip of an iceberg problem. The article documents all the food-borne parasitic zoonoses reported in Malaysia. An epidemiological assessment of the diseases with research needs is highlighted.
The prevalence of human skeletal muscle sarcocystosis in Malaysia was determined by serial examination of formalin-fixed, paraffin-embedded sections of tongue tissues obtained from consecutive, routine autopsies of subjects aged 12 years or more. Of 100 tongues examined, 21% were found to contain Sarcocystis; 66 cysts were found. The number of cysts per case varied from 1 to 13. In one case, 5 cysts were found in a single tissue section. The age range of positive cases was from 16 to 57 years (mean 37.7 years). Prevalence did not differ with regard to race, sex or occupation. The prevalence of human muscular sarcocystosis in our study was higher than that reported elsewhere. Preferential localization of Sarcocystis in tongue or head and neck and/or genuinely high prevalence in south-east Asia are possible explanations for this observation.
Sarcocystosis, an unusual parasitic zoonotic disease, is caused by coccidian/ apicomplexan protozoa in humans and animals. The parasites usually develop in a heteroxenous predator-prey life-cycle involving final (carnivore) and intermediate (omnivore/herbivore) hosts. Besides the intestinal, non-invasive form of the disease in which humans and animals are the definitive hosts for certain Sarcocystis spp., the invasive form has come to recent attention. In the latter, humans and animals serve as intermediate host harbouring sarcocysts in their muscle tissue. Already in 1991 sarcocystosis was seen as a potential emerging food borne zoonosis in Malaysia, and in 2011 and 2012 the largest cluster of symptomatic human muscular sarcocystosis world-wide was reported from Tioman Island, Pahang state. In this review, we focus on invasive sarcocystosis in humans and animals in Malaysia, review the recorded cases and epidemiology, and present hypotheses.
An outbreak of fever associated with myalgia and myositis occurred in 2012 among 89 of 92 college students and teachers who visited Pangkor Island, Malaysia. The Sarcocystis nesbitti 18S rRNA gene and sarcocysts were obtained from muscle tissues of 2 students. Our findings indicate emergence of S. nesbitti infections in humans in Malaysia.
A number of methods have been used for the detection of the presence of microsarcocysts in animals, but little information exists on the value between the various methods. This study therefore examined for Sarcocystis spp. using three different methods in 105 samples of skeletal muscle collected from goats slaughtered in an abattoir in Selangor, Malaysia from January to February 2014. Three methods were used, direct light microscopy of squashed fresh muscle tissues; histological examination of fixed, sectioned, and hematoxylin and eosin (H&E)-stained samples of muscle; and molecular identification by polymerase chain reaction (PCR). Of the 105 tissue samples, 55 (52.38 %) were positive by light microscopy (LM), 46 (43.8 %) by histology, and 95 (90.48 %) by PCR. Only 29 (27.6 %) and 5 (4.76 %) samples were positive and negative, respectively, by all three methods. The cysts were elongated to a spindle shape with a mean size of 393.30 × 81.6 μm and containing banana-shaped bradyzoites of size 12.32 × 2.08 μm. The wall of the cyst was radially striated with a thickness of 2.83 μm. Samples were tested for the presence of Sarcocystis-specific 18S rRNA and were identified as Sarcocystis capracanis. Of the three methods used, the PCR test appears to be the most useful method for the diagnosis of sarcocystosis especially for species identification.
Travels, migration and circulation of goods facilitate the emergence of new infectious diseases often unrecognized outside endemic areas. Most of emerging infections are of viral origin. Muscular Sarcocystis infection, an acute illness acquired during short trips to Malaysia, and Chagas disease, a chronic illness with long incubation period found among Latin American migrants, are two very different examples of emerging parasitic diseases. The former requires a preventive approach for travelers going to Malaysia and must be brought forth when they return with fever, myalgia and eosinophilia, while the latter requires a proactive attitude to screen Latin American migrant populations that may face difficulties in accessing care.
