Diagnosis of Clostridium difficile infection using real-time PCR.

Clostridium difficile is known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Toxinogenic strains of the bacterium produce toxins A (TcdA) and B (TcdB), which are associated with the pathogenicity. The standard methods for diagnosis of C.

Comparison of molecular typing methods applied to Clostridium difficile.

Since the 1980s the epidemiology of Clostridium difficile infection (CDI) has been investigated by the application of many different typing or fingerprinting methods. To study the epidemiology of CDI, a typing method with a high discriminatory power, typeability, and reproducibility is required. Molecular typing methods are generally regarded as having advantages over phenotypic methods in terms of the stability of genomic markers and providing greater levels of typeability.

Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing.

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.

Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study.

In this prospective multicentre study, an enzyme-linked fluorescent assay (VIDAS CDA2; bioMérieux), an enzyme-linked assay [Premier Toxins A and B (PTAB); Meridian] and an in-house real-time PCR amplifying the tcdB gene were compared with the cell cytotoxicity assay used as the 'gold standard' for diagnosis of Clostridium difficile-associated diarrhoea (CDAD). Faecal samples from patients with a request for C. difficile diagnosis and samples from patients with diarrhoea hospitalized for at least 72 h were collected for 3 consecutive months from four university medical centres in The Netherlands.

Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis.

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other.

Clostridium difficile ribotype 027, toxinotype III, the Netherlands.

Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain.

Prospective multicenter evaluation of a new immunoassay and real-time PCR for rapid diagnosis of Clostridium difficile-associated diarrhea in hospitalized patients.

In a prospective multicenter study, 367 fecal samples from 300 patients with diarrhea were tested for Clostridium difficile-associated diarrhea (CDAD) with a new immunochromatography assay for toxins A and B (ICTAB), a real-time PCR on the toxin B gene, and the cell cytotoxicity assay. Twenty-three (6.2%) of the 367 fecal samples were positive by the cell cytotoxicity assay.

Coexistence of multiple PCR-ribotype strains of Clostridium difficile in faecal samples limits epidemiological studies.

Clostridium difficile is an important cause of antibiotic-associated diarrhoea. The simultaneous presence of different strains in individual faecal samples has not yet been established, but is important for epidemiological studies. Recurrences of Clostridium difficile-associated diarrhoea (CDAD) are observed in 15-20 % of patients and have been reported as relapses or reinfections with a new strain.

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains. Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods.