The pitfalls of laboratory diagnostics of Clostridium difficile infection
Authors: M. Krutova, M.H. Wilcox, E.J. Kuijper
Journal: Clinical Microbiology and Infection (2018)

An update of the diagnostic guidance document for Clostridium difficile infection (CDI) issued by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) [[1]Crobach M.J. Planche T. Eckert C. Barbut F. Terveer E.M. Dekkers O.M. et al.European society of clinical microbiology and infectious diseases: update of the diagnostic guidance document for Clostridium difficile infection.Clin Microbiol Infect. 2016; 22: S63-S81https://doi.org/10.1016/j.cmi.2016.03.010Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar] was recently reviewed by Gateau et al. [[2]Gateau C. Couturier J. Coia J. Barbut F. How to: diagnose infection caused by Clostridium difficile.Clin Microbiol Infect. 2018; 24: 463-468Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. The aim of these publications was to optimize and improve CDI laboratory diagnostic on a global level by defining sample selection, testing algorithms, and interpreting laboratory results [1Crobach M.J. Planche T. Eckert C. Barbut F. Terveer E.M. Dekkers O.M. et al.European society of clinical microbiology and infectious diseases: update of the diagnostic guidance document for Clostridium difficile infection.Clin Microbiol Infect. 2016; 22: S63-S81https://doi.org/10.1016/j.cmi.2016.03.010Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Gateau C. Couturier J. Coia J. Barbut F. How to: diagnose infection caused by Clostridium difficile.Clin Microbiol Infect. 2018; 24: 463-468Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. We highlight some recently identified pitfalls that do not affect CDI laboratory diagnosis overall, but could have a significant impact on individual patients. To speed up the identification of cultured C. difficile strains, a chromogenic agar was developed (ChromID® agar, bioMérieux). However, as was recently shown for strains from Spain and Northern Ireland, isolates of PCR ribotype 023 may fail to produce black colonies, a presumptive identification of C. difficile [3Connor M.C. Fairley D.J. McKenna J.P. Marks N.J. McGrath J.W. Clostridium difficile ribotype 023 lacks the ability to hydrolyze esculin, leading to false-negative results on chromogenic agar.J Clin Microbiol. 2016; 54: 1404-1405https://doi.org/10.1128/JCM.00234-16Crossref PubMed Scopus (8) Google Scholar, 4Reigadas E. Alcalá L. Marín M. Martín A. Bouza E. C. difficile PCR-ribotype 023 might go undetected when using ChromId C. difficile agar.Anaerobe. 2017; 44: 34-35https://doi.org/10.1016/j.anaerobe.2017.01.008Crossref PubMed Scopus (4) Google Scholar], because C. difficile PCR ribotype 023 strains cannot hydrolyse aesculin [[3]Connor M.C. Fairley D.J. McKenna J.P. Marks N.J. McGrath J.W. Clostridium difficile ribotype 023 lacks the ability to hydrolyze esculin, leading to false-negative results on chromogenic agar.J Clin Microbiol. 2016; 54: 1404-1405https://doi.org/10.1128/JCM.00234-16Crossref PubMed Scopus (8) Google Scholar]. The C. difficile PCR ribotype 023 was one of the ten most frequent ribotypes identified in Eastern and Western Europe during the period 2012–2013 [[5]Davies K.A. Ashwin H. Longshaw C.M. Burns D.A. Davis G.L. Wilcox M.H. EUCLID study group. Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.Euro Surveill. 2016; 21 (pii=30294)https://doi.org/10.2807/1560-7917.ES.2016.21.29.30294Crossref Scopus (137) Google Scholar], which highlights the need for awareness among clinical microbiologists that PCR ribotype 023 strains produce atypical (colourless) colonies on ChromID® agar. The clinical significance of C. difficile PCR ribotype 033 in humans was recently reported in Australia, France and Italy [6Androga G.O. Hart J. Foster N.F. Charles A. Forbes D. Riley T.V. Infection with toxin A-negative, toxin B-negative, binary toxin-positive Clostridium difficile in a young patient with ulcerative colitis.J Clin Microbiol. 2015; 53: 3702-3704https://doi.org/10.1128/JCM.01810-15Crossref PubMed Scopus (33) Google Scholar, 7Eckert C. Emirian A. Le Monnier A. Cathala L. De Montclos H. Goret J. et al.Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B..New Microbe. New Infect. 2014; 3: 12-17https://doi.org/10.1016/j.nmni.2014.10.003Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Grandesso S. Arena F. Eseme F.E. Panese S. De Angelis Henrici Spigaglia P. et al.Clostridium difficile ribotype 033 colitis in a patient following broad-spectrum antibiotic treatment for KPC producing Klebsiella pneumoniae infection, Italy.New Microbiol. 