Clostridioides difficile – Antigen, PCR, Susceptibility, and Typing

Testing Indications

As per the  Infectious Diseases Society of America and Society for Health Epidemiology America (IDSA/SHEA ) guidelines, C. difficile testing is indicated for individuals with unexplained diarrhea (e.g. ≥ three unformed feces per day without underlying diarrheal condition such as laxative use) and with risk factors of C. difficile disease (e.g. systemic antibiotic therapy, hospitalization, advanced age, impaired immunity, gastrointestinal surgery).¹

Important: C. difficile testing is not indicated for asymptomatic individuals due to the possibility of clinically insignificant toxigenic C. difficile excretion in healthy individuals. Similarly, testing is not routinely indicated in patients under 12 months of age with diarrheal symptoms due to the high rate of concomitant C. difficile colonization in healthy newborns and infants. Repeat testing to monitor treatment response (or ‘test of cure’) is also not indicated, as test results may remain positive for weeks to months despite resolution of infection.

In addition to routine testing, PHO can perform additional fecal culture for susceptibility and typing; this additional testing is restricted to local health unit requests for case management and cluster/outbreak investigations.

Acceptance/Rejection Criteria

Routine testing requests will be rejected in the following scenarios:

• Formed fecal specimens (refer to the stool consistency chart for acceptable stool types)
• Rectal swabs
• Specimens in transport or preservative media (e.g. Cary-Blair, formalin, sodium acetate-acetic acid- formaldehyde -SAF, polyvinyl alcohol- PVA)
• Specimens from patients under 12 months of age or if date or birth not indicated
• Request for ‘test of cure’
• Insufficient specimen volume submitted (> 10 ml of feces required)
• Leaking specimen
• Multiple specimens collected on the same date from the same patient
• Specimens collected greater than 5 days from date of collection and not frozen
• Date of collection not indicated

In addition, testing requests specifically for susceptibility and typing will be rejected if received from submitters other than local health units.

Storage and Transport

Place the sealed specimen container in a biohazard bag and properly seal bag. Specimens should be stored at 2-8°C and shipped to PHO on ice packs within 48 hours of collection. If specimens might not be able to arrive at PHO within 48 hours of collection, they should be stored frozen (- 20°C or lower) and shipped on ice pack as soon as possible. Avoid repeated freezing and thawing of the specimen. All specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

The following fecal specimen collection method is recommended:

1. Uncap the empty sterile container.
2. During a bowel movement, collect a sufficient amount of unformed* feces in a clean and dry receptable (e.g. Chamber pot, wide-mouth jar, pie plate, or cellophane wrapped around toilet bowl) while avoiding urine or water coming in contact with the feces. (*Note: refer to the stool consistency chart for acceptable stool types.)
3. Using a disposable clean stick or spoon, transfer a minimum* of 10 ml of unformed feces. (*Note: do not overfill).
4. Recap the container tightly and shake vial to check that there is no leakage.
5. Label the container with the patient’s full name, date of collection, and date of birth or health card number.

Test Frequency and Turnaround Time (TAT)

Routine C. difficile antigen testing and confirmatory molecular testing are performed Monday to Saturday at PHO. Turnaround time is up to 24 hours for antigen testing and up to 3 business days for confirmatory molecular testing from receipt date at PHO’s laboratory, Toronto location.

Additional susceptibility testing is performed upon request at PHO. Turnaround time is up to 15 days from receipt at PHO. Additional typing is performed upon request at the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time is up to 28 days from receipt at PHO.

STAT and Critical Specimens Testing

Priority testing for cluster/outbreak investigations is available upon request. If needed, contact PHO’s Laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

C. difficile antigen testing is performed by enzyme immunoassay (EIA) using either the TechLab C. DIFF CHEK-60 (PHO’s laboratory, Toronto location) or the TechLab C. DIFF QUIK CHEK COMPLETE (all other locations) for the detection of glutamate dehydrogenase (GDH) (CHEK-60) or GDH in combination with toxins A and B (QUIK CHEK COMPLETE). The difference in test methodology for antigen detection was selected based on the volume of specimens received at PHO.

C. difficile confirmatory molecular testing is performed by qualitative PCR using the BD MAX™ Cdiff assay for the detection of the tcdB (toxin B) gene.

