Science. Evaluation of Glutamate Dehydrogenase Immunoassay Screening with Toxin Confirmation for the Diagnosis of Clostridium difficile Infection

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1 Evaluation of Glutamate Dehydrogenase Immunoassay Screening with Toxin Confirmation for the Diagnosis of Clostridium difficile Infection Steve Miller, MD, PhD, 1 * Arun Wiita, MD, PhD, 1 Carolyn Wright, MT(ASCP), 1 Helen Reyes, MT(ASCP), 1 and Catherine Liu, MD 2 ABSTRACT Clostridium difficile infections (CDIs) cause significant morbidity and increased hospital stays. Rapid and sensitive detection algorithms are needed to properly manage patients with suspected CDI. To determine the best algorithm for detection of CDI, we evaluated 2 assays for glutamate dehydrogenase (GDH) bacterial detection and toxin confirmation by enzyme immunoassay (EIA), cell culture neutralization (CCNA), and toxin B gene polymerase chain reaction (PCR). The 2 GDH screening methods had 76% and 90% sensitivity, respectively, for PCR-positive stool samples. Sensitivity and specificity compared with CCNA were 66% and 99% for toxin B EIA and 98% and 75% for PCR. Discrepancy analysis using toxigenic culture indicated a revised specificity for PCR of 93%. Review of the medical records of 8 patients with GDH-negative, PCR-positive stools showed that 1 patient had a false-negative result in 1 of 2 GDH assays, and data from 7 patients were believed not to represent true CDI cases. Use of a sensitive GDHscreening assay followed by PCR toxin confirmation is an efficient algorithm for detection of CDI. Keywords: Clostridium difficile infection, antibiotic-associated diarrhea, glutamate dehydrogenase screening, PCR toxin confirmation DOI: /LM31ZX1PFRZTGJUI Abbreviations CDI, Clostridium difficile infection; GDH, glutamate dehydrogenase; CDT, C difficile toxin; ADP, adenosine diphosphate; PCR, polymerase chain reaction; CCNA, cell culture neutralization assay; UCSF, University of California, San Francisco; IRB, institutional review board; EIA, enzyme immunoassay; CPE, cytopathic effect; TSB, tryptic soy broth; CI, confidence interval; IgG, immunoglobulin G Department of Laboratory Medicine 1 and Department of Medicine, Division of Infectious Diseases, 2 University of California, San Francisco *To whom correspondence should be addressed. Steve.Miller@ucsfmedctr.org Clostridium difficile infection (CDI) has become a major cause of diarrhea in hospitalized and nursing-home patients, leading to substantial morbidity and increased medical costs. The best approach to diagnosis of CDI has been a continuing subject of debate as newer test methods have been developed and the number and severity of cases increases. The accepted reference method of toxigenic culture requires several days to complete because C difficile bacteria are first isolated and then tested for toxin production. Several authors 1,2,3 have suggested 2- or 3-step algorithms in an effort to maintain sensitivity while offering a reasonable turnaround time. Most of these algorithms have relied on screening bacterial glutamate dehydrogenase (GDH) enzyme with subsequent toxin confirmation to rule out nontoxigenic C difficile strains. Therefore, the sensitivity of GDH screening and specificity of toxin confirmatory tests are key elements for optimal diagnosis of CDI. C difficile strains can produce 2 key virulence factors, toxin A and toxin B, that act by inactivating GTP-binding proteins of the Rho subfamily, leading to cellular apoptosis. 4 Some strains also produce C difficile toxin (CDT), a binary protein toxin with adenosine diphosphate (ADP) ribosylating activity; however, the clinical significance of this toxin is unclear. 5 Most strains produce toxin A and toxin B, with a number of reports describing toxigenic A - /B + strains 6 ; however, there have been no reports, to our knowledge, of toxin A + /B - strains causing clinical disease. This indicates that detection of toxin B or its gene in a patient with diarrhea is a specific marker for CDI. More recently, polymerase chain reaction (PCR) methods have been developed that detect the C difficile toxin genes. Most of these target the toxin B gene (GenBank Summer 2013 Volume 44, Number 3 Lab Medicine e65