2016; 39: 235-236PubMed Google Scholar]. PCR ribotype 033 clusters with ribotypes 045, 066, 078, 126 and 193 in sequence type 11 [[9]Knetsch C.W. Terveer E.M. Lauber C. Gorbalenya A.E. Harmanus C. Kuijper E.J. et al.Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages.Infect Genet Evol. 2012; 12: 1577-1585https://doi.org/10.1016/j.meegid.2012.06.003Crossref PubMed Scopus (72) Google Scholar] and encodes binary toxin genes, but also contains a truncated pathogenicity locus (part of the tcdA gene); however, neither of the large clostridial toxins are produced [[10]Rupnik M. Brazier J.S. Duerden B.I. Grabnar M. Stubbs S.L. Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes.Microbiology. 2001; 147: 439-447Crossref PubMed Scopus (111) Google Scholar], and thus this ribotype cannot be detected by commercial tests for the detection of free toxins A/B in faeces. Depending on the chosen primers, nucleic acid amplification tests (NAATs) can reveal a positive result for the detection of binary toxin gene(s) and/or for the presence of the tcdA gene, but this information is lacking for most of the commercially available NAATs. While PCR ribotype 033 CDI did not result in diarrhoea and death in a hamster model [[11]Geric B. Carman R.J. Rupnik M. Genheimer C.W. Sambol S.P. Lyerly D.M. et al.Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters.J Infect Dis. 2006; 193: 1143-1150Crossref PubMed Scopus (161) Google Scholar], there is evidence for its ability to cause CDI in humans [6Androga G.O. Hart J. Foster N.F. Charles A. Forbes D. Riley T.V. Infection with toxin A-negative, toxin B-negative, binary toxin-positive Clostridium difficile in a young patient with ulcerative colitis.J Clin Microbiol. 2015; 53: 3702-3704https://doi.org/10.1128/JCM.01810-15Crossref PubMed Scopus (33) Google Scholar, 7Eckert C. Emirian A. Le Monnier A. Cathala L. De Montclos H. Goret J. et al.Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B..New Microbe. New Infect. 2014; 3: 12-17https://doi.org/10.1016/j.nmni.2014.10.003Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Grandesso S. Arena F. Eseme F.E. Panese S. De Angelis Henrici Spigaglia P. et al.Clostridium difficile ribotype 033 colitis in a patient following broad-spectrum antibiotic treatment for KPC producing Klebsiella pneumoniae infection, Italy.New Microbiol. 2016; 39: 235-236PubMed Google Scholar]. PCR ribotype 033 has also been recovered from domestic animals and livestock [12Janezic S. Zidaric V. Pardon B. Indra A. Kokotovic B. Blanco J.L. et al.International Clostridium difficile animal strain collection and large diversity of animal associated strains.BMC Microbiol. 2014; 14: 173https://doi.org/10.1186/1471-2180-14-173Crossref PubMed Scopus (101) Google Scholar, 13Schneeberg A. Neubauer H. Schmoock G. Grossmann E. Seyboldt C. Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and 045 in diarrhoeic calves in Germany.J Med Microbiol. 2013; 62: 1190-1198https://doi.org/10.1099/jmm.0.056473-0Crossref PubMed Scopus (44) Google Scholar]. Furthermore, a newly described four-gene insertion, which affects trehalose metabolism and may be associated with increased virulence, has been found in only a few C. difficile PCR ribotypes, including 033 [[14]Collins J. Robinson C. Danhof H. Knetsch C.W. van Leeuwen H.C. Lawley T.D. et al.Dietary trehalose enhances virulence of epidemic Clostridium difficile.Nature. 2018; 553: 291-294https://doi.org/10.1038/nature25178Crossref PubMed Scopus (213) Google Scholar]. These observations mean that further research is needed on the role of PCR ribotype 033 in human CDI. The simultaneous detection of glutamate dehydrogenase (GDH) and toxins A/B by enzyme immunoassays, which include both of these targets in one assay, is an alternative to a two-step CDI testing algorithm. A GDH-negative and toxin A/B-positive test result is considered as invalid, with a need for stool sample retesting [1Crobach M.J. Planche T. Eckert C. Barbut F. Terveer E.M. Dekkers O.M. et al.European society of clinical microbiology and infectious diseases: update of the diagnostic guidance document for Clostridium difficile infection.Clin Microbiol Infect. 2016; 22: S63-S81https://doi.org/10.1016/j.cmi.2016.03.010Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Gateau C. Couturier J. Coia J. Barbut F. How to: diagnose infection caused by Clostridium difficile.Clin Microbiol Infect. 