P1-30 Detection of Cyclospora cayetanensis on Fresh Produce: Assessment of Improved Produce Wash, DNA Extraction, and Molecular Detection Procedures

Sunday, July 26, 2015
Exhibit Hall (Oregon Convention Center)
Seulgi Lee , Food and Drug Administration , Laurel , MD
Alexandre DaSilva , U.S. Food and Drug Administration , Laurel , MD
Helen Murphy , Food and Drug Administration , Laurel , MD
Introduction: Cyclospora cayetanensis is a coccidian parasite causing foodborne diarrheal disease linked to consumption of contaminated fresh produce such as leafy greens and raspberries. During the U.S. outbreaks of 2013 and 2014 a total of 935 cases of cyclosporiasis were confirmed in several states.  In both outbreaks, some illnesses were linked to the consumption of imported fresh cilantro.

Purpose: Detection of Cyclospora cayetanensis on produce relies heavily on efficient oocyst recovery and sensitive molecular methods since this organism cannot be enriched or cultured. The objective of this study was to evaluate new methods of detection for Cyclospora cayetanensis on produce for future implementation into FDA regulatory analyses.

Methods: Samples of cilantro (25 g) and raspberries (50 g) were seeded with oocysts. Six or more replicates (n) at each seeding level were tested. Oocysts were recovered from seeded samples using a detergent wash solution and DNA was extracted using a commercial kit.  Molecular detection was performed using a conventional nested PCR assay currently used in FDA labs and a new optimized qPCR assay.

Results: Nested PCR detected C. cayetanensis DNA in 100%, 94%, 44%, and 38% of cilantro replicates seeded with 200 (n=16), 10 (n=16), 5 (n=16), and 2 (n=8) oocysts, respectively. Using qPCR, detection rates were 100%, 94%, 69%, and 63%, respectively. Detection rates using nested PCR for raspberries seeded with 200 (n=18), 10 (n=16), 5 (n=16), and 2 (n=6) oocysts were 100%, 63%, 19%, and 33%, respectively, and 100%, 75%, 31%, and 33%, respectively, using qPCR.

Significance: The low detection limit for C. cayetanensis was 10 or fewer oocysts on two commodities, which is the presumed infective dose for this organism. In addition, qPCR provides a robust, streamlined, and faster alternative to nested PCR. These results support public health and the FDA mission by providing improved detection methods for the foodborne parasite, Cyclospora cayetanensis.