2 accession number: X ) encoding for toxin B, which is required for clinical pathogenicity, although a few include toxin A and others probe for tcdc, a marker of the hypervirulent NAP1/BI/027 strain that produces a binary toxin. This study was performed to evaluate the diagnostic efficiency of laboratory testing for CDI, including GDH screening immunoassays and toxin detection by immunoassay, cell culture neutralization (CCNA), and PCR. We describe a 2-step algorithm that allows for effective CDI diagnosis and we discuss alternate testing strategies and their implications for clinical practice and infection control. Materials and Methods Specimens Analyzed This study was performed at the University of California, San Francisco (UCSF) Medical Center between January and June 2010, under institutional review board (IRB) approval. We analyzed 381 stool specimens that were submitted for C difficile testing to the clinical laboratory. Specimens were rejected if they did not conform to the shape of the container, and repeat specimens were rejected if the patient had been previously tested within 48 hours. All 4 methods were used to test 106 samples; 275 samples were screened for the presence of bacterial GDH by enzyme immunoassay (EIA) and lateral flow immunoassay. If the samples tested GDH-positive by either method, they were further tested via CCNA and PCR and frozen at -70ºC for further analysis. An additional 71 samples were tested using GDH and PCR methods, with results used to evaluate GDH sensitivity. A total of 31 samples with discrepant results were selected for confirmatory testing by toxigenic culture (culturing C difficile bacteria and testing isolates for the presence of toxin by CCNA). added to 1 well, and plates were incubated at 37º C and checked twice daily for cytopathic effect (CPE). A positive result was determined by CPE in the well containing the diluted stool specimen that was neutralized in the presence of antitoxins. PCR for the C difficile toxin B gene was performed using the GeneXpert real-time PCR instrument (Cepheid, Sunnyvale CA) according to the manufacturer s instructions. For toxigenic culture CCNA testing, C difficile bacteria were isolated from the stool and broth culture supernatant and applied to CCNA plates. Briefly, frozen stool specimens were treated with an equal volume of 95% ethanol for 1 hour to select for bacterial spores, inoculated to Brucella agar, and incubated anaerobically. Suspect C difficile colonies were identified by RapID ANA panel (Thermo Fisher Scientific Inc, Waltham, MA) and grown in tryptic soy broth (TSB) used for CCNA testing for toxin production. Patient results were reported using the C. DIFF CHEK-60 assay for GDH screening followed by CCNA for toxin confirmation. Samples negative for GDH were reported as negative for CDI approximately 4 hours after receipt in the laboratory, and samples positive for GDH were tested by CCNA with incubation for 3 days before reporting a negative result. Analysis The laboratory standard for diagnosis of CDI was a positive result for CCNA or toxin B PCR. Medical records review was performed for patients with stool samples that tested GDH-negative but PCR-positive to determine the progression of CDI. Patients who were given metronidazole or vancomycin for CDI or whose diarrhea did not improve within 1 week were considered to have true CDI, whereas patients who were not treated or had been treated before the positive test result and had resolution of diarrhea within 1 week were considered not to have clinical CDI. Laboratory Methods GDH EIA testing was performed using the C. DIFF CHEK- 60 EIA assay (hereafter, CHEK-60, Techlab, Blacksburg, VA) according to the manufacturer s instructions. A combination of GDH and toxin A/B antigen detection was performed using the C. DIFF QUIK CHEK Complete lateral flow immunoassay (hereafter, QUIK, Techlab) according to the manufacturer s instructions. A CCNA was performed by diluting stool specimens 1:4 in phosphate-buffered saline and applying the diluted mixture to 16-well plates containing human foreskin fibroblast monolayers (Diagnostics Hybrids Inc, Athens OH). Antisera to toxins A and B were Results Comparison of C difficile Detection Algorithms We tested 244 stool samples using 5 methods: 2 that detected GDH antigen and 3 that detecting toxin production or toxic B presence. An additional 137 stool samples were screened for GDH antigen and toxin by lateral flow immunoassay; due to their negative results, these samples were not subjected to the other tests. The results for these samples are shown in Table 1. In comparison to CCNA, the QUIK lateral flow EIA assay Downloaded e66 Lab Medicine Summer 2013 Volume 44, Number 3

