Standardising molecular detection methods to IMprove risk assessment capacity for foodborne protozoan Parasites, using Cryptosporidium in ready-to-eat salad as a model

Protozoan parasites such as Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are important causes of human and animal disease. Between them, these three parasites were estimated in 2010 to cause about 47 million cases of illness (incidence 185 cases per 100,000 population) in Europe. Outbreaks are increasingly recognised and attributed to consumption of food; fresh produce and leafy greens in particular. To date, there is a lack of readily-applicable standardised methods for testing food and feedstuffs, which could provide additional evidence in risk assessments and validating control points and interventions. The aim of the IMPACT project was to increase the European-level capacity for risk assessment of foodborne protozoa using Cryptosporidium and ready-to-eat salad leaves as a model example. A review of procedures for the detection of Cryptosporidium in fresh produce has led to the production of guidance for undertaking artificial contamination studies to verify and validate detection methods, and as well as to the selection of a real-time PCR to help improve detection. These were achieved through literature review, a market survey of oocyst producers and suppliers, questionnaires, e-meetings and a workshop of experts. A SOP for Cryptosporidium molecular detection of oocysts in leafy greens by real-time PCR was developed and evaluated in two laboratories and rolled out to four other recipient laboratories for optimisation. Video tutorials were compiled to facilitate SOP implementation. Validation of the SOP through a ring trial involved five partners from the consortium and seven further European laboratories.

This is a short summary of the project results.For detailed information, please refer to the report obtainable under https://www.efsa.europa.eu/de/supporting/pub/en-7195
Differences in artificial contamination protocols, and the lack of reporting of the efficiency and performance of methods used in surveys of fresh produce has made comparisons and understanding of the occurrence of parasites such as Cryptosporidium difficult. Through a combination of surveys, consultations, and a workshop, we have developed guidance that we hope will improve the consistency and comparability of assays reporting the results of artificial-contamination experiments and lead to improved method development and reporting of sample surveys. The guidance developed in this project should be adopted by those carrying out artificial contamination studies.The guidance has been proposed for adoption by ISO/TC 34/SC 9/WG 3 ‘Method validation’ for incorporation in to Microbiology of the food chain — Method validation — Part #: Protocol for the validation of alternative methods against a reference method for viruses, and parasites and other difficult to culture microorganisms.Although many papers describing molecular methods for Cryptosporidium detection were identified by the systematic reviews and questionnaire, only a few of the methods described were fully validated and as such fulfilled our expectations. Most were found inappropriate either due to cross-reactivity (particularly in environmental samples), difficulty in interpretation of the results, inapplicability to all Cryptosporidium species and or insufficient validation. The qPCR presented by Kristin Elwin from Public Health Wales (PHW) at the IMPACT workshop targeted all Cryptosporidium species, had high analytical sensitivity, high specificity, and good discriminatory power for Cryptosporidium species by Sanger sequencing and was thus selected as the appropriate method to be used for the purpose of the IMPACT project.A comparative and iterative experimental approach was applied for the evaluation and optimization of the three different steps in the procedure to detect C. parvum oocyst DNA in leafy greens. A SOP was compiled and could be implemented in all partner laboratories, even with limited or no expertise in the subject, providing that at least one online training was offered. The ring trial results of the trained laboratories mirrored the results found during the validation/ implementation stage of the project and a detectability of 10 oocysts was reached. The SOP proved to be robust, reaching the expected performance independently from reagents or equipment used. Based on the ring trial results of the external laboratories, it became clear that when implementing the SOP in a new laboratory, practice and verification steps are clearly needed. Especially the selection of the master mix and determining the Ct cut-off value are necessary steps. We also recommend to plan a training in expert laboratory or to access to online training resources (i.e. video tutorials prepared in this project) prior implementation.To assess the impact of the project on both consortium members and external participants and also identify the potential future use of the guidance document and/or the SOP in the participating laboratories, an online questionnaire was administered . In total, 16 of 21 persons answered the questionnaire within the allocated time. Ten were consortium members, six were external partners. 56% of all participants classified themselves as having little experience/ proficiency in the detection of Cryptosporidium oocysts from fruits and/or vegetables prior to the IMPACT project, 38% rated their experience as middle and only one person defined their prior experience as high.The areas where more than half of the participants stated that their confidence increased significantly included 1) performing method evaluation/ implementation, 2) implementing a new SOP in the laboratory, 3) understanding quality control measures needed for artificial contamination experiments with protozoa, 4) working with leafy greens as a food matrix, 5) working with Cryptosporidium oocysts, 6) performing leafy green sample preparation steps up to DNA extraction, 7) comparing results from contamination experiments and 8) participating in a ring trial.
The two areas the consortium members gained most experience were 1) performing leafy green sample preparation steps up to DNA extraction and 2) understanding quality control measures needed for artificial contamination experiments with protozoa, whereas the external partners benefitted most in the areas 1) participating in a ring trial and 2) understanding quality control measures needed for artificial contamination experiments with protozoa.