Laboratory Diagnosis of Clostridium difficile Infections: There Is Light at the End of the Colon

INVITED ARTICLE CLINICAL PRACTICE Ellie J. C. Goldstein, Section Editor Laboratory Diagnosis of Clostridium difficile Infections: There Is Light at the End of the Colon Stephen M. Brecher, 1,2 Susan M. Novak-Weekley, 3 and Elisabeth Nagy 4 1 Pathology and Laboratory Medicine, VA Boston Healthcare System, West Roxbury, and 2 Department of Pathology, Boston University School of Medicine, Massachusetts; 3 Southern California Permanente Medical Group, Kaiser Permanente, North Hollywood; and 4 Institute of Clinical Microbiology, University of Szeged, Hungary Single molecular or multistep assays (glutamate dehydrogenase, toxin A/B, ± molecular) are recommended for the diagnosis of CDI in patients with clinically significant diarrhea. Rapid and accurate tests can improve resource allocations and improve patient care. Enzyme immunoassay (EIA) for toxins A/B is too insensitive for use as a stand-alone assay. This guideline will examine the use of molecular tests and multitest algorithms for the diagnosis of Clostridium difficile infection (CDI). These new tests, alone or in a multistep algorithm consisting of >1 assay, are more expensive than the older EIA assays; however, rapid and accurate testing can save money overall by initiating appropriate treatment and infection control protocols sooner and by possibly reducing length of hospital stay. We recommend testing only unformed stool in patients with clinically significant diarrhea by a molecular method or by a 2- to 3-step algorithm. Keywords. Clostridium difficile; laboratory design of C. difficile; Brecher guidelines Diagnostic assays to detect Clostridium difficile infection (CDI) have improved dramatically in the past 3 years. Enzyme immunoassays (EIAs) for toxins A/B are no longer recommended as primary, stand-alone tests due to poor sensitivity. The current discussion is focused on selecting the molecular assay ( polymerase chain reaction [PCR]/loop-mediated isothermal amplification) or test algorithm (2-step algorithms ± PCR or PCR ± toxin assay) that best fits the needs of patients at risk for CDI. This practice guideline is not meant to be a comprehensive review of the literature on test performance. For interested readers, there are many excellent reviews of this type available [1 3]. There are also other guidelines that review CDI including testing strategies [4, 5]. In Europe, the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines are also available [6, 7]. The issues that will be considered in this practice guideline are: 1. Who, what, and when to test (Why do we think this patient has CDI? How do we rule out CDI? Do we need to test multiple samples? Do we need to perform a test of cure?). 2. Acceptance or rejection of specimens (loose vs solid stool). 3. Accuracy of current assays (Does this test work well enough to rely on the results?). 4. Sufficiency of detection of the gene(s) for toxin production for the diagnosis of CDI (Do we need to confirm positive gene assays with a toxin assay?). 5. Cost and value (Can we save money with a more expensive test, and how do we measure cost/value in a complex medical system?). Received 26 March 2013; accepted 8 June 2013; electronically published 20 June 2013. Correspondence: Stephen M. Brecher, PhD, Pathology and Laboratory Medicine, VA Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA 02132 (stephen.brecher@va.gov). Clinical Infectious Diseases 2013;57(8):1175 81 The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. DOI: 10.1093/cid/cit424 WHO, WHAT, AND WHEN TO TEST The goals of testing patients with diarrhea are to identify cases of CDI and to rule out CDI, as diarrhea is quite common in hospitalized and long-term care patients for a variety of infectious and noninfectious reasons [8]. It is important to limit CDI testing to patients with clinically CLINICAL PRACTICE CID 2013:57 (15 October) 1175

