Development of an Asymmetric Five-Primer Loop-Mediated Isothermal Amplification for the Detection of Sarcocystis spp. in Humans

Introduction: Sarcocystis comprises Apicomplexan species with a two-host life cycle and humans may be both intermediate and definitive hosts for some of these species. Sarcocystis undergoes sexual reproduction in the intestinal epithelium and oocysts are subsequently shed in host stool in definitive hosts. At least two species, namely, Sarcocystis hominis and Sarcocystis suihominis, exist with humans as definitive hosts and hence as potential causes of human intestinal sarcocystosis, with cattle and pigs as intermediate hosts, respectively. Notwithstanding the potential for intestinal sarcocystosis to afflict a large number of people, extent and importance of human sarcocystosis remain largely unknown.

Purpose: The objective of this study has been the development  of a new molecular diagnostic tool (LAMP method) to facilitate the detection of the parasites in stool samples and the understanding of the epidemiology and clinical significance of Sarcocystis infections in humans.

Methods: A set of five primers were designed according to the conserved region of the 18S rRNA gene of S. hominis and S. suihominis by using the software PrimerExplorer V4. Alignment of Cystoisospora belli and Cryptosporidium hominis were used to ensure the specificity of the assay. The LAMP products were monitored using 2% agarose gel electrophoresis and also visually detected by adding 2 μL of the amplification product to 1:1000 diluted fluorescent dye EuroSafe to the reaction tubes, visualized under UV light. Samples from human stools (n=10), positive to S. hominis, and pig muscular tissue harbouring S. suihominis (n=5) , were used as positive samples. Stool samples that tested negative with the conventional PCR for Sarcocystis spp.(n=20) were included as negative samples.

Results: The new LAMP protocol confirmed the results of the conventional PCR for Sarcocystis spp., demonstrating, at least, the same sensitivity and specificity.

Significance: The new assay is therefore a rapid, simple and specific method for the detection of zoonotic Sarcocystis in humans.