1887

Abstract

In this study, a total of 650 stool samples were tested to show that our method is capable of detecting four genes; , , encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin transferase genes ( and/or ) encoding CDT toxin. Besides detecting the targeted genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID™ , can be applied directly for screening -associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent strains.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.058339-0
2013-09-01
2020-01-25
Loading full text...

Full text loading...

/deliver/fulltext/jmm/62/9/1350.html?itemId=/content/journal/jmm/10.1099/jmm.0.058339-0&mimeType=html&fmt=ahah

References

  1. Bacci S., Mølbak K., Kjeldsen M. K., Olsen K. E. P.. ( 2011;). Binary toxin and death after Clostridium difficile infection. . Emerg Infect Dis 17:, 976–982. [CrossRef][PubMed]
    [Google Scholar]
  2. Bélanger S. D., Boissinot M., Clairoux N., Picard F. J., Bergeron M. G.. ( 2003;). Rapid detection of Clostridium difficile in feces by real-time PCR. . J Clin Microbiol 41:, 730–734. [CrossRef][PubMed]
    [Google Scholar]
  3. Bowman R. A., Riley T. V.. ( 1988;). Laboratory diagnosis of Clostridium difficile-associated diarrhoea. . Eur J Clin Microbiol Infect Dis 7:, 476–484. [CrossRef][PubMed]
    [Google Scholar]
  4. Carroll K. C., Loeffelholz M.. ( 2011;). Conventional versus molecular methods for the detection of Clostridium difficile.. J Clin Microbiol 49: (9 Suppl), S49–S52. [CrossRef]
    [Google Scholar]
  5. Cheng A. C., Ferguson J. K., Richards M. J., Robson J. M., Gilbert G. L., McGregor A., Roberts S., Korman T. M., Riley T. V..Australasian Society for Infectious Diseases ( 2011;). Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection. . Med J Aust 194:, 353–358.[PubMed]
    [Google Scholar]
  6. Cohen S. H., Gerding D. N., Johnson S., Kelly C. P., Loo V. G., McDonald L. C., Pepin J., Wilcox M. H..Society for Healthcare Epidemiology of AmericaInfectious Diseases Society of America ( 2010;). 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 31:, 431–455. [CrossRef][PubMed]
    [Google Scholar]
  7. de Boer R. F., Wijma J. J., Schuurman T., Moedt J., Dijk-Alberts B. G., Ott A., Kooistra-Smid A. M., van Duynhoven Y. T.. ( 2010;). Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools. . J Microbiol Methods 83:, 59–65. [CrossRef][PubMed]
    [Google Scholar]
  8. Drudy D., Fanning S., Kyne L.. ( 2007;). Toxin A-negative, toxin B-positive Clostridium difficile.. Int J Infect Dis 11:, 5–10. [CrossRef][PubMed]
    [Google Scholar]
  9. Eastwood K., Else P., Charlett A., Wilcox M.. ( 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. [CrossRef][PubMed]
    [Google Scholar]
  10. Geric B., Johnson S., Gerding D. N., Grabnar M., Rupnik M.. ( 2003;). Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins. . J Clin Microbiol 41:, 5227–5232. [CrossRef][PubMed]
    [Google Scholar]
  11. Geric B., Carman R. J., Rupnik M., Genheimer C. W., Sambol S. P., Lyerly D. M., Gerding D. N., Johnson S.. ( 2006;). Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters. . J Infect Dis 193:, 1143–1150. [CrossRef][PubMed]
    [Google Scholar]
  12. Grando D., Said M. M., Mayall B. C., Gurtler V.. ( 2012;). High resolution melt analysis to track infections due to ribotype 027 Clostridium difficile.. J Microbiol Methods 89:, 87–94. [CrossRef][PubMed]
    [Google Scholar]
  13. Guilbault C., Labbé A. C., Poirier L., Busque L., Béliveau C., Laverdière M.. ( 2002;). Development and evaluation of a PCR method for detection of the Clostridium difficile toxin B gene in stool specimens. . J Clin Microbiol 40:, 2288–2290. [CrossRef][PubMed]
    [Google Scholar]