2018; 24: 463-468Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. We have confirmed that five stool samples, sent from different Czech hospitals, repeatedly showed such GDH-negative and toxin A/B-positive test results. However, when these five stool samples were anaerobically cultured, the C. difficile colonies tested were positive for both GDH and toxins A/B with the same combined assay. Moreover, PCR ribotyping of the cultured C. difficile colonies revealed five different PCR ribotypes (012, 014, 020, 070 and 176). Although there is no explanation at present for the failure to detect GDH, it suggests that a third diagnostic step may be needed in order to confirm a GDH-negative toxin A/B-positive result. NAATs that target tcdB or tcdA of toxigenic C. difficile are an alternative first-step option in CDI diagnostics due to their high negative predictive value. NAATs can also be used as a third-step option to distinguish toxigenic from non-toxigenic C. difficile strains in GDH-positive and free toxin A/B-negative stool samples [1Crobach M.J. Planche T. Eckert C. Barbut F. Terveer E.M. Dekkers O.M. et al.European society of clinical microbiology and infectious diseases: update of the diagnostic guidance document for Clostridium difficile infection.Clin Microbiol Infect. 2016; 22: S63-S81https://doi.org/10.1016/j.cmi.2016.03.010Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Gateau C. Couturier J. Coia J. Barbut F. How to: diagnose infection caused by Clostridium difficile.Clin Microbiol Infect. 2018; 24: 463-468Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. Some NAATs, e.g. Xpert® C. difficile (Cepheid), GenoType® Cdiff (Hain Lifescience), Verigene C. difficile Test (Nanosphere), can also detect the binary toxin gene(s) cdtA/cdtB and the specific deletion at position 117 of the tcdC gene, which is assumed to indicate the presence of C. difficile PCR ribotype 027. However, this marker has recently been identified in other PCR ribotypes [[15]Krutova M. Nyc O. Matejkova J. Kuijper E.J. Jalava J. Mentula S. The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027.J Microbiol Immunol Infect. 2018; 51: 344-351Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]. Thus, the interpretation of 'presumptive ribotype 027-positive' results should be correlated with the local CDI epidemiology. Definitive ribotype identification requires capillary electrophoresis-based PCR ribotyping. Though most frequently associated with CDI, a wide differential diagnosis should be considered when pseudomembranous colitis is diagnosed endoscopically and laboratory tests are negative. Other bacterial, viral, and parasitic pathogens have also been implicated in pseudomembranous colitis, including Escherichia coli O157:H7, cytomegalovirus and Entamoeba histolytica. Additionally, pseudomembranous colitis can occur in inflammatory bowel diseases and ischaemic colitis, and also can be caused by several chemicals such as cisplatin and cyclosporine A [[16]Farooq P.D. Urrunaga N.H. Tang D.M. von Rosenvinge E.C. Pseudomembranous colitis.Dis Mon. 2015; 61: 181-206https://doi.org/10.1016/j.disamonth.2015.01.006Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. There is a wide choice of assays available to facilitate the rapid laboratory diagnosis of CDI. It is crucial, however, that microbiology laboratories select appropriate test combinations to optimize CDI diagnosis and provide clear local guidance on sample selection for CDI testing [1Crobach M.J. Planche T. Eckert C. Barbut F. Terveer E.M. Dekkers O.M. et al.European society of clinical microbiology and infectious diseases: update of the diagnostic guidance document for Clostridium difficile infection.Clin Microbiol Infect. 2016; 22: S63-S81https://doi.org/10.1016/j.cmi.2016.03.010Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Gateau C. Couturier J. Coia J. Barbut F. How to: diagnose infection caused by Clostridium difficile.Clin Microbiol Infect. 2018; 24: 463-468Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. Furthermore, given the complexity of CDI diagnosis, the interpretation and communication of test results is at least as important as the result itself. In the case of an inconclusive test result, particularly when associated with an unwell patient or if suboptimal assay performance is suspected, the national reference or central laboratory for C. difficile should be contacted. All authors report no conflict of interest relevant to this article. No financial support was received for this study.