Testing for C. difficile infection (CDI)

Who should be tested?

Only patients with obvious signs and symptoms of CDI should be tested. C. difficile toxin tests have been studied and validated in patients with a high pretest probability and clinical symptoms of CDI. The sensitivity, specificity, and negative and positive prediction values of antigen and toxin assays are unknown for asymptomatic patients.¹

It is not clinically useful to test asymptomatic patients.⁶

Therefore, it is particularly important to:

• Limit testing to those patients who:
  • Exhibit the clinical symptoms of CDI, including watery or loose diarrhea three times a day or more⁶
  • Have risk factors for CDI, such as recent or current antibiotic therapy, age, and concomitant illnesses
• Test only diarrhea specimens (those specimens that take the shape of the container)

Testing for C. difficile: an historical perspective

The traditional “gold standard” for identifying toxinogenic Clostridium difficile is the cell cytotoxicity test. A laboratory cell line is exposed to toxin B in fecal eluate. C. diff is confirmed when an antitoxin reverses the effects of the toxin on the cells. Alternatively, C. diff can be cultured and then tested for the presence of toxins.²

Today, many laboratories do not perform cytotoxicity or toxinogenic culture for C. difficile due to the time and technical skills required and the need to maintain cell lines for cytotoxicity assay. Most labs now rely on enzyme immunoassay tests to expedite results to clinicians in order to assist them in making accurate diagnoses.

Tests for C. diff toxins A and B (EIA)

Enzyme immunoassay (EIA) detects the presence of C. diff toxins A and B in stool. These assays are relatively inexpensive, easy to perform, and can provide accurate, rapid results. It is important to recognize that the performance of immunoassays can vary widely by product and can also be affected by protocol deviations or improper technique or specimen handling. To optimize testing results with immunoassay testing, select the highest performing toxin immunoassays, practice careful specimen handling, and adhere to published testing protocols. 

Download the Meridian ImmunoCard^® Toxins A&B Package Insert

Download the Meridian Premier™ Toxins A&B Package Insert

Common antigen (common enzyme or GDH) test

This assay detects the presence of the enzyme, glutamate dehydrogenase (GDH), which is produced by all C. difficile organisms. GDH is also produced by other bacteria. All positive antigen results must be combined with a toxin test to confirm the presence of C. difficile toxin.

Download the Meridian ImmunoCard^® C. difficile PI

Toxigenic anaerobic culture test

Fecal specimens are shocked with heat or alcohol to kill all C. diff except spores. The spores are then incubated anaerobically on selective media. In 24-48 hours colonies will grow and fluoresce chartreuse on blood agar and give off a manure-like odor. Colonies are placed into chopped meat carbohydrate broth and incubated for up to 5 days. The culture is tested for C. diff with the cell culture cytotoxin neutralization assay (see below) or an enzyme immunoassay (EIA).

Tissue cytotoxin neutralization assay

The cell culture cytotoxin neutralization assay detects the presence of C. diff toxin B in culture. Diluted fecal supernate is placed on a monolayer of human or other mammalian cells in culture. If C. diff cytotoxin B is present, a cytopathic effect (CPE) causes the mammalian cells to round up and slough off the monolayer. If the CPE is then reversed by antitoxin, the test is positive for C. diff toxin B. The assay takes 24-48 hours.

While cytotoxicity assays are very sensitive and specific, there are drawbacks,including:

• Slow turnaround (usually 48 hours for a negative)
• Variability due to cell lines chosen for testing
• Critical procedural parameters requiring significant experience and great care
• Significant procedural variability from lab to lab
• Variability of interpreting the results

Choosing a C. difficile test strategy for your laboratory

The following factors should be weighed when deciding which test(s) to use:

• Size of the healthcare facility
• Age range of the patient population
• Number of C. diff diagnoses per year
• General experience level of laboratory personnel
• Cost per test

Molecular Diagnostic Testing

Molecular testing for C. difficile has become commercially available. Currently marketed assays detect the presence of toxinogenic forms of C. diff through a target on the C. diff toxin B gene (tcdB) in human stool from symptomatic patients. The sensitivity and specificity of these assays vary. PCR requires special equipment that must be purchased to perform extractions and/or amplifications.  A newer molecular technology, LAMP, is also now available. illumigene™ is the first commercial assay to use this technology by utilizing Loop Mediated Isothermal Amplification (LAMP) which detects and amplifies a pathogenic DNA region common to all toxin-producing strains of C. difficile.  This technology eliminates the need for costly capital equipment.

Proper specimen handling and transport is vital

The accuracy of all tests depends upon proper specimen handling and transport. The following rules should be followed when collecting samples:

• Stool samples should be freshly passed within 1-2 hours
• 10-20 mL of watery, soft, or unformed stool should be collected
• Stool should be passed into a clean, dry container

For transport:
Place sample in a sterile, leak-proof, wide-mouth container of >5 mL

Maintenance time and temperatures:

• ≤1 hr at room temperature (20°-27° C)
• 1-24 hrs at 2°-8°C
• >24 hrs at -20°C or colder