Sarcocystosis outbreaks in Tioman and Pangkor islands of Malaysia between 2011 and 2014 have raised the need to improve Sarcocystis species detection from environmental samples. In-house works found that published primers amplifying the 18S rRNA gene of Sarcocystis either could not produce the target from environmental samples or produced Sarcocystis DNA sequence that was insufficient for species identification. Using the primer pair of 18S S5 F (published) and 28S R6 R (new), this study improved the PCR amplification of Sarcocystidae to overcome these two difficulties. The PCR product spanned from the 18S to 28S rRNA genes, providing more information for species identification. The long DNA sequence allowed comparison between the "Ident" and "Query Cover" sorting in GenBank identity matching. This revealed the ambiguity in identity matching caused by different lengths of reference DNA sequences, which is seldom discussed in the literature. Using the disparity index test, a measurement of homogeneity in nucleotide substitution pattern, it is shown that the internal transcribed spacer (ITS)1-5.8S-ITS2 and 28S genes are better than the 18S gene in indicating nucleotide variations, implying better potentials for species identification. The example given by the handful of Sarcocystidae long DNA sequences reported herein calls for the need to report DNA sequence from the 18S to the 28S rRNA genes for species identification, especially among emerging pathogens. DNA sequence reporting should include the hypervariable 5.8S and ITS2 regions where applicable, and not be limited to single gene, per the current general trend.
The prevalence of sarcocystosis in cattle and water buffaloes from peninsular Malaysia was investigated in abattoirs in Selangor state, February, 2011, to March, 2012. Fresh muscle samples were collected from the tongue, heart, oesophagus, diaphragm and skeletal muscles of 102 cattle and 18 water buffaloes. Each sample was initially screened by light microscopy and then fixed for further histopathological analysis. Out of 120 animals examined, 49 (40.8%) harboured the microscopic type of Sarcocystis spp. The positivity rate for cattle was 36.2% and for water buffaloes 66.7%. In cattle, the organs highly infected were the skeletal muscles and diaphragm (27% each), followed by tongue and esophagus (24.3% each), and the heart (8%). In water buffaloes, the heart was most often infected (66.7%), followed by the oesophagus (50%) and skeletal muscle (33.3%); no sarcocysts were detected in the tongue and diaphragm. The shape of the sarcocyst was fusiform to oval with a mean cyst size of 151.66 x 75.83 μm and wall thickness of 2.47 μm in cattle, and 114 x 50.81 μm cyst size and the wall thickness of 1.11 μm in water buffaloes, consistent with Sarcocystis cruzi and Sarcocystis levinei, respectively. Remaining tissue from cattle was subjected to parasite specific 18S rRNA gene PCR and Sarcocystis cruzi was confirmed, at least exemplarily. The peripheral metrocytes and the banana-shaped bradyzoites (15.23 x 2.2 μm in cattle and 11.49 x 2.45 μm in water buffalo hosts) were easily recognized. In conclusion, a high positivity rate was found in Malaysian meat-producing animals with possible implications for meat consumption and human health.
Soil-transmitted helminth (STH) infections have been documented among these minority groups since 1938. However the prevalence of STH is still high among these communities. Most studies tend to consider the Orang Asli (indigenous) as a homogenous group. In contrary, different subtribes have their own cultural practices. To understand this variation better, we studied the prevalence and associated factors of STH and other gut parasitic infections among two common subtribes (i.e. Temuan and Temiar). Results showed that the prevalence of the overall STH infections was higher in the Temuan subtribe (53.2% of 171) compared to the Temiar subtribe (52.7% of 98). Trichuris trichiura (46.2%) was the most prevalent parasite in the Temuan subtribe, followed by Ascaris spp. (25.7%) and hookworm (4.1%). In contrast, Ascaris spp. (39.8%) was more prevalent among the Temiar subtribe, preceded by T. trichiura (35.7%) and finally hookworm (8.3%). There were also co-infections of helminthiasis and intestinal protozoa among both Temuan and Temiar subtribes with rates being three times higher among the Temiar compared to Temuan. The most common co-infection was with Entamoeba histolytica/dispar/moshkovskii (n = 24; 24.5%, 16.0-33.0), followed by Giardia spp. (n = 3; 3.1%, -0.3-6.5). In Temuan, STH infection individuals were also infected with Entamoeba histolytica/dispar/moshkovskii (n = 11; 6.4%, 5.0-13.8), Cryptosporidium spp. (n = 3, 1.8%, -0.2-3.8) and Giardia spp. (n = 2, 1.2%, -0.4-2.8). In comparison, there was no Cryptosporidium spp. detected among the Temiar. However, it was interesting to note that there was an occurrence of co-infection of intestinal helminthiasis and sarcocystosis (intestinal) in a Temiar individual. The last report of sarcocystosis (muscular) among the Orang Asli was in 1978. The present study highlighted the importance of understanding the variation of infections amongst the different Orang Asli subtribes. It is vital to note these differences and use this knowledge to customise effective control measures for the various subtribes.