Hot Topic: Clostridium difficile 9/1/2012
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1 Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speaker for this program is Dr. Jon Rosenblatt, a Professor of Medicine and Microbiology at Mayo Clinic, as well as a consultant in the Division of Clinical Microbiology. Dr. Rosenblatt provides an update on the laboratory diagnosis of Clostridium difficile infection. Thank you, Dr. Rosenblatt. Thank you, Sarah. 1
2 I have no disclosures. 2
3 Clostridium difficile is an anaerobic spore forming gram-positive bacillus. It is not part of the normal stool flora but may be present in many normal infants and up to 5% to 10% of asymptomatic adults. Both toxigenic, that is enterotoxin-producing, and nontoxigenic strains exist. 3
4 This slide depicts on the left a Gram stain of clostridia which are typically large grampositive bacilli. On the right-hand side of the slide we see a spore stain illustrating the oval subterminal spores of clostridia which are an integral part of the ability of this organism to persist in the environment and in colonized or infected patients. 4
5 C difficile is the most important cause of antibiotic-associated diarrhea and pseudomembranous colitis that are now collectively termed Clostridium difficile infection or CDI. The development of CDI requires both acquisition of a toxigenic strain of C difficile as well as alteration of the normal bowel flora allowing the organism to proliferate which is usually related to antimicrobial use. 5
6 This slide illustrates the pathogenic result of severe Clostridium difficile infection. On the left-hand side and upper right are endoscopic views of the pseudomembranes in the colon. On the bottom right, we see a toxic megacolon which can result in the necessity for colectomy or removal of large parts of the colon. 6
7 Virtually all antimicrobials may be associated with CDI, usually in a healthcare setting. However, increasingly, CDI may be community acquired and may not necessarily be related to recent use of antimicrobials. In addition, proton pump inhibitor use has also been recognized as a predisposing factor for CDI. 7
8 Disease is due to toxins A or B and possibly a recently recognized binary toxin. Recent outbreaks of severe CDI have been related to a toxin hyper producing strain of C difficile which is known as North American pulse field type 1 (NAP 1) or also as ribotype 027. Many 027 strains contain binary toxin and are also resistant to quinolone antimicrobials. 8
9 The pathogenicity locus or so-called PA-Loc region of the Clostridium difficile genome contains the genes for toxins A and B which are called tcda and tcdb and also the tcdc gene which actually modulates or down regulates toxin production. There are nucleic acid base pair deletions and a mutation in the tcdc gene which are thought to be associated with increased toxin production. 9
10 This is a graphic illustration of the PA-Loc region of the C difficile genome showing the locations of the genes and indicating, at the bottom, the deletions and mutation in the tcdc gene. 10
11 The 027 strain of C difficile has a single nucleotide mutation at position 117 which results in a truncation of the tcdc gene product. This is thought to be the major factor in the hyperproduction of C difficile toxins associated with this strain. 11
12 The laboratory diagnosis of CDI depends upon several important principles One should test specimens only from patients who actually have diarrhea, as defined as having 3 or more unformed stools within 24 hours. The specimen should take the shape of the container. There's no need to test more than 1 specimen when using a sensitive and specific method. After a positive test result, do not repeat the test unless the patient does not improve on treatment after 5 to 7 days or relapses after improvement. In other words, it is not necessary to perform a test of cure. 12
13 Culture of stool on cycloserine cefoxitin fructose agar (or CCFA medium) and toxin testing of the isolates recovered is the gold standard method for detecting C difficile. This is a sensitive and specific method but is too complex and time consuming for routine use. The cell culture cytotoxicity neutralization assay or CCNA was widely used in the past but is less sensitive than toxigenic culture and is also complex and time consuming. It is also dependant on subjective interpretation of the result. 13
14 There is an immunoassay for glutamate dehydrogenase (or GDH), the so-called common antigen found in all strains of C difficile. This is a sensitive test but it is not specific for toxigenic strains. Numerous enzyme linked immunoassays or EIAs for toxins A and B are available and 1 version is combined with the GDH test. However the EIAs are only 30 to 50% as sensitive as toxigenic culture and are not suitable as definitive tests for the diagnosis of CDI. 14
