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Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

Toxoplasma gondii is a ubiquitous foodborne protozoan that can infect humans at low dose and displays different prevalences among countries in the world. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there are no regulations for monitoring oocysts in food, mainly because of a lack of standardized methods to detect them. The objectives of this study were (i) to develop a reliable method, applicable in biomonitoring, for the rapid detection of infectious oocysts by cell culture of their sporocysts combined with quantitative PCR (sporocyst-CC-qPCR) and (ii) to adapt this method to blue and zebra mussels experimentally contaminated by oocysts with the objective to use these organisms as sentinels of aquatic environments. Combining mechanical treatment and bead beating leads to the release of 84% ± 14% of free sporocysts. The sporocyst-CC-qPCR detected fewer than ten infectious oocysts in water within 4 days (1 day of contact and 3 days of cell culture) compared to detection after 4 weeks by mouse bioassay. For both mussel matrices, oocysts were prepurified using a 30% Percoll gradient and treated with sodium hypochlorite before cell culture of their sporocysts. This assay was able to detect as few as ten infective oocysts. This sporocyst-based CC-qPCR appears to be a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts directly in water and also using biological sentinel mussel species. This method offers a new perspective to assess the environmental risk for human health associated with this parasite. [ABSTRACT FROM AUTHOR]

Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp. is responsible for several food and waterborne disease outbreaks worldwide. Healthier lifestyles attract consumers to eat, notably, fresh food like fruits and vegetables. The consumption of raw or under-cooked food increases the risk of foodborne transmission of Cryptosporidiosis. The assessment of the consumer's exposure to Cryptosporidium danger is crucial for public health. Still, the standardized method to detect this parasite in fresh leafy greens and berry fruits has only been available since 2016 and suffers from weaknesses. Consequently, in this study, we propose a method with minimum processing steps that combines cell culture and the quantitative PCR (CC–qPCR) for detecting infectious C. parvum oocysts recovered from lamb's lettuce. This CC–qPCR is a rapid and easy method that can detect up to one oocyst, whereas it is undetectable by classic qPCR. [ABSTRACT FROM AUTHOR]