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Rapid detection of Clostridium difficile toxins and laboratory diagnosis of Clostridium difficile infections

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Abstract

Background

Clostridium difficile is an anaerobic, spore-forming and Gram-positive bacillus. It is the major cause of antibiotic-associated diarrhea prevailing in hospital settings. The morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Because of the poor clinical different between CDI and other causes of hospital-acquired diarrhea, laboratory test for C. difficile is an important intervention for diagnosis of CDI.

Objective

Laboratory tests for CDI can broadly detect either the organisms or its toxins. Currently, several laboratory tests are used for diagnosis of CDI, including toxigenic culture, glutamate dehydrogenase detection, nucleic acid amplification testing, cell cytotoxicity assay, and enzyme immunoassay towards toxin A and/or B. This review focuses on the rapid testing of C. difficile toxins and currently available methods for diagnosis of CDI, giving an overview of the role that the toxins rapid detecting plays in clinical diagnosis of CDI.

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References

  1. Bartlett JG, Moon N, Chang TW, Taylor N, Onderdonk AB. Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis. Gastroenterology. 1978;75:778–82.

    CAS  PubMed  Google Scholar 

  2. McFarland LV, Mulligan ME, Kwok RY, Stamm WE. Nosocomial acquisition of Clostridium difficile infection. N Engl J Med. 1989;320:204–10. doi:10.1056/NEJM198901263200402.

    Article  CAS  PubMed  Google Scholar 

  3. Steele SR, McCormick J, Melton GB, Paquette I, Rivadeneira DE, Stewart D, et al. Practice parameters for the management of Clostridium difficile infection. Dis Colon Rectum. 2015;58:10–24. doi:10.1097/DCR.0000000000000289.

    Article  PubMed  Google Scholar 

  4. Kelly CP, LaMont JT. Clostridium difficile infection. Annu Rev Med. 1998;49:375–90. doi:10.1146/annurev.med.49.1.375.

    Article  CAS  PubMed  Google Scholar 

  5. Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev. 2005;18:247–63. doi:10.1128/CMR.18.2.247-263.2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, 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. doi:10.1086/651706.

    Article  PubMed  Google Scholar 

  7. Kelly CP, LaMont JT. Clostridium difficile—more difficult than ever. N Engl J Med. 2008;359:1932–40. doi:10.1056/NEJMra0707500.

    Article  CAS  PubMed  Google Scholar 

  8. Carter GP, Rood JI, Lyras D. The role of toxin A and toxin B in the virulence of Clostridium difficile. Trends Microbiol. 2012;20:21–9. doi:10.1016/j.tim.2011.11.003.

    Article  CAS  PubMed  Google Scholar 

  9. Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J, et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet. 2005;366:1079–84. doi:10.1016/S0140-6736(05)67420-X.

    Article  CAS  PubMed  Google Scholar 

  10. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, et al. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med. 2005;353:2442–9. doi:10.1056/NEJMoa051639.

    Article  CAS  PubMed  Google Scholar 

  11. Pepin J, Valiquette L, Alary ME, Villemure P, Pelletier A, Forget K, 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. doi:10.1503/cmaj.1041104.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Popoff MR, Boquet P. Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. Biochem Biophys Res Commun. 1988;152:1361–8. doi:10.1016/S0006-291X(88)80435-2.

    Article  CAS  PubMed  Google Scholar 

  13. Popoff MR, Rubin EJ, Gill DM, Boquet P. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect Immun. 1988;56:2299–306.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Eckert 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 Microbes New Inect. 2014;3:12–7. doi:10.1016/j.nmni.2014.10.0003.

    Article  Google Scholar 

  15. Gerding DN, Johnson S, Rupnik M, Aktories K. Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes. 2014;5:15–27. doi:10.4161/gmic.26854.

    Article  PubMed  Google Scholar 

  16. Bartlett JG, Gerding DN. Clinical recognition and diagnosis of Clostridium difficile infection. Clin Infect Dis. 2008;46:S12–8. doi:10.1086/521863.

    Article  PubMed  Google Scholar 

  17. Bagdasarian N, Rao K, Malani PN. Diagnosis and treatment of Clostridium difficile in adults: a systematic review. JAMA. 2015;313:398–408. doi:10.1001/jama.2014.17103.

    Article  PubMed  Google Scholar 

  18. Pepin J, Alary ME, Valiquette L, Raiche E, Ruel J, Fulop K, et al. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec. Canada Clin Infect Dis. 2005;40:1591–7. doi:10.1086/430315.

