Clostridioides difficile Toxin A & B
The Clostridioides difficile Toxin A&B test detects C. difficile toxin proteins (or toxin genes, depending on assay methodology) in unformed stool specimens, providing microbiological confirmation of CDI as the cause of clinically significant diarrhoea. Testing is only appropriate in patients with ≥3 unformed stools in 24 hours — formed stool testing and asymptomatic testing are not indicated.
2
Biomarkers
Stool
Sample
1–2 business days
Turnaround
No
Fasting
What is the C. difficile Toxin A&B test?
The C. difficile Toxin A&B test detects the presence of Clostridioides difficile toxin proteins (or toxin gene products, depending on assay methodology) in stool, providing microbiological confirmation of CDI as the cause of clinically significant diarrhoea. Clostridioides difficile infection is the leading cause of healthcare-associated infectious diarrhoea in developed countries and a clinically significant cause of community-acquired diarrhoea, particularly in patients who have recently received antibiotics, been hospitalised, are elderly, or are immunocompromised. Detection of C. difficile toxins in stool from a symptomatic patient with ≥3 unformed stools in 24 hours is the microbiological cornerstone of CDI diagnosis and is required before specific antimicrobial therapy (oral vancomycin, fidaxomicin, or metronidazole) is initiated. The appropriate clinical use of this test requires adherence to a fundamental testing discipline: C. difficile toxin testing must only be performed on unformed (liquid or soft) stool specimens from patients with clinically significant diarrhoea — defined as ≥3 unformed stools in a 24-hour period. Testing of formed stool, asymptomatic patients, or post-treatment surveillance in the absence of new symptoms is not indicated and produces a high rate of clinically misleading positive results — particularly from NAAT-based assays that detect toxin gene carriage (colonisation) rather than active toxin-mediated disease. C. difficile colonisation without active toxin production occurs in approximately 4–15% of hospitalised adults and does not require treatment. Repeat testing within 7 days of a prior negative result is not recommended due to low additional yield. The test should be ordered once per episode of clinically significant diarrhoea.
What does the C. difficile Toxin A&B test measure?
Clostridioides difficile (formerly Clostridium difficile) is a gram-positive, anaerobic, spore-forming bacillus that produces two primary virulence toxins in disease-causing strains — Toxin A (TcdA, enterotoxin, 308 kDa) and Toxin B (TcdB, cytotoxin, 270 kDa). Both toxins are glucosyltransferases that inactivate Rho GTPases within colonocytes, disrupting the actin cytoskeleton and tight junctions, leading to colonic epithelial cell death, fluid secretion, and mucosal inflammation. Toxin A was historically considered the primary enterotoxic driver, but toxin B is now established as the dominant virulence determinant — B-only-producing strains cause clinically indistinguishable disease from A+B-producing strains, and some hypervirulent strains (ribotype 027) produce a binary toxin (CDT) in addition to TcdA and TcdB. Toxin genes tcdA and tcdB are carried on the pathogenicity locus (PaLoc) of toxigenic strains; non-toxigenic strains can colonise the gut without causing disease.
Detection of Toxin A in stool confirms the presence of free active toxin — the direct mediator of colonic mucosal damage — in a symptomatic patient, supporting the diagnosis of Clostridioides difficile infection (CDI) requiring treatment. EIA-based toxin A detection has relatively low sensitivity (70–85%) compared to NAAT but high specificity — a positive EIA toxin A result in a patient with clinically significant diarrhoea is highly specific for active CDI. A negative toxin A EIA does not exclude CDI if toxin B or NAAT is not also tested, as approximately 3–5% of C. difficile strains produce toxin B only.
