Pyrosequencing for rapid molecular identification of Schistosoma japonicum and S. mekongi eggs and cercariae.

Schistosomiasis, which is caused by Schistosoma japonicum and S. mekongi, is a chronic and dangerous widespread disease affecting several countries in Asia. Differentiation between S. japonicum and S. mekongi eggs and/or cercariae via microscopic examination is difficult due to morphological similarities. It is important to identify these etiological agents isolated from animals and humans at the species or genotype level. In this study, a pyrosequencing assay designed to detect S. japonicum and S. mekongi DNA in fecal samples and infected snails was developed and evaluated as an alternative tool to diagnose schistosomiasis. New primers targeting the 18S ribosomal RNA gene were designated for specific amplification. S. japonicum and S. mekongi were identified using a 43-nucleotide pattern of the 18S ribosomal RNA gene and were differentiated using 7 nucleotides within this region. S. japonicum and S. mekongi-infected snails and fecal samples derived from infected mice and rats were differentially detected within a short period of time. The analytical sensitivity of the method enabled the identification of as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and 2 eggs inoculated in 100mg of non-infected fecal sample. To evaluate the comparative efficacy of the assay, identical samples were also analyzed via microscopy and Sanger sequencing. The pyrosequencing technique was found to be superior to the microscopy method and more rapid than the Sanger sequencing method. These results suggest that the pyrosequencing assay is rapid, simple, sensitive and accurate in identifying S. japonicum and S. mekongi in intermediate hosts and fecal samples of the final host.