Test Id : CDIFS

When this test is ordered, the reflex tests may be performed and charged separately. Isolates of Clostridioides (Clostridium) difficile (1,2) will automatically have susceptibility testing performed and billed as appropriate. Antimicrobial agents appropriate to the organism and specimen source will be tested according to Mayo’s practice and the laboratory’s standard operating procedures.

See Anaerobic Bacteria Antimicrobials in Special Instructions to review the table which provides a listing of the antimicrobials routinely tested in our laboratory as well as antimicrobials that may be tested upon request. If the antimicrobial of interest is not listed in this table, contact Mayo Clinic Laboratories at 800-533-1710 and ask to speak to the Bacteriology Anaerobe Laboratory.

Anaerobe susceptibilities battery will routinely be performed at an additional charge. If fewer than 3 antibiotics will be reported, then anaerobe susceptibilities battery will be canceled and anaerobe susceptibilities per agent will be charged per antibiotic tested. Based on susceptibility criteria, anaerobe susceptibilities per agent may be performed at an additional charge.

Culture is not the preferred diagnostic test for Clostridioides difficile. For routine diagnostic testing, order CDPCR / Clostridioides difficile Toxin, PCR, Feces

Specimen source is required.

Submit only 1 of the following specimens:

Patient Preparation: Patient should not use antacids, barium, bismuth, antidiarrheal medication, zinc oxide paste, Vagisil cream or oily laxatives prior to specimen collection.

Preferred:

Specimen Type: Preserved feces

Supplies: Culture and Sensitivity Stool Transport Vial (T058); Stool Collection Kit, Random (T635)

Container/Tube: Commercially available transport system specific for recovery of enteric pathogens from fecal specimens (15 mL of non-nutritive transport medium containing phenol red as a pH indicator, either Cary-Blair or Para-Pak C and S [T058])

Specimen Volume: Representative portion of feces; 5 mL

Collection Instructions:

1. Collect 1 gram or 5 mL fresh fecal specimen and submit in container with transport medium.

2. Place feces in preservative within 2 hours of collection.

Additional Information: Only diarrheal (ie., unformed) feces should be tested. Testing formed feces for C difficile is not clinically indicated.

Specimen Stability Information: Ambient (preferred) 96 hours/Refrigerated 96 hours/Frozen 7 days

Acceptable:

Specimen Type: Unpreserved feces

Supplies: Stool container, Small (Random), 4 oz (T288); Stool Collection Kit, Random (T635)

Specimen Volume: Representative portion of stool

Collection Instructions: Collect fresh stool and submit representative sample in stool container.

Specimen Stability Information: Ambient (preferred) 72 hours/Frozen 7 days

Additional Information: Only diarrheal (i.e., unformed) stool should be tested. Testing formed stool for C difficile is not clinically indicated.

Specimen Stability Information: Ambient (preferred) 72 hours/Frozen 7 days

Specimen Type: Fresh tissue or biopsy

Sources: Colon

Supplies: Anaerobe Transport Tube (T588)

Specimen Volume: Entire collection, 1-2 cm(3)

Collection Instructions: Aseptically collect a 1-2 cm(3) piece of tissue whenever possible. In general, a larger piece of tissue is preferred. Submit in an anaerobic transport tube.

Specimen Stability Information: Ambient 72 hours

• Anaerobic Bacteria Antimicrobials

Stool: 1 gram or 5 mL

Tissue: 5 mm(3)

Providing an isolate suitable for antimicrobial susceptibility testing to direct antimicrobial therapy of extraluminal infections and in cases of treatment failure

When this test is ordered, the reflex tests may be performed and charged separately. Isolates of Clostridioides (Clostridium) difficile (1,2) will automatically have susceptibility testing performed and billed as appropriate. Antimicrobial agents appropriate to the organism and specimen source will be tested according to Mayo’s practice and the laboratory’s standard operating procedures.

See Anaerobic Bacteria Antimicrobials in Special Instructions to review the table which provides a listing of the antimicrobials routinely tested in our laboratory as well as antimicrobials that may be tested upon request. If the antimicrobial of interest is not listed in this table, contact Mayo Clinic Laboratories at 800-533-1710 and ask to speak to the Bacteriology Anaerobe Laboratory.

Anaerobe susceptibilities battery will routinely be performed at an additional charge. If fewer than 3 antibiotics will be reported, then anaerobe susceptibilities battery will be canceled and anaerobe susceptibilities per agent will be charged per antibiotic tested. Based on susceptibility criteria, anaerobe susceptibilities per agent may be performed at an additional charge.

Clostridioides difficile can cause diarrhea and may cause pseudomembranous colitis. Overgrowth of toxin-producing C difficile in the colon leads to the production of toxins A and/or B by the organism, and subsequent diarrhea. C difficile infection should be suspected in patients with symptoms of diarrhea with risk factors such as current or recent use of antibiotics, history of C difficile infection, current or recent hospitalization or placement in a nursing home or long-term care facility, over 65 years of age, and gastric acid suppression. C difficile infection is the most common cause of diarrhea in hospitalized patients and may lead to serious complications, including sepsis, bowel perforation, and increased overall mortality (especially in elderly patients). The incidence of C difficile infection has risen in the community and in healthcare settings. While culture is not the preferred means to diagnose C difficile-associated diarrhea, culture for C difficile provides an isolate suitable for antimicrobial susceptibility testing.

