Detection of human Sarcocystosis using dried blood on filter papers: An Immunofluorescent Antibody Test

@#Sarcocystosis, a parasitic infection caused by a protozoa belonging to the genus Sarcocystis, is found worldwide in both and animals. Sarcocystis spp., require two animal hosts to complete their life cycle. The infection has gathered more global attention after recent outbreaks, especially amongst wester travellers to Malaysia. Other than sporadic cases and the current outbreaks, little information is available regarding human Sarcocystis infection in Malaysia. The present study aims to determine the prevalence of sarcocystosis among humans using an immunofluorescent antibody (IFA) test applied to dried blood on filter papers. A total of 200 blood samples were collected on filter papers from autopsy cases at two Malaysian hospitals: Sungai Buloh Hospital (peninsular Malaysia) and Queen Elizabeth Hospital (Malaysian Borneo). Antigens were prepared from bradyzoites harvested from positive goats’ muscle samples. Of the 200 samples, 32 (16%) had Sarcocystis antibodies that showed positive fluorescence reactions on filter papers. There was no significant difference (t-test, p value > 0.05) in prevalence rates between samples collected from autopsies at peninsular Malaysia and Malaysian Borneo. The results demonstrated that the filter paper technique can be used as one of the alternative serological tests in the diagnostic of human sarcocystosis.

A quadriplex PCR assay for rapid detection of diarrhoeacausing parasitic protozoa from spiked stool samples

@#Diarrhoea is a leading killer of children, accounting for 9% of all deaths among children under age 5 worldwide and 3% in Malaysia in 2015. A large proportion of diarrhoea illnesses among children in developing countries are ascribed to an unknown etiology because microscopic examination was the only available technique which has low detection limits. The proposed study aimed to evaluate a new quadriplex PCR assay to detect parasitic pathogens namely E. histolytica, G. lamblia and C. parvum which considered responsible for the majority of human infections. Three set of specific primer pairs were designed for detection of parasitic pathogens. Quadriplex PCR assay was optimized and an internal amplification control was incorporated to check for PCR inhibitors in samples. The PCR assay was evaluated using spiked stool samples. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity of the quadriplex PCR at the DNA level was found to be 50 ng DNA. The analytical specificity was evaluated with 11 reference protozoal and bacterial strains and was found to be 100%. We concluded that the developed quadriplex PCR assay was rapid and gave results within 5 hours which is essential for the identification of parasitic pathogen and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of parasite that cause diarrhoea.

Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia

One thousand and forty-five tissue samples of skeletal muscles, tongue, heart, diaphragm and esophagus were collected from 209 animals (43 sheep, 89 goats and 77 cattle) from an abattoir in Selangor between February and October, 2013. Each sample was divided into three pieces with each piece measuring 2-3 mm3. Each piece was then squeezed between two glass slides and examined microscopically at x 10 magnification for the presence of sarcocystosis. Three positive samples from each animal species were then fixed in 10% formalin for histological processing. Seven positive samples collected from each animal species were preserved at -80°C or 90% ethanol for gene expression studies. Microsarcocysts were detected in 114 (54.5%) animals by light microscopy (LM). The infection rates in sheep, goat and cattle were 86, 61.8 and 28.6% respectively. The highest rate of infection was in the skeletal muscles of sheep (64.9%) and goats (63.6%) and in the heart of cattle (63.6%). The cysts were spindle to oval in shape and two stages were recognized, the peripheral metrocytes and centrally located banana-shaped bradyzoites. 18S rRNA gene expression studies confirmed the isolates from the sheep as S. ovicanis, goats as S. capracanis and cattle as S. bovicanis. This, to the best of our knowledge, is the first molecular identification of an isolate of S. ovicanis and S. capracanis in Malaysia. Further studies with electron microscopy (EM) are required in the future to compare the features of different types of Sarcocysts spp.