Expires: September 7, 2023
Audrey Schuetz, M.D.
Professor of Laboratory Medicine and Pathology
Division of Clinical Microbiology
Mayo Clinic, Rochester, Minnesota
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Thank you for joining us for this “Hot Topic” on the laboratory diagnosis of C. difficile. My name is Audrey Schuetz, and I direct the initial processing laboratory and co-direct the Bacteriology Laboratory at Mayo Clinic –Rochester in Mayo Clinic Laboratories.
I do not have any relevant disclosures to share.
Before we get started, I wanted to update you on a name change for C. difficile. Bacterial names change based on updated genotypic and phylogenetic information highlighting evolutionary relatedness between organisms. In 2016, the genus name of this particular bacterial species—Clostridium difficile—was updated to Clostridioides. Not all the genera of Clostridium species were updated to Clostridioides, and the name changes are species specific. Thankfully, the genus name retained “C” as the first letter, so the disease can still be referred to as C. difficile infection (CDI) or C. difficile associated disease (CDAD).
Clostridioides difficile has received a lot of attention in the last two decades due to the increasing burden it has placed on our patients and our health care system. C. difficile is the most commonly reported pathogen causing health care-associated infections in U.S. hospitals, having surpassed methicillin-resistant Staphylococcus aureus, or MRSA, some years ago. Although health care-associated CDI cases have been decreasing somewhat since 2015 in the U.S., community-associated CDI cases have not. Since 2013, the National Healthcare Safety Network (NHSN) has mandated institutional reporting of CDI for hospitals or institutions participating in the Centers for Medicare and Medicaid Services (CMS) program.
Accurate and rapid diagnosis of CDI is important. Upon diagnosis, providers begin therapy with an appropriate antimicrobial agent such as oral vancomycin, metronidazole, fidaxomicin, or others and discontinue antimicrobial agents that may predispose to CDI. Infection control precautions are put into place in order to curb the spread of spores.
C. difficile disease presents with a range of clinical findings, from diarrhea to pseudomembranous colitis to toxic megacolon. Pseudomembranes in the colon may be seen on flexible sigmoidoscopy; they are adherent thick layers of inflammatory cells and mucus.
The most significant risk factor for CDI is antibiotic exposure. Although clindamycin, broad-spectrum cephalosporins, and ampicillin have most often been implicated in CDI, any antibiotic can cause this. When a patient takes antibiotics, beneficial bacteria in the intestine are destroyed or impaired for a period of time, increasing the likelihood that C. difficile leads to infection.
C. difficile is an obligate anaerobe that is a spore-forming Gram-positive rod. Note the pale-staining, empty areas of the bacterial rods on the Gram stain photo on the right, indicating the spores. In the 1930s, it was originally named Bacillus difficilis due to difficulty isolating this bacterium in the laboratory.
C. difficile spores are ubiquitous in the natural environment including seawater, rivers, and soil. The organism is spread by the fecal-oral route, person to person. When spores are ingested from the environment, they can then germinate in the intestine and produce toxins.
In hospitals, spores are present on many environmental surfaces such as commodes and bed curtains, as well as on the hands of caregivers. They have even been found in animals, but there has been no definitive evidence to date of zoonotic transmission from animals to humans.
The spores are resistant to alcohol gels and many hospital disinfectants and can persist on inanimate surfaces for several months if surfaces are inadequately cleaned.
The epidemiology of C. difficile has been changing since the year 2000, with a rise in disease severity and changes in ribotypes. One factor associated with this evolving epidemiology is the NAP1/B1/027 strain, also referred to as the hypervirulent strain. NAP1 stands for North American pulsed-field gel electrophoresis type-1 and 027 refers to PCR ribotype number. Compared to non-NAP1 strains, this strain has been more often associated with increased CDI frequency and more severe disease and complications. NAP1 has also shown higher rates of fluoroquinolone resistance. In addition, NAP1 strains were found to produce binary toxin as well as toxins A and B. Although fluoroquinolone resistance does not affect management of CDI, because this class of antimicrobials is not used for CDI treatment, resistance to fluoroquinolones may provide the NAP1 strain with a survival advantage over susceptible strains in health care facilities where these antibiotics are commonly used. However, non-NAP1 strains have also been associated with increased severity of CDI and production of binary toxin.
The prevalence of various C. difficile strains differs according to geographical region and patient subsets, and differentiation of the NAP1 strain in particular has not become yet commonplace in C. difficile diagnosis.
There are several C. difficile toxins that are involved in disease. Toxin A (encoded by the tcdA gene) is an enterotoxin that causes fluid accumulation in the bowel. Toxin B (encoded by the tcdB gene) is cytopathic to (causes distortion of) cells when cultured in the laboratory. The tcdC gene regulates toxin A and B production. Genes cdtA and cdtB encode the binary toxin. Molecular assays target a variety of these genes.
