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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2010, p. 4347–4353 0095-1137/10/$12.00 doi:10.1128/JCM.02028-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 12

POINT-COUNTERPOINT What Is the Current Role of Algorithmic Approaches for Diagnosis of Clostridium difficile Infection?䌤 With the recognition of several serious outbreaks of Clostridium difficile infection in the industrialized world coupled with the development of new testing technologies for detection of this organism, there has been renewed interest in the laboratory diagnosis of C. difficile infection. Two factors seem to have driven much of this interest. First, the recognition that immunoassays for detection of C. difficile toxins A and B, for many years the most widely used tests for C. difficile infection diagnosis, were perhaps not as sensitive as previously believed at a time when attributed deaths to C. difficile infections were showing a remarkable rise. Second, the availability of FDA-approved commercial and laboratory-developed PCR assays which could detect toxigenic strains of C. difficile provided a novel and promising testing approach for diagnosing this infection. In this point-counterpoint on the laboratory diagnosis of C. difficile infection, we have asked two experts in C. difficile infection diagnosis, Ferric Fang, who has recently published two articles in the Journal of Clinical Microbiology advocating the use of PCR as a standalone test (see this author’s references 12 and 28), and Mark Wilcox, who played a key role in developing the IDSA/SHEA guidelines on Clostridium difficile infection (see Wilcox and Planche’s reference 1), along with his colleague, Tim Planche, to address the following question: what is the current role of algorithmic approaches to the diagnosis of C. difficile infection?

POINT

Appropriate determination of C. difficile test accuracy. A drawback to debating the optimal laboratory diagnostic approach to CDI remains the uncertainty surrounding what is the best gold standard method for use in comparative studies. Put simply, if toxin detection is the prime goal of laboratory diagnosis, then cytotoxin testing is the appropriate standard bearer. Conversely, if detection of toxigenic strains is the aim, then culture with demonstration of toxigenic potential of the isolate is the appropriate reference method (toxigenic culture). It is known that toxigenic culture is more commonly positive than toxin detection. However, more is not always better, and depending on your point of view, either culture lacks specificity or toxin detection lacks sensitivity. Unhelpfully, there is also no universally accepted method for either cytotoxin or cytotoxigenic culture testing. This unsatisfactory scenario has led some authors to use composite gold standards and/or discrepant analysis of results, each having potential drawbacks. These issues are crucial to the correct interpretation of studies comparing the accuracies of different C. difficile detection methods. Misleading interpretations may result if tests that do not target equivalent endpoints are compared. A typical C. difficile test evaluation is carried out on ⬃300 samples collected in a single institution, with ⬃20 to 50 positive and ⬃2 to 6 false-positive and false-negative samples (10). Point estimates of sensi-

The optimal laboratory diagnosis of Clostridium difficile infection (CDI) remains an area of controversy. The availability of multiple tests with different C. difficile targets (the bacterium, toxin(s), toxin gene, glutamate dehydrogenase enzyme [GDH]) both reflects and contributes to this uncertainty. The common tests, toxin enzyme immunoassays (EIA), have both poor sensitivity and specificity (2, 10). At low prevalence, the positive predictive values (PPV) of these tests may be as low as 50% (2, 10). Also, EIA sensitivities may be as low as 60% and are rarely above 90%. These deficiencies have led to the development of algorithms that combine two and sometimes three tests to improve diagnostic accuracy (3, 4, 5, 11, 12, 14). Although multistage laboratory diagnostic algorithms are commonplace in the diagnosis of HIV and syphilis, such approaches are less than ideal from a practicability standpoint, but are they necessary? Recent guidelines note that PCR tests that target a C. difficile toxin gene (PCRCDT) “appear to be rapid, sensitive, and specific and may ultimately address testing concerns. More data on utility are necessary before this methodology can be recommended for routine testing” (1).



