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Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia Coli Directly From Stool Samples by Real-Time PCR in Comparison to Culture, Enzyme Immunoassay and Vero Cell Cytotoxicity Assay by Andreas Gerritzen, Johann-Wolfgang Wittke, Dietmar Wolff

Background: From May until July 2011 a large outbreak of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) occurred in Germany. More than 800 patients suffered from hemolytic uremic syndrome (HUS), and 49 fatal cases were reported. Obviously, a mandatory requirement for such a clinical situation is the availability of rapid and reliable STEC tests from the investigating laboratory. The standard methods like enzyme immu-noassay (EIA), vero cell cytotoxicity assay (VCA), and microbiological culture are, however, hampered by a lack of sensitivity and specificity unless a prior, time consuming broth enrichment step is employed. In order to acelerate the laboratory diagnosis, we evaluated an in-house real-time PCR assay for the detection of the Stx genes (stx1 and stx2) directly from stool specimens without the need of broth enrichment procedures.
Methods: 754 faecal samples were collected from 481 predominantly hospitalised patients with diarrhea from May 23 to June 10, 2011 at the Medical Laboratory Bremen, Germany. The samples were analysed with a direct stx real-time PCR and compared to EIA, VCA and culturing on enterohemolysin, ESBL, and CPS agar after broth enrichment. In addition, artificial samples (n = 12) from three official EHEC/STEC PCR quality proficiency panels (INSTAND, Germany, September 2006, September 2007, and April 2008) were analysed by real-time PCR only.
Results: The real-time PCR produced reliable, distinct melting profiles with characteristic peaks for the stx1 and stx2 PCR products. The quality proficiency panels revealed a detection limit of 10 CFU/PCR per reaction. 112, 86, 99, and 122 of 754 clinical samples were positive for culture, EIA, VCA, and real-time PCR, respectively. 121 of 122 PCR samples were positive only for stx2. Compared to culture as the gold standard, sensitivities of EIA, VCA, and real-time PCR were 76.8 %, 83.9 %, and 96.4 % and specificities were 99.4 %, 99.2 %, and 97.8 %, respectively.
Conclusions: The direct fecal stx real-time PCR proved superior to enrichment based VCA and EIA and can be recommended as a quick and sensitive tool for the early diagnosis of STEC infection in addition to microbiological culture.

DOI: Clin. Lab. 2011;57:993-998