Clostridioides difficile Testing

Clostridioides difficile is an anaerobic, spore forming Gram positive bacillus that colonises the gut in 2 to 5% of healthy adults and up to 15 to 20% of hospital inpatients. When antibiotics disturb the normal flora, toxigenic strains multiply and release toxin A (TcdA) and toxin B (TcdB), which damage the colonic mucosa and cause diarrhoea, pseudomembranous colitis and toxic megacolon. A binary toxin and the hypervirulent ribotype 027 are associated with severe disease.

Testing is done on unformed stool in a patient with a compatible illness. No single test is both sensitive and specific, so laboratories use a combination: glutamate dehydrogenase (GDH) antigen, toxin enzyme immunoassay (EIA), and nucleic acid amplification (NAAT or PCR) for toxin genes. Toxigenic culture and cell cytotoxicity neutralisation assays are reference methods.

Clostridioides difficile infection (CDI) causes about 450 000 infections and 29 000 deaths a year in the United States, and is the leading cause of healthcare associated infectious diarrhoea. Recurrence occurs in 15 to 25% after a first episode and 40 to 60% after further episodes. Community acquired infection is rising, including in young people with no antibiotic exposure.

Indications. Three or more unformed stools in 24 hours (or equivalent) with a risk factor: antibiotic use in the last 3 months (clindamycin, cephalosporins, fluoroquinolones and broad spectrum penicillins are highest risk), hospital or care home stay, age over 65 years, proton pump inhibitors, chemotherapy, inflammatory bowel disease, or prior CDI. Also ileus or toxic megacolon with suspected CDI.

Specimen. Fresh liquid stool that takes the shape of the container. Formed stool is rejected, except in ileus. The sample is tested within 2 hours or kept at 2 to 8 °C for up to 48 to 72 hours, since toxin degrades at room temperature. Do not test for cure, or repeat within 7 days.

Test results.

• GDH positive, toxin positive: CDI confirmed.
• GDH negative: CDI unlikely, with negative predictive value over 99%.
• GDH positive, toxin negative: indeterminate, and a NAAT or toxigenic culture settles it. A positive NAAT with negative toxin suggests colonisation or mild disease with low toxin levels.
• NAAT alone: very sensitive (over 95%) but cannot separate infection from colonisation, so it is used in patients with a good pre-test probability.
• Toxin EIA alone: specific (95 to 99%) but only 60 to 85% sensitive.

Severity grading. Used to select treatment.

• Non-severe: white cell count 15 x 10^9/L or under and serum creatinine under 133 micromol/L (1.5 mg/dL).
• Severe: white cell count over 15 x 10^9/L or creatinine over 133 micromol/L (1.5 mg/dL), or serum albumin under 30 g/L.
• Fulminant: hypotension or shock, ileus or megacolon, with lactate over 2.2 mmol/L, white cell count over 30 x 10^9/L, or temperature over 38.5 °C.

Causes of a false positive diagnosis. Colonisation (asymptomatic carriage, especially in infants under 12 months, older adults in care homes and those on repeated hospital admission), recent laxatives, enteral feeds, and inflammatory bowel disease with another cause of diarrhoea.

Critical values and findings needing immediate action.

• A positive test with abdominal distension, absent bowel sounds, fever, rising white cell count or lactate, hypotension or abdominal pain with a falling stool volume (ileus): fulminant disease, which needs urgent surgical and intensive care review.
• A rising creatinine or albumin under 25 g/L.
• Isolation precautions and contact precautions from the moment CDI is suspected, with soap-and-water hand hygiene (alcohol gel does not kill spores).
• An outbreak on a ward (two or more cases within a short period).

Interferences and pitfalls.

• Do not test formed stool, children under 2 years (colonisation is common), or asymptomatic patients.
• Do not repeat testing within 7 days, since repeat tests mostly give the same result and increase false positives.
• Do not use a test of cure. Toxin and PCR remain positive for weeks after clinical recovery.
• NAAT overdiagnoses CDI when used without clinical selection, and leads to unnecessary treatment.
• Toxin EIA misses cases with low toxin levels.
• Laxatives and enemas in the preceding 48 hours cause diarrhoea that mimics CDI.
• Recurrence, defined as a return of symptoms within 8 weeks, needs a new test only if symptoms recur after treatment is complete, and a positive result is read with caution.
• Stopping the inciting antibiotic, if possible, is the first measure.

Special groups.

• Children: colonisation is common under 2 years, so testing is reserved for older children with severe disease and risk factors.
• Older adults: higher mortality (30-day mortality 10 to 20%), and recurrence more likely.
• Immunocompromised and inflammatory bowel disease patients: severe disease, and colonoscopy may be needed when stool tests fail.
• Pregnancy and peripartum: CDI occurs, and the drugs used in adults apply.
• Regions: ribotype 027 and other epidemic strains have spread across continents, and testing capacity for toxins is limited in many settings.

Follow up tests. Full blood count, creatinine, albumin, lactate, abdominal X-ray or CT for megacolon, flexible sigmoidoscopy for pseudomembranes when testing is negative and suspicion remains high, stool culture and ribotyping in outbreaks, and faecal calprotectin to separate inflammatory bowel disease.