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FIG. 1. Schema of the possible methods of combining C. difficile tests.

tivity and specificity are only as good as their overall precision. For this reason, 95% confidence intervals (95% CI), particularly the lower 95% CI, which gives reassurance that the test will perform at least that well, are more important than point estimates of sensitivity or specificity. Most published studies are only sufficiently powered to estimate a lower 95% CI of sensitivity between 40 and 85%, and specificity may be as low as 96% (10). Sensitivity and specificity are interrelated; thus, if a test cutoff is low, then the test will have a high sensitivity and a low specificity. Conversely, if a higher test cutoff is applied, then the specificity will be low and the sensitivity high. Comparing assays or algorithms only according to sensitivity is clearly not ideal, and composite measures, such as the area under the ROC curve (AUCROC) or the diagnostic odds ratio, should be used to compare tests. Another key issue when comparing results of multiple test evaluations is the prevalence of CDI in the cohort under study. As CDI prevalence decreases, the PPV of a test result is reduced; since the great majority of patients tested generally will not have CDI, the negative predictive value (NPV) will remain relatively high. A systematic review of 28 C. difficile toxin EIA kit evaluations found widely varying positivity (prevalence) rates (range, 6 to 53%; median, 15%) (10). Normally, ⬃5 to 10% of fecal samples, depending on CDI endemicity and sampling protocols, test positive for C. difficile toxin. Thus, PPVs and NPVs should be recalculated taking account of the local prevalence of disease to reduce misleading interpretations of test accuracy. Also, as local CDI prevalence changes (for example, as CDI is successfully controlled in an institution), then the PPV for a particular test within the same institution will decrease. C. difficile test algorithms. There are multiple potential two- and three-step algorithms for C. difficile testing, and crucially, the practicability of these will vary according to local expertise, facilities, and finance (3, 4, 5, 11, 12, 14). Multistep algorithms may increase laboratory

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costs or add delays, although interim results can be issued to minimize the latter. Improved diagnosis may conceivably lead to lower total CDI health care costs for CDI. There are a number of possible methods of combining assays (Fig. 1). Most reported algorithms rely on a strategy of performing an initial test (usually a GDH assay due to its high sensitivity) followed by a confirmatory assay on positive samples. Manufacturers’ cutoffs are nearly always used, but these are optimized for use in single-stage assays. The performance of this simple type of algorithm can be summarized by the following formulae: algorithm sensitivity ⫽ senstest1 ⫻ senstest2 and algorithm specificity ⫽ spectest1 ⫹ [spectest 2 ⫻ (1 ⫺ spectest1)], where “sens” is sensitivity and “spec” is specificity. It is clear from these formulae that for any test combination using this type of algorithm, there is an overall increase in specificity and decrease in sensitivity unless a reference test is performed as the confirmatory assay. Figure 1 shows alternative ways to combine tests that may be superior to those generally reported. Possibilities include optimizing assay cutoffs for use in two-stage algorithms. Alternatively, two tests performed in parallel may be combined into a single result using a formula derived by logistic regression, in a manner almost identical to that of the combination of antigen and antibody assays that are used in fourth-generation HIV assays. Thus, promising methods of combining C. difficile tests have yet to be fully explored. Also, there is a lack of evaluations comparing different algorithms and, notably, studies of sufficient power to determine true differences in accuracy. Disadvantages of PCRCDTs. There are potential Achilles heels for PCRCDTs centering on the fact that they do not detect the presence of fecal toxin, which is considered by many to be sine qua non for CDI. Elderly inpatients typically have C. difficile toxigenic culture positive rates of 10 to 20%. For example, 14% of 271 patients were colonized with C. difficile on admission, including 18 who were asymptomatic carriers. A further 47 patients (17%) became colonized during the hospital stay, 19 of whom remained asymptomatic (7). Furthermore, colonization by C. difficile is actually protective against the development of CDI when accompanied by an antitoxin antibody response (7). As diarrhea is such a common symptom, especially in elderly inpatients, a sizeable proportion of whom will have received antibiotics or laxatives or have been exposed to other pathogens (notably norovirus), there is therefore considerable potential to detect toxigenic C. difficile as an innocent (possibly protective) bystander. Second, up to a quarter of primary CDI cases experience a recurrence of symptoms following antibiotic treatment. However, long-term C. difficile gut colonization is also frequent; in an endemic CDI set-