3 Table 1.Results of Clostridium difficile Testing a C. DIFF C. DIFF QUIK C. DIFF QUIK Samples, Positive Results Via CHEK-60 GDH CHEK GDH CHEK ToxB b CCNA PCR No. c Toxigenic Culture, % Neg Neg Neg Neg Neg 73 ND Neg Neg Neg ND ND 137 ND Pos Pos Neg Neg Neg 31 ND Pos Neg Neg Neg Neg 11 ND Neg Pos Neg Neg Neg 1 ND Pos Pos Pos Neg Neg 1 ND Neg Neg Neg Pos Neg 1 0 Neg Neg Neg Neg Pos 3 ND Pos Pos Pos Pos Pos 57 ND Pos Pos Neg Pos Pos 27 ND Pos Pos Neg Neg Pos Pos Pos Neg Pos Neg Pos Neg Neg Neg Pos Neg Pos Neg Pos Pos 1 0 Neg Pos Neg Neg Pos Abbreviations: GDH, glutamate dehydrogenase; CCNA, cell culture neutralization assay; PCR, polymerase chain reaction;neg, negative; Pos, positive; ND, not determined. a When available, samples with discrepant results were tested by toxigenic culture.c. DIFF CHEK-60 EIA assay and C. DIFF QUIK CHEK Complete are manufactured by Techlab (Blacksburg, VA). b GenBank accession number: X c Total = 381. results yielded sensitivity of 66% and specificity of 99% for toxigenic C difficile. Comparison of PCR to CCNA showed sensitivity of 98% and specificity of 75%. However, testing of PCR-positive, CCNA-negative samples via toxigenic culture showed that 22 of 28 (79%) had positive results. Extrapolating this true positivity rate yields an adjusted specificity of 93% for the GeneXpert PCR assay. Table 2 shows a comparison of 2 algorithmic approaches using GDH screening for diagnosis of CDI. Compared with our previous clinical approach using CHEK-60 GDH followed by CCNA, an algorithm using QUIK GDH followed by PCR testing had sensitivity of 99% and specificity of 74%. Most of the difference in positivity rates is due to the fact that more samples have toxin B detected by PCR and test negative for toxins via CCNA. Overall, PCR detected an additional 40 samples that had tested negative via CCNA, which constitutes 10% of total samples and 24% of GDHpositive samples. Table 3 describes the sensitivity and specificity of various testing algorithms for the diagnosis of CDI, using a reference standard of a positive result via CCNA or PCR. GDH detection methods, by themselves, had sensitivities of close to 95% but lower specificities due to their detection of nontoxigenic C difficile strains. The addition of toxin B EIA led to high specificity but poor sensitivity compared to the CCNA/PCR standard. GDH testing followed by CCNA Table 2. Comparison of 2 Algorithms Involving GDH Screening for Diagnosis of Clostridium difficile Infection a C. DIFF CHEK-60 GDH CCNA b C. DIFF QUIK CHEK GDH PCR Positive Negative Total Positive Negative Total Abbreviations: GDH, glutamate dehydrogenase; PCR, polymerase chain reaction; CCNA, cell culture neutralization assay. a C. DIFF CHEK-60 EIA assay and C. DIFF QUIK CHEK Complete are manufactured by Techlab (Blacksburg, VA). b Previous clinical algorithm. yielded high specificity but lower sensitivity than GDH testing followed by PCR. We analyzed samples with discrepant results via CCNA and PCR by toxigenic culture. Of the 2 samples that had tested CCNA-positive but PCR-negative, 1 tested positive via toxigenic culture. Both of these stool samples were from the same patient, who had been previously treated for CDI; PCR testing of the bacterial isolate for toxin B was positive. Toxigenic cultures yielded positive results for 22 of 28 (79%) of the CCNA-negative, PCR-positive samples. Summer 2013 Volume 44, Number 3 Lab Medicine e67

4 Table 3. Sensitivity and Specificity of Test Algorithms for Diagnosis of Clostridium difficile Infection a,b C. DIFF C. DIFF QUIK C. DIFF QUIK CHEK GDH + C. DIFF CHEK-60 C. DIFF QUIK C. DIFF CHEK C. DIFF QUICK Variable CHEK-60 GDH CHEK GDH C. DIFF QUIK CHEK ToxB c GDH + CCNA CHEK GDH + CCNA GDH + PCR CHEK GDH + PCR Sensitivity 94.4% 96.1% 44.9% 66.1% 67.7% 92.9% 96.1% Specificity 83.1% 87.0% 99.6% 100% 100% 100% 100% Abbreviations: GDH, glutamate dehydrogenase; CCNA, cell culture neutralization assay; PCR, polymerase chain reaction. a Reference standard is positive for CCNA or PCR. Samples that were tested by GDH methods only, which yielded negative results, are presumed to be negative via CCNA and PCR for the purposes of this analysis. b C. DIFF CHEK-60 EIA assay and C. DIFF QUIK CHEK Complete are manufactured by Techlab (Blacksburg, VA). c GenBank accession number: X One sample that tested CCNA- and PCR-positive tested negative via toxigenic culture, which demonstrates the difficulty in applying even the accepted