Toxin B (TcdB) is a potent glucosyltransferase cytotoxin that inactivates Rho, Rac, and Cdc42 GTPases in colonocytes via UDP-glucose transfer, producing irreversible actin cytoskeletal depolymerisation, tight junction disruption, colonic epithelial apoptosis, and intense inflammatory neutrophil recruitment to the mucosa. Toxin B is 1,000-fold more potent than Toxin A in in vitro cell cytotoxicity assays and is now recognised as the dominant virulence factor in CDI pathogenesis. Hypervirulent ribotype 027 strains produce elevated quantities of both TcdA and TcdB via mutations in the tcdC negative regulatory gene, contributing to their association with severe and refractory CDI outbreaks.
Toxin B detection is clinically the more sensitive and critical toxin target in C. difficile diagnostics. Its presence in stool from a symptomatic patient with clinically significant diarrhoea confirms active toxin-mediated colonic injury. Detection of Toxin B in the absence of Toxin A — occurring in approximately 3–5% of toxigenic C. difficile strains — would be missed by Toxin A-only assays, supporting the clinical rationale for combined A&B testing. In the context of EIA-based testing, a positive Toxin B result alongside or without Toxin A has high specificity for CDI and supports treatment initiation per IDSA/SHEA guidelines.
Why is the C. difficile Toxin A&B test ordered?
- Clinically significant new-onset diarrhoea — ≥3 unformed stools in 24 hours — occurring during or within 8 weeks of completion of antibiotic therapy, which disrupts the protective colonic microbiome and markedly increases CDI susceptibility
- New-onset clinically significant diarrhoea in hospitalised or recently hospitalised patients — healthcare-associated CDI remains prevalent despite infection control measures, and any inpatient with unexplained diarrhoea warrants C. difficile testing before empirical antidiarrhoeal therapy is prescribed
- Community-acquired diarrhoea with CDI risk factors — proton pump inhibitor (PPI) use, recent hospitalisation within 3 months, age over 65, inflammatory bowel disease, immunosuppression, or chemotherapy, even without recent antibiotic exposure
- Moderate-to-severe diarrhoea with systemic features — fever, abdominal pain, elevated leucocyte count, or elevated creatinine — where fulminant CDI must be excluded promptly given its mortality risk
- Inflammatory bowel disease flare evaluation — CDI superinfection is a common and clinically important trigger of IBD exacerbation that mimics primary inflammatory flare; C. difficile testing should be performed before escalating immunosuppressive therapy in any IBD patient with new or worsening diarrhoea
- Recurrent diarrhoea following completed CDI treatment — CDI recurrence occurs in approximately 15–30% of treated patients; C. difficile toxin testing is indicated when diarrhoea returns after symptom resolution at least 2–8 weeks following treatment completion
- Integrative and functional medicine practitioners evaluating gut dysbiosis and post-antibiotic microbiome disruption — C. difficile toxin testing provides direct microbiological evidence of CDI as a cause of ongoing gastrointestinal symptoms before gut restoration protocols are initiated
- Nursing home and long-term care facility resident assessment — institutionalised elderly patients have the highest CDI incidence and severity; any resident with new diarrhoea warrants prompt C. difficile testing given the outbreak risk and high case fatality rate in this population
Sample collection and turnaround
Sample type
Stool
Fasting required
No
Collection method
Patient self-collection
Turnaround
1–2 business days
Collection notes
Patient collects a fresh unformed stool sample into the Access Medical Laboratories stool collection container immediately after defecation. The specimen must be unformed — liquid, watery, or soft/mushy consistency (Bristol Stool Scale types 5–7). Formed stool must not be submitted and will be rejected by the laboratory as unsuitable for C. difficile testing. No rectal swabs — stool specimen only. Collect at least 2–5 mL (approximately a teaspoon) of stool into the container. Do not contaminate the specimen with urine or water from the toilet bowl. Specimen must be refrigerated at 2–8°C immediately after collection and transported to Access Medical Laboratories within the specified timeframe — do not freeze. Practitioners should instruct patients to collect the specimen before initiating any antibiotic therapy if possible. Document on the requisition form: (1) symptom onset date and severity — number of unformed stools per 24 hours; (2) recent and current antibiotic use — agents, duration, and date of last dose; (3) hospitalisation or healthcare exposure within the preceding 3 months; (4) current PPI or antidiarrhoeal medication use; (5) IBD or immunosuppressed status. # REVIEWER FLAG: Confirm Access Medical Laboratories stool collection container type (preservative-free or with fixative), minimum required volume, maximum transit time from collection to receipt, refrigeration requirements, and formed-stool rejection policy before publication.