Where applicable, C. difficile susceptibility testing is performed by selective culture for isolation of the organism followed by gradient strip minimal inhibitory concentration (MIC) measurement following the Clinical and Laboratory Standards Institute (CLSI) M100.² C. difficile virulence typing is performed by laboratory-developed cdtB (binary toxin subunit B), tcdA (toxin A), tcdB (toxin B), and tpi (triose phosphate isomerase) PCR testing on the isolate at PHO.³ C. difficile cluster subtyping is performed by whole genome sequencing (WGS) using a research use protocol.⁴

Performance and limitations
GDH antigen testing evaluates the potential presence of C. difficile bacteria in feces. It has a reported sensitivity of 94-96% and specificity of 92-95% compared with reference methods (e.g., cell cytotoxin assay or cytotoxigenic culture)⁵. Negative results usually rule out CDI but positive results may represent nontoxigenic C. difficile excretion and therefore require confirmation with either toxin antigen testing or toxin gene PCR testing.

Toxin antigen testing evaluates the presence of toxin A and/or B in feces but does not distinguish between the two toxins. It has a reported sensitivity of 58-83% and specificity of 99% compared with reference methods. Positive results usually correlate well with clinically significant CDI but negative results do not rule out C. difficile due to the limited standalone assay sensitivity. When combined with parallel GDH antigen testing without PCR testing, it has a reported sensitivity of 58-82% and specificity of 99.5%.

Toxin B PCR testing evaluates the presence of toxigenic C. difficile genetic material in feces. When following a positive GDH antigen test result, it has a reported combined sensitivity of 91-96% and specificity of 96-98% compared with reference methods. In some instances, positive results may represent toxigenic C. difficile excretion without clinically significant toxin production, therefore results should always be correlated with clinical findings and the absence of other potential diarrheal illness etiologies.

Algorithm

If susceptibility and typing is requested for cluster/outbreak investigations: C. difficile susceptibility is available for vancomycin and metronidazole on the first four isolates of the cluster. Both virulence typing and cluster subtyping are available on a subset of cases most closely linked by epidemiological assessment.

Interpretation

The following table provides possible routine test results with associated interpretations:

If susceptibility testing is performed for cluster/outbreak investigation: Results will be provided for each antimicrobial agent tested as either “susceptible”, “susceptible dose-dependent”, “intermediate”, or “resistant” according to the applicable CLSI clinical breakpoint for systemic therapy use.

If typing is performed for cluster/outbreak investigation: The isolate’s cdtB, tcdA, tcdB, and tpi gene status will be reported. WGS results will be reported based on a range of analytical methods including single nucleotide variant (SNV) and/or core genome multilocus sequence typing (cgMLST) differences.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Note: positive results are not routinely reported to the Medical Officer of Health (MOH), therefore submitters suspecting a C. difficile outbreak in their hospital facility have the responsibility to report the outbreak to their facility’s MOH.

References

1. McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, Dubberke ER, Garey KW, Gould CV, Kelly C, Loo V, Shaklee Sammons J, Sandora TJ, Wilcox MH. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018 Mar 19;66(7):e1-e48. doi: 10.1093/cid/cix1085.
2. Clinical & Laboratory Standards Institute. M100 Performance Standards for Antimicrobial Susceptibility Testing. Wayne, PA.
3. Lemee L, Dhalluin A, Testelin S, Mattrat MA, Maillard K, Lemeland JF, Pons JL. Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol. 2004 Dec;42(12):5710-4. doi: 10.1128/JCM.42.12.5710-5714.2004. PMID: 15583303; PMCID: PMC535266.
4. Alfa MJ, Kabani A, Lyerly D, Moncrief S, Neville LM, Al-Barrak A, Harding GK, Dyck B, Olekson K, Embil JM. Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea. J Clin Microbiol. 2000 Jul;38(7):2706-14. doi: 10.1128/JCM.38.7.2706-2714.2000. PMID: 10878068; PMCID: PMC87004.
5. Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, O'Connor L, Oakley SJ, Pope CF, Wren MW, Shetty NP, Crook DW, Wilcox MH. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis. 2013 Nov;13(11):936-45.