What is being tested?

This test detects the presence of the toxin produced by Clostridium difficile in fresh or frozen faecal samples. This bacterium is a component of the normal gastrointestinal bacterial flora in up to 65% of healthy infants but only 3% of healthy adults. Clostridium difficile most commonly causes diarrhoea in people who have recently received antibiotics. Antibiotics disrupt the normal gastrointestinal bacterial flora, allowing Clostridium difficile to replicate and over-grow in the bowel. Other risk factors for acquisition of C. difficile infection include advanced age, being immunocompromised, recent gastrointestinal surgery, chemotherapy or the use of acid suppressive medications most commonly used for gastroesophageal reflux.

About 75 per cent of C. difficile produces two main toxins - toxin A and toxin B. The combination of an overgrowth of C. difficile and toxin production can damage the lining of the colon causing diarrhoea. Dead tissue, fibrin, and numerous white blood cells can form a layer over the inflamed bowel, which is referred to as pseudomembranous colitis. C. difficile toxins are detected from stools of 15-25% of patients with antibiotic-associated diarrhoea and in stools of more than 95% of patients with pseudomembranous colitis.

C. difficile toxin is a common cause of diarrhoea in hospitalised patients. Most infections are acquired whilst in hospital. A minority of infections are acquired in the community. Many patients may have C. difficile in their intestine on hospital admission without any specific symptoms, and it only becomes a problem after they are treated with antibiotics.

The disease manifestations of C. difficile infection vary. In the simplest form, C. difficile causes a mild diarrhoeal illness. The more severe manifestations are a severe diarrhoeal illness associated with fever, dehydration, septicaemia and in rare circumstances marked distension of the bowel wall called toxic megacolon. Treatment typically consists of stopping the original antibiotic and administering specific oral antibiotic therapy to which the C. difficile is susceptible. Most patients improve as the normal flora re-establishes itself, but about 20 per cent of patients relapse, occurring usually within a week of completion of therapy. Multiple relapses are possible in a minority of patients.

Recently, a new, more dangerous variety of C. difficile, called ribotype 027 strain, has been implicated in hospital-acquired infection outbreaks in North America and Europe, which have been associated with increased morbidity and mortality. There have been isolated cases of this severe strain in Australia but overall it is uncommon.

How is it used?

The Clostridium difficile toxin test is used to diagnose antibiotic-associated diarrhoea and pseudomembranous colitis that is caused by C. difficile. It may also be ordered to detect recurrent disease.

If the patient has a positive toxin test, the doctor will typically discontinue any antibiotics that the patient may be taking and prescribe an appropriate treatment of oral antibiotic, such as metronidazole or vancomycin, to eliminate the C. difficile bacteria.

When is it requested?

Early diagnosis is key to preventing complications from severe C. difficile infection as well as preventing transmission to other people. Stool tests for C. difficile infection may be considered in the following circumstances:

• Hospitalised patients with frequent loose stools +/- abdominal pain +/- fever, particularly if these symptoms follow (within 6-8 weeks) a course of antibiotics or if the patient has recently undergone gastrointestinal surgery or chemotherapy.
• Community dwelling patients with unexplained frequent loose stool motions, particularly if symptoms follow antimicrobial exposure.
• Relapse of frequent loose stools shortly after treatment for a confirmed episode of Clostridium difficile infection.

What does the result mean?

If the C. difficile toxin test is positive, it is likely that the patient’s diarrhoea and related symptoms are due to an overgrowth of toxin-producing C. difficile. Occasionally, false positives may be seen with grossly (visibly) bloody faecal samples.

If the test is negative but the diarrhoea continues, another sample needs to be tested. The rapid C. difficile toxin tests detect less than 85% of cases, so the toxin may have been missed the first time. Since the toxin breaks down at room temperature, a negative result may also indicate that the sample was not transported, stored, or processed promptly. A negative test result may also mean that the diarrhoea and other symptoms are being caused by something other than C. difficile.

Is there anything else I should know?

There are a number of tests that are available to detect the infection and to determine if the strain that is present produces toxin. Exact procedures between different laboratories vary but in most circumstances a combination of tests are performed on the stool sample to optimise the sensitivity and specificity of the test. Some of the commonly used tests include:

• C. difficile toxin B, or toxins A and B, by enzyme immunoassay (EIA) tests are some of the most common tests used by laboratories. Results are typically available within 1 to 4 hours. Though these tests are rapid and widely available, they are not sensitive enough to detect many infections; they miss up to 30% of cases. Therefore, they are not recommended for use by some professional organisations.
• A glutamate dehydrogenase (GDH) test detects an antigen that is produced in high amounts by C. difficile, both toxin and non-toxin producing strains. It may be used as a first step to rule out an infection with C. difficile, but it should not be used alone. Since it is not very specific for toxin-producing C. difficile, it is often used in combination with a test for toxin by EIA or a cytotoxicity culture.
• A relatively new molecular PCR (polymerase chain reaction) test can rapidly detect the C. difficile toxin B gene (tcdB) in a stool sample. This test is sensitive and has become increasingly available in Australian laboratories.
• Tissue culture to detect the C. difficile toxin is a test that looks for the effects of the cytotoxin on human cells grown in culture. It is a more sensitive method to detect toxin, but it requires 24 to 48 hours to get the test result.
  Toxigenic stool culture, which requires growing the bacteria in a culture and detecting the presence of the toxins, is the most sensitive test for C. difficile, and it is still considered to be the gold standard. However, it can take 2 to 3 days for results. A culture will also not distinguish between C. difficile colonisation and overgrowth/infection.

A commonly employed testing algorithm is to use a screening test such as the glutamate dehydrogenase test and follow this up with a confirmatory test with either enzyme immunoassays for stool toxins or PCR for toxins.

Common questions

• What else can cause diarrhoea?

Diarrhoea can be due to a pathogenic bacterial infection, a viral infection, a parasite, food intolerance, certain medications, chronic bowel disorders such as IBS (irritable bowel syndrome), or malabsorption disorders (such as coeliac disease). Diarrhoea may also be caused or exacerbated by psychological stresses.

• Why must the stool sample be fresh?

For C. difficile toxin testing, the faecal sample must be fresh because the toxin breaks down in one to two hours at room temperature and be undetectable within 2 hours after collection. Samples tested after this time has passed may occasionally result in a false negative test. Nevertheless, nucleic acid testing (NAT) would still be appropriate in this scenario.

• Why shouldn’t I take an over the counter anti-diarrhoea medicine when I have diarrhoea caused by C. difficile?

Anti-diarrhoea medicine can slow down the passage of stool through the gastrointestinal tract, increasing the length of time that the colon is exposed to the toxin and increasing tissue damage and inflammation.

• Once I’ve had a C. difficile infection, can I be re-infected?

Yes, but relapses of symptoms soon after the cessation of treatment for C. difficile infection are thought to be related to re-population of the bowel by Clostridium difficile spores (spores are a dormant form of the bacterium) rather than re-infection.

• Are some antibiotics more likely to cause antibiotic-related diarrhoea?

Almost any antibiotic may lead to diarrhoea since the drugs alter the normal population of good bacteria in the bowel. Broad-spectrum antibiotics, which kill many different types of bacteria, are more likely to wipe out normal bowel flora and allow C. difficile to overgrow and produce toxin.

More information

RCPA Manual: Clostridium difficile detection