Clinical Infectious Diseases Advance Access published July 2, 2015


Correspondence Fecal-Free Toxin Detection Remains the Best Way to Detect Clostridium difficile Infection TO THE EDITOR—Rao et al report that toxin enzyme immunoassay (EIA) positivity of fecal samples is not associated with severity of Clostridium difficile infection (CDI) or related mortality [1]. This appears to contradict our findings that detection of free toxin in feces using the reference assay (cell cytotoxicity assay [CCTA]) was associated with increased mortality [2]. Notably, others have confirmed that toxin in feces is significantly more predictive of true CDI than is finding a toxigenic strain in the absence of free toxin [3]. This issue is of great importance as it determines which assays should optimally be used to detect true infection. Rao et al used the C. DIFF QUIK CHEK COMPLETE EIA, which comprises 2

Table 1. Comparison of Studies Relating to Role of Clostridium difficile Toxin Planche et al Number of patients Duration of study, mo Test rate per 10 000 bed-days Toxin assay Fecal sample Effect of free toxin Selection of fecal sample Samples toxin assay positive Samples toxin assay negative & Nucleic Acid Amplification Test positive Negative samples

4689 10

Rao et al 2590 27

142.7 Cell cytotoxicity assay

Unknown Membrane enzyme immunoassay

Undiluted Associated with fatal outcome

Cary–Blair transport medium No association with fatal outcome

All unformed fecal samples 435 (9.3%)

Physician request only 426 (16%)

311 (6.6%)

3943 (84.1%)

718 (28%)

1446 (56%)

Overall 30-d patient fatality rate

92/642 (14.3%)

89/1144 (7.8%)

Toxin positive, 30-d patient fatality rate

72/435 (16.6%)

33/426 (7.7%)

Age, y (standard deviation)

69 (20)

57 (18)

Prevalence ribotype 027



younger patients who were not as ill, with a lower fatality rate (7.8% vs 14.3%), reducing power to detect genuine independent associations. The sample positivity rate was also more than double that reported by Planche et al, but the number of cases detected monthly was about 3 times lower. Rao et al do not report the number of hospital beds or the testing rate, but these are likely to be considerably less than the 142.7 tests/10 000 bed-days reported by Planche et al. Studies across Europe show that CDI is often missed by failure to test samples and by relying upon physician requesting only [7, 8], possibly leading to ascertainment bias, in turn, reducing power further. Lack of power could also explain why Rao et al failed to identify a statistically significant effect of detectable stool toxin independently of ribotype 027, with which it has been associated. Finally, Rao et al state that clinical studies investigating associations between ribotype 027 and CDI mortality have reached different conclusions [1]. They did not cite both the largest study performed in this area, which clearly showed that ribotypes 027 and 078 are independently associated with excess mortality, and correspondence highlighting the key contribution of lack of study power [9, 10]. Given the low sensitivity of the assays used and the low rates of testing and potential selection bias reducing power, we feel reassured that there is no reason to reassess the conclusion that detecting free toxin in feces, either by CCTA or a more sensitive EIA, is the best way to detect true CDI. Without free fecal toxin, a samples toxin assay negative & Nucleic Acid Amplification Test positive indicates potential C. difficile carriage [2, 3].




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components: glutamate dehydrogenase and toxin detection. While this assay is simple to perform and provides rapid results, when compared with CCTA, the toxin component has a reported sensitivity of 0.50 (95% confidence interval [CI], 0.30–.70) [4] or 0.73 (95% CI, .48–.89) [5]. Additionally, specimens were transported in Cary–Blair medium. While permitted by the manufacturer, this dilutes specimens and may lower sensitivity further. Previous studies with other EIAs using samples in Cary–Blair medium did not compare with reference assays performed directly on undiluted feces [6]. Given the suboptimal sensitivity of the assay used by Rao et al, it could be expected that this study would fail to find an effect detected using the gold standard toxin method, that is, CCTA. Table 1 compares 2 studies [1, 2]. The Rao et al study was of considerably



Tim Planche,1 Mark Wilcox,2 and A. Sarah Walker3,4 1

St. George’s University of London, Cranmer Terrace, 2Microbiology, Leeds Teaching Hospitals, University of Leeds & Public Health England, 3National Institute of Health Research Biomedical Research Centre, Oxford, and 4 Nuffield Department of Medicine, University of Oxford, United Kingdom




1. Rao K, Micic D, Natarajan M, et al. Clostridium difficile ribotype 027: relationship to age, detectability of toxins A or B in stool with rapid testing, severe infection, and mortality. Clin Infect Dis 2015; doi:10.1093/cid/civ254. 2. Planche TD, Davies KA, Coen PG, et al. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C. difficile infection. Lancet Infect Dis 2013; 13:936–45. 3. 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. 4. Ota KV, McGowan KL. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population. J Clin Microbiol 2012; 50:1185–8. 5. Swindells J, Brenwald N, Reading N, Oppenheim B. Evaluation of diagnostic tests for Clostridium difficile infection. J Clin Microbiol 2010; 48:606–8. 6. Brown NA, Lebar WD, Young CL, Hankerd RE, Newton DW. Diagnosis of Clostridium difficile infection: comparison of four methods





Correspondence: Tim Planche, MD, MRCP, FRCPath, St. George’s University of London, Cranmer Terrace, London SW17 0RE, UK ([email protected]). Clinical Infectious Diseases® © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. [email protected]. DOI: 10.1093/cid/civ479

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Potential conflicts of interest. M. W. reports grants and personal fees from Actelion, Cubist, Astellas, Merck, Sanofi-Pasteur, Summit, Biomerieux, and Qiagen and personal fees from Optimer, Seres, and Alere during the conduct of the study. M. W. also reports grants and personal fees from Cerexa, Abbott, and the European Tissue Symposium; personal fees from Astra-Zeneca, Durata, Nabriva, Pfizer, Roche, The Medicines Company, VH Squared, and Basilea; and grants from Da Volterra outside the submitted work. T. P. has received grant support and fees for lectures from Astellas outside the submitted work. All other authors report no potential 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.

on specimens collected in Cary-Blair transport medium and tcdB PCR on fresh versus frozen samples. Infect Dis Rep 2011; 3:e5. Davies KA, Longshaw CM, Davis GL, et al. Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis 2014; 14:1208–19. Alcala L, Martin A, Marin M, et al. The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem? Clin Microbiol Infect 2012; 18:E204–13. Walker AS, Eyre DW, Crook DW, Wilcox MH, Peto TE. Regarding “Clostridium difficile ribotype does not predict severe infection.” Clin Infect Dis 2013; 56:1845–6. Walker AS, Eyre DW, Wyllie DH, et al. Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection. Clin Infect Dis 2013; 56:1589–600.

Fecal-Free Toxin Detection Remains the Best Way to Detect Clostridium difficile Infection.

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