significant diarrhea (CSD), because asymptomatic colonization with C. difficile has been reported [4, 9, 10]. CSD is defined as 3 unformed stool samples within 24 hours in patients who also have risk factors for CDI (Table 1). Dubberke et al emphasized the importance of obtaining clinical information from patients with diarrhea [9]. The number of positive test results varied considerably when patients were classified as having CSD when compared to test results alone. In their study, 19% of patients were on laxatives and 36% did not have CSD. The accurate diagnosis of CDI begins with obtaining appropriate specimens from patients with diarrhea and risk factors for CDI. With respect to stool specimens, the Brecher guidelines (Table 2) should be followed: only loose or liquid stool should be tested for CDI. Strict adherence to specimen consistency will help eliminate false positives in molecular-based assays due to carriage of toxigenic C. difficile. In patients with ileus and a strong suspicion for CDI, stool of any consistency, including rectal swabs, can be tested [11]. An important side issue here is that many physicians insist that the laboratory test solid and semisolid specimens. It is imperative for laboratories to create and enforce specimen guidelines. Appropriate specimen collection is paramount to CDI detection in any clinical laboratory today. Because individuals can be colonized with C. difficile, testing of formed stool can result in falsepositive tests, which may result in unnecessary treatments. DO NOT PERFORM A TEST OF CURE Patients who recover from CDI can become carriers of C. difficile and shedders of C. difficile spores [12, 13]. Performing a test of cure is not recommended, and physicians or healthcare providers should not order a test-of-cure assay on patients whose symptoms have resolved. If symptoms have not resolved following a full treatment course, or if diarrhea returns after treatment, additional testing for CDI is appropriate. Treatment failure occurs in 10% 25% of cases, and recurrent infection in 10% 25% of cases, depending on the specific treatment antibiotics used and host factors [4, 5]. Clinical predictors for recurrent CDI include age >65, severe underlying disease, and continued use of antibiotics for non-cdi treatment [14]. LABORATORY TESTING: TARNISHED GOLD STANDARDS It is generally accepted that toxigenic culture is the gold standard for CDI. Unfortunately, as a method, toxigenic culture is not standardized and thus can incorporate bias into data analysis. When diagnostic manufacturers submit an assay to the Food and Drug Administration (FDA), enriched toxigenic culture is used as the gold standard and comparator assay. Historically, as mentioned previously, a multitude of assays are Table 1. Risk Assessment: Does My Patient Have Clostridium difficile Infection? Risk Factor Significance Comment Currently on antibiotics Antibiotics in last 2 mo[45] 3 UF BMs per 24 h High Moderate Case definition available for C. difficile testing. Emerging data following the introduction of PCR in the past few years have highlighted the lack of sensitivity of many of the tests that have been used previously to detect CDI, and comparisons with different iterations of nonstandardized gold standards have not helped to separate the good from the bad. CYTOTOXICITY ASSAYS Certain antibiotics have greater risk than others All recent antibiotic use important As CDI severity increases, number of UF BMs increases Leukocytosis High As CDI severity increases, WBC usually increases Creatinine 1.5 times premorbid level Moderate Renal disease associated with CDI Decreasing albumin Moderate Protein loss associated with CDI Abbreviations: BM, bowel movement; CDI, Clostridium difficile infection; UF, unformed; WBC, white blood cell. The first diagnostic tests for CDI detected the presence of toxin B directly in stool specimens by its cytopathic effects on cells in cell or tissue culture followed by specific neutralization with antibody. These were time consuming (2 3 days), and cell/tissue culture technology was not within the scope of practice of a typical clinical microbiology laboratory. These tests are only rarely performed today as a routine diagnostic procedure, although they were originally the gold standard. In a comprehensive study by Planche et al, these tests performed poorly when compared to toxigenic culture [15]. TOXINS A AND B BY ENZYME IMMUNOASSAY EIA for toxins A and B became widely available around the mid-1980s and quickly became the routinely used diagnostic Table 2. The Brecher Guidelines Observation Look at the stool specimen Put a thin lab grade stick in the specimen a Refers to a single stool specimen. Response If it ain t loose, it s of no use If the stick stands, the test is banned If the stick falls, test them all a 1176 CID 2013:57 (15 October) CLINICAL PRACTICE

