At a Glance

Why Get Tested?

To detect the presence of an infection caused by toxin-producing Clostridium difficile bacteria

When To Get Tested?

When a person has mild, moderate, or severe diarrhea that persists for several days, without another explanation, especially in people who have recently been on antibiotic therapy

Sample Required?

A fresh liquid or unformed stool sample is collected in a sterile container. The stool sample should not be contaminated with urine or water. Once it has been collected, the stool should be taken to the laboratory immediately or refrigerated and taken to the lab as soon as possible.

Test Preparation Needed?


What is being tested?

Clostridium difficile (commonly called C. difficile or C. diff) is a type of bacteria that is associated with diarrhea resulting from antibiotic use. C. difficile testing and C. difficile toxin tests identify the presence of these bacteria, genes associated with toxin production, and/or detect the toxins produced by them.

Clostridium difficile has been recently reclassified and renamed as Clostridioides difficile, but since many people still use the former name, it will be used for the purposes of this article.

C. difficile may be present as part of the normal bacterial flora in the digestive tract of up to 65% of healthy infants and 3% of healthy adults. Sometimes, when broad-spectrum antibiotics are used to treat other infections, typically for an extended period, the balance of the normal flora in the digestive tract is disrupted. Normal bacterial flora that are susceptible to the antibiotic are eliminated from the digestive tract, while C. difficile that are resistant to the antibiotic remain and begin to overgrow, or new types (strains) of C. difficile are acquired.

C. difficile usually produced two toxins: toxin A and toxin B. The resulting combination of decreased normal flora, overgrowth of C. difficile, and toxin production can damage the lining of the lower portion of the digestive tract (colon, bowel) and lead to severe inflammation of the colon and prolonged diarrhea. Dead tissue, fibrin, and numerous white blood cells can form a lining over the surface of the inflamed bowel (a pseudomembrane), a condition that is referred to as pseudomembranous colitis.

C. difficile infection is the most common cause of diarrhea in people who develop diarrheal symptoms while hospitalized. C. difficile toxin is detected in the stools of up to 20-30% of those with antibiotic-associated diarrhea and greater than 95% of those with pseudomembranous colitis. While the organism is frequently carried by infants, it does not usually cause diarrhea in this population.

The risk of having symptoms increases with age and increases in those who have weakened immune systems, have acute or chronic colon conditions, have been previously affected by C. difficile, or who have had recent gastrointestinal surgery or chemotherapy. C. difficile-associated diarrhea usually occurs in people who have been taking antibiotics for several days, but it can also occur several weeks after treatment is completed.

C. difficile-associated disease is a spectrum of illness ranging from mild diarrhea to a more severe colitis, or to toxic megacolon or perforated bowel, which can result in sepsis and death. Signs and symptoms may include frequent loose stools, abdominal pain and cramps, nausea, fever, dehydration, fatigue, and high white blood cell count (leukocytosis). Treatment typically involves discontinuing use of the original antibiotic and administering specific oral antibiotic therapy targeting C. difficile. Most people improve as the normal bacterial flora re-colonize the gastrointestinal tract, but about 12-24% of those affected may have a second episode within two months.

Common Questions

How is the test used?

Tests to detect Clostridium difficile (C. difficile or C. diff for short), the genes associated with toxin production, and its toxins are used to help diagnose diarrhea and other conditions and complications caused by toxin-producing C. difficile. There are many infectious and non-infectious causes of acute and chronic diarrhea and this testing may be used to help determine the cause.

A number of tests are available to detect C. difficile and to determine if the strain that is present produces toxin.

The Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) 2017 guideline and the American College of Gastroenterology (ACG) 2013 guideline have both proposed using either a one-step or a multi-step testing process. The one-step process includes:

  • C. difficile toxin gene testing—this tests for the toxin genes using nucleic acid amplification tests (NAAT), typically a PCR method. These tests are rapid and very sensitive methods to confirm the presence of C. difficile toxin gene. Not all laboratories have the capability of performing these molecular tests, but they have recently become much more widely available. False positives with this method can be minimized by only testing those people who are symptomatic and likely to have C. difficile infection.

The multi-step process includes:

  • Perform an initial screen on stool samples using a test for a C. difficile antigen called glutamate dehydrogenase (GDH). This test detects an antigen that is produced in high amounts by C. difficile, both toxin and non-toxin producing strains. It is considered to be very sensitive, but it is not very specific for toxin-producing C. difficile. This test indicates if C. difficile is present but not if the bacteria are producing toxins.
  • Follow-up positive screening results with a test to confirm and to detect the presence of toxins:
    • Toxins, by enzyme immunoassay (EIA) tests; these tests are rapid but less sensitive.
    • In some laboratories, a positive GDH test with a negative toxin EIA test leads to a NAAT.
  • As an alternative multi-step process, SHEA/IDSA list performing a NAAT toxin gene test followed by a toxin test (rather than a NAAT test by itself).

