Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective.

Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal-oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.

Development of a Molecular Marker Based on the Mitochondrial Genome for Detection of Cyclospora cayetanensis in Food and Water Samples.

Cyclospora cayetanensis is a coccidian parasite that causes diarrheal illness outbreaks worldwide. The development of new laboratory methods for detection of C. cayetanensis is of critical importance because of the high potential for environmental samples to be contaminated with a myriad of microorganisms, adversely impacting the specificity when testing samples from various sources using a single molecular assay. In this study, a new sequencing-based method was designed targeting a specific fragment of C. cayetanensis cytochrome oxidase gene and developed as a complementary method to the TaqMan qPCR present in the U.S. FDA BAM Chapter 19b and Chapter 19c. The comparative results between the new PCR protocol and the qPCR for detection of C. cayetanensis in food and water samples provided similar results in both matrices with the same seeding level. The target region and primers in the protocol discussed in this study contain sufficient Cyclospora-specific sequence fidelity as observed by sequence comparison with other Eimeriidae species. The sequence of the PCR product appears to represent a robust target for identifying C. cayetanensis on samples from different sources. Such a sensitive method for detection of C. cayetanensis would add to the target repertoire of qPCR-based screening strategies for food and water samples.

Analysis of enterotoxigenic Bacillus cereus strains from dried foods using whole genome sequencing, multi-locus sequence analysis and toxin gene prevalence and distribution using endpoint PCR analysis.

Bacillus cereus strains were isolated from dried foods, which included international brands of spices from South East Asia, Mexico and India purchased from several retail stores, samples of powdered infant formula (PIF), medicated fish feed and dietary supplements. The genetic diversity of 64 strains from spices and PIF was determined using a multiplex endpoint PCR assay designed to identify hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Thirteen different B. cereus toxigenic gene patterns or profiles were identified among the strains. Randomly selected B. cereus strains were sequenced and compared with reference Genomic Groups from National Center Biotechnology Information using bioinformatics tools. A comprehensive multi-loci sequence analysis (MLSA) was designed using alleles from 25 known MLST genes specifically tailored for use with whole genome assemblies. A cohort of representative genomes of strains from a few FDA regulated commodities like dry foods and medicated fish feed was used to demonstrate the utility of the 25-MLSA approach for rapid clustering and identification of Genome Groups. The analysis clustered the strains from medicated fish feed, dry foods, and dietary supplements into phylogenetically-related groups. 25-MLSA also pointed to a greater diversity of B. cereus strains from foods and feed than previously recognized. Our integrated approach of toxin gene PCR, and to our knowledge, whole genome sequencing (WGS) based sequence analysis, may be the first of its kind that demonstrates enterotoxigenic potential and genomic diversity in parallel.