O0127 Validation of a multiplex real-time PCR gastrointestinal parasite panel Rachel Lau1, Jason Kwan2, Ruben Cudiamat1, Min Qun Ellen Chen1, Amanda Wang1, Krista Orejana1, Filip Ralevski1, Andrea Boggild*3,4,1

1 Public Health Ontario Laboratory, Toronto, Canada, 2 McMaster University, Hamilton, Canada, 3 University of Toronto, Department of Medicine, Toronto, Canada, 4 Toronto General Hospital, Tropical Disease Unit, Toronto, Canada Background: Microscopy is the conventional method for identification of gastrointestinal parasitic pathogens, however, it requires high technical expertise and prolonged turnaround time. Molecular methods provide higher throughput and potentially higher sensitivity and specificity. We validated a commercial multiplex parasitic real time PCR panel detecting 6 protozoal pathogens: Blastocystis hominis (Bh), Cryptosporidium, Cyclospora, Dientamoeba fragilis (Df), Entamoeba histolytica (Eh), and Giardia lamblia (Gl) at Public Health Ontario, Canada. Materials/methods: We analyzed 489 fecal specimens including: 131 banked frozen specimens containing Bh (n=13), Cryptosporidium (n=15), Cyclospora (n=13), Df (n=10), Eh (n=15), Gl (n=13), 13 mixed infections, and 39 negatives; and 358 fresh prospective specimens randomly selected from fecal parasitology submissions containing Bh (n=24), Cryptosporidium (n=4), Cyclospora (n=6), Df (n=7), Eh (n=1), Gl (n=2), 10 mixed infections, and 304 negatives. A panel of Entamoeba dispar, , Strongyloides stercoralis, , Diphyllobothrium latum, Enterobius vermicularis, hookworm, Taenia spp., mansoni, and positive specimens and human DNA were used for cross-reactivity evaluation. DNA extraction and PCR were conducted with the Hamilton Starlet automated platform and Seegene’s extraction and PCR kits. Microscopy was the reference standard for all organisms with stool ELISA as an additional reference assay for Eh. Results: Sensitivity, specificity, positive predictive, and negative predictive values for combined frozen and fresh specimens were: 93%, 94%, 67%, and 99% for Bh; 100%, 100%, 96%, 100% for Cryptosporidium; 85%, 100%, 100%, and 99% for Cyclospora; 86%, 99%, 83%, and 99% for Df; 82%, 100%, 100%, and 99% for Eh; and 96%, 99%, 83% and 100% for Gl, respectively. No cross-reactivity was observed with other protozoa or helminths. Limit of detection (parasites/g stool) was 8 for Bh, 9 for Cryptosporidium, 38 for Cyclospora, 697 for Df, 47 for Eh and 22 for Gl. Conclusions: The platform had high sensitivity for detection of Blastocystis hominis, Cryptosporidium, Cyclospora, Dientamoeba fragilis, and Giardia lamblia, but suboptimal sensitivity for Entamoeba histolytica which is most likely due to the low number of Eh-containing prospective specimens available. This enteric panel provides a useful diagnostic tool to complement microscopic helminth detection.

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