assay for CDI in clinical microbiology laboratories. EIA testing predominated for many years despite poor sensitivity. Historically, the use of these poorly performing assays for the diagnosis of CDI resulted in physicians ordering the test multiple times from the same patient. It became, in fact, standard of practice in many facilities to order C. difficile EIA 3, which delayed diagnosis and increased the cost of testing. If only 1 of the 3 specimens was positive, the patient was considered positive for CDI. Sensitivity for EIA assays averages about 60%; specificity is better, but 1 study reported false positives when EIA values were in the low positive range [16 18]. Therefore, it is strongly recommended that EIA testing for toxins A/B should not be performed as the initial diagnostic assay for CDI because of unacceptable sensitivity (Table 3). GLUTAMATE DEHYDROGENASE ENZYME IMMUNOASSAY Glutamate dehydrogenase (GDH), a metabolic enzyme, is found in all ribotypes of C. difficile [19]. An EIA assay for GDH can be used as a reasonable screening assay for the detection of the organism but cannot differentiate toxin-positive from toxin-negative strains and, therefore, cannot be used as a standalone test to confirm the presence of toxigenic strains. Tenover and colleagues reported that the GDH assay had lower sensitivity with specimens positive for ribotypes other than 027 [20]. This variation in test performance in regard to ribotype supports the varied reports seen in the literature regarding GDH sensitivity [20]. The sensitivity of GDH ranges from 75% to >90% in documented studies [21, 22]. If institutions are unaware of the ribotypes circulating in their hospital, GDH performance could effect overall positivity rate and detection of CDI. TWO- TO 3-STEP ALGORITHMS A combination 2-step EIA assay for GDH and toxins A/B became available around 2007 [23]. If both tests were positive, or both were negative, the test results were acceptable. There are several publications using 2-step algorithms that document the wide range in sensitivity of GDH in combination with various confirmatory tests [23 27]. Two publications [24, 26] reported that 12% 13% of the samples required additional testing due to discrepant results (1 positive, 1 negative). A third test, either molecular or cell culture, was then performed for a definitive result (Table 3). This algorithm has replaced toxins A/B in many laboratories but still requires 2 3 steps to get a final result. Goldenberg et al published a small study that promotes GDH as the initial screen assay but found no value performing toxins A/B as the second step [28]. A more sensitive PCR was performed after the initial GDH screening. Step testing that successfully screens out negative specimens can significantly reduce the number of specimens that require a more expensive next-step test (eg, molecular test). As molecular tests become more universally available, it is suspected they will replace multistep algorithms for the diagnosis of CDI, especially if the cost is reduced. In the meantime, the 2- to 3-step testing is a much preferred alternative to toxins A/B EIA alone. MOLECULAR ASSAYS FDA-approved molecular tests became available in the United States around 2010. There are now 8 FDA-approved assays available in the United States (Tables 4 and 5). Five are PCR based and detect the toxin B gene. Two of the PCR assays can also detect other genes including the tcdc gene (found in NAP1/BI/O27strains and associated with increased toxin production). Three of the assays are isothermal based; 2 detect the toxin A gene, 1 detects the toxin B gene. Four of the assays (Cepheid GeneXpert, BD GeneOhm, Prodesse ProGastro Cd, and Meridian Bioscience Illumigene) have very good sensitivity and specificity for CDI [27, 29 33]. The other assays, Great Basin Portrait, Focus Diagnostics Simplexa, Quidel AmpliVue, and Nanosphere Verigene SP are newer to the market and have limited published literature available [34]. Molecular-based Table 3. Performance Characteristics: Results for Different Testing Strategies Assay(s) Sensitivity, % Specificity, % References a Toxin A/B alone 32 98.7 84 100 [16, 23, 46 50] GDH and toxin 41 92 94 100 [24, 46, 51] A/B EIA GDH/toxin A/B EIA 68 100 97 100 [26, 27, 51] and molecular Molecular alone 73 100 91 100 [27, 46, 51 56] Abbreviations: EIA, enzyme immunoassay; GDH, glutamate dehydrogenase. a Reference list is not inclusive of all published studies. Table 4. Food and Drug Administration Approved Polymerase Chain Reaction Assays for Clostridium difficile Company Gene Target(s) Time (min) BD GeneOhm tcdb 75 120 Prodesse ProGastro CD tcdb 180 200 Cepheid GeneXpert tcdb +/ tcdc Binary toxin 30 45 Focus Diagnostics Simplexa tcdb 60 90 Nanosphere Verigene SP tcda tcdb tcdc 110 120 CLINICAL PRACTICE CID 2013:57 (15 October) 1177

