Testing times for C. difficile

C. difficile is increasingly common, requiring improved detection and testing.

Clostridium difficile is a spore-forming Gram-positive bacterium that can be found in stool specimens of many healthy children aged under two years, and some adults. Following antimicrobial treatment, toxin-producing strains of C. difficile are a common cause of antibiotic-associated diarrhoea. 

C. difficile is a major nosocomial pathogen causing a range of symptoms from mild to severe diarrhoea and is the aetiological agent of pseudomembranous colitis. Most toxigenic strains produce two cellular cytotoxins, A and B, responsible for the clinical features of infection.

Rising Incidence

There are several possible explanations for the rise in C. difficile disease over the past three decades. Better detection methods have almost certainly contributed to the increase in reported cases.

High-frequency use of antibiotics and chemotherapeutics increases the likelihood of acquiring C. difficile-associated disease. Also, as the disease frequency has increased, hospitals have become contaminated with spores of C. difficile, making infection of susceptible patients more likely. 

The incidence and severity of C. difficile has increased in both hospital and long-term facilities due in part to the emergence of several novel strains classed as hypervirulent. These strains of C. 

difficile include ribotypes 027, 078, 014, 001 and 106. Hypervirulent strains express increased production of toxins, so that spore production is increased. They are also resistant to fluoroquinolones and third-generation cephalosporins. 

The worldwide emergence of hypervirulent strains of C. difficile has caused extensive outbreaks in hospitals and the community, with significant mortality, especially in the elderly. 

Laboratory Testing

Testing algorithms must be adjusted to enable detection of hypervirulent strains. Historically, the cell culture cytotoxicity assay (CCCA) that detects cytotoxin production has been the gold standard for C. difficile detection. However, cell culture is labour-intensive and results are not available rapidly enough to be clinically useful. 

Accordingly, many laboratories have adopted other testing methods, such as enzyme immunoassays (EIAs) for toxins A and B, which are easier and faster to perform than CCCA. Recent reports, however, have highlighted the lack of sensitivity of such EIAs for toxins A and B. Sensitivities may be as low as 48%, meaning  more than half the cases are not detected by this method. 

Other approaches to improve both the sensitivity and the cost-effectiveness of C. difficile testing have been introduced. Laboratories can use an assay for a C. difficile-specific enzyme, glutamate dehydrogenase (GDH). This assay has a higher sensitivity (detects more cases) but lacks specificity (may have more false positives), because GDH assays detect antigen present in both toxigenic and non-toxigenic strains. 

In recent times, higher sensitivity and specificity of polymerase chain reaction (PCR) assays means that PCR has emerged as a potential replacement for the less-sensitive EIA and less-specific GDH assays. This test has a high negative predictive value, which can be useful to exclude infection.

Methods for C. difficile testing in hospitals have evolved due to the need for more accurate assays to detect those patients with C. difficile infections (CDI). The GDH assay can be used as a screening test and then GDH-positive results can be confirmed with one of the following tests: toxin A and toxin B EIA, CCCA or PCR. 

Although these multi-test algorithms improve the specificity of the GDH assay, they delay the reporting of confirmatory results to the clinician, particularly if CCCA or PCR is used for confirmation. 

Many clinicians start empirical therapy when a patient is suspected of having CDI, but some will wait for the results before stopping other antibiotics and initiating specific CDI therapy (e.g. metronidazole or vancomycin). 

This delay, especially in combination with the low sensitivity of the EIA, can facilitate the spread of C. difficile to other patients and further contaminate the environment, increasing the potential for the patient to develop more severe disease. 

C. difficile can be detected in the stool of infected patients by using tests that are commonly available in most hospital laboratories. 

Identifying hypervirulent strains and their ability to produce toxins is complex and performed by only a limited number of Australian laboratories. Patient outcome should be considered when deciding what approach to C. difficile testing is required.

Effective infection control and antibiotic policy may help prevent the establishment of hypervirulent strains in healthcare facilities. 

1. Eastwood et al JCM Oct 2009 p.3211-3217

2. Ballard and Voth JCM Apr 2005 p.247-263

3. Weekley et al JCM Mar 2010 p.889-893

4. MacCannell et al JCM Jun 2006 p.2147-2152

5. NSW Health Dec 2010 – Clostridium difficile resources for clinicians and patients. 

Dr Hudson is a clinical microbiologist and infectious diseases physician

Co-author: Tom KaragiannisSenior hospital scientist, Microbiology Department, Pacific Laboratory Medicine Services (PaLMS)