Shiga Toxin Detection in Stool

Shiga toxin producing Escherichia coli (STEC) have been identified as the major cause of bloody diarrhea and life-threatening Hemolytic Uremic Syndrome (HUS).

STEC-associated HUS predominantly afflicts children under the age of 5 and is a leading cause of acute renal failure in this age group. Mortality is reported to be between 2-7% and there is a significant risk of residual organ damage. In persons above 65 years, the incidence of hemorrhagic colitis is greatest and the mortality is between 15-23%. STEC has also been recognized as an important cause of common watery diarrhea (i.e. gastroenteritis).

E coli 0157:H7 is the most commonly implicated serotype, but infections involving non 0157 serotypes are increasingly common. The infectious dose is very low (in the order of 100 bacteria) and outbreaks have been linked to contaminated food, particularly undercooked ground beef (hamburgers) and unpasteurized milk, and recently to a contaminated community water supply. Contaminated drinking and swimming water, including chlorinated swimming pools, plus lakes and ocean water, have also been linked to outbreaks. Person-to-person fecal-oral transmission is common and is the predominant route of infection in daycare facilities. The organism has also been isolated in farm animals where healthy cattle act as the primary reservoir. During food processing, the bacteria are effectively distributed in a large lot of product.

The major virulence factor for E.coli 0157 and other serotypes, is the production of one or more Shiga toxins, also known as verocytotoxins. Shiga toxin1 is indistinguishable from the shiga toxin produced by Shigella dysenteriae type 1, while type 2 is a more divergent molecule. Both toxins consist of five B subunits and a single A subunit and are encoded on a temperate bacteriophage inserted into the E.coli chromosome.

E.coli 0157, the first of the STEC E. coli to be involved in outbreaks, is unique in the E.coli family in its lack of fermentation of sorbitol. Commercially prepared sorbitol agars, that display pH mediated color changes with sorbitol fermentation, form the basis of our current diagnosis in the microbiology lab. The laboratory technologist looks for sortibol-negative E. coli, and then chooses these colonies for specific identification. The disadvantage of this method is that any other organism or E.coli serotypes, that may indeed produce a shiga toxin, will not be identified. Culture technique requires between 24 and 48 hours, only detects E.coli 0157, and does not assay for the toxin itself.

A new, reliable and rapid test for the detection of shiga toxin 1 and 2 is now available. The Shiga Toxin E. coli (STEC) Microplate Assay is an enzyme immunoassay which allows direct detection of the two shiga toxins in stool specimens. The basic outline of the test is: Rabbit polyclonal anti-Shiga toxin 1 and 2 antibodies are bound to microplate wells to which diluted specimens is added. After incubation the enzyme conjugate (monoclonal mouse anti-Shiga Toxins 1 and 2 labeled with horseradish peroxidase enzyme) is added. The second incubation period is followed by an addition of enzyme substrate and then visual or spectrophotometric color detection.

The test is best done on fresh stool specimens because toxin degradation occurs after several hours. Evaluation of this test was performed at sites in the United States, Canada and Germany. It was compared to a cytotoxin assay and all positive or discrepant results were confirmed by PCR. The summation surveys confirmed a sensitivity of 87% and a specificity of 96.6%. The negative predictive value was constantly above 99% in all surveys. The Shiga Toxin E.coli (STEC) Microplate Assay detects a lower limit of 52pg/ml and 126pg/ml of shiga toxins 1 and 2 respectively. Because HUS is a clinical syndrome, the detection of STEC does not mean systemic disease. Also a correlation between the amount of toxin and the clinical presentation has not been established. This direct toxin test will replace our sorbitol agar method, which had required overnight incubation and subsequent confirmation. The Clinical Microbiology laboratory will perform this test routinely on all stool specimens. The test will be performed Monday through Friday with same day results, provided specimens reach the lab by noon. On other days, please call the Clinical Microbiology Laboratory, at 688-2460 or the Laboratory Medicine Resident.

Sanjivi Wadhwa, M.D.
Marissa Wilck, M.D.
Stephen Edberg, Ph.D., A.B.M.M.

References:

1. Karch H, Brelaszewska M, Brizon M, Schmidt. Epidemiology and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Diagn Microbiol Infect Dis 1999; 34: 229-243.

2. Mead P, Griffin P. Escherichia coli 0157:H7. The Lancet 1998; 352: 1207-1212 Alexon-Trend ProspecT Shiga Toxin E.coli (STEC) Microplate Assay , Package insert.