Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli from Nonenriched Stool Specimens by Real-Time PCR in Comparison to Enzyme Immunoassay and Culture

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of food-borne outbreaks of diarrhea (15). Disease caused by STEC is characterized by abdominal pain and bloody diarrhea, and 5 to 15% of those individuals infected with serotype O157:H7 develop hemolytic uremic syndrome (HUS), a potentially life-threatening condition consisting of hemolytic anemia, thrombocytopenia, and kidney failure caused primarily by Stx (8). STEC may carry genes for one or both types of Stx, Stx1 and Stx2 (17).Although STEC strains are a diverse group of pathogens, up to the present, the most common serotype in the United States has been O157:H7. A common association is that of E. coli O157:H7 contaminating ground beef (3, 7), but recent large outbreaks have involved a variety of other foods, including leafy greens (6, 29). The diversity of potentially contaminated food means that patients may acquire STEC infection from many foodstuffs, far beyond the stereotypical risk of undercooked ground beef. The common denominator of tainted food products seems to be direct or indirect contamination from bovine feces. To best detect infected patients and potential outbreaks, clinical laboratories must have tools to quickly and accurately detect STEC in stool specimens. Culture on sorbitol MacConkey agar is an inexpensive, effective, and widely used method based on lack of sorbitol fermentation by E. coli O157:H7. Several drawbacks limit the utility of culture, including slow turnaround, false-negative results in antibiotic-treated patients, and false-negative results due to emerging serotypes of non-O157 STEC that ferment sorbitol (1, 14, 16, 29). Alternatively, a method that is increasingly utilized is detection of Stx antigen from stool, either directly or after broth enrichment. Our experience concurs with enzyme immunoassay (EIA) product insert data that optimal sensitivity and specificity are achieved only when a broth enrichment step is employed; this results in slow turnaround.Here, we describe a real-time PCR assay that can detect STEC using nucleic acid extracts of stool specimens. We evaluated the performance of this assay using both archived stool specimens and prospectively collected specimens and compared the results to those of culture and Stx antigen detection.(This study was presented in part at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)