Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

Abstract

To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in 'ready to eat' (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin.

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

“…oocysts or Giardia duodenalis cysts, there are no standardized methods available for detection of T. gondii oocysts in water and food samples. Current methods are mainly based on PCR (Villena et al 2004;Karanis et al 2012;Wells et al 2015;Marchioro et al 2016;Cong et al 2017;Lalle et al 2018) and microscopy (Al-Megrin 2010; Harito et al 2017;Caradonna et al 2017); however, they do not provide information regarding the viability of the detected parasites. Interest for molecular-based methods to assess viability of food and waterborne parasites is growing.…”

A Molecular Tool for Rapid Detection and Traceability of Cyclospora cayetanensis in Fresh Berries and Berry Farm Soils

“…To improve sensitivity in PCR-based detection of C. cayetanensis, two strategies were implemented; first, a nested PCR approach was designed because this pathogen is found at low concentrations in fruits and environmental samples [13]. Second, PCR assays were designed to amplify the 18S rRNA locus because multi-copy genes increase PCR sensitivity [30].…”

“…Importantly, these primer sets have been included in a new U.S. Food and Drug Administration method developed for detection of C. cayetanensis on cilantro and raspberries [31]. To improve sensitivity in PCR-based detection of C. cayetanensis, two strategies were implemented; first, a nested PCR approach was designed because this pathogen is found at low concentrations in fruits and environmental samples [13]. Second, PCR assays were designed to amplify the 18S rRNA locus because multi-copy genes increase PCR sensitivity [30].…”

“…This level of sensitivity could be attributed to the inclusion of bovine serum albumin in each PCR reaction to overcome the effects of PCR-inhibitory substances found in environmental samples [32]. Thus, this nested PCR protocol could be a useful tool for the rapid and accurate detection of C. cayetanensis in environmental samples where oocyst concentrations are considered low; for example, 10 2 to 10 3 per gram of produce [13], 10 3 per liter of river water [6], 10 1 per liter of tap water [11], and 10 2 to 10 5 per gram of stool [33]. Because an infective dose of C. cayetanensis has been estimated between 10 1 and 10 2 oocysts [34,35], this PCR assay is sensitive enough to detect these levels of contamination in berries and environmental samples.…”

“…However, this technique is time-consuming, non-specific, and lacks 2 of 11 sensitivity [9]. To overcome these issues, molecular methods have been developed to detect C. cayetanensis in clinical and environmental samples [10][11][12]; however, the food producing industry requires a molecular method able to detect a low oocyst concentration (40-1500 oocyst per gram) as found in food and environmental samples [13]. Also, it is important to have a tool available to perform molecular traceability and identification of contamination sources.…”