Description of the test
The test consists of:
- testing for toxins in stool or culture by enzymatic immuno-assays.
- evaluate the toxigenic potential of C. difficile by cytopathogenic effect on cell cultures.
- to simultaneously detect with a multiplex the genes for toxin B (tcdB), toxin A (tcdA), binary toxin (cdtA and cdtB) and the deletions of 18bp, 39bp and 54bp in the regulatory gene tcdC.
Purpose of the test
The main virulence factor of C.difficile is cytotoxin B. Most pathogenic strains are toxin A- and toxin B-positive (A+B+) or, more rarely, toxin A-negative and toxin B-positive (A-B+). A small number of C.difficile strains also produce a binary toxin. The binary toxin locus contains two different genes (cdtA and cdtB). In hypervirulent strains such as 027/NAP1/BI, in addition to the A&B and binary toxin genes, mutations in the toxin downregulation gene (tcdC), namely a deletion of 18bp and of a nucleotide at position 117, can be shown. These would affect the amount of toxin the strain can produce.
Criteria for performing this test in the context of reference activities.
After contacting the laboratory.
Instructions for samples
The strain should be supplied as pure culture and freshly inoculated on a suitable medium. Suitable media are: deep gelose, platelet anaerobically packed type Anaerogen Compact (Oxoid), Anaerocult p (Merck) or other such methods, reduced media, thioglycolate or ‘ port a cul ’ type tubes. Although it is recommended to send cultures, the NRC also accepts stool samples.
Instructions for transport
- Transport is best done under anaerobic conditions. Transport at room temperature if the sample arrives at the lab within 6 hours of collection, or refrigerated within 72 hours (after that, stool samples can be frozen at -20°C). Make sure the tubes are tightly closed to prevent ‘leaking’; to this end, you can wrap the closed tube in parafilm.
- The sample is packed with absorbent material in a transport blister pack. This secondary packaging can also be a plastic zip-lock bag. This is then placed in an envelope or box.
- According to the applicable guidelines, the following symbol must appear on the outer packaging: UN3373 Biological substance category B.
- The NRC does not provide sample collection equipment.
Unacceptable requests
- Samples damaged or leaked in the packaging.
- Samples with missing information.
Turn around time (and frequency of analysis)
Analyses are performed on request (non-surveillance form). For urgent requests, contact the laboratory before sending strains.
- Maximum 10 working days for detection of toxin in stool or culture by enzyme-linked immunosorbent assay.
- Maximum 42 working days for testing for cytopatheogenic effect and virulence factors by multiplex PCR.
Reporting of test results
- By post.
- If specifically requested on the application form, by fax or e-mail.