Table 5. Food and Drug Administration Approved Isothermal Amplification Based Tests for Clostridium difficile Company Gene Time (min) Meridian BioScience illumigene tcda 45 60 Quidel AmpliVue tcda 80 90 Great Basin Portrait tcdb 90 assays can require relatively expensive instrumentation, which, in combination with the cost of the individual assay, has slowed the widespread acceptance of this technology. However, they are rapidly gaining acceptance because of the increased sensitivity compared to the toxin A/B immunoassays. Of the 3 isothermal assays, 2 of the companies provide an inexpensive reader for clients who use their assay and the other assay does not require instrumentation. Isothermal-based assays may appeal to laboratories with low to moderate test loads (Tables 4 and 5). DOES A POSITIVE MOLECULAR TEST NEED A TOXIN ASSAY? A current topic of debate is whether a stool sample that was positive by a molecular assay needs to be tested with a confirmatory toxin assay. This issue was raised by Planche and others who presented data that a positive PCR assay should be confirmed with a toxin A/B assay [15]. They reported increased mortality in PCR-positive patients who also were toxin A/B positive. A 2-step protocol used by the National Health Service Laboratories in England uses either a GDH EIA for screening or a nucleic acid amplification test for toxin gene detection. If either is positive, the stool sample is tested by EIA for toxins A/ B[35]. In addition, a recent study by Longtin et al demonstrated that PCR alone was associated with a >50% increase in CDI incidence rate when compared to a 3-step assay that included GDH and toxin A/B detection. Of interest, toxin-positive patients had increased complications [36]. However, a recent study by Humphries et al did not show a correlation between toxin levels and severity of disease [37]. Limiting testing to loose or liquid stool in patients with risk factors for CDI can minimize the issue of colonization vs infection. An unmet need at this time is the absence of a highly sensitive and specific toxin assay. MOLECULAR CHARACTERIZATION OF ISOLATES It is well beyond the capacity of the routine clinical microbiology laboratory to grow and molecularly type individual patient isolates. There are limited data that clinical response to certain antibiotics may be related to specific ribotypes [38, 39]. The diversity of ribotypes circulating at a specific institution within the patient population may be important with respect to outcomes, but it is still beyond the scope of most laboratories to provide ribotype or other molecular characterization within a useful time span. That said, most laboratories are unaware of the ribotypes circulating within their patient population and how those ribotypes might impact assay performance and patient outcomes. Ribotyping and other molecular characterization studies are necessary for understanding the complex epidemiology and patient outcomes following infection with C. difficile but are not part of the routine diagnostic flow in a clinical microbiology laboratory [40]. A BREAKTHROUGH RATHER THAN AN OUTBREAK In facilities that have switched from EIA to multistep algorithms or molecular strategies, the number of detected cases of CDI increased [9, 36, 41]. These cases represented previously missed cases rather than an actual increase in the incidence of CDI. It will be important in the future to understand the true incidence of CDI in healthcare institutions as there could be Medicare penalties (United States) for nosocomial cases or financial penalties for increases in CDI cases (England) as reported by Goldenberg et al [42]. GATEKEEPERS To overcome the old habit of ordering C. diff 3, laboratories that change to molecular assays or multistep algorithms should limit testing to 1 specimen per patient per week [43]. Changing this ordering pattern will be difficult but not impossible; it will require focused education to physicians and to nurses on improved test performance and on who, why, and when to test. Pecking order, politics, and ego are hard to overcome. However, education and data go a long way. In fact, it may be time to form Team CDIFF to guide and educate staff as changes are made (Table 6). For example, The Boston VA Healthcare System switched from a toxins A/B EIA to a PCR assay in 2010 and went from 4000 assays per year to an average of 1400 per year for the past 3 years (S.M. Brecher, unpublished data). This reduction in test volume was achieved through educational seminars and meetings with the medical/surgical staff. Kaiser Permanente in Southern California took a similar approach and rejects duplicate specimens on patients that are submitted within 7 days of each other (S. M. Novak-Weekley, written personal communication, March 2013). VALUE, COST, AND MURAL DYSLEXIA Laboratories today, perhaps more than ever, are not only being asked to maintain a high level of quality but are also being asked to cut budgets and be more cost effective. More data are 1178 CID 2013:57 (15 October) CLINICAL PRACTICE