gold standard test to diagnosis of CDI. GDH Sensitivity for PCR-Positive Stools For calculations of GDH sensitivity, we excluded samples that had been screened by GDH and subject them to further testing only if they tested positive for GDH. A total of 176 samples were tested initially via GDH and PCR, yielding 29 PCR-positive results. Of these 29 stool samples, 22 tested GDH-positive via the CHEK-60 GDH method (76% sensitivity; 95% confidence interval [CI], 56%-89%) and 26 tested GDH-positive via the QUIK GDH method (90% sensitivity; 95% CI, 72%-97%). Patient Characteristics for GDH-Negative, PCR-Positive Results Of the 8 samples that tested GDH-negative and toxin B PCR-positive, 4 tested negative via CHEK-60 GDH only, 1 tested negative via QUIK GDH only, and 3 tested GDHnegative via both assays, representing 8 patients total. Although 1 of the samples that had tested negative via CHEK-60 GDH also tested positive via CCNA, all 8 results were reported as negative for CDI using the previous clinical algorithm (CHEK-60 GDH followed by cytotoxin assay). The patient with a negative result via CHEK-60 GDH only but with a positive result via CCNA (reported as having tested negative for CDI) was clinically retested 12 days later and she was reported to have a positive result for CDI. This patient had multiple medical morbidities, including type-2 diabetes mellitus, end-stage renal disease, aortic insufficiency, and endocarditis; she was admitted for posterior spinal fusion surgery. On admission, she was noted to have diarrhea and a dilated sigmoid colon and was empirically started on metronidazole, which was discontinued when her test results returned with negative results for CDI. On hospital day 12, the patient developed fever and leukocytosis and tested positive at that time for CDI. She was treated for CDI with oral vancomycin and metronidazole, resulting in resolution of her diarrhea. The original and repeat specimens from this patient would have been reported as positive for CDI using the revised algorithm (QUIK GDH followed by PCR). Of the other 7 patients with positive PCR results but negative GDH results (6 patients: negative via CHEK-60 GDH; 4 patients: negative by QUIK GDH), none were treated for CDI. All had resolution of their diarrhea or an alternate diagnosis for their symptoms. The 4 samples that were negative via CHEK-60 GDH only would have been reported as positive for CDI using the revised algorithm (QUIK GDH followed by PCR), whereas the other 4 would be reported as negative for CDI using the revised algorithm. Patient Characteristics for CCNA-Positive, PCR-Negative Results Two samples were CCNA-positive and PCR-negative; both were from the same patient. The first sample had tested GDH-positive via both assays (reported as positive for CDI) and positive via toxigenic culture CCNA, whereas the other had tested negative via both GDH assays (reported as negative for CDI) and negative via toxigenic culture CCNA. The patient had had 2 previous samples reported as having positive results for CDI 5 days before the first study sample. The patient had been admitted for consolidation chemotherapy for acute myelogenous leukemia and was being treated for CDI before discharge; the diarrhea resolved after submission of the second sample. Downloaded e68 Lab Medicine Summer 2013 Volume 44, Number 3

5 Discussion Comparison of GDH Screening Methods Our results showed high concordance of GDH screening methods, with 96% agreement between the 2 assays tested. Whereas the CHEK-60 GDH detected positivity in more stool samples overall, compared with the QUIK GDH (42.5% vs. 40.7%), it had a lower ability to detect samples that were positive for toxin B via PCR (76% vs. 90%). This discrepancy could occur due to differing ability of these GDH assays to detect locally circulating toxigenic strains of differing ribotypes. The lateral flow immunoassay QUIK Complete has the advantages of allowing for rapid individual testing while yielding a result for C difficile toxin; however, it is more expensive than the batched well EIA CHEK-60 GDH test. Sensitivity of GDH Screening It is well accepted 7 that GDH lacks specificity, so that positive results require confirmation via another method, such as CCNA or PCR. However, there is considerable debate on the ability of GDH screening to detect cytotoxin-positivity in stool samples for diagnosis of CDI. Previous studies 1,8,9,10 have described GDH sensitivity ranging from 76% to 