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

Abstract

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.

A summary of cryptosporidiosis outbreaks reported in France and overseas departments, 2017–2020

“…Before 2017, only 6 cryptosporidiosis outbreaks in France were reported in the literature ( Beaudeau et al, 2008 ; Dalle et al, 2003 ; Mosnier et al, 2018 ). Many reasons could explain the increased number of reported cases of cryptosporidiosis outbreaks in France: i) the proactive monitoring of the CNR-LE-Cryptosporidiosis, ii) the growing number of laboratories in the CNR-LE-Cryptosporidiosis network, iii) the democratisation of syndromic and multiplex molecular methods allowing parasitological investigations which were not initially suspected, and iv) the improvement of food and environmental screening methods ( de Roubin et al, 2002 ; Razakandrainibe et al, 2020 ; Utaaker et al, 2015 ).…”

“…Since 2016, an international standardardized method exists for the detection and enumeration of Cryptosporidium (and Giardia ) in fresh leafy green vegetables and berry fruits: ISO 18744:2016. However, the recovery of Cryptosporidium oocysts varies according to food matrices, sometimes demonstrating weak performances (especially with saponin-rich matrices) ( Razakandrainibe et al, 2020 ; Utaaker et al, 2015 ). Thus, Cryptosporidium screening from food matrices appears very complicated and sensitivity is low.…”

Protozoan parasites and free-living amoebae contamination in organic leafy green vegetables and strawberries from Spain

“…Several modifications to this standard method for recovering these parasites to increase recovery efficiency as well as lower costs have been published by Utaaker et al (2015) . On spinach leaves, Razakandrainibe et al (2020) also reported that the recovery of Cryptosporidium from saponin-rich leaves, such as spinach ( Spinacia oleraceae ), can be increased by using an alkaline solution instead of the acidic 1 M glycine buffer recommended in ISO 18744:2016. In the present study, an alkaline detergent elution buffer at pH 8 has been employed; however, Cryptosporidium oocysts counts on spinach leaves remained zero.…”

Detection of Infectious Cryptosporidium parvum Oocysts from Lamb’s Lettuce: CC–qPCR’s Intake

“…After incubation, the oocyst recovery was realized according to the ISO 18744:2016 "Microbiology of the food chain -Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits" method with some modifications. The pH of 1 M glycine elution buffer (5.5) was adjusted to 3 for better recovery (data not shown), and for each step, samples were centrifuged at 3000× g for 30 min at 4 • C. Before oocyst purification by IMS, pellets were resuspended, and 2 mL of a detergent solution containing 1% SDS (Sigma), 0.1% Tween 80 (Sigma), and 0.001% antifoam Y (Sigma) were added as described by Razakandrainibe et al [27]. After centrifugation, pellets were resuspended in 7 mL of PBS, and oocysts were recovered by IMS (Isolate, TCS Biosciences, Buckingham, UK).…”