Multi-Laboratory Evaluation of Modified BAM Chapter 19b and Long-Term Detection of Cyclospora cayetanensis in Fresh Salsa/Pico de Gallo

Cyclospora cayetanensis is a foodborne parasite that causes the human diarrheal disease cyclosporiasis. Multiple outbreaks of C. cayetanensis have been linked to Mexican-style dishes containing uncooked cilantro. The Bacteriological Analytical Manual (BAM) Chapter 19b method for the detection of C. cayetanensis has been validated in cilantro, but not in complex foods containing cilantro, such as fresh salsa/pico de gallo. In a previous study, specific modifications of the BAM method were performed in fresh salsa/pico de gallo for optimal detection of C. cayetanensis. In the present study, further evaluation of those modifications in fresh salsa/pico de gallo were performed using the updated BAM Chapter 19b mitochondrial-based qPCR (Mit1C qPCR). Six FDA laboratories analyzed six blind-coded salsa/pico de gallo test samples each. These samples included two unseeded samples, three samples seeded with five oocysts, and one sample seeded with 200 oocysts. All laboratories performed the updated washing and DNA extraction modification steps. The overall C. cayetanensis detection rates across laboratories for fresh salsa/pico de gallo samples inoculated with 200 oocysts, five oocysts, and un-inoculated samples were 100% (6/6), 100% (18/18), and 0% (0/12), respectively. The method showed extremely high sensitivity and high reproducibility across all laboratories. Un-inoculated samples were all negative for each laboratory, indicative of high C. cayetanensis specificity. This is the first evaluation of the performance of detection of C. cayetanensis using the updated BAM Chapter 19b (Mit1C qPCR) in a complex matrix, e.g., fresh salsa/pico de gallo. A second study evaluated the presence of the parasite in a long-term study in fresh salsa/pico de gallo samples inoculated with C. cayetanensis and analyzed up to three weeks after inoculation. Samples were positive for the detection of the parasite at each time-point of the study, indicating that the method could be used to detect the parasite in leftovers of fresh salsa/pico de gallo up to three weeks old in outbreak investigations. Evaluation and deploying effective methods to detect C. cayetanensis in high-risk fresh produce and prepared dishes are critical to monitor and respond to active outbreaks.