100%. A recent study 11 showed that algorithms using GDH screening had comparable sensitivity (91%) to PCR for ribotype 027 strains (also known as NAP1/BI or hypervirulent strains) but reduced sensitivity (69%) for non-027 strains. Although this study does not describe the loss of sensitivity that occurred due to GDH screening versus the rest of the testing algorithm, most of the reduction in sensitivity occurred due to GDH screening (David Persing, MD, PhD, personal oral communication). We found that different GDH assays can have varying sensitivity for toxin-positive stool samples; the CHEK-60 GDH test had 76% sensitivity, whereas the QUIK GDH method had 90% sensitivity for stool samples that had positive results via PCR testing. Our study is limited by the relatively low number of positive samples but its results are consistent with those of a recent meta-analysis that demonstrated greater than 90% sensitivity for GDH, compared with CCNA or toxigenic culture, as the gold standard. 12 The optimal testing approach taken by laboratories for diagnosis of C difficile infection is unclear. No test is perfectly sensitive; each approach has its benefits and drawbacks. Direct PCR screening will identify more potential CDI cases; however, in some of these, diarrhea will have other causes because the patient s body is merely colonized with the C difficile bacteria, such that treatment could be ineffective while the true cause of diarrhea is left uninvestigated. However, GDH screening may miss some patients with true CDI, particularly for non-027 ribotype strains. We performed medical record review for 8 patients with positive toxin B PCR results but negative GDH screening results and found that 1 of them had a false-negative CHEK-60 GDH result early in the disease course. There were no clinically significant cases of CDI with negative results via QUIK GDH. Laboratories should be aware of the limits of GDH screening, as noted in one of our cases, and allow for confirmatory testing with PCR in high-suspicion cases with negative GDH results. Infection control practices at each institution should be aligned with its choice of C difficile test algorithm. Generally, contact precautions are initiated for inpatients with diarrhea while C difficile testing results are being determined. Some hospitals will discontinue contact precautions based on a negative result of a C difficile toxin assay; for these institutions, a sensitive method that can detect low-level carriers, such as direct PCR, may be appropriate. In other hospitals such as ours, discontinuation of contact precautions is primarily symptom-based, so GDH screening algorithms can allow for improved clinical diagnostic specificity while patients with diarrhea that has no alternate explanation and who may carry low levels of toxigenic C difficile bacteria remain isolated. Patient Clinical Courses and Specificity of CDI Testing Asymptomatic carriage of C difficile is not uncommon for hospitalized inpatients, with as many as half of patients who test positive for the bacteria remaining asymptomatic. In a study by Kyne et al, 13 79% of asymptomatic C difficile carriers had evidence of cytotoxin production; also, carriers had elevated levels of anti-toxin immunoglobulin G (IgG) antibodies, which suggests that low-level carriage may actually protect against development of CDI. In this study, 8 patients with diarrhea tested positive for toxin B detected by PCR but tested negative for this toxin via GDH assays, indicating probable low-level bacterial carriage. Seven of these patients were not treated for CDI; they had improved or had alternate explanations for their diarrhea. One patient subsequently developed CDI that responded to treatment; this patient would have been diagnosed earlier if the QUIK GDH test had been in clinical use at the time. Although these patient numbers are low, our results suggest that most of the GDH-negative but PCR-positive Summer 2013 Volume 44, Number 3 Lab Medicine e69