Table 6. Assemble a Multidisciplinary Team: Team CDIFF a Clostridium difficile Infection Management Team Representative member Infectious disease physician Gastroenterologist Microbiologist Pharmacy representative Infection control nurse/epidemiologist Building management specialist Hospital administration Combined role/mission Communication Education Test selection Infection control policies Antibiotic stewardship Cost and value Documentation needed to tie laboratory testing to outcomes in the hospital setting. It is possible that improved laboratory testing, even at a higher cost, will result in overall savings to the hospital due to decreased length of stay, decreased use of antibiotics, and appropriate use of infection control measures. However, this flexibility in resource allocation requires a symbiotic relationship between the hospital and the laboratory, which historically has not existed in most institutions. The value of an assay can be measured by the impact of the test results on both the patient and on the facility. It is important to remember that no laboratory test is 100% accurate. A clinically correct result impacts patient care, length of stay, and infection control protocols. The cost of a test can be low, but it is of little value if the result is inaccurate and has to be repeated 3 times over several days to get an accurate result. Mural dyslexia is the inability to see the big picture and is common in hospitals that have departments that do not work together. The cost of a PCR is significantly greater than that of an EIA but the value of the PCR is high as determined by patient outcomes. In other words, a molecular assay may increase laboratory costs but the hospital can significantly reduce other costs by improved patient care. There is little or no value to continue using a poorly performing, albeit inexpensive, assay. Remember, the most expensive test is one that does not work. Rapid, accurate diagnosis of CDI is essential to control nosocomial CDI. Lucado et al [44] reported that patients who acquire a secondary diagnosis of CDI are 6 times more likely to die and 4 times more likely to be at risk of dying. Also, from the same report, the mean length of stay was almost 3 times longer for patients with nosocomial CDI. The cost for patients with nosocomial CDI was 3 4 times higher than for non-cdi Table 7. patients and 3 times higher than for patients with primary CDI. Prevention is always less expensive than treatment, and a significant part of a prevention program is rapid and accurate diagnosis of CDI. CONCLUSIONS Toxin A/B EIA is not recommended as a stand-alone assay due to poor sensitivity. One-step molecular assays have high sensitivity and specificity and are recommended. As cost of these tests is an issue, algorithms involving screening with an EIA for GDH followed by a toxin assay and/or a molecular test are also recommended. Repeat testing and test for cure are not recommended (Table 7). Diagnostic tests for the accurate and rapid detection of CDI have improved dramatically over the past 3 years. There is light at the end of the colon. Note Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References Summary and Recommendations Test only loose (Brecher guidelines) or liquid stool specimens Only test stool from patients with clinically significant diarrhea Perform a single molecular test or a 2- to 3-step algorithm Test 1 stool sample per patient per week unless there are clinically compelling reasons to test another sample Do not perform a test of cure Create a Clostridium difficile infection management team 1. Tenover FC, Baron EJ, Peterson LR, Persing DH. Laboratory diagnosis of Clostridium difficile infection can molecular amplification methods move us out of uncertainty? J Mol Diagn 2011; 13:573 82. 2. Carroll KC, Bartlett JG. Biology of Clostridium difficile: implications for epidemiology and diagnosis. Annu Rev Microbiol 2011; 65:501 21. 3. Jones AM, Kuijper EJ, Wilcox MH. Clostridium difficile: a European perspective. J Infect 2013; 66:115 28. 4. Cohen SH, Gerding DN, Johnson S, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol 2010; 31:431 55. 5. Surawicz CM, Brandt LJ, Binion DG, et al. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am J Gastroenterol 2013; 108:478 98. 6. Crobach MJ, Dekkers OM, Wilcox MH, Kuijper EJ. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): data review and recommendations for diagnosing Clostridium difficile-infection (CDI). Clin Microbiol Infect 2009; 15:1053 66. 7. Bauer MP, Kuijper EJ, van Dissel JT. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): treatment guidance CLINICAL PRACTICE CID 2013:57 (15 October) 1179

