Untersuchung zur Etablierung einer Real-time-PCR zum schnellen Screening auf Clostridium difficile aus Patienten- und Umgebungsproben

Clostridium difficile ist der häufigste Erreger von Antibiotika-assoziierten Diarrhoen und hat in den letzten Jahren als nosokomialer Problemkeim stark an Bedeutung gewonnen. Das Bakterium ist grampositiv, stäbchenförmig, obligat anaerob und bildet Sporen aus. Bei einer Antibiotikatherapie wird d...

Full description

Saved in:
Bibliographic Details
Main Author: Albrecht, Ulla Maria
Contributors: Mutters, Reinier (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2011
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Table of Contents: Clostridium difficile is the leading cause of antibiotic associated diarrhoe. Over the last years clostridium difficile became more important because of the occurence of new more toxigenic and hyper virulent strains. Clostridium difficile is a gram positive, anaerobe, rod-shaped and spore-forming bacteria. In case of an antibiotic therapy the normal flora of the intestine is deranged and Clostridium difficile colonize the intestine. The main transmission of Clostridium difficile is faecal-oral. The bacteria formes three different toxines, toxin A, toxine B and the binary toxine. The binary toxine is often present in the new more toxigenic and hypervirulent strains, for example in the strain Nap1/027. Infected patients excret a lot of spores that persist for months in a clinical environment if they are not eliminated by spore-effective desinfection. The spores can be the origin of the infection of other patients. For this reason, a quick and sensitive diagnostic tool to detect Clostridium difficile is necessary in order to take measures against the spreading of spores. In this study at first a Real-time PCR Assay for detecting the 16S rRNA of Clostridium difficile was established. After that, 242 faecal samples from two patient-groups were tested by the Real-time PCR Assay detecting the 16SrRNA of Clostridium difficile. The first group contains faecal samples, which were tested positive in an toxinimmunoassay, detecting toxine A and B. The second group contains faecal samples which were tested negative in an ELISA test detecting toxine A and B. The Real-time PCR Assay reached high levels of sensitivity and specificity and it was shown that the Real-time PCR Assay was a quick and reliable method to detect the 16 SrRNA of Clostridium difficile in faecal samples. All samples were also tested by a conventional PCR Assay detecting toxine A, B and the binary toxine. In most cases the result of the conventional PCR Assay detecting toxine A and B confirms the result of the toxinimmunoassay. In ten samples the binary toxine was detected which shows that there are new strains of Clostridium difficile also at the university clinic of Marburg. In a second study 531 hospital environmental samples werte examined by the Real-time PCR assay detecting the 16 S rRNA of Clostridium difficile. A first group contains samples from the environment of patients infected by Clostridium difficile, a second group contains patients not infected with Clostridium difficile but present at the same ward as the infected patient. A third group contains samples from wards without the detection of Clostridium difficile within the last six months. The study revealed that not only the direct surrounding of an infected patient is contaminated by the spores of Clostridium difficile but the entire ward. In a correlation-analysis it became evident that the hands of the staff are the predominant way of transmission of spores of Clostridium difficile. Thus it is evident that the most important way to prevent the transmission of Clostridium difficile are hand-hygiene and desinfection activities.