Laboratory Diagnosis of Clostridium difficile [Hot Topic]

Clostridium difficile is an important cause of health-care-associated infections. C. difficile infections (CDI) present with a wide range of symptoms, from diarrhea to toxic megacolon. Accurate and rapid diagnosis is important for two reasons: 1. Begin therapy with an appropriate antimicrobial agent, and 2. Discontinue antimicrobial agents that may be predisposing the patient to CDI.

There are several methods for diagnosing CDI, including molecular NAATs, EIAs, and culture. The information provided about the relative strengths and weaknesses of these methods can help you interpret test results in light of the clinical presentation.

Audrey Schuetz, M.D.


Audrey Schuetz, M.D., is the Director of Initial Processing, Co-Director of Bacteriology, and a Senior Associate Consultant in the Clinical Microbiology Laboratory at Mayo Clinic in Rochester, Minnesota.


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I have no relevant disclosures.

Utilization Message

Let’s start with some test utilization messages. As you view this presentation, consider the following important points regarding laboratory testing for Clostridium difficile:

  • Various testing methodologies are available.
  • Laboratories may use more than one testing platform in reflexive or algorithmic approaches in order to reach a diagnosis.

Burden of Clostridium difficile Disease

Clostridium difficile has received a lot of attention in the last two decades due to the increasing burden it has been placing on our patients and our health care system. C. difficile is the most commonly reported pathogen causing health-care-associated infections in U.S. hospitals, recently surpassing methicillin-resistant Staphylococcus aureus, or MRSA. Since 2013, the National Healthcare Safety Network (NHSN) has mandated reporting of C. difficile infections for hospitals participating in the Centers for Medicare and Medicaid Services (CMS) program.

Accurate and rapid diagnosis of CDI is important for the patient and the health care environment. Upon diagnosis, providers begin therapy with an appropriate antimicrobial agent such as metronidazole or oral vancomycin and discontinue antimicrobial agents that may be predisposing to CDI. Infection control precautions are instituted in order to curb spread of the spores.

Clinical Disease

C. difficile disease presents with a range of clinical findings from diarrhea to pseudomembranous colitis to toxic megacolon. Pictured are pseudomembranes in the colon as seen on flexible sigmoidoscopy in a patient with CDI. Pseudomembranes are adherent thick layers of inflammatory cells and mucus.

The most significant risk factor for CDI is antibiotic exposure. Although clindamycin, broad-spectrum cephalosporins, and ampicillin have most often been implicated in CDI, any antibiotic can cause this. When a patient takes antibiotics, beneficial bacteria in the intestine are destroyed or impaired for a period of time, increasing the likelihood that C. difficile can lead to infection.


C. difficile is an obligate anaerobe that is a spore-forming Gram-positive rod. In the 1930s, it was originally named Bacillus difficilis due to difficulty isolating this bacterium in the laboratory.

C. difficile spores are ubiquitous in the natural environment, including seawater, rivers, and soil. The organism is spread by the fecal-oral route. When spores are ingested from the environment, they can then germinate in the intestine and produce toxins.

In hospitals, spores are present on many environmental surfaces such as commodes and bed curtains and on the hands of caregivers. They have even been found in pet animals, but there has been no evidence to date of zoonotic transmission from animals to humans.

The spores are resistant to alcohol gels and many hospital disinfectants and can persist on inanimate surfaces for several months if inadequately cleaned.

Changing Face of C. difficile

The epidemiology of C. difficile has been changing since 2000, with a rise in incidence and severity.

One factor associated with this evolving epidemiology is recognition of the NAP1/BI/027 strain, also referred to as the hypervirulent strain. NAP1 stands for North American pulsed-field gel electrophoresis type-1 and 027 refers to PCR ribotype number. Compared to non-NAP1 strains, this strain has been more often associated with severe disease, has shown higher rates of fluoroquinolone resistance, and produces more toxins A and B. In addition, NAP-1 strains were found to produce binary toxin. Although fluoroquinolone resistance does not affect management of CDI, because this class of antimicrobials is not used for CDI treatment, resistance to fluoroquinolones may provide the NAP1 strain with a survival advantage over susceptible strains in health care facilities where these antibiotics are commonly used. However, non-NAP1 strains have also been associated with high severity and production of binary toxin.