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ting, 56% of patients were asymptomatic C. difficile carriers 1 to 4 weeks after treatment cessation (13). Thus, the significance of a positive C. difficile PCRCDT after a primary episode of CDI is evidently unclear. These examples emphasize the importance of appropriate specimen selection and indeed clear clinical details, without which accurate interpretations of test result cannot be made. Furthermore, the lower 95% CI of PCRCDTs is ⬃80 to 85% in most studies, with a lower 95% CI for specificity as low as 93% (6, 8, 9). These are clearly unacceptably low performance characteristics for a standalone test. Comparisons of PCRCDTs and algorithms. A review of three published evaluations of PCRCDTs compared with C. difficile test algorithms shows drawbacks similar to those highlighted above. Novak-Weekley et al. compared Xpert C. difficile testing with a combination of GDH and toxin EIA with or without cytotoxin testing but used frozen fecal samples, against the manufacturer’s instructions (9). The PCRCDT had the highest sensitivity (predictable from the above equations) but had a PPV of only 84% (hence almost 1 in 6 positives were false) and was not superior to the algorithm. Larson used an in-house real-time PCRCDT as part of a three-step algorithm (with GDH and toxin EIAs) (8). True test accuracy remains uncertain as not all of the tests under evaluation were carried out on all samples. Also, a gold standard of cytotoxin testing and PCRCDT was used to compare the performances of individual tests and the threestep algorithm, which likely introduced bias. Notably, the calculated adjusted sensitivity of the GDH EIA was 86.3%, which is markedly lower than those in some but not all studies (2, 8, 14). Kvach and colleagues compared the BD GeneOhm C. difficile assay with a toxin EIA and a two-step algorithm (GDH EIA followed by cytotoxin neutralization) (6). Specimens with discrepant results were tested by toxigenic culture as a gold standard. This definition explains why the “calculated” PPV for PCRCDT was 100%. Not surprisingly, the PCRCDT was more sensitive than the toxin EIA (91.4% versus 66.0%; P ⬍ 0.0001). However, the difference between PCRCDT and the two-step algorithm was not significant (P ⫽ 0.12). All these studies compared only sensitivities and specificities individually and did not compare AUCROC. All the studies used toxigenic culture or a composite score as a reference standard and used the manufacturers’ cut-off values for single tests in the two-stage algorithms (6, 8, 9). Only one study tested all samples by reference methods (9). Although the majority of these studies found that the sensitivity of the PCRCDT was greater than that of the algorithm, this is predictable from the design of the studies. Given these limitations in methodology and the sizes of the study populations, it is difficult to draw firm conclusions.