Susceptibility testing routinely includes metronidazole and vancomycin. Routine antimicrobial susceptibility testing of C difficile isolates associated with intestinal infection is not suggested.(4,5) Recent Infectious Diseases Society of America and Society for Healthcare Epidemiology of America guidelines provide treatment guidance for C difficile infection based on clinical course and history rather than measured minimal inhibitory concentrations of isolates.(6) Susceptibility testing of C difficile isolates may be warranted in cases of treatment failure, or when used to guide therapy of extraluminal infections.

Note that this test does not differentiate between toxin-producing and nontoxigenic strains of C difficile.

No growth of Clostridioides difficile.

Susceptibility results are reported as minimal inhibitory concentration (MIC) in mcg/mL. Breakpoints (also known as clinical breakpoints) are used to categorize an organism as susceptible, intermediate, or resistant according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.(3)

In some instances, an interpretive category cannot be provided based on available data and the following comment will be included: "There are no established interpretive guidelines for agents reported without interpretations."

Susceptible (S):

A category defined by a breakpoint that implies that susceptibility of an isolate depends on the dosing regimen that is used in the patient. To achieve levels that are likely to be clinically effective against isolates for which the susceptibility testing results (either MICs or zone diameters) are in the susceptible-dose dependent (SDD) category, it is necessary to use a dosing regimen (ie, higher doses, more frequent doses, or both, or extended infusion) that results in higher drug exposure than that achieved with the dose that was used to establish the susceptible breakpoint. Consideration should be given to the maximum, literature-supported dosage regimen, because higher exposure gives the highest probability of adequate coverage of an SDD isolate. The drug label should be consulted for recommended doses and adjustment for organ function.

Intermediate (I):

A category defined by a breakpoint that includes isolates with MICs or zone diameters within the intermediate range that approach usually attainable blood and tissue levels and/or for which response rates may be lower than for susceptible isolates.

Note: The intermediate category also includes a buffer zone for inherent variability in test methods, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.

Resistant (R):

A category defined by a breakpoint that implies that isolates with an MIC at or above or a zone diameter at or below the resistant breakpoint are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or that demonstrate MICs or zone diameters that fall in the range in which specific microbial resistance mechanisms are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

Epidemiological Cutoff Value (ECV):

The MIC that separates microbial populations into those with and without phenotypically detectable resistance (non-wild-type or wild-type, respectively). The ECV defines the highest MIC for the wild type population of isolates. ECVs are based on in vitro data only, using MIC distributions. ECVs are not clinical breakpoints, and the clinical relevance of ECVs for a particular patient has not yet been identified or approved by CLSI or any regulatory agency. 

When an ECV is reported, the following comment will be included: "This MIC is consistent with the Epidemiological Cutoff Value ECV observed in isolates [with or without] acquired resistance; however, correlation with treatment outcome is unknown."(CLSI: Performance Standards for Antimicrobial Susceptibility Testing. 32nd. CLSI Supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2023)

European Committee on Antimicrobial Susceptibility Testing (EUCAST) Interpretive Category Definitions:

S - Susceptible, standard dosing regimen: A microorganism is categorized as "Susceptible, standard dosing regimen", when there is a high likelihood of therapeutic success using a standard dosing regimen of the agent.

I - Susceptible, increased exposure*: A microorganism is categorized as "Susceptible, Increased exposure*" when there is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection.

R - Resistant: A microorganism is categorized as "Resistant" when there is a high likelihood of therapeutic failure even when there is increased exposure*.

*Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution and excretion of the antimicrobial agent will influence the infecting organism at the site of infection.

(The European Committee on Antimicrobial Susceptibility Testing. Available at www.eucast.org.)

A positive result indicates the presence of viable Clostridioides difficile in stool.

A positive culture may be found with asymptomatic C difficile colonization with a toxin-producing or non-toxin-producing strain, or with C difficile-associated diarrhea.

A negative result indicates the absence of C difficile growth in culture.

Isolation of C difficile does not differentiate between toxin-producing and non-toxin-producing strains.

Refer to the Reference Values section for interpretation of various antimicrobial categories.

The assay must be performed on fresh feces, fresh-frozen feces, or feces in transport medium. Only diarrheal (ie, unformed) feces should be tested.

Submission of more than 1 specimen for testing is not recommended.

Repeated testing during a single episode of diarrhea is not recommended.

Testing of asymptomatic patients (ie, without diarrhea) or for test of cure is not recommended.

Patients may asymptomatically carry C difficile.

Testing of specimens collected by colostomy, ileostomy, or colonoscopy has not been validated.

When antimicrobial susceptibilities are performed, in vitro susceptibility does not guarantee clinical response. Therefore, the decision to treat with a particular agent should not be based solely on the antimicrobial susceptibility testing result.