document for Clostridium difficile infection (CDI). Clin Microbiol Infect 2009; 15:1067 79. 8. Polage CR, Solnick JV, Cohen SH. Nosocomial diarrhea: evaluation and treatment of causes other than Clostridium difficile. Clin Infect Dis 2012; 55:982 9. 9. Dubberke ER, Han Z, Bobo L, et al. Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol 2011; 49:2887 93. 10. Riggs MM, Sethi AK, Zabarsky TF, Eckstein EC, Jump RL, Donskey CJ. Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents. Clin Infect Dis 2007; 45:992 8. 11. Kundrapu S, Sunkesula VC, Jury LA, Sethi AK, Donskey CJ. Utility of perirectal swab specimens for diagnosis of Clostridium difficile infection. Clin Infect Dis 2012; 55:1527 30. 12. Sethi AK, Al-Nassir WN, Nerandzic MM, Bobulsky GS, Donskey CJ. Persistence of skin contamination and environmental shedding of Clostridium difficile during and after treatment of C. difficile infection. Infect Control Hosp Epidemiol 2010; 31:21 7. 13. McFarland LV, Elmer GW, Surawicz CM. Breaking the cycle: treatment strategies for 163 cases of recurrent Clostridium difficile disease. Am J Gastroenterol 2002; 97:1769 75. 14. Kelly CP. Can we identify patients at high risk of recurrent Clostridium difficile infection? Clin Microbiol Infect 2012; 18 (suppl 6):21 7. 15. Planche T, Davis KA, Coen PG, et al. Clinical validation of Clostridium difficile infection (CDI) diagnostics: importance of toxin detection. In: 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 9 12 September 2012. Abstract 160. 16. Sloan LM, Duresko BJ, Gustafson DR, Rosenblatt JE. Comparison of real-time PCR for detection of the tcdc gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection. J Clin Microbiol 2008; 46:1996 2001. 17. Alcala L, Sanchez-Cambronero L, Catalan MP, et al. Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens. J Clin Microbiol 2008; 46: 3833 5. 18. Brecher SM, O Brien C, Strymish J, Gupta K. Clostridium difficile EIA and PCR testing of EIA high positive, low positive and high negative stool samples. In: 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 15 September 2009. Abstract 1422. 19. Carman RJ, Wickham KN, Chen L, et al. Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes. J Clin Microbiol 2012; 50:1425 6. 20. Tenover FC, Novak-Weekley S, Woods CW, et al. Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches. J Clin Microbiol 2010; 48:3719 24. 21. Planche T, Aghaizu A, Holliman R, et al. Diagnosis of Clostridium difficile infection by toxin detection kits: a systematic review. Lancet Infect Dis 2008; 8:777 84. 22. Shetty N, Wren MW, Coen PG. The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis. J Hosp Infect 2011; 77:1 6. 23. Gilligan PH. Is a two-step glutamate dehyrogenase antigen-cytotoxicity neutralization assay algorithm superior to the premier toxin A and B enzyme immunoassay for laboratory detection of Clostridium difficile? J Clin Microbiol 2008; 46:1523 5. 24. Quinn CD, Sefers SE, Babiker W, et al. C. Diff Quik Chek complete enzyme immunoassay provides a reliable first-line method for detection of Clostridium difficile in stool specimens. J Clin Microbiol 2010; 48:603 5. 25. Swindells J, Brenwald N, Reading N, Oppenheim B. Evaluation of diagnostic tests for Clostridium difficile infection. J Clin Microbiol 2010; 48:606 8. 26. Sharp SE, Ruden LO, Pohl JC, Hatcher PA, Jayne LM, Ivie WM. Evaluation of the C.Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of Clostridium difficile disease. J Clin Microbiol 2010; 48:2082 6. 27. Novak-Weekley SM, Marlowe EM, Miller JM, et al. Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms. J Clin Microbiol 2010; 48:889 93. 28. Goldenberg SD, Cliff PR, French GL. Glutamate dehydrogenase for laboratory diagnosis of Clostridium difficile infection. J Clin Microbiol 2010; 48:3050 1. 29. Terhes G, Urban E, Soki J, Nacsa E, Nagy E. Comparison of a rapid molecular method, the BD GeneOhm Cdiff assay, to the most frequently used laboratory tests for detection of toxin-producing Clostridium difficile in diarrheal feces. J Clin Microbiol 2009; 47:3478 81. 30. Stamper PD, Babiker W, Alcabasa R, et al. Evaluation of a new commercial TaqMan PCR assay for direct detection of the Clostridium difficile toxin B gene in clinical stool specimens. J Clin Microbiol 2009; 47:3846 50. 31. Lalande V, Barrault L, Wadel S, Eckert C, Petit JC, Barbut F. Evaluation of a loop-mediated isothermal amplification assay for diagnosis of Clostridium difficile infections. J Clin Microbiol 2011; 49:2714 6. 