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Conclusions. Accurate diagnosis of CDI is vital for patient management, infection control, and the attainment of reliable surveillance data. The ongoing dilemmas concerning the accurate laboratory diagnosis of CDI highlight the need for sufficiently powered studies that incorporate both alternative gold standards and clinical interpretative data to resolve these uncertainties. C. difficile toxin EIAs are not suitable as standalone tests for CDI. The availability of commercial PCRCDTs is welcome, but their use as wholesale replacements for other tests is premature. Commercial PCRCDTs can deliver relatively rapid results, usually with high NPV, but specificity remains an issue. We believe that the evidence points to the use of algorithm testing for CDI, with prudent test selection according to local resources and needs, and good clinical acumen. C. difficile was named with good reason; accurate diagnosis of CDI remains an open and challenging book. REFERENCES 1. Cohen, S. H., D. N. Gerding, S. Johnson, C. P. Kelly, V. G. Loo, L. C. McDonald, J. Pepin, and M. H. Wilcox, Society for Healthcare Epidemiology of America, and Infectious Diseases Society of America. 2010. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Health care Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect. Control Hosp. Epidemiol. 31:431–455. 2. Eastwood, K., P. Else, A. Charlett, and M. Wilcox. 2009. Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J. Clin. Microbiol. 47:3211–3217. 3. Fenner, L., A. F. Widmer, G. Goy, S. Rudin, and R. Frei. 2008. Rapid and reliable diagnostic algorithm for detection of Clostridium difficile. J. Clin. Microbiol. 46:328–330. 4. Gilligan, P. H. 2008. Is a two-step glutamate dehydrogenase antigen-cytotoxicity neutralization assay algorithm superior to the Premier toxin A and B enzyme immunoassay for laboratory detection of Clostridium difficile? J. Clin. Microbiol. 46:1523–1525. 5. Goldenberg, S. D., P. R. Cliff, S. Smith, M. Milner, and G. L. French. 2010. Two-step glutamate dehydrogenase antigen real-time polymerase chain reaction assay for detection of toxigenic Clostridium difficile. J. Hosp. Infect. 74:48–54. 6. Kvach, E. J., D. Ferguson, P. F. Riska, and M. L. Landry. 2010. 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. 48:109–114. 7. Kyne, L., M. Warny, A. Qamar, and C. P. Kelly. 2000. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N. Engl. J. Med. 342:390–397. 8. Larson, A. M., A. M. Fung, and F. C. Fang. 2010. Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile. J. Clin. Microbiol. 48:124–130. 9. Novak-Weekley, S. M., E. M. Marlowe, J. M. Miller, J. Cumpio, J. H. Nomura, P. H. Vance, and A. Weissfeld. 2010. Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms. J. Clin. Microbiol. 48:889–893. 10. Planche, T., A. Aghaizu, R. Holliman, P. Riley, J. Poloniecki, A. Breathnach, and S. Krishna. 2008. Diagnosis of Clostridium difficile infection by toxin detection kits: a systematic review. Lancet Infect. Dis. 8:777–784. 11. Reller, M. E., C. A. Lema, T. M. Perl, M. Cai, T. L. Ross, K. A. Speck, and K. C. Carroll. 2007. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for the detection of toxigenic Clostridium difficile. J. Clin. Microbiol. 45:3601–3605. 12. Schmidt, M. L., and P. H. Gilligan. 2009. Clostridium difficile testing algorithms: what is practical and feasible? Anaerobe 15:270–273. 13. Sethi, A. K., W. N. Al-Nassir, M. M. Nerandzic, G. S. Bobulsky, and C. J. Donskey. 2010. Persistence of skin contamination and environmental shedding of Clostridium difficile during and after treatment of C. difficile infection. Infect. Control Hosp. Epidemiol. 31:21–27. 14. Wren, M. W. D., M. Sivapalan, R. Kinson, and N. P. Shetty. 2009. Laboratory diagnosis of Clostridium difficile infection. An evaluation of tests for faecal toxin, glutamate hydrogenase, lactoferrin and toxigenic culture in the diagnostic laboratory. Br. J. Biomed. Sci. 66:1–5.

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TECHNIQUES FOR DIAGNOSIS OF C. DIFFICILE INFECTION Mark H. Wilcox* Microbiology, Leeds Teaching Hospitals and University of Leeds, Old Medical School, Leeds General Infirmary, Leeds LS1 3EX, W. Yorkshire, United Kingdom. Tim Planche Centre for Infection, Division of Cellular and Molecular Medicine, St. George’s University of London, Cranmer Terrace, London SW17 0RE, United Kingdom.

* Phone: 44 (0) 113 392 6818. Fax: 44 (0) 113 392 2696. E-mail: [email protected]

