Sarcocystis (sarco = flesh; cystis = cyst-forming) are intracellular protozoan parasites characterised by their ability to invade muscle tissue, where they mature into sarcocysts. The life-cycle of Sarcocystis involves an obligate prey-predator relationship with a definitive host (the predator) and an intermediate host (the prey). Upon ingestion of sarcocyst-infected muscle tissue from the intermediate host, sexual stages develop within the small intestine of the definitive host, and then sporulated oocysts containing sporocysts are expelled in the faeces. Ingestion of sporocysts by an intermediate host, i.e. the faecal-oral route, enables the asexual stages of Sarcocystis to develop, resulting in intramuscular sarcocysts [1].
Humans are the definitive host for at least two known Sarcocystis species, S. hominis and S. suihominis, and ingestion of undercooked beef and pork, respectively, that contain sarcocysts leads to intestinal sarcocystosis, which can induce symptoms such as nausea, vomiting and enteritis. Most cases, however, are presented as mild or asymptomatic [2]. Conversely, only one Sarcocystis species is known to utilize humans as intermediate hosts. This species is S. nesbitti and it was acknowledged to be involved in the largest known acute muscular sarcocystosis (AMS) outbreak, which occurred in 2011 and 2012 on Tioman Island, Malaysia [3].
In 2011 and 2012, GeoSentinel and TropNet reported a total of 100 cases involving foreigners travelling to Tioman Island during the summer months, i.e. from July to August. All patients exhibited two distinct symptoms, relapsing fever and myalgia, whilst a few reported suffering from arthralgia, asthenia, headache, cough and diarrhoea [3]. Additionally, facial swelling was reported in a few of the affected individuals. Histological examination of muscle biopsy samples that were obtained from six patients, stained with hematoxylin and eosin (H&E), revealed sarcocysts within the muscle fibres. Observation of the sarcocyst wall via electron microscopy and molecular characterisation via amplification of the 18S rRNA gene region revealed that S. nesbitti was the cause of the AMS outbreak [4].
Sarcocystis nesbitti utilises a snake-primate life-cycle [5–7], where humans are considered an aberrant host presenting intermediate host-like symptoms. As transmission of muscular sarcocystosis occurs via the faecal-oral route, infection probably occurred via ingestion of either food or water contaminated with S. nesbitti sarcocysts. Most water supplied to residents of Tioman Island comes from untreated environmental sources, i.e. gravity feed systems and tube wells [8]. It was therefore hypothesised that water contamination was the most probable cause of the AMS outbreak. A surveillance study conducted in November 2011, however, reported that all water samples tested negative for presence of S. nesbitti [8]. This was attributed to the water samples being too dilute, making it impossible to detect sporocysts via microscopy.
To overcome the diluting factor, this study screened water samples indirectly by obtaining sediment from respective water sources. As sporocysts are purified using floatation techniques [1], it was assumed that sporocysts in water would sink and be trapped in the sediment of water tanks and rivers with relatively stagnant water. In addition to microscopy, samples were screened via PCR due to its greater sensitivity when compared to microscopy alone.
In previous studies, the 18S rRNA gene has proven to be a suitable candidate for species identification [5, 9]. In this study, therefore, amplification of the 18S rRNA gene with subsequent BLASTn and phylogenetic analyses was conducted in order to identify potential S. nesbitti sources, and to also determine what other Sarcocystis species are present on the island.