Clostridium difficile Detection and Identification Methods
Gram stain of Clostridium difficile - Courtesy CDC
- Obligate anaerobe
- Commonest identifiable cause of antibiotic associated diarrhea
- Toxins A & B can cause illness independantly
Recently, outbreaks of
Clostridium difficile Associated Diarrhea (CDAD) have made the news
headlines. C. difficile is the commonest identifiable cause of
antibiotic associated diarrhea (AAD).
Following changes in the gut flora
associated with the administration of broad spectrum antibiotics, the colon becomes colonised with
this opportunistic organism which can produce two toxins: A and B.
Toxin A is an enterotoxin that causes fluid secretion, mucosal damage and
internal inflammation. Toxin B is a more potent cytotoxin than toxin A, but is
not enterotoxic. The toxins cause a characteristic mucosal damage consisting of
plaque-like lesions that may lead to the formation of a pseudomembrane and
pseudo-membranous colitis, severe cases may be fatal. Not all strains of C.
difficile produce both toxins and some are not toxigenic at all and
therefore do not cause any illness.
The discovery of C. diff strains (initially in North America and subsequently Europe) that have increased virulence has emphasised the
need for effective screening and control measures.
for these items:
lateral flow assays
Isolation of C. difficile from stool samples is not diagnostic of CDAD but is
important for the epidemiological typing of strains. Isolation is best
performed after alcohol shock treatment. Stool samples are treated with an equal
volume of methylated spirit or absolute alcohol, homogenised using a vortex
mixer and left at room temperature for 1 hour, then inoculated on to
Clostridium difficile selective agars e.g. CDSA with 7% horse blood; Cycloserine, Cefoxitin, egg yolk agar (CCEY), and
incubated anaerobically for 40 - 48hrs.
Following primary selective culture, rapid latex agglutination tests allow identification
of presumptive colonies of C. difficile within minutes. The latex particles are coated with
antibodies specific for C. difficile somatic antigen. These screening tests
can be used to reduce the number of samples that need to be progressed onto a
toxin assay. If required confirmation may be done by biochemical identification tests.
For diagnosis of CDAD it is necessary to demonstrate the presence of
C. difficile toxins. Toxin B may be detected by the production of a
cytopathic effect (CPE) of faecal extracts on Vero cell cultures that is neutralised by C. sordellii antitoxin. It takes between 24 and 48 hours
for a final result to be obtained. A CPE that is not neutralised by C.
sordellii antitoxin may indicate that another pathogen is present in the
faeces e.g. C. perfringens or Vero-toxic E. coli. Many labs use
locally developed assays but commercial cytotoxity kits are available..
Direct testing of stool sample extracts for toxin presence using a variety of immunoassay techniques has largely replaced the culture method. The toxin is unstable at room temperature so
the sample needs to be processed quickly or held refrigerated. Both toxins of C. difficile may be detected by these tests and there are a
range of commercially available kits in different formats. As strains which
only produce either toxin A or B have been reported as pathogenic, methods which detect
both are preferable.
For the high volume laboratory, microtitre plate or automated methods are available.
Assays can also be presented in a lateral flow format as single tests which
give faster results, normally within a few minutes and with less operator skill
requirements. Some assays are designed to detect C. difficile common antigen (glutamate
dehydrogenase or GDH) produced by both toxigenic and non-toxigenic strains, and can also have a toxin test included in the panel so both toxin and organism may be revealed.
Studies using molecular detection of toxin A and B genes have been performed but they
remain technically more demanding and hence more expensive and do not confirm
expression of toxin. They do however allow identification of hypervirulent strains of C. difficile e.g. PCR ribotype 027 [or NAP1 in North Americas].
C. difficile infections are mainly healthcare associated, however there are increasing reports of community acquired infections. The need for the implementation effective infection control measures is clear.
Hypervirulent strains are of increasing concern.
The need for epidemilogical and antibiotic resistance data would appear to encourage a return to cultural techniques - at least in some targeted clinical situations.
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