Specimen requirements
Minimum 2–5 mL (approximately 2–5 grams) of unformed stool in Access Medical Laboratories collection container. Refrigerate immediately at 2–8°C; transport cold to the laboratory. Do not freeze. Do not submit formed stool. # REVIEWER FLAG: Confirm exact minimum volume and maximum acceptable transit time with Access Medical Laboratories.
What can affect C. difficile Toxin A&B results?
C. difficile testing should only be performed on unformed (liquid to soft/mushy) stool — Bristol Stool Scale types 5–7. Formed stool submitted for C. difficile testing produces unreliable results regardless of assay type and should be rejected by the laboratory. More importantly, a patient producing formed stool does not have clinically meaningful CDI-related diarrhoea, making the test clinically inappropriate independent of specimen quality. Most laboratories, including Access Medical Laboratories, should apply a formed-stool rejection criterion. # REVIEWER FLAG: Confirm Access Medical Laboratories' formed-stool rejection policy and whether the laboratory applies a Bristol Stool Scale assessment or consistency rejection criterion.
C. difficile toxins A and B are proteolytically labile and degrade at room temperature — prolonged delay between stool collection and laboratory processing reduces the detectable toxin concentration in EIA-based assays, producing falsely negative results. Specimens should be refrigerated at 2–8°C immediately after collection and processed within 48–72 hours. Freezing at −70°C preserves toxin activity for research purposes but is not required for routine clinical testing. # REVIEWER FLAG: Confirm Access Medical Laboratories maximum acceptable transit time and required temperature for C. difficile stool specimens.
Oral vancomycin, fidaxomicin, or metronidazole directly suppress C. difficile toxin production and reduce the number of vegetative toxin-producing organisms in the colon. Testing performed after even a single dose of appropriate anti-CDI therapy can produce falsely negative toxin results despite active pre-treatment CDI. Specimen collection should ideally occur before antibiotic initiation — if clinical urgency requires treatment before testing, document antibiotic timing and interpret a negative result with this caveat.
Chronic PPI use alters gastric acid secretion, reduces colonocyte defence mechanisms, and shifts the colonic microbiome toward a composition that increases susceptibility to C. difficile colonisation and toxin production. PPI use is a documented independent risk factor for CDI and is associated with higher C. difficile positivity rates in tested patients. This is a biological risk factor rather than an assay interference — PPI use should be documented and factored into clinical risk assessment.
Severely dilute stool specimens — from high-volume secretory diarrhoea or significant water intake — may contain toxin concentrations below the EIA detection threshold despite active CDI, producing false-negative results. When clinical suspicion for CDI is high despite a negative EIA toxin result, NAAT testing for toxin genes (tcdB) provides superior sensitivity and should be requested as a reflex or concurrent test. # REVIEWER FLAG: Confirm whether Access Medical Laboratories offers reflex NAAT testing following negative toxin EIA, or whether the combined algorithm includes NAAT as a primary or reflex component.
Severely immunocompromised patients — particularly those on high-dose chemotherapy, haematopoietic stem cell transplant recipients, or patients with HIV — may produce atypical CDI presentations including less prominent diarrhoeal volume and reduced inflammatory response. The threshold of ≥3 unformed stools per 24 hours may not always be met despite significant mucosal toxin-mediated damage in these patients, requiring clinical judgement about testing appropriateness below the standard diarrhoeal threshold.
What do C. difficile Toxin A&B results mean?
