Cyclospora cayetanensis is an apicomplexan protozoan and is one of the most common pathogens causing chronic diarrhea worldwide. Eight stool samples with diarrheal symptom out of 18 Korean residents who traveled to Nepal were obtained, and examined for 25 enteropathogens including 16 bacterial species, 5 viral species, and 4 protozoans in stool samples as causative agents of water-borne and food-borne disease. Only C. cayetanensis was detected by nested PCR, and 3 PCR-positive samples were sequenced to confirm species identification. However, the oocysts of C. cayetanensis in fecal samples could not be detected by direct microscopy of the stained sample. As far as we know, this is the first report of a group infection with C. cayetanensis from a traveler visiting Nepal, and the second report of a traveler’s diarrhea by C. cayetanensis imported in Korea.
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This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler’s diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAMTM, HEXTM, Cy5TM, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler’s diarrhea.
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