Clostridium (Clostridioides) difficile Diagnostic Testing

C difficile is a potentially toxin-producing bacterial pathogen of the gastrointestinal tract and is the most common cause of healthcare-associated diarrhea. C difficile causes antibiotic-associated diarrhea and can escalate to pseudomembranous colitis or toxic megacolon, a potentially fatal complication. C difficile is estimated to cause almost half a million infections in the United States each year.¹  

Disease-causing C difficile strains produce 1 or both of 2 toxins: toxin A is an enterotoxin and toxin B is a cytotoxin. Other strains produce neither toxin and are thought to colonize the colon without causing disease. A hypervirulent strain (027/NAP1/B1) is associated with a higher recurrence rate and higher production of toxin. 

Infections are commonly seen in patients with the following risk factors:

• Recent or current treatment with antibiotics (especially fluoroquinolones, third or fourth generation cephalosporins, clindamycin, and carbapenems)
• Gastrointestinal surgery or manipulation
• Long length of stay in healthcare settings
• A serious underlying illness
• Immunocompromising conditions
• Advanced age

Testing should be considered for patients with recent or current antibiotic use and ≥3 episodes of non-formed stool within 24 hours. Other symptoms may include fever, stomach tenderness or pain, loss of appetite and nausea.

For children ages 1 to 3 with diarrhea, testing can be considered, but other causes (eg. viral) should be considered first. Routine testing in children <1 should be avoided given the high carriage rates of C difficile. For children >3, testing considerations are the same as for adults. Although C difficile can colonize neonates/infants, they may lack the cellular machinery to bind and process toxins.²

Repeat testing to determine a cure is not recommended.

The American College of Gastroenterology³ and the American Society for Microbiology⁴ guidelines recommend utilizing 2 different testing options for the detection of C difficile infection:

1. Nucleic acid amplification tests (NAAT) for C difficile toxin genes (test code 16377)
2. 2- or 3-step algorithm that includes assessment of C difficile toxin and glutamate dehydrogenase (GDH), as well as detection of the C difficile toxin B gene (test code 91664)

The Infectious Disease Society of America/The Society for Healthcare Epidemiology of America (IDSA/SHEA) guidelines, which are posted on the CDC website, recommend gene detection for confirmation when the GDH test is positive and toxin is not detected.

They also recommend gene detection when the GDH test is negative and toxin is detected. PCR or another nucleic acid amplification test (NAAT) can be used.^1,5

The first step is an immunoassay to simultaneously assess for toxin and GDH presence. If toxin (either A or B or both) and GDH are present, the specimen is considered positive for toxigenic C difficile infection. If both toxin and GDH are absent, then the specimen is considered negative (please see Test Algorithm Figure (TC 91664) below).

Specimens with discordant results (ie, GDH-positive but toxin-negative or GDH-negative but toxin-positive) proceed to the second step: reflex (at additional charge and additional CPT code) to a PCR C difficile gene detection test. According to Quest validation studies, discordant results occur in about 6% of cases.

If the gene is detected, results will include genotype information that indicates if the hypervirulent strain (ribotype 027/NAP1/toxinotype III) is present. Table 1 details how the results will be reported and how to interpret them. 

The C difficile cytotoxicity assay has served as a historical gold standard for diagnosing clinically significant disease caused by C difficile; however, it is not timely for diagnosis. This assay is best utilized as a reference method or epidemiologic tool.³

The newer testing methods are faster than culture and have higher sensitivity and specificity than GDH testing alone. The 2-step algorithm increases the sensitivity to 90.5% and the specificity to 93%.⁶ The sensitivity of the stand-alone NAAT is 93.4% and the specificity is 90.9%.⁷

Quest Diagnostics offers multiple tests to help identify C difficile infections (Table 2). Information pertaining to test selection can be found in Questions 3 and 5.