32. Boyanton BL Jr., Sural P, Loomis CR, et al. Loop-mediated isothermal amplification compared to real-time PCR and enzyme immunoassay for toxigenic Clostridium difficile detection. J Clin Microbiol 2012; 50:640 5. 33. Pancholi P, Kelly C, Raczkowski M, Balada-Llasat JM. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/epi, and Illumigene C. difficile assays. J Clin Microbiol 2012; 50:1331 5. 34. Hicke B, Pasko C, Groves B, et al. Automated detection of toxigenic Clostridium difficile in clinical samples: isothermal tcdb amplification coupled to array-based detection. J Clin Microbiol 2012; 50:2681 7. 35. Wilcox MH. Overcoming barriers to effective recognition and diagnosis of Clostridium difficile infection. Clin Microbiol Infect 2012; 18(suppl 6):13 20. 36. Longtin Y, Trottier S, Brochu G, et al. Impact of the type of diagnostic assay on Clostridium difficile infection and complication rates in a mandatory reporting program. Clin Infect Dis 2013; 56:67 73. 37. Humphries RM, Uslan DZ, Rubin Z. Performance of Clostridium difficile toxin enzyme immunoassay and nucleic acid amplification tests stratified by patient disease severity. J Clin Microbiol 2013; 51:869 73. 38. Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med 2011; 364:422 31. 39. Cornely OA, Crook DW, Esposito R, et al. Fidaxomicin versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis 2012; 12:281 9. 40. Wilcox MH, Shetty N, Fawley WN, et al. Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England. Clin Infect Dis 2012; 55:1056 63. 41. Kaltsas A, Simon M, Unruh LH, et al. Clinical and laboratory characteristics of Clostridium difficile infection in patients with discordant diagnostic test results. J Clin Microbiol 2012; 50:1303 7. 42. Goldenberg SD, Price NM, Tucker D, Wade P, French GL. Mandatory reporting and improvements in diagnosing Clostridium difficile infection: an incompatible dichotomy? J Infect 2011; 62:363 70. 43. Luo RF, Banaei N. Is repeat PCR needed for diagnosis of Clostridium difficile infection? J Clin Microbiol 2010; 48:3738 41. 44. Lucado J, Gould C, Elixhauser A. HCUP Statistical Brief 124. Rockville, MD: Agency for Healthcare Research and Quality, Centers for Disease Control and Prevention, 2012; 124:1 12. 45. Pepin J, Valiquette L, Alary ME, et al. Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ 2004; 171:466 72. 1180 CID 2013:57 (15 October) CLINICAL PRACTICE

46. Peterson LR, Mehta MS, Patel PA, et al. Laboratory testing for Clostridium difficile infection: light at the end of the tunnel. Am J Clin Pathol 2011; 136:372 80. 47. Stamper PD, Alcabasa R, Aird D, et al. Comparison of a commercial real-time PCR assay for tcdb detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing Clostridium difficile in clinical samples. J Clin Microbiol 2009; 47:373 8. 48. Musher DM, Manhas A, Jain P, et al. Detection of Clostridium difficile toxin: comparison of enzyme immunoassay results with results obtained by cytotoxicity assay. J Clin Microbiol 2007; 45:2737 9. 49. Ticehurst JR, Aird DZ, Dam LM, Borek AP, Hargrove JT, Carroll KC. Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin. J Clin Microbiol 2006; 44:1145 9. 50. Eastwood K, Else P, Charlett A, Wilcox M. Comparison of nine commercially available Clostridium difficile toxin detection assays, a realtime PCR assay for C. difficile tcdb, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J Clin Microbiol 2009; 47:3211 7. 51. Walkty A, Lagace-Wiens PR, Manickam K, et al. Evaluation of an algorithmic approach in comparison with the Illumigene assay for laboratory diagnosis of Clostridium difficile infection. J Clin Microbiol 2013; 51:1152 7. 52. Chapin KC, Dickenson RA, Wu F, Andrea SB. Comparison of five assays for detection of Clostridium difficile toxin. J Mol Diagn 2011; 13:395 400. 53. Noren T, Alriksson I, Andersson J, Akerlund T, Unemo M. Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard. J Clin Microbiol 2011; 49:710 1. 54. Kvach EJ, Ferguson D, Riska PF, Landry ML. Comparison of BD GeneOhm Cdiff real-time PCR assay with a two-step algorithm and a toxin A/B enzyme-linked immunosorbent assay for diagnosis of toxigenic Clostridium difficile infection. J Clin Microbiol 2010; 48: 109 14. 55. O Horo JC, Jones A, Sternke M, Harper C, Safdar N. Molecular techniques for diagnosis of Clostridium difficile infection: systematic review and meta-analysis. Mayo Clin Proc 2012; 87:643 51. 56. Gyorke CE, Wang S, Leslie JL, Cohen SH, Solnick JV, Polage CR. Evaluation of Clostridium difficile fecal load and limit of detection during a prospective comparison of two molecular tests, the illumigene C. difficile and Xpert C. difficile/epi tests. J Clin Microbiol 2013; 51:278 80. CLINICAL PRACTICE CID 2013:57 (15 October) 1181