Evaluation of a modified method for the detection of Cryptosporidium oocysts on spinach leaves

Despite the infection risk associated with the consumption of contaminated food, techniques for recovering and detecting Cryptosporidium oocysts from fruit and vegetables are generally inadequate due to the variable recovery efficiencies and high reagent costs, such as those presented by ISO 18744:2016 "Microbiology of the food chain -Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits". Although an improved method for recovering these parasites from Iceberg lettuce, which reported increased recovery efficiency as well as lower costs, has been published, it appears to have limitations for the recovery of Cryptosporidium from saponin-rich leaves such as spinach (Spinacia oleraceae), which have previously been implicated in Cryptosporidium parvum outbreaks. In this study, we refined the method to improve its use with these more challenging samples. The use of alkaline elution buffer (1 M glycine) of different pH values was evaluated for their effectiveness in removing C. parvum from spinach leaves. The refinement of Utaaker's method showed, from spinach leaves inoculated with 100 oocysts, an increased oocyst recovery rate with an overall mean recovery rate of 33.79% ± 2.82%. The emergence of parasitic foodborne illnesses and outbreaks associated with the consumption of fresh produce demonstrates the need for the development of an optimal recovery process for parasites from suspected foods. Results showed that refinement of existing protocols could improve the retrieval of Cryptosporidium oocysts from these more challenging leafy greens.