C. difficile has also been increasingly reported outside of acute-care facilities in nursing homes and community home settings.

C. difficile Toxins

There are several C. difficile toxins. Toxin A (encoded by the tcdA gene) is an enterotoxin that causes fluid accumulation in the bowel. Toxin B (encoded by the tcdB gene) is cytopathic to (causes distortion of) cells when cultured in the laboratory. Genes encoding for toxins A and B are present on the pathogenicity locus.

The tcdC gene regulates toxin A and B production.

Genes cdtA and cdtB are located at an unknown distance from the pathogenicity locus and encode the binary toxin.

C. difficile Colonization

Asymptomatic carriage or colonization can occur with nontoxigenic, or nontoxin-producing, strains as well as toxigenic strains. This colonization state complicates the clinical diagnosis of infection due to C. difficile.

Colonization with nontoxigenic strains ranges from 0.4% to 6.9% of adults (2% of nonhospitalized adults are colonized). Infant intestinal cells do not appear to have receptors for toxins A and B; therefore, a much higher percentage of neonates may have detectable C. difficile in their stools but do not manifest with disease.

Therefore, only unformed stools should be tested when assessing infection due to C. difficile. Dr. Stephen Brecher coined CDI submission guidelines for stools shown in the table. Formed stools may be tested, however, in cases of ileus or toxic megacolon when stool is often not passed.

Enzyme Immunoassay (EIA)

Enzyme immunoassays, or EIAs, may detect either toxins A/B and/or glutamate dehydrogenase (GDH).

Toxin A/B EIAs are rapid tests and take minutes to perform. However, toxin EIAs are somewhat less sensitive than other methods in diagnosing infection.

GDH EIAs detect the enzyme that is produced by both toxigenic and nontoxigenic strains of C difficile. GDH is produced at much higher levels than toxins A and B. Advantages of the GDH test include rapidity, and recent metanalyses have shown high sensitivity for these assays. The disadvantage is that this test cannot be used as a stand-alone test for CDI. A confirmatory test for the presence of toxin is needed. These assays have been used in algorithmic approaches to diagnosis in combinations of testing involving toxin EIA, molecular assays, or cytotoxicity assays.

An EIA is also available that simultaneously detects C. difficile GDH, and toxins A and B, and the sensitivity of this assay is high.


Culture is a highly sensitive method of recovering the organism when selective culture media is used. Recovery of strains allows for further molecular typing studies (such as for comparison of relatedness of strains) or for antimicrobial susceptibility testing when indicated.

However, nontoxigenic strains can be recovered in culture, so further testing is required to confirm toxigenicity. Time to results is typically 24 to 48 hours.

The chromogenic medium CHROMagar C difficile (bioMérieux) is utilized by Mayo Clinic in Rochester for culture upon request, which is a recent addition to our testing capabilities. C. difficile colonies will grow on this media and will fluoresce under ultraviolet light, which is pictured. Other bacteria will be inhibited from growing or will not fluoresce.

Nucleic Acid Amplification Tests (NAATs)

Nucleic acid amplification tests (NAATs) are molecular-based assays that detect the genes encoding for the toxins rather than the toxins themselves. Various NAATs are available to laboratories. At Mayo Clinic in Rochester, C. difficile PCR targeting the tcdC gene is used. NAATs are more sensitive than enzyme immunoassays.


A macrolide agent, fidaxomicin, was FDA-approved in May 2011 for the treatment of CDI. It was the second agent after vancomycin to be approved by the FDA for CDI.

It is bactericidal, and oral administration leads to high fecal concentrations that exceed the minimum inhibitory concentrations

Mayo Clinic offers metronidazole and vancomycin susceptibility testing for C. difficile from intestinal sources.

Testing Guidelines

We’ll wrap up this talk with general testing guidelines.

Repeat testing for use as a test of cure is not acceptable. Toxins can be detected in stool as long as 30 days after resolution of symptoms.

Formed stools should not be tested when assessing for C. difficile infection.

Testing should not be performed on children under 1 year of age.

In summary, there are a variety of testing methodologies available, and laboratories may use more than one testing platform in reflexive or algorithmic approaches when assessing C. difficile diagnosis.


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This post was developed by our Education and Technical Publications Team.