Outbreak of Clostridium difficile 027 infection in Vienna, Austria 2008-2009.

From November 2008 to 15 April 2009, 36 isolates of CD027 identified in Austria, all originating from four hospitals in Vienna. All isolates were positive for toxin A, toxin B and the binary toxin, and showed a characteristic 18 bp deletion in the tcdC gene. Clostridium difficile is an anaerobic spore-forming bacterium. Some strains may cause diarrhoea due to formation of toxins. Symptomatic C. difficile infection (CDI) is primarily linked with hospital admission and antibiotic treatment, although antibiotic exposure is neither necessary nor sufficient for CDI [1,2]. In Belgium, for instance, one third of CDI cases reported in the hospital surveillance system are not hospital-associated [3]. Symptoms range from mild diarrhoea to serious manifestations such as pseudomembranous colitis, toxic megacolon or perforation of the colon. C. difficile challenges hygiene standards as it is forms spores. The risk of infection rises with increasing age, underlying disease and immunodeficiency [4]. In recent years, a particularly virulent strain, ribotype 027 (CD027), has emerged in a number of countries, particularly in connection with hospital outbreaks, but also in community-acquired diarrhoea cases [5]. The risk of serious disease and death associated with CD027 exceeds that of other C. difficile strains. The classical CD027 is characterised - among other things - by an increased production of toxins A and B, production of a binary toxin and resistance to newer fluoroquinolones such as moxifloxacin. The first three Austrian cases of CD027 occurred in 2006 and in March 2008 [6,7]. Since August 2006, the Austrian National Reference Centre for C. difficile has ribotyped approximately 2,700 human C. difficile isolates received from all nine Austrian provinces. In recent months, a drastic increase in CD027 cases has been noted, all originating from four hospitals in Vienna. From November 2008 to 15 April 2009, 36 isolates of CD027 were received at the National Reference Centre. The Figure summarises these C. difficile 027 cases by month of reception of the sample at the reference centre.

Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping.

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006-2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.