ABSTRACT
Both the human enteric viruses and the parasitic protozoa are now recognized as significant causes of human disease, perhaps being responsible for as much as 68% and 3% of all foodborne illness in the U.S., respectively (1). Although they have been recognized for years, the human enteric viruses and parasitic protozoa could be considered “emerging” agents of foodborne disease, largely because scientists have only recently been able to detect these pathogens. In fact, prior to the advent of molecular
biological techniques, epidemiological criteria were the primary means by which cases of enteric viral and parasitic illness were recognized. Unfortunately, epidemiology had several limitations including the fact that the diseases caused by most gastrointestinal viruses and parasites were (and are) not reportable in the U.S.; only the largest, most severe, and/or most widespread outbreaks were (and are) investigated, leaving smaller outbreaks and sporadic disease underestimated; and early detection capabilities, even for clinical (fecal and blood) specimens, were severely limited. These early detection methods, which sought to
Doris H. D’Souza, Julie Jean, and Lee-Ann Jaykus Department of Food Science, College of Agriculture and Life Sciences, North Carolina State University
I. Introduction ......................................................................................................................................................188-1 II. General Detection Considerations....................................................................................................................188-3
III. Sampling ..........................................................................................................................................................188-3 IV. Pathogen Concentration....................................................................................................................................188-3
A. Principles of Virus Concentration in Foods..............................................................................................188-3 1. Virus Concentration Methods for Shellfish ......................................................................................188-5 2. Virus Concentration Methods for Other Foods ................................................................................188-6
B. Principles of Parasitic Protozoa Concentration in Foods ........................................................................188-6 1. Parasitic Protozoa Concentration Methods for Foods ......................................................................188-7
V. Nucleic Acid Extraction ..................................................................................................................................188-9 A. Nucleic Acid Extraction of Food Concentrates — Viruses......................................................................188-9 B. Nucleic Acid Extraction of Food Concentrates — Parasitic Protozoa ..................................................188-10
VI. Detection ........................................................................................................................................................188-10 A. RT-PCR Detection of Viruses in Foods ..................................................................................................188-11 B. PCR Detection of Parasitic Protozoa in Foods ......................................................................................188-14 C. Alternative Nucleic Acid Amplification Methods ..................................................................................188-14
VII. Confirmation ..................................................................................................................................................188-14 A. Real-Time Detection ..............................................................................................................................188-15
VIII. Detection of Viruses and Parasitic Protozoa in Field and Foodborne Disease Outbreak Specimens Using Molecular Methods ..............................................................................................................................188-15
IX. Discussion and Conclusions ..........................................................................................................................188-16 Acknowledgments ......................................................................................................................................................188-17 References ..................................................................................................................................................................188-17
oocyst, were based mostly on some form of microscopy. Later methods relied on detection of antigen (enzyme immunoassay) in the stool, or alternatively, on seroconversion, i.e., a rise in specific antibody titer against the pathogen. The microscopic methods had poor sensitivity, and the reagents necessary for the serological methods were not always available to clinical laboratories. Among other factors, the absence of dependable detection methods contributed to an underestimate of the true scope and significance of foodborne viral and parasitic protozoan infections.
