Description of the test
The ribotyping technique is based on the existence of multiple ribosomal RNA operons in the bacteria. These code for the 16S-23S-5S genes. The 16S and 23S genes are separated by an intergenic (non-coding) region of variable size. The ribotyping technique consists of amplifying these intergenic regions, which thus vary in number and length, via PCR. The chosen primers allow amplification from the segment of the 16S gene to that of the 23S gene. The obtained amplicons are analysed by capillary electrophoresis on a sequencer. Work on nomenclature harmonisation is ongoing in collaboration with several European teams. More than 600 different profiles have already been identified at our reference centre. When the European nomenclature (Brazier) is used, the ribotype is denoted by the prefix ‘EU’, while the other ribotypes have the prefix ‘UCL’.
Purpose of the test
In the context of the increasing incidence of C. difficile infections (CDI) and the emergence of hypervirulent strains, Sciensano, in collaboration with the NRC, is organising a national surveillance.
The aims of the surveillance are
- To monitor the incidence of CDI in hospitals and at the national level
- Identify the microbiological characteristics of strains isolated in Belgian hospitals and monitor the emergence of hypervirulent and/or multi-resistant strains
- From July 2007 to December 2014, participation was mandatory for six months (6 months) per year (Royal Decree of 19 June 2007) for all general hospitals, except Specialised and Geriatric hospitals and wards with less than 150 beds, Specialised palliative care hospitals and wards and burn wards. The first version of the protocol was published in July 2006. Since 2015, specific participation in this protocol is no longer mandatory.
Criteria for performing this test in the context of reference activities.
The isolated strains from (maximum) 5 consecutive patients collected during the surveillance period are sent to the reference laboratory, together with the print-out of the registration form on the NSIHweb site, as proof of registration in the epidemiological surveillance system. The case registration does not have to be complete (e.g., typing can be requested before knowing the evolution of the patient) but the form should contain at least the following data: code of the submitting laboratory, unique patient code, the automatic code and the NISS number.
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 material for sample collection.
Unacceptable requests
- Samples that are damaged or have leaked packaging.
- Samples with missing information.
Turn around time (and frequency of analysis).
Maximum 42 days, analysis is performed every 3 to 4 weeks as part of surveillance. For non-surveillance requests, contact the laboratory before sending strains.
Reporting of test results
- By post.
- If specifically requested on the request form, by fax or e-mail.