Laboratory diagnosis can be difficult for C. difficile, in part due to the colonization state. Asymptomatic carriage or colonization can occur with nontoxigenic, or non-toxin-producing, strains as well as toxigenic strains; however, carriage with toxigenic strains is more common than with nontoxigenic strains.
Colonization with C. difficile ranges from 0.4% to 15% of adults from the general population, but the percentage increases with the presence of particular risk factors such as elderly age, inpatient status, residency in a long-term care facility, and others. On the other hand, up to 90% of infants are colonized. The intestinal cells of neonates do not appear to have receptors for toxins A and B; a much higher percentage of neonates may have detectable C. difficile in their stools, but do not manifest with disease.
Therefore, the sole presence of C. difficile toxins is insufficient for a diagnosis of CDI. As a result, only unformed stools should be tested when assessing CDI. Formed stools may be tested, however, in cases of ileus or toxic megacolon when stool is not passed.
There are various strategies for the laboratory diagnosis of C. difficile. The most commonly employed assays are those outlined in the red box. Nucleic acid amplification tests (NAATs) are molecular-based assays that detect the genes encoding the toxins rather than the toxins themselves. There are various commercial and lab-developed NAATs. They are highly sensitive but may pick up the colonization state if the proper stool specimen is not sent to the laboratory for testing. NAATs are more sensitive than enzyme immunoassays (EIAs).
EIAs may detect either toxins A/B and/or glutamate dehydrogenase (GDH). Toxin A/B EIAs are rapid tests and take minutes to perform; however, they are less sensitive than other methods. GDH EIAs detect the enzyme that is produced by both toxigenic and nontoxigenic strains of C. difficile. GDH is produced at much higher levels than toxins A and B. Advantages of the GDH test include rapidity, and recent studies have shown high sensitivity for these assays. The disadvantage is that this test cannot be used as a stand-alone test for CDI. A confirmatory test for the presence of toxin is needed. These assays have been used in algorithmic diagnostic approaches using combinations of tests such as toxin A/B EIA coupled with GDH EIA, molecular assays, and/or cytotoxicity assays.
Culture is a highly sensitive method of recovering the organism when selective culture media is used. Recovery of strains allows for further molecular typing studies (such as for comparison of relatedness of strains) or for antimicrobial susceptibility testing when indicated.
However, nontoxigenic strains can be recovered in culture, so further testing would be required to confirm toxigenicity. Resultantly, C. difficile culture should not be used as the primary diagnostic method for CDI. Time to results is typically 24 to 48 hours.
The chromogenic medium CHROMagar by bioMérieux for C. difficile is utilized by Mayo Clinic – Rochester for culture, when requested. C. difficile colonies will grow on this medium and will fluoresce under ultraviolet light, as pictured. Other bacteria will be inhibited from growth or will not fluoresce. Confirmation of toxigenicity is not performed at Mayo Clinic – Rochester with the C. difficile culture.
Clinical practice guidelines for CDI in adults and children were published in 2018 by a joint group of experts from the Infectious Diseases Society of America (IDSA) and the Society for Healthcare Epidemiology of America (SHEA). In these guidelines, there were two important updates regarding diagnosis of CDI as it relates to the microbiology laboratory. The first question and recommendation is as follows. What is the most sensitive method of diagnosis of CDI in stool specimens from patients likely to have CDI based on clinical symptoms? Their recommendation is to use NAAT alone or a multistep algorithm for testing (i.e., GDH plus toxin; GDH plus toxin, arbitrated by NAAT; or NAAT plus toxin) rather than a toxin test alone when there are pre-agreed institutional criteria for patient stool submission. For institutions which provide clear guidance on sending only liquid stools in the appropriate clinical scenario, either of these two testing approaches may be used.
Their second question is: What is the best-performing method (i.e., in use positive and negative predictive value) for detecting patients at increased risk for clinically significant C. difficile infections in commonly submitted stool specimens? Their recommendation for settings in which there are no pre-agreed institutional criteria for patient stool submission is to use a stool toxin test as part of a multistep algorithm rather than an NAAT alone.
A macrolide agent, fidaxomicin, was approved by the U.S. Food and Drug Administration (FDA) in May 2011 for the treatment of CDI. It was the second agent after vancomycin to be approved by the FDA for CDI. It is bactericidal, and oral administration leads to high fecal concentrations that exceed the minimal inhibitory concentrations (MICs). Mayo Clinic – Rochester currently offers metronidazole and vancomycin susceptibility testing for C. difficile from intestinal sources.
I’ll wrap up this talk with general testing guidelines. First, repeat testing for use as a test of cure is not acceptable. Toxins can be detected in stool as long as 30 days after resolution of symptoms. Second, formed stools should not be tested when assessing for C. difficile infection. Third, testing should not be performed on children under one year of age.
In summary, several testing approaches are available, and laboratories may use more than one testing platform in reflexive or algorithmic approaches when assessing CDI. The choice of which assay to use to detect CDI is dependent upon many factors, including the population tested and the laboratory’s testing capabilities with that particular testing platform or platforms.
Thank you for listening. My references follow.