To facilitate the harmonized surveillance and investigation of cyclosporiasis outbreaks in the US and Canada, we adapted and verified the US-FDA’s BAM 19b method and employed it in a national produce survey. Performance was verified by spiking 200, 10, 5 or 0 C. cayetanensis oocysts onto berries (50 ± 5 g, n = 85) and 200, 10 or 0 oocysts onto green onions (25 ± 3 g, n = 24) and leafy greens (25 ± 1 g, n = 120) and testing these samples by the BAM method on Bio-Rad CFX96. Method robustness was assessed by aging (0 or 7 days) and freezing the produce and washes prior to testing, then implementing the method for the surveillance testing of 1759 imported leafy green, herb and berry samples. Diagnostic sensitivity was 100/44% and 93/30% for berries and leafy greens spiked with 200/10 oocysts, respectively. The diagnostic and analytical specificity were 100% for all matrices and related parasites tested. The proportion positive was unaffected (p = 0.22) by age or condition of produce (7d, fresh, frozen) or wash concentrate (3d, fresh, frozen); however, the Cq values were higher (p = 0.009) for raspberries aged 7d (37.46 ± 0.29) compared to fresh (35.36 ± 0.29). C. cayetanensis was detected in berries (two), herbs (two) and leafy greens (one), representing 0.28% of the tested survey samples. These results independently verified the reported performance characteristics and robustness of the BAM method for the detection of C. cayetanensis in a variety of matrices, including under adverse sample conditions, using a unique detection platform and demonstrating its routine diagnostic use in our Canadian Food Inspection Agency (CFIA) laboratory.

BackgroundZoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp.MethodsWe compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites.ResultsThe protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile.ConclusionThe new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Giardia duodenalis is one of the most common food and water-borne intestinal parasites of humans and animals worldwide. Fresh, ready-to-eat produce such as leafy greens and salad mixes are considered potential transmission vehicles for Giardia infection in humans. Therefore, a specific, sensitive, and reliable method for Giardia detection in leafy greens is needed. We optimized washing procedures for the recovery of Giardia cysts from leafy greens and adapted and validated an existing EF1α LAMP assay for the detection of Giardia DNA to support routine diagnostic surveillance and disease outbreak investigations. Four leafy green types (35 ± 1 g) were spiked with 100 Giardia cysts and we compared washing by shaking with 1 M glycine ( n = 20) or 0.1% Alconox ( n = 20). DNA was extracted from washes, tested by LAMP and melt curve analysis, and time to positive (TTP) values compared. The detection limit was determined by spiking 10 ( n = 40) Giardia cysts onto these same types of leafy greens and processing as above with 0.1% Alconox. Method robustness was assessed by subjecting spring mix ( n = 45 total) to aging (1, 3 or 7 days) and washes to aging and freezing conditions prior to testing. Assay repeatability and specificity were evaluated, and an artificial positive control (APC) distinguishable by melt temperature (Tm) from DNA of Giardia spiked on leafy greens was designed to rule out cross-contamination from the control. Giardia detection rates were higher and TTP was lower ( P < 0.05) for 0.1% Alconox (19/20, 8.85 ± 0.3 min) compared with 1 M glycine (15/20, 14.53 ± 7.2 min). The LAMP assay detected 10 Giardia cysts spiked on leafy greens in 13–34 min in 14/40 samples tested. Robustness assessment showed that TTP was higher ( P < 0.0001) when spiked produce was stored for 7 days (13.09 ± 1.14 min) compared to fresh (9.72 ± 0.43 min). No unspiked samples were positive by LAMP, and the Tm for DNA of Giardia spiked on leafy greens was higher ( P < 0.0001, 87.43 ± 0.05 °C) than the APC (86.43 ± 0.12 °C). Within-assay repeatability co-efficient of variation (CV) for TTP was 5.4% and no cross-contamination occurred when spiked and un-spiked samples were processed in alternate order. The optimized sample processing procedure combined with the EF1α LAMP assay is a sensitive, specific, labour-saving, and rapid method for the detection of Giardia cysts in leafy greens.