    Article  CAS  PubMed  Google Scholar 

  19. Vardakas KZ, Polyzos KA, Patouni K, Rafailidis PI, Samonis G, Falagas ME. Treatment failure and recurrence of Clostridium difficile infection following treatment with vancomycin or metronidazole: a systematic review of the evidence. Int J Antimicrob Agents. 2012;40:1–8. doi:10.1016/j.ijantimicag.2012.01.004.

    Article  CAS  PubMed  Google Scholar 

  20. Chen S, Sun C, Wang H, Wang J. The role of rho GTPases in toxicity of Clostridium difficile toxins. Toxins (Basel). 2015;7:5254–67. doi:10.3390/toxins7124874.

    Article  CAS  Google Scholar 

  21. Steiner C, Barrett M, Sun Y, Weiss A. HCUP projections: Clostridium difficile hospitalizations 2003–2014. HCUP projections report #2014-03. (U.S Agency for Healthcare Research and Quality.). 2014. http://www.hcup-us.ahrq.gov/reports/projections/2014-03.pdf. Accessed 19 Nov 2014.

  22. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, et al. Burden of Clostridium difficile infection in the United States. N Engl J Med. 2015;372:825–34. doi:10.1056/NEJMoa1408913.

    Article  CAS  PubMed  Google Scholar 

  23. Ricciardi R, Rothenberger DA, Madoff RD, Baxter NN. Increasing prevalence and severity of Clostridium difficile colitis in hospitalized patients in the United States. Arch Surg. 2007;142:624–31. doi:10.1001/archsurg.142.7.624 (discussion 31).

    Article  PubMed  Google Scholar 

  24. Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol. 2009;7:526–36. doi:10.1038/nrmicro2164.

    Article  CAS  PubMed  Google Scholar 

  25. Shields K, Araujo-Castillo RV, Theethira TG, Alonso CD, Kelly CP. Recurrent Clostridium difficile infection: from colonization to cure. Anaerobe. 2015;34:59–73. doi:10.1016/j.anaerobe.2015.04.012.

    Article  PubMed  PubMed Central  Google Scholar 

  26. McFarland LV. Epidemiology of infectious and iatrogenic nosocomial diarrhea in a cohort of general medicine patients. Am J Infect Control. 1995;23:295–305.

    Article  CAS  PubMed  Google Scholar 

  27. Garey KW, Graham G, Gerard L, Dao T, Jiang ZD, Price M, et al. Prevalence of diarrhea at a university hospital and association with modifiable risk factors. Ann Pharmacother. 2006;40:1030–4. doi:10.1345/aph.1H028.

    Article  PubMed  Google Scholar 

  28. Planche T, Wilcox MH. Diagnostic pitfalls in Clostridium difficile infection. Infect Dis Clin North Am. 2015;29:63–82. doi:10.1016/j.idc.2014.11.008.

    Article  PubMed  Google Scholar 

  29. Bartlett JG. Clinical practice. Antibiotic-associated diarrhea. N Engl J Med. 2002;346:334–9. doi:10.1056/NEJMcp011603.

    Article  PubMed  Google Scholar 

  30. Poutanen SM, Simor AE. Clostridium difficile-associated diarrhea in adults. CMAJ. 2004;171:51–8.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Freeman J, Baines SD, Jabes D, Wilcox MH. Comparison of the efficacy of ramoplanin and vancomycin in both in vitro and in vivo models of clindamycin-induced Clostridium difficile infection. J Antimicrob Chemother. 2005;56:717–25. doi:10.1093/jac/dki321.

    Article  CAS  PubMed  Google Scholar 

  32. Riley TV, Brazier JS, Hassan H, Williams K, Phillips KD. Comparison of alcohol shock enrichment and selective enrichment for the isolation of Clostridium difficile. Epidemiol Infect. 1987;99:355–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol. 1992;30:514–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hink T, Burnham CA, Dubberke ER. A systematic evaluation of methods to optimize culture-based recovery of Clostridium difficile from stool specimens. Anaerobe. 2013;19:39–43. doi:10.1016/j.anaerobe.2012.12.001.

    Article  PubMed  Google Scholar 

  35. George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979;9:214–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Wren M. Clostridium difficile isolation and culture techniques. Mehtods Mol Biol. 2010;646:39–52. doi:10.107/978-1-60327-365-7_3.