  14. Huang H., Weintraub A., Fang H., Nord C. E.. ( 2009;). Comparison of a commercial multiplex real-time PCR to the cell cytotoxicity neutralization assay for diagnosis of clostridium difficile infections. . J Clin Microbiol 47:, 3729–3731. [CrossRef][PubMed]
    [Google Scholar]
  15. Jalal H., Stephen H., Curran M. D., Burton J., Bradley M., Carne C.. ( 2006;). Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction. . J Clin Microbiol 44:, 206–213. [CrossRef][PubMed]
    [Google Scholar]
  16. Kuehne S. A., Cartman S. T., Heap J. T., Kelly M. L., Cockayne A., Minton N. P.. ( 2010;). The role of toxin A and toxin B in Clostridium difficile infection. . Nature 467:, 711–713. [CrossRef][PubMed]
    [Google Scholar]
  17. Luna R. A., Boyanton B. L. Jr, Mehta S., Courtney E. M., Webb C. R., Revell P. A., Versalovic J.. ( 2011;). Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children’s hospital. . J Clin Microbiol 49:, 851–857. [CrossRef][PubMed]
    [Google Scholar]
  18. Lyras D., O’Connor J. R., Howarth P. M., Sambol S. P., Carter G. P., Phumoonna T., Poon R., Adams V., Vedantam G.. & other authors ( 2009;). Toxin B is essential for virulence of Clostridium difficile.. Nature 458:, 1176–1179. [CrossRef][PubMed]
    [Google Scholar]
  19. McEllistrem M. C., Carman R. J., Gerding D. N., Genheimer C. W., Zheng L.. ( 2005;). A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes. . Clin Infect Dis 40:, 265–272. [CrossRef][PubMed]
    [Google Scholar]
  20. Perelle S., Gibert M., Bourlioux P., Corthier G., Popoff M. R.. ( 1997;). Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. . Infect Immun 65:, 1402–1407.[PubMed]
    [Google Scholar]
  21. Peterson L. R., Manson R. U., Paule S. M., Hacek D. M., Robicsek A., Thomson R. B. Jr, Kaul K. L.. ( 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. [CrossRef][PubMed]
    [Google Scholar]
  22. Riley T. V.. ( 2004;). Nosocomial diarrhoea due to Clostridium difficile.. Curr Opin Infect Dis 17:, 323–327. [CrossRef][PubMed]
    [Google Scholar]
  23. Salari M. H., Badami N., Sadeghifard N., Harati F. A.. ( 2008;). Investigation of various tissue culture monolayers sensitivity in detection of Clostridium difficile toxin. . Iran J Public Health 37:, 99–102.
    [Google Scholar]
  24. Schwan C., Stecher B., Tzivelekidis T., van Ham M., Rohde M., Hardt W. D., Wehland J., Aktories K.. ( 2009;). Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. . PLoS Pathog 5:, e1000626. [CrossRef][PubMed]
    [Google Scholar]
  25. Spigaglia P., Mastrantonio P.. ( 2002;). Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. . J Clin Microbiol 40:, 3470–3475. [CrossRef][PubMed]
    [Google Scholar]
  26. Stubbs S. L., Brazier J. S., O’Neill G. L., Duerden B. I.. ( 1999;). PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. . J Clin Microbiol 37:, 461–463.[PubMed]
    [Google Scholar]
  27. Tenover F. C., Novak-Weekley S., Woods C. W., Peterson L. R., Davis T., Schreckenberger P., Fang F. C., Dascal A., Gerding D. N.. & other authors ( 2010;). Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches. . J Clin Microbiol 48:, 3719–3724. [CrossRef][PubMed]
    [Google Scholar]
  28. Voth D. E., Ballard J. D.. ( 2005;). Clostridium difficile toxins: mechanism of action and role in disease. . Clin Microbiol Rev 18:, 247–263. [CrossRef][PubMed]
    [Google Scholar]
  29. Wroblewski D., Hannett G. E., Bopp D. J., Dumyati G. K., Halse T. A., Dumas N. B., Musser K. A.. ( 2009;). Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens. . J Clin Microbiol 47:, 2142–2148. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.058339-0
Loading
/content/journal/jmm/10.1099/jmm.0.058339-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error