COUNTERPOINT “Fast is fine but accuracy is everything.” —Wyatt Earp Methods of CDI diagnosis. Chekhov wryly observed, “If many remedies are prescribed for an illness, you may know the illness is incurable” (4). The corollary is, if many tests are recommended for an illness, you may know the illness is difficult to diagnose. Clinicians seeking a diagnosis of Clostridium difficile infection (CDI) can order a stool culture, cytotoxin neutralization assay, immunoassay for glutamate dehydrogenase (GDH), immunoassay for toxin A or B, molecular assay for the toxin B or tcdC genes, or some combination of the above. It is time to simplify. CDI is a toxin-mediated illness. Cytotoxin (toxin B) encoded by the tcdB gene is both essential and sufficient for disease (14). While some pathogenic strains lack the tcdA gene, encoding toxin A (2, 10), the presence of the tcdB gene appears to be an excellent surrogate for toxigenicity. Strains producing only toxin A are extremely rare (5, 16, 24), and their virulence has not been firmly established, whereas strains producing only toxin B are clearly able to cause disease. The diagnosis of CDI requires both clinical assessment and the laboratory detection of toxigenic C. difficile. Not all patients carrying toxigenic C. difficile are symptomatic (11, 17, 22). The clinical laboratory can place the perpetrator (C. difficile) and weapon (toxin B) at the scene of the crime, but only the clinician can establish whether a crime (CDI) has taken place. Thus, laboratory testing should be limited to patients who exhibit clinical syndromes compatible with CDI, and the goal of the laboratory is to quickly and accurately ascertain whether toxigenic C. difficile is present. A tarnished gold standard. The “gold standard” for CDI diagnosis has long been considered to be the cytotoxin neutralization assay, which uses tissue culture to detect a cytotoxic phenotype directly from diluted stool specimens. Antibody neutralization makes this assay highly specific. Looking for cytotoxicity would seem to make sense since toxin B is responsible for the clinical manifestations of CDI. However, numerous studies have

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shown that the cytotoxin neutralization assay is only 65 to 80% sensitive (9, 19) in comparison to toxigenic culture, which is performed by isolating C. difficile on selective media and demonstrating cytotoxin production by the cultured organism. One possible factor contributing to the insensitivity of the direct cytotoxin neutralization assay in clinical practice is the instability of cytotoxin activity in fecal specimens at room temperature, which is even more pronounced at body temperature (3). Enzyme immunoassays are worse. The cytotoxin neutralization assay was replaced years ago in most clinical laboratories with an enzyme immunoassay (EIA) for toxin B (with or without toxin A), a test that is highly specific, inexpensive, convenient, and rapid. Unfortunately, toxin EIAs are even less sensitive than the cytotoxin neutralization assay (19, 30). Reliance on the insensitive “gold standard” of cytotoxin neutralization has lulled microbiologists into believing that their assays are better than they truly are. As more sensitive standards for comparison have been employed, it has become apparent that EIAs can miss half or more of CDI cases (12, 25)—clearly an unacceptable situation. There is no question that patients can experience severe and even fatal CDI despite negative C. difficile cytotoxin neutralization assays or EIAs, and the failure of the clinical laboratory to detect toxigenic C. difficile is a risk factor for lifethreatening fulminant CDI (6, 13). Toxigenic culture is accurate but slow. Why not use toxigenic culture to diagnose CDI? Unfortunately, the culture of C. difficile and subsequent demonstration of toxigenicity take too long to be useful for patient care. Nevertheless, toxigenic culture remains useful for the validation of new assay methods and for epidemiological studies. It is important to note that even toxigenic culture itself varies in sensitivity, depending upon factors such as the medium used and whether heat or alcohol shock procedures are used to induce spore germination. For optimal yield, procedures to detect both spores and vegetative cells must be employed in parallel (15). Molecular assays for C. difficile. In recent years, the CDI diagnostic conundrum has been dramatically transformed by the development of molecular assays for toxigenic C. difficile that utilize real-time PCR to directly detect the tcdB gene encoding toxin B from fecal specimens (1, 8, 12, 18, 20, 25, 26, 31). Home-brew assays have been succeeded by FDA-approved commercial assays, with options currently including the GeneOhm (BD), GeneXpert (Cepheid), and proGastro (Prodesse) platforms. Insufficient data are available at this time to indicate whether these assays are equivalent in sensitivity, but all are more sensitive than the toxin EIAs, as well as being highly specific. Laboratories may opt for one or another molecular assay depending on consideration of such factors as cost, sample prep time, volume, workflow, and the need for other assays using the same plat-

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TABLE 1. Diagnostic assays for toxigenic Clostridium difficilea Assay