Fifty fecal specimens in Cary Blair transport media that were previously determined as positive for Clostridioides difficile by toxin PCR were subcultured directly onto CHROMagar C difficile plates. Plates were incubated under anaerobic conditions at 37 degrees C for 24 hours in accordance with the manufacturer's recommendation. C difficile identification was performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly from bacterial colony growth on CHROMagar plates. Using this method, C difficile was identified from 47 of 50 fecal specimens, corresponding to a 94% recovery rate. Two specimens that did not yield C difficile on CHROMagar media also failed to produce growth on conventional C difficile selective media (taurochocolate, cycloserine, cefoxitin, fructose agar; TCCFA). One specimen was recovered by the TCCFA media, but not by the CHROMagar C difficile media. Organisms other than C difficile were not recovered on CHROMagar, corresponding to 100% analytical specificity.

Metronidazole clinical breakpoints are interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. For C difficile, an epidemiological cutoff value (ECV) of less than or equal to 2 mcg/mL (wild-type) and greater than or equal to 4 mcg/mL (non-wild type) is applied for vancomycin, according to CLSI guidelines. If C difficile strains were to acquire a resistance gene or undergo gene mutation resulting in reduced susceptibility, vancomycin minimal inhibitory concentration (MIC) values of greater than or equal to 4 mcg/mL would be expected. When considering vancomycin therapy for C difficile infection, clinical breakpoints have not been established due to lack of sufficient data on clinical outcomes by MIC.(7,8) It is suggested that susceptibility testing of C difficile isolates and interpretation of results be discussed with appropriate clinical specialists (eg, infectious diseases and/or pharmacy).

1. Lawson PA, Citron DM, Tyrrell KL, Finegold SM. Reclassification of clostridium difficile as clostridioides difficile (Hall and O'Toole 1935) Prevot 1938. Anaerobe. 2016;40;95-99. doi:10.1016/j.anaerobe.2016.06.008

2. Oren A, Garrity GM. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol, 2016;66(7):2463-2466. doi:10.1099/ijsem.0.001149

3. CLSI: Performance Standards for Antimicrobial Susceptibility Testing. 32nd edition. CLSI Supplement M100. Wayne, PA. Clinical and Laboratory Standards Institute. 2023

4. McDonald LC, Gerding DN, Johnson S, et al. 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;66(7):987-994)

5. Schuetz AN, Carpenter DE: Susceptibility test methods: anaerobic bacteria. In: Carroll KC, Pfaller MA, eds. Manual of Clinical Microbiology. 12th ed. ASM Press; 2019:1377-1397

6. Johnson S, Lavergne V, Skinner AM, et al. Clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA): 2021 focused update guidelines on management of clostridioides difficile infection in adults. Clin Infect Dis. 2021;73(5):e1029-e1044

7. Snydman DR, McDermott LA, Jacobus NV, et al. U.S.-Based National Sentinel Surveillance Study for the epidemiology of clostridium difficile-associated diarrheal isolates and their susceptibility to fidaxomicin. Antimicrob Agents Chemother. 2015;59(10):6437-6443. doi:10.1128/AAC.00845-15

8. Goldstein EJ, Citron DM, Tyrrell KL, Merriam CV. Comparative in vitro activities of SMT19969, a new antimicrobial agent, against clostridium difficile and 350 gram-positive and gram-negative aerobic and anaerobic intestinal flora isolates. Antimicrob Agents Chemother. 2013;57(10):4872-4876. doi:10.1128/AAC.01136-13

6. Johnson S, Lavergne V, Skinner AM, et al. Clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA): 2021 focused update guidelines on management of clostridioides difficile infection in adults. Clin Infect Dis. 2021;73(5):e1029-e1044

Specimens are directly inoculated onto a chromogenic C difficile plate which is incubated anaerobically at 35 to 37 degrees C for 24 hours. Plates are observed for characteristic fluorescence using ultraviolet light at 365 nm. Fluorescent colonies are identified by MALDI-TOF MS or 16S ribosomal RNA gene sequencing.(Instruction manual: CHROMagar C. difficile, NT-EXT-077, Version 9, Available at www.CHROMagar.com)

The agar dilution method is used for routine susceptibility testing. The antimicrobial is added to agar in various concentrations depending upon levels attainable in serum. A standardized suspension of the organism is applied to the agar plates, which are incubated anaerobically for 42 to 48 hours at 35 to 37 degrees C. The end point is that in which a marked reduction occurs in the appearance of growth on the test plate as compared to that of growth on the control plate. Examples of marked change include a change from confluent growth to a haze, less than 10 tiny colonies, or 1 to 3 normal-sized colonies.(CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. 9th ed. CLSI standard M11. Wayne PA, CLSI, 2018)

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This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

87081-C. difficile Culture

87076-Anaerobe Ident (if appropriate)

87076-Id MALDI-TOF Mass Spec Anaerobe (if appropriate)

87153-Anaerobe Ident by Sequencing (if appropriate)

87181-Anaerobe Susceptibility per agent (if appropriate)

87181 x 3-Antimicrobial Susceptibility, Anaerobic Bacteria, MIC (if appropriate)