Elevated
A positive C. difficile Toxin A and/or B result in a patient with clinically significant diarrhoea (≥3 unformed stools in 24 hours) and a compatible clinical risk profile confirms CDI and warrants treatment initiation per current IDSA/SHEA guidelines. First-line treatment for non-severe CDI is oral vancomycin (125 mg four times daily for 10 days) or fidaxomicin (200 mg twice daily for 10 days) — metronidazole is no longer recommended as first-line therapy per current guidelines due to inferior efficacy. Severe CDI (leucocytes ≥15 ×10³/µL, creatinine ≥1.5 mg/dL) and fulminant CDI (hypotension, ileus, toxic megacolon) require escalated treatment and urgent surgical assessment. Positive results must never be used as a standalone diagnosis without concurrent clinical assessment — a positive toxin in a patient without genuine diarrhoea or with formed stool is more likely to represent laboratory colonisation detection than active CDI.
Normal
A negative result in a patient whose clinical presentation was initially concerning but who is now improving spontaneously, has formed stool at the time of testing, or in whom an alternative diagnosis has been confirmed does not require further C. difficile investigation. Repeat testing within 7 days of a negative result is not recommended — the incremental diagnostic yield is below 5% and positive results within this window typically reflect colonisation rather than new active infection.
Reduced
A negative C. difficile Toxin A&B EIA result has limited negative predictive value in high-clinical-suspicion presentations due to EIA sensitivity of approximately 70–85%. In a patient with high clinical probability of CDI — recent broad-spectrum antibiotics, hospitalisation, significant diarrhoea, fever, and elevated leucocytes — a negative EIA toxin result should prompt NAAT/PCR testing for tcdB gene detection, which has sensitivity above 95%. A negative NAAT result in a high-suspicion clinical context warrants alternative gastrointestinal diagnoses — other infectious diarrhoeas, inflammatory bowel disease, microscopic colitis, ischaemic colitis, or medication-induced diarrhoea.
Note: The most critical clinical caveat is the distinction between C. difficile colonisation and active CDI — NAAT-based assays (PCR) detect toxin gene carriage in both states and cannot independently distinguish active toxin-mediated disease from asymptomatic colonisation. A positive NAAT in a patient with formed stool, minimal symptoms, or a non-CDI cause of diarrhoea is more likely to represent colonisation than active CDI and should not trigger antibiotic treatment without clinical correlation. EIA-based toxin detection is more specific for active CDI because it detects free toxin protein — the direct disease mediator — rather than gene carriage, but at the cost of reduced sensitivity. The methodology used by Access Medical Laboratories determines which of these interpretive frameworks applies — confirm assay type before applying clinical guidance. Test-of-cure after treatment is NOT recommended, as NAAT can remain positive for weeks following clinical resolution of CDI due to persistent colonisation without active toxin production.
Frequently asked questions
C. difficile Toxin A&B testing is appropriate only in patients with clinically significant diarrhoea — defined as ≥3 unformed stools in a 24-hour period — in the context of relevant risk factors including recent antibiotic use, hospitalisation within 3 months, age over 65, PPI use, immunosuppression, or inflammatory bowel disease. Testing asymptomatic patients, patients with formed stool, or repeat testing within 7 days of a prior negative result is not indicated and produces clinically misleading results.
How to access the C. difficile Toxin A&B test
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Published: May 23, 2026
Last reviewed: May 23, 2026
- McDonald LC, et al. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America and Society for Healthcare Epidemiology of America. Clin Infect Dis. (2018)
- Crobach MJT, et al. European Society of Clinical Microbiology and Infectious Diseases: Update of the Diagnostic Guidance Document for Clostridium difficile Infection. Clin Microbiol Infect. (2016)
- Surawicz CM, et al. Guidelines for Diagnosis, Treatment, and Prevention of Clostridium difficile Infections. Am J Gastroenterol. (2013)
- Leffler DA, Lamont JT. Clostridium difficile Infection. N Engl J Med. (2015)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.