Sensitivity

Specificity

Speed

Convenience

Economy

Cytotoxin neutralization Toxigenic culture Toxin EIA GDH-based algorithm Molecular assay (PCR, LAMP)

⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹⫹

⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹

⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹/⫹⫹⫹⫹ ⫹⫹⫹

⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹

a

Each feature is rated from least (⫹) to most (⫹⫹⫹⫹) desirable.

form. A new molecular assay called Illumigene (Meridian) based on loop-mediated isothermal amplification has just received FDA clearance and provides yet another alternative. Initial data suggest that the Illumigene assay has sensitivity and specificity comparable to those of PCR-based assays (33). Loop-mediated isothermal amplification does not require expensive capital equipment, which may make this option particularly attractive for laboratories not currently performing molecular testing. GDH-based algorithms. Given the expense and limited availability of molecular assays, various multistep algorithms have been devised in which evaluation is limited to specimens positive for the C. difficile common antigen (glutamate dehydrogenase [GDH]) by EIA, an assay reported to have high sensitivity and NPV (21, 23, 27, 29). This approach is more sensitive than the toxin EIA and has been rapidly gaining in popularity. As most specimens are negative, the GDH screening step substantially reduces the number of specimens that require evaluation with more-specific methods. Since both toxigenic and nontoxigenic C. difficile strains express GDH, a positive GDH EIA requires follow-up testing with a toxin EIA and/or a sensitive assay for toxin B (i.e., a molecular assay). Overall performance of a GDH-based algorithm depends on the secondary tests used to follow up a positive GDH result, and turnaround time may be prolonged for some specimens if they must be referred to a reference lab. The reporting of results can become complicated, since multiple assays are used and provide results at different times. GDH immunoassays may miss cases of CDI. A major concern is that some laboratories, including ours, have found the GDH EIA to miss approximately 15% of CDI (12, 18, 25, 28), and this may depend on the predominant strains at one’s institution. In a recent multicenter study, GDH-based algorithms were able to detect C. difficile ribotype 027 with a sensitivity comparable to that of PCR assays, but the sensitivity of GDH was only 72.2% for non-027 strains (P ⫽ 0.001) (28). After being burned by instances in which repeated false-negative GDH assays delayed the diagnosis of C. difficile infection, with serious clinical consequences (12), our laboratory recently switched to performing a molecular assay on all specimens submitted for C. difficile testing. We

decided that the cost of missing the diagnosis of CDI is simply too great. If molecular methods are impractical at your institution, a multistep GDH-based algorithm may be a reasonable option, but one must accept the possibility of reduced sensitivity, the problem of resolving GDH-positive/toxin-negative isolates, and a delayed turnaround time for some specimens if a molecular assay for toxigenic C. difficile is not available on site. Molecular assays are the simplest approach to C. difficile diagnosis. A prompt and accurate diagnosis of CDI saves money (7) and almost certainly saves lives as well. Although the cost of molecular testing is a concern for many laboratories, the rapid detection of additional CDI cases can offset laboratory expenses by reducing hospitalization and C. difficile transmission, along with the need for repeat testing (12, 32). Molecular assays for CDI combine sensitivity, specificity, speed, and convenience in a single test (Table 1). Prevention of C. difficile infections and the treatment of relapsing or fulminant colitis remain tres difficile. But with molecular methods, the diagnosis of C. difficile, c’est simple! The Harborview Medical Center Clinical Microbiology Laboratory participated in a clinical trial of the Cepheid GeneXpert C. difficile assay. I thank the members of the Clinical Microbiology Laboratory at Harborview Medical Center, in particular Ann Larson, medical technologist (ASCP), for her review of the manuscript prior to submission. REFERENCES 1. Belanger, S. D., M. Boissinot, N. Clairoux, F. J. Picard, and M. G. Bergeron. 2003. Rapid detection of Clostridium difficile in feces by real-time PCR. J. Clin. Microbiol. 41:730–734. 2. Borriello, S. P., B. W. Wren, S. Hyde, S. V. Seddon, P. Sibbons, M. M. Krishna, S. Tabaqchali, S. Manek, and A. B. Price. 1992. Molecular, immunological, and biological characterization of a toxin A-negative, toxin Bpositive strain of Clostridium difficile. Infect. Immun. 60:4192–4199. 3. Chang, T. W., M. Lauermann, and J. G. Bartlett. 1979. Cytotoxicity assay in antibiotic-associated colitis. J. Infect. Dis. 140:765–770. 4. Chekhov, A. 1965. The Cherry Orchard. Allyn and Bacon, Boston, MA. 5. Cohen, S. H., Y. J. Tang, B. Hansen, and J. J. Silva. 1998. Isolation of a toxin B-deficient mutant strain of Clostridium difficile in a case of recurrent C. difficile-associated diarrhea. Clin. Infect. Dis. 26:410–412. 6. Dallal, R. M., B. G. Harbrecht, A. J. Boujoukas, C. A. Sirio, L. M. Farkas, K. K. Lee, and R. L. Simmons. 2002. Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann. Surg. 235:363–372. 7. Dubberke, E. R., K. A. Reske, M. A. Olsen, L. C. McDonald, and V. J. Fraser. 2008. Short- and long-term attributable costs of Clostridium difficile-associated disease in nonsurgical inpatients. Clin. Infect. Dis. 46:497–504. 8. Eastwood, K., P. Else, A. Charlett, and M. Wilcox. 2009. Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J. Clin. Microbiol. 47:3211–3217. 9. Fang, F. C., and N. E. Madinger. 1994. Clostridium difficile colitis. N. Engl. J. Med. 330:1754–1755.

