C-223. Development of a Rapid Screening Assay for the Detection of Zoonotic Cryptosporidium Species in Human Faeces

A. Alagappan1, N. A. Tujula1, M. L. Power1, P. L. Bergquist1,2, B. C. Ferrari1;
1Macquarie Univ., Sydney, AUSTRALIA, 2Univ. of Auckland Med. Sch., Auckland, NEW ZEALAND.

Background: Cryptosporidiosis is a major diarrhoeal illness caused by the protozoan parasite Cryptosporidium. In humans, 7 Cryptosporidium species are potentially pathogenic but the majority of infections can be attributed to 2 species; the zoonotic species C. parvum and the host-specific species C. hominis. Current methods for Cryptosporidium detection used by diagnostic pathology laboratories provide presence/absence data only, but risk management requires species information. Species identification is only possible using DNA-based research tools that are cumbersome, time-consuming, and expensive and therefore, not conducive to routine applications. Hence rapid and sensitive detection methods that can aid in species identification possess a huge potential. Methods: A simple and rapid multi-colour Fluorescence in situ Hybridisation (FISH) assay was developed for the identification of the dominant species of Cryptosporidium. The assay uses two fluorescent probes, each labeled with a different fluorescent dye (FITC and Cy3) that specifically target the 18S rRNA of C. parvum and C. hominis, enabling differentiation between the two species. Probe specificity was validated against PCR-RFLP of the 18S rRNA gene using 33 human faecal samples positive for Cryptosporidium. Results: The FISH assay indicated that the occurrence of C. parvum and C. hominis in human faeces was 60% and 37% respectively. A PCR-RFLP method indicated that 59.3% of samples were C. parvum and 39.4% as C. hominis. The correlation coefficient between PCR-RFLP and FISH was 0.994, indicating a strong correlation between the methods. Conclusion: Species identification using FISH was as reliable as the results obtained from PCR-RFLP which is commonly used in research for Cryptosporidium species identification. The microscopy-based FISH assay is simple, cost-effective and a quick screening tool for differentiating C. hominis and C. parvum in faecal samples, water and other environmental samples.