Development of a sensitive detection system for Cryptosporidium in environmental samples

The identification of Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition. Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of Cryptosporidium parasites in environmental samples.