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10. Johnson, S., S. P. Sambol, J. S. Brazier, M. Delme´e, V. Avesani, M. M. Merigan, and D. N. Gerding. 2003. International typing study of toxin Anegative, toxin B-positive Clostridium difficile variants. J. Clin. Microbiol. 41:1543–1547. 11. Kyne, L., M. Warny, A. Qamar, and C. P. Kelly. 2000. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N. Engl. J. Med. 342:390–397. 12. Larson, A. M., A. M. Fung, and F. C. Fang. 2010. Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile. J. Clin. Microbiol. 48:124–130. 13. Lashner, B. A., J. Todorczuk, D. F. Sahm, and S. B. Hanauer. 1986. Clostridium difficile culture-positive toxin-negative diarrhea. Am. J. Gastroenterol. 81:940–943. 14. Lyras, D., J. R. O’Connor, P. M. Howarth, S. P. Sambol, G. P. Carter, T. Phumoonna, R. Poon, V. Adams, G. Vedantam, S. Johnson, D. N. Gerding, and J. I. Rood. 2009. Toxin B is essential for virulence of Clostridium difficile. Nature 458:1176–1179. 15. Marler, L. M., J. A. Siders, L. C. Wolters, Y. Pettigrew, B. L. Skitt, and S. D. Allen. 1992. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J. Clin. Microbiol. 30:514–516. 16. Mathis, J. N., L. Pilkinton, and D. E. McMillin. 1999. Detection and transcription of toxin DNA in a nontoxigenic strain of Clostridium difficile. Curr. Microbiol. 38:324–328. 17. McFarland, L. V., G. W. Elmer, W. E. Stamm, and M. E. Mulligan. 1991. Correlation of immunoblot type, enterotoxin production, and cytotoxin production with clinical manifestations of Clostridium difficile infection in a cohort of hospitalized patients. Infect. Immun. 59:2456–2462. 18. Novak-Weekley, S. M., E. M. Marlowe, J. M. Miller, J. Cumpio, J. H. Nomura, P. H. Vance, and A. Weissfeld. 2010. Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms. J. Clin. Microbiol. 48:889–893. 19. Peterson, L. R., and P. J. Kelly. 1993. The role of the clinical microbiology laboratory in the management of Clostridium difficile-associated diarrhea. Infect. Dis. Clin. North Am. 7:277–293. 20. Peterson, L. R., R. U. Manson, S. M. Paule, D. M. Hacek, A. Robicsek, R. B. Thomson, Jr., and K. L. Kaul. 2007. Detection of toxigenic Clostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficile-associated diarrhea. Clin. Infect. Dis. 45:1152–1160. 21. Reller, M. E., C. A. Lema, T. M. Perl, M. Cai, T. L. Ross, K. A. Speck, and K. C. Carroll. 2007. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difficile. J. Clin. Microbiol. 45:3601–3605. 22. Riggs, M. M., A. K. Sethi, T. F. Zabarsky, E. C. Eckstein, R. L. Jump, and C. J. Donsky. 2007. Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents. Clin. Infect. Dis. 45:992–998. 23. Sharp, S. E., L. O. Ruden, J. C. Pohl, P. A. Hatcher, L. M. Jayne, and W. M. Ivie. 2010. 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. 48:2082–2086. 24. Shin, B. M., E. Y. Kuak, E. J. Lee, and J. G. Songer. 2009. Algorithm

