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Validating the Updated BAM Chapter 19b Method for the Detection of Cyclospora cayetanensis in Outbreak-Linked Fresh
1Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742, USA.
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Cyclospora cayetanensis is a human-specific protozoan parasite that induces the diarrheal illness cyclosporiasis. This study evaluated the ability to detect C. cayetanensis in artificially contaminated fresh produce commodities previously linked to C. cayetanensis outbreaks. Parsley, basil, and broccoli were evaluated using the updated, highly specific Bacteriological Analytical Manual (BAM) Chapter 19b method. To our knowledge, this is the first performance evaluation of any method for C. cayetanensis detection in broccoli. Individual samples (25 g) of each commodity were seeded with 5 and 200 C. cayetanensis oocysts. Unseeded produce samples of each commodity were used as negative controls. The method included a produce washing procedure, followed by C. cayetanensis DNA concentration and extraction, and molecular detection of the C. cayetanensis mitochondrial Cox3 gene (Mit1C qPCR). The method was slightly modified for broccoli at the washing step. As few as five oocysts were detected in each of the analyzed produce. No statistically significant differences were observed in any detection parameters between parsley and basil. However, average Mit1C C. cayetanensis CT values were significantly higher in broccoli compared to both parsley and basil in high-level (200 oocysts) seeded samples (p < 0.05). In addition, lower percentages of positive rates were observed in broccoli compared to the two herbs in samples seeded with five oocysts (83.3%, 75.0%, and 41.7% for basil, parsley, and broccoli, respectively). The average Mit1C C. cayetanensis CT values were also significantly higher in broccoli compared to parsley and basil in samples seeded with five oocysts (p < 0.05). We speculate that the florets of broccoli may trap oocysts and make washing less efficient. The differences observed in fresh produce detection confirm previous studies showing the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions. These studies will ensure effective detection of C. cayetanensis in basil, parsley, and broccoli in future surveillance and outbreak investigations.
