Development of New Methods for Potential Detection of Blastocystis sp. in Fresh Produce

Introduction: The zoonotic parasite, Blastocystis sp., has been regularly detected in surface water and on leafy vegetables. Surveillance studies revealed that Blastocystis infection is very common among food handlers worldwide. Prevalence of Blastocystis in food industry personnel ranged from 4.87% in Turkey, to 35.5% in Egypt, 38.7% in Venezuela, 41.5% in Thailand, and 41.7% in Mexico. In the US, Blastocystis is one of the most frequently found parasites in humans and is prevalent in livestock.

Purpose: To develop immunofluorescence microscopy and molecular techniques to facilitate detection and identification of Blastocystis sp. in produce. 

Methods: Conserved regions of published nucleotide SSU rDNA sequences of Blastocystis (available from GenBank) were used to develop a set of primers for PCR. A new immunoflourescence microscopic test for Blastocystis sp. (Boulder Diagnostics) was evaluated for sensitivity and specificity by conducting Blastocystis detection in biologic samples containing multiple zoonotic protozoan pathogens such as Giardia duodenalis, Cryptosporidium spp., and Enterocytozoon bieneusi.

Results: A specific pair of PCR primers was successfully developed for the identification and subtyping of Blastocystis. The specificity of these primers was confirmed by the successful amplification of DNA from all Blastocystis specimens and their inability to amplify DNA from other protist parasites present in the sample. For the first time the successful immunofluorescence detection of Blastocystis in complex poly-protist samples was completed.

Significance: Development and application of superior detection and identification procedures for Blastocystis are needed to evaluate the true prevalence as well as routes of parasite transmission and contamination of fresh produce. The methods described herein are potentially applicable to screening of fresh produce.