Detection of protozoan parasites in ready-to-eat packaged salads

Protozoan parasites commonly infect humans and animals worldwide, and Giardia, Cryptosporidium Cyclospora and Toxoplasma have been found in irrigation water, soil and vegetables. These products are covered by EU law only with regard to bacterial pathogens. The aim of this study was to investigate possible contamination by protozoans in ‘ready to eat’ (RTE) salads on sale in Italy. From March 2015 to February 2016, 486 packages of RTE salads produced by industrial brands and by local companies were purchased from supermarkets and grocery shops, respectively. Nine individual packages (100g each) were collected per sampling month (n=9), pooled and processed. After concentration, the pellets were subjected to microscopy, immunofluorescence, RealTime-PCR and sequencing. A total of 54 pooled samples were tested i.e., 243 industrial brand packages and 243 local brand packages. Four (7.4%, 95% CI 0.4-14.4) pools tested positive to Blastocystis hominis and 1 (1.8%, 95% C.I. 0-5.4) to Dientamoeba fragilis, by microscopy; whereas Giardia duodenalis, Cyclospora cayetanensis and Toxoplasma gondii were detected in 6 (11.5%, 95% CI 2.8-20.2), 7 (13.5%, 95% CI 4.1-22.7) 2 (3.8%, 95% CI 0-9.1) samples, respectively, but only molecularly. Mixed contamination was found in 4 (7.7%, 95% CI 0.5-14.9) samples. The mean number of protozoa contamination was 0.4 protozoa species/pool in industrial brands and 0.3 in local brands. This work represents the first large-scale study on packaged salads in Europe. These results show that 'ready to eat' salads are contaminated by one or more emerging protozoan pathogens and that the sanitation process (from harvesting to packaging) cannot guarantee a product free from protozoans of fecal origin. These results indicate the need for additional research on the sources of contamination of these foods and determination of parasite viability and infectivity, and for improvement of Hazard Analysis and Critical Control Points efficiency to reduce possible risks for human health.