    CAS  Google Scholar 

  37. Burnham CA, Carroll KC. Diagnosis of Clostridium difficile infection: an ongoing conundrum for clinicians and for clinical laboratories. Clin Microbiol Rev. 2013;26:604–30. doi:10.1128/CMR.00016-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wilcox MH, Planche T, Fang FC, Gilligan P. What is the current role of algorithmic approaches for diagnosis of Clostridium difficile infection? J Clin Microbiol. 2010;48:4347–53. doi:10.1128/JCM.02028-10.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Planche T, Wilcox M. Reference assays for Clostridium difficile infection: one or two gold standards? J Clin Pathol. 2011;64:1–5. doi:10.1136/jcp.2010.080135.

    Article  CAS  PubMed  Google Scholar 

  40. Lashner BA, Todorczuk J, Sahm DF, Hanauer SB. Clostridium difficile culture-positive toxin-negative diarrhea. Am J Gastroenterol. 1986;81:940–3.

    CAS  PubMed  Google Scholar 

  41. Barbut F, Braun M, Burghoffer B, Lalande V, Eckert C. Rapid detection of toxigenic strains of Clostridium difficile in diarrheal stools by real-time PCR. J Clin Microbiol. 2009;47:1276–7. doi:10.1128/JCM.00309-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Eastwood K, Else P, Charlett A, Wilcox M. 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. 2009;47:3211–7. doi:10.1128/JCM.01082-09.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Peterson LR, Manson RU, Paule SM, Hacek DM, Robicsek A, Thomson RB Jr, et al. 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. 2007;45:1152–60. doi:10.1086/522185.

    Article  CAS  PubMed  Google Scholar 

  44. Stamper PD, Alcabasa R, Aird D, Babiker W, Wehrlin J, Ikpeama I, 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. doi:10.1128/JCM.01613-08.

    Article  CAS  PubMed  Google Scholar 

  45. de Jong E, de Jong AS, Bartels CJ, van der Rijt-van den Biggelaar C, Melchers WJ, Sturm PD. Clinical and laboratory evaluation of a real-time PCR for Clostridium difficile toxin A and B genes. Eur J Clin Microbiol Infect Dis. 2012;31:2219–25. doi:10.1007/s10096-012-1558-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Landry ML, Ferguson D, Topal J. Comparison of Simplexa universal direct PCR with cytotoxicity assay for diagnosis of Clostridium difficile infection: performance, cost, and correlation with disease. J Clin Microbiol. 2014;52:275–80. doi:10.1128/JCM.02545-13.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Murray PR, Weber CJ. Detection of Clostridium difficile cytotoxin in HEp-2 and CHO cell lines. Diagn Microbiol Infect Dis. 1983;1:331–3.

    Article  CAS  PubMed  Google Scholar 

  48. Doern GV, Coughlin RT, Wu L. Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays. J Clin Microbiol. 1992;30:2042–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. George WL, Rolfe RD, Finegold SM. Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions. J Clin Microbiol. 1982;15:1049–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. George WL, Rolfe RD, Harding GK, Klein R, Putnam CW, Finegold SM. Clostridium difficile and cytotoxin in feces of patients with antimicrobial agent-associated pseudomembranous colitis. Infection. 1982;10:205–8.

    Article  CAS  PubMed  Google Scholar 

  51. Gerding DN, Olson MM, Peterson LR, Teasley DG, Gebhard RL, Schwartz ML, et al. Clostridium difficile-associated diarrhea and colitis in adults. A prospective case-controlled epidemiologic study. Arch Intern Med. 1986;146:95–100.

    Article  CAS  PubMed  Google Scholar 

  52. Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, 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. doi:10.1016/S1473-3099(13)70200-7.

    Article  PubMed  PubMed Central  Google Scholar 

  53. van den Berg RJ. Bruijnesteijn van Coppenraet LS, Gerritsen HJ, Endtz HP, van der Vorm ER, Kuijper EJ. Prospective multicenter evaluation of a new immunoassay and real-time PCR for rapid diagnosis of Clostridium difficile-associated diarrhea in hospitalized patients. J Clin Microbiol. 2005;43:5338–40. doi:10.1128/JCM.43.10.5338-5340.2005.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Le Guern R, Herwegh S, Grandbastien B, Courcol R, Wallet F. Evaluation of a new molecular test, the BD Max Cdiff, for detection of toxigenic Clostridium difficile in fecal samples. J Clin Microbiol. 2012;50:3089–90. doi:10.1128/JCM.01250-12.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Novak-Weekley SM, Marlowe EM, Miller JM, Cumpio J, Nomura JH, Vance PH, et al. Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms. J Clin Microbiol. 2010;48:889–93. doi:10.1128/JCM.01801-09.