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Ferric C. Fang Departments of Laboratory Medicine and Microbiology, University of Washington School of Medicine, Seattle, WA 98195-7242. Phone: (206) 221-6770. Fax: (206) 616-1575. E-mail: [email protected]

SUMMARY Points of Agreement: ● ●





Detection of C. difficile toxins A and B by enzyme-linked immunosorbent assay (EIA) is no longer an adequate standalone diagnostic test for the detection of C. difficile infection. Despite many years of study, the “gold standard” for diagnosis for C. difficile infection has still not been resolved, although published data indicate that detection of either toxin in stool by tissue culture cytotoxicity neutralization or the demonstration of toxigenic organisms in stools is the best current method. Neither is routinely used in clinical settings because the methods are too slow, cumbersome, and technically difficult. The detection of C. difficile glutamate dehydrogenase (GDH) by EIA has enhanced sensitivity for detection of C. difficile compared to C. difficile toxin A and B EIA but lacks specificity, so if used in a diagnostic setting, a confirmatory test should be used. The accuracy of this testing algorithm is highly dependent on the confirmatory test that is used. Detection of the gene encoding C difficile toxin B, tcdB, using FDA-approved PCR and perhaps loop-mediated isothermal amplification (LAMP) techniques has very high sensitivity and specificity when compared with toxigenic C. difficile culture.

Issues to be Resolved: ●

Until there is agreement on what the reference method is for detection of C. difficile infection, the optimal testing approach will be defined by what is believed to be the “correct” reference method used when evaluating specific testing approaches. Both reference methods currently in use are flawed; detection of C. difficile toxin in feces by

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POINT-COUNTERPOINT

tissue culture neutralization is not as sensitive as toxigenic culture. However, toxigenic culture is not as specific as toxin detection. Additionally, there is no standard method for performing either of these diagnostic tests. Toxigenic culture is particularly problematic in a testing situation where high levels of toxigenic C. difficile carriage can be anticipated, as outlined by Wilcox and Planche. Since PCR correlates closely with toxigenic C. difficile culture, this is a patient population that may prove particularly problematic for laboratories using a nucleic acid amplification test (NAAT)-only testing approach. If GDH detection is used as a screening test in a testing algorithm, does its sensitivity approach 100%, as some studies have suggested, or could it be as low as 70% depending upon the clonal mix of C. difficile strains in an institution, as some recently published data (Fang’s reference [28]) suggest? Published data show that NAAT for detection of C. difficile infection either as a confirmatory test for positive GDH results or as a standalone test is a significant improvement over the still widely used EIAs for C. difficile toxins A and B. However, little has been published about the performance of NAAT in situations where patients do not respond to therapy or have recurrence of infection, an increasingly common event in this patient population. In laboratories that have adopted a NAAT approach only, how specimens from these patients should be evaluated for C. difficile needs to be determined since the length of time nucleic acid can be detected after the initiation of C. difficile targeted therapy in the absence of actively dividing and thus toxin-producing organisms is not known. Peter Gilligan Editor, Point-Counterpoint

The views expressed in this feature do not necessarily represent the views of the journal or of ASM.

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