Other tests that may sometimes be performed to detect C. difficile include:

  • Gastrointestinal (GI) pathogen panels—these are used to simultaneously test for the presence of multiple disease-causing (pathogenic) viruses, bacteria, and/or parasites in a stool sample and help diagnose an infection of the digestive system (GI tract). Since there are many causes of GI infections, including C. difficile, a GI pathogen panel may be used in conjunction with other tests, such as a stool culture or an ova and parasite exam (O&P), to help establish a diagnosis.
  • Cell cytotoxicity test is a tissue culture to detect the C. difficile toxin. It is a test that looks for the effects of the cytotoxin (cytotoxicity) on human cells grown in culture. It is a sensitive method to detect toxin, but it requires 24 to 48 hours to get the test result. It is rarely performed in clinical microbiology laboratories.
  • Toxigenic stool culture, which requires growing the bacteria in a culture and a second step to detect the presence of the toxins, is a very sensitive test for C. difficile. It is still considered to be the gold standard. However, it can take 2 to 3 days for results. It is rarely performed in clinical microbiology laboratories.

When is it ordered?

C. difficile testing may be ordered when a person hospitalized for more than three days has frequent watery stools, abdominal pain, fever, and/or nausea during or following a course of antibiotics or following a recent gastrointestinal surgery. Testing may be ordered for outpatients when someone develops these symptoms within 6-8 weeks after taking antibiotics, several days after chemotherapy, or when a person has a chronic digestive tract disorder that a healthcare practitioner suspects is being worsened by a C. difficile infection. Tests for C. difficile may be ordered to help diagnose the cause of diarrhea when no other infectious or non-infectious causes have been detected.

When a person treated for antibiotic-associated diarrhea or colitis relapses and symptoms reoccur, C. difficile testing may be ordered to confirm the presence of the toxin. Testing should not be ordered to monitor the effectiveness of treatment or for those who are asymptomatic. A subsiding of symptoms (diarrhea ceases and stools are formed) indicates a cure from infection. Repeat testing for C. difficile after a positive result is not advised and provides no useful clinical information. Molecular tests may remain positive for weeks after someone is cured and asymptomatic.

What does the test result mean?

If tests for C. difficile toxin gene and C. difficile toxin are positive, it is likely that the diarrhea and related symptoms are due to the presence of toxin-producing C. difficile.

A positive result for C. difficile bacteria or C. difficile antigen (GDH) but a negative C. difficile toxin result means that the bacteria are present in the digestive tract but are not producing a detectable level of toxin.

A negative test result for the C. difficile toxin gene likely indicates that the person’s diarrhea and related symptoms are not due to toxin-producing C. difficile.

Negative test results for both the bacteria and the toxin may mean that the diarrhea and other symptoms are being caused by something other than C. difficile. Since the toxin breaks down at room temperature within two hours, a negative result may also indicate that the sample was not transported, stored, or processed promptly. If there is a concern that a stool specimen has not been collected and processed properly, a second specimen may be submitted for testing after discussion with the laboratory.

Is there anything else I should know?

If a person has positive C. difficile test results, the healthcare practitioner will typically discontinue any antibiotics that the person is taking and prescribe an appropriate treatment of oral antibiotic, such as vancomycin or fidaxomicin, to eliminate the C. difficile bacteria. Recently, so-called stool transplants have been investigated as an effective treatment for people with recurrent C. difficile infections.

An endoscopic procedure can be used to diagnose C. difficile colitis. A specialist (gastroenterologist) can examine the colon and biopsy any characteristic pseudomembranous lesions that may be present.

What else can cause diarrhea?

Diarrhea can be caused by a number of infections and other conditions. It may be due to a pathogenic bacterial infection (commonly caused by Salmonella, Shigella, Campylobacter or Escherichia coli), a viral infection, a parasitic infection, food intolerance, certain medications, chronic bowel disorders such as irritable bowel syndrome (IBS), or malabsorption disorders (such as celiac disease). Diarrhea may also be caused or worsened by psychological stresses.

Why must the stool sample be fresh?

For Clostridium difficile toxin testing, the sample must be fresh because the toxin will break down in one to two hours and may result in a false-negative test.

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

Anti-diarrhea medication can slow down the passage of stool through the digestive 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, if symptoms reoccur shortly after the initial infection, it is generally a case of recurrence of overgrowth and toxin production by the same strain rather than an infection with a new strain. This happens because the normal bacterial flora has not re-colonized the digestive tract yet. A person who has had C. difficile diarrhea may also be at an increased risk of developing a new infection with future courses of antibiotics.

Are some antibiotics more likely to cause antibiotic-related diarrhea?

Almost any antibiotic may lead to diarrhea 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.


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