    Article  PubMed  PubMed Central  Google Scholar 

  56. 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. doi:10.1128/JCM.00032-08.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Han Z, McMullen KM, Russo AJ, Copper SM, Warren DK, Dubberke ER. A Clostridium difficile infection “intervention”: change in toxin assay results in fewer C difficile infection cases without changes in patient outcomes. Am J Infect Control. 2012;40:349–53. doi:10.1016/j.ajic.2011.04.002.

    Article  PubMed  Google Scholar 

  58. Planche T, Aghaizu A, Holliman R, Riley P, Poloniecki J, Breathnach A, et al. Diagnosis of Clostridium difficile infection by toxin detection kits: a systematic review. Lancet Infect Dis. 2008;8:777–84. doi:10.1016/S1473-3099(08)70233-0.

    Article  PubMed  Google Scholar 

  59. Fang YS, Chen SY, Huang XJ, Wang LS, Wang HY, Wang JF. Simple approach for ultrasensitive electrochemical immunoassay of Clostridium difficile toxin B detection. Biosens Bioelectron. 2014;53:238–44. doi:10.1016/j.bios.2013.09.063.

    Article  CAS  PubMed  Google Scholar 

  60. Song L, Zhao M, Duffy DC, Hansen J, Shields K, Wungjiranirun M, et al. Development and validation of digital enzyme-linked immunosorbent assays for ultrasensitive detection and quantification of Clostridium difficile toxins in stool. J Clin Microbiol. 2015;53:3204–12. doi:10.1128/JCM.01334-15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Wren B, Clayton C, Tabaqchali S. Rapid identification of toxigenic Clostridium difficile by polymerase chain reaction. Lancet. 1990;335:423.

    Article  CAS  PubMed  Google Scholar 

  62. Kato N, Ou CY, Kato H, Bartley SL, Brown VK, Dowell VR Jr, et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol. 1991;29:33–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Gumerlock PH, Tang YJ, Meyers FJ, Silva J Jr. Use of the polymerase chain reaction for the specific and direct detection of Clostridium difficile in human feces. Rev Infect Dis. 1991;13:1053–60.

    Article  CAS  PubMed  Google Scholar 

  64. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol. 1993;31:507–11.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Longtin Y, Trottier S, Brochu G, Paquet-Bolduc B, Garenc C, Loungnarath V, 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. doi:10.1093/cid/cis840.

    Article  PubMed  Google Scholar 

  66. 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. doi:10.1128/JCM.44.3.1145-1149.2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Fenner L, Widmer AF, Goy G, Rudin S, Frei R. Rapid and reliable diagnostic algorithm for detection of Clostridium difficile. J Clin Microbiol. 2008;46:328–30. doi:10.1128/JCM.01503-07.

    Article  PubMed  Google Scholar 

  68. Reller ME, Lema CA, Perl TM, Cai M, Ross TL, Speck KA, et al. 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. 2007;45:3601–5. doi:10.1128/JCM.01305-07.

    Article  PubMed  PubMed Central  Google Scholar 

  69. Wren MW, Sivapalan M, Kinson R, Shetty NR. Laboratory diagnosis of Clostridium difficile infection. An evaluation of tests for faecal toxin, glutamate dehydrogenase, lactoferrin and toxigenic culture in the diagnostic laboratory. Br J Biomed Sci. 2009;66:1–5.

    Article  CAS  PubMed  Google Scholar 

  70. 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. doi:10.1128/JCM.00129-10.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by Grants from the Innovation Foundation for Science and Technology from the Department of Education of Guangdong Province (2013KJCX0013), Natural Science Foundation of Guangdong Province (2015A030310322), and the Guangdong Special Grant Program for High-level Talents 2013.

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    1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China

      Shuyi Chen, Huawei Gu, Chunli Sun, Haiying Wang & Jufang Wang

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    S. Chen and H. Gu contributed equally to this work.

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    Chen, S., Gu, H., Sun, C. et al. Rapid detection of Clostridium difficile toxins and laboratory diagnosis of Clostridium difficile infections. Infection 45, 255–262 (2017). https://doi.org/10.1007/s15010-016-0940-9

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