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Evaluation of a new LightCycler approach for detection of diarrhoeagenic Escherichia coli

Abstract number: 1733_95

Ljung R., Eysturskard J., Olesen B., Bruun B., Hansen D.S.

Objectives: Modern laboratory diagnosis of infectious gastroenteritis comprises the classical diarrhoeagenic E. coli: verotoxin-producing E. coli (VTEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC).

Our objective was to compare a novel LightCycler (LC) real-time PCR approach to our standard identification performed by a reference laboratory (ref. lab.).

Methods: During 2 two-months periods, October-November 2005 and May-June 2006, stool samples examined for diarrhoeagenic E. coli were investigated by two different approaches: 1) the standard procedure which was sending the sample to a reference laboratory for analysis by conventional multiplex PCR, and 2) a new LC real-time PCR approach, detecting the eae, vtx1, vtx2, LTI, STIa, STIb, ipaH, and 16S rDNA (internal amplification control) genes from over night cultures. We used the 1.5 LC with 4.0 software, Master Mix from Roche Diagnostics, and primers and hydrolysis probes from TIBMolbiol, Berlin. All protocols used were previously described in the literature except for the ipaH and 16S rDNA genes which were developed in our laboratory.

Results: A total of 371 stool samples were analysed. The number of positive samples and patients in our laboratory and at the ref. lab. were 49 and 34, and 35 and 22, respectively. From 9 of the 14 samples initially not found positive at the ref. lab., 8 E. coli strains were isolated that consequently confirmed our results when examined by the reference laboratory. For one patient our results could be verified, as another sample from the patient was found positive at the ref. lab. Four samples could not be verified as we were unable to isolate the bacteria resulting in the positive PCR result. The number of diarrhoeagenic E. coli found in our laboratory and at the ref. lab. (in brackets), respectively, was: 5 (3) VTEC, 6 (5) EPEC, 11 (9) ETEC, 3 (2) EIEC and 24 (16) intimin producing E. coli (eae positive, but not belonging to the classical EPEC O-groups).

Conclusions: A positive rate of 7% shows that detection of the classical diarrhoeagenic E. coli is diagnostically important. Additionally 6% of samples were positive for intimin producing E. coli, whose clinical importens is disputed. Our results demonstrate that the new LC real-time PCR approach is at least as sensitive (and specific) as standard identification in a reference laboratory, identifying totally 40% more positive samples and 55% more patients.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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