title: 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.
abstract: 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 immunoassay (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.
authors.affiliation: Medical Laboratory Bremen, Bremen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gerritzen
authors.forename: Johann-Wolfgang
authors.initials: JW
authors.lastname: Wittke
authors.forename: Dietmar
authors.initials: D
authors.lastname: Wolff
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques; D002522:Chlorocebus aethiops; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D003967:Diarrhea / Q000209:etiology / Q000382:microbiology; D004196:Disease Outbreaks; D042241:Early Diagnosis; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology*; D005243:Feces / Q000382:microbiology*; D005858:Germany / Q000453:epidemiology; D006463:Hemolytic-Uremic Syndrome / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology; D006801:Humans; D007124:Immunoenzyme Techniques; D007297:Inpatients; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D014709:Vero Cells
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2