6 patients do not go on to develop clinical CDI. However, we recommend that patients with a high index of suspicion and negative results on initial testing undergo further testing via a sensitive method, such as direct PCR with stool samples. In our study, 2 samples tested CCNA-positive but PCRnegative. Both samples were from the same patient and were taken 4 days apart. The patient was immunocompromised and being treated for CDI; repeat samples were sent to evaluate treatment efficacy. Although guidelines do not recommend retesting after a diagnosis of CDI, many physicians continue to do so for reasons of infection control and discharge planning. In this case, GDH results correlated with toxigenic culture CCNA results and yielded an earlier negative report than direct CCNA testing would have allowed. Use of a more sensitive test for CDI might delay patient discharge when repeat testing is used for this purpose. Further study is required to determine the kinetics of the disappearance of C difficile bacteria and toxins from patients being treated for CDI. Algorithm for C difficile Diagnosis Our previous diagnostic algorithm for C difficile disease using CHEK-60 GDH screening followed by CCNA allowed same-day turnaround time for GDH-negative samples, whereas GDH-positive samples required as many as 3 additional days to obtain the final result. Many patients with suspected CDI were treated empirically before results were reported; also, the known insensitivity of CCNA led to the ordering of multiple tests on patients suspected to harbor C difficile. Implementation of a new testing algorithm consisting of screening stool samples with QUIK Complete allowed for more rapid turnaround time with increased sensitivity for C difficile toxin detection. Our results showed that 74.5% of samples had concordant GDH and toxin results on the QUIK Complete and that the test results could be obtained within 1 hour of receipt in the laboratory. Samples with discordant results for GDH and toxin on the QUIK Complete (primarily GDH-positive and toxin-negative) were subject to PCR for toxin B, and results could be obtained in approximately 2 hours on all shifts due to the rapid and random-access nature of the GeneXpert instrument. Another approach to testing could be to perform PCR only, which would slightly increase the overall sensitivity but would add significant cost for the laboratory. Compared with our previous algorithm, use of the QUIK Complete, followed by PCR testing, yielded 99% sensitivity and 74% specificity. The difference in specificity occurred primarily due to CCNA-negative samples that tested positive for toxic B via PCR. Of these samples, 79% tested positive via toxigenic culture, indicating that most are true positives and are likely associated with CDI. Screening using QUIK GDH would not have missed any patients harboring toxin B-positive strains and with PCR-positive results who had subsequently developed clinical CDI. Using a combination CCNA/PCR reference standard, the QUIK GDH plus PCR algorithm yielded 96.1% sensitivity and 100% specificity, making it the optimal approach for CDI diagnosis. In conclusion, the C difficile testing algorithm presented herein allows for rapid, cost-effective diagnosis of CDI with improved sensitivity compared with conventional methods. LM Acknowledgements This work was supported by the UCSF Department of Laboratory Medicine. C. DIFF QUIK CHEK Complete EIA cartridges were supplied by Techlab for evaluation purposes. GeneXpert C difficile PCR cartridges were supplied by Cepheid for evaluation purposes. Author Disclosures None reported. References 1. 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Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile.j Clin Microbiol. 2004;42: van den Berg RJ, Claas ECJ, Oyib DH, et al. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. JClinMicrobiol. 2004;42: Schmidt ML, Gilligan PH. Clostridium difficile testing algorithms: What is practical and feasible? Anaerobe. 2009;15: Downloaded e70 Lab Medicine Summer 2013 Volume 44, Number 3

7 8. Snell H, Ramos M, Longo S, John M,Hussain Z. Performance of the TechLabC. DIFF Chek-60 enzyme immunoassay (EIA) in combination with the C. difficiletox A/B II EIA kit, the Triage C. difficilepanel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile associated diarrhea. JClinMicrobiol. 2004;42: 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.jclinmicrobiol. 2008;46: Chapin KC, Dickenson RA, Wu F and Andrea SB. Comparison of five assays for detection of Clostridium difficile toxin. JMolDiagn. 2011;13: 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. JClinMicrobiol. 2010;48: Shetty N, Wren MWD,Coen PG. The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis. JHosp Infect. 2011;77: Kyne L, Warny M, Qamar A, Kelly CP. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. NEngl J Med. 2000;342: Summer 2013 Volume 44, Number 3 Lab Medicine e71