15 Polymerase chain reaction (or PCR) assays have recently been developed which detect genetic markers in the PaLoc region of C difficile including tcda, and/or tcdb or tcdc and/or the binary toxin and the 117 mutation. A number of these are available commercially including the BD genome assay, Cepheid Xpert, Prodesse Gastro and Meridian Illumigene assay. At the Mayo Clinic we use a laboratory-developed PCR method which uses fluorescence resonance energy transfer (or FRET) probes in a real-time format performed on the LightCycler. It detects the presence of tcdc. PCR assays are approximately 90% as sensitive as toxigenic culture. 15
16 A recent study at the Mayo clinic compared a number of these PCR assays with our own laboratory-developed assay for detection of toxigenic Clostridium difficile. In detection of 36 true positives and 310 true negatives, the PCR assays were essentially equivalent. Other published studies have reported similar results. 16
17 This slide illustrates an algorithmic approach to the testing for toxigenic C difficile which some investigators have advocated. It starts with screening for the GDH common antigen using a single test or a version which also includes a toxin EIA. If the GDH, antigen test is negative, one can report as negative for C difficile. If the GDH test is positive, then one considers the result of the toxin EIA test, which, if positive, can result in a presumptive positive report. If the EIA is negative, one must perform a more sensitive confirmatory test, either PCR or CCNA. If positive, the diagnosis of CDI is confirmed, or if negative CDI is not present and presumably only a nontoxigenic strain of C difficile was detected in the specimen. 17
18 The introduction of a commercial PCR which identifies the 027 strain of C difficile raises the question of how important it is to identify this particular strain in the clinical laboratory. This assay is 87% sensitive for the presumptive identification of the 027 strain but actually is not a typing method; it presumptively detects 027 by a PCR which detects tcdb, the 117 mutation, and the binary toxin. It is not FDA approved to guide therapy but only for epidemiologic use. 18
19 For background purposes we should recognize that the antimicrobials fidaxomicin (which costs $2800 per course of treatment) and vancomycin have similar cure rates for CDI, which are however lower for 027 strain infections than for non-027 infections. Also fidaxomicin treatment has been reported to have lower recurrence rates for CDI but only for infection with non-027 strains. It has therefore been suggested that because of this information, it would be helpful to report the presence or absence of the 027 strain. 19
20 It is also important to recognize that a recent report from Chicago, shows that the 027 strain is the most common strain in that area and also probably is in other parts of the country. Moreover, the spectrum of disease caused by this strain varies from mild to severe. Worldwide strains other than 027 are known to cause severe disease outbreaks and severe disease. Guidelines from SHEA, the Society for Hospital Epidemiology and the Infectious Diseases Society of America indicate that treatment and infection control should be based on disease severity and not on a particular strain type. 20
21 A potential problem associated with identifying a strain as 027 (or not 027) is that clinicians may misinterpret the significance of the result. This could result in excessive use of fidaxomicin or other agents and in unnecessarily aggressive infection control measures. Moreover, in an outbreak situation, it is preferable to perform actual strain typing rather than simply presumptive identification of 027, so that the outbreak strain can be definitively identified and further studied. 21
22 In summary CDI may be caused by any of several toxin producing strains. Clostridium difficile culture with isolate toxin testing is the gold standard for diagnosis. PCR is the most efficient direct method for lab diagnosis of CDI although some laboratories may prefer the algorithm approach using PCR as a confirmatory test for the presence of toxin. The Mayo standard of care for diagnosis is direct testing by PCR because of the low cost of our laboratory-developed test and the efficiency of its use in our high test volume setting. 22
23 This slide lists some references which may be of particular interest on this topic. Thank you. 23
24 24
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