1887

Abstract

This study demonstrates a novel detection assay able to identify and subtype strains of . Primers carefully designed for melting curve analysis amplify DNA from three genes, , and , during quantitative (q)PCR. The gene allows for confirmation of organism presence, whilst the and genes allow for differentiation of virulence status, as deletions in the gene and the concurrent presence of the gene, which produces binary toxin, are associated with hypervirulence. Following qPCR, subtyping is then achieved by automated, inline melting curve analysis using only a single intercalating dye and verified by microchip electrophoresis. This assay represents a novel means of distinguishing between toxigenic and hypervirulent strains NAP1/027/BI and 078 ribotype, which are highly prevalent hypervirulent strains in humans. This methodology can help rapidly detect and identify strains that impose a significant health and economic burden in hospitals and other healthcare settings.

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2016-01-01
2020-04-06
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References

  1. Angione S. L., Sarma A. A., Novikov A., Seward L., Fieber J. H., Mermel L. A., Tripathi A.. 2014; A novel subtyping assay for detection of Clostridium difficile virulence genes. J Mol Diagn16:244–252 [CrossRef][PubMed]
    [Google Scholar]
  2. Blossom D. B., McDonald L. C.. 2007; The challenges posed by reemerging Clostridium difficile infection. Clin Infect Dis45:222–227 [CrossRef][PubMed]
    [Google Scholar]
  3. Burnham C.-A.D., Carroll K. C.. 2013; Diagnosis of Clostridium difficile infection: an ongoing conundrum for clinicians and for clinical laboratories. Clin Microbiol Rev26:604–630 [CrossRef][PubMed]
    [Google Scholar]
  4. Carter G. P., Lyras D., Allen D. L., Mackin K. E., Howarth P. M., O'Connor J. R., Rood J. I.. 2007; Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator. J Bacteriol189:7290–7301 [CrossRef][PubMed]
    [Google Scholar]
  5. Cockerill F. R. III. 2003; Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Arch Pathol Lab Med127:1112–1120[PubMed]
    [Google Scholar]
  6. Curry S. R., Marsh J. W., Muto C. A., O'Leary M. M., Pasculle A. W., Harrison L. H.. 2007; tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile. J Clin Microbiol45:215–221 [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. D., van Duynhoven Y.T.H.P.. 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 Methods83:59–65 [CrossRef][PubMed]
    [Google Scholar]
  8. de Jong E., de Jong A. S., Bartels C. J. M., van der Rijt-van den Biggelaar C., Melchers W. J. G., Sturm P. D. J.. 2012; Clinical and laboratory evaluation of a real-time PCR for Clostridium difficile toxin A and B genes. Eur J Clin Microbiol Infect Dis31:2219–2225 [CrossRef][PubMed]
    [Google Scholar]
  9. Dubberke E. R., Han Z., Bobo L., Hink T., Lawrence B., Copper S., Hoppe-Bauer J., Burnham C.-A.D., Dunne W. M. Jr. 2011; Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol49:2887–2893 [CrossRef][PubMed]
    [Google Scholar]
  10. Dupuy B., Govind R., Antunes A., Matamouros S.. 2008; Clostridium difficile toxin synthesis is negatively regulated by TcdC. J Med Microbiol57:685–689 [CrossRef][PubMed]
    [Google Scholar]
  11. 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 Microbiol47:3211–3217 [CrossRef][PubMed]
    [Google Scholar]
  12. Espy M. J., Uhl J. R., Sloan L. M., Buckwalter S. P., Jones M. F., Vetter E. A., Yao J. D. C., Wengenack N. L., Rosenblatt J. E., other authors. 2006; Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev19:165–256 [CrossRef][PubMed]
    [Google Scholar]
  13. Fletcher K. R., Cinalli M.. 2007; Identification, optimal management, and infection control measures for Clostridium difficile-associated disease in long-term care. Geriatr Nurs28:171–181 quiz 182 [CrossRef]
    [Google Scholar]
  14. Goorhuis A., Bakker D., Corver J., Debast S. B., Harmanus C., Notermans D. W., Bergwerff A. A., Dekker F. W., Kuijper E. J.. 2008; Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis47:1162–1170 [CrossRef][PubMed]
    [Google Scholar]
  15. 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 Methods89:87–94 [CrossRef][PubMed]
    [Google Scholar]
  16. Guion C. E., Ochoa T. J., Walker C. M., Barletta F., Cleary T. G.. 2008; Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR. J Clin Microbiol46:1752–1757 [CrossRef][PubMed]
    [Google Scholar]
  17. Houser B. A., Hattel A. L., Jayarao B. M.. 2010; Real-time multiplex polymerase chain reaction assay for rapid detection of Clostridium difficile toxin-encoding strains. Foodborne Pathog Dis7:719–726 [CrossRef][PubMed]
    [Google Scholar]
  18. Hundsberger T., Braun V., Weidmann M., Leukel P., Sauerborn M., von Eichel-Streiber C.. 1997; Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur J Biochem244:735–742 [CrossRef][PubMed]
    [Google Scholar]
  19. Janezic S., Indra A., Allerberger F., Rupnik M.. 2011; Use of different molecular typing methods for the study of heterogeneity within Clostridium difficile toxinotypes V and III. J Med Microbiol60:1101–1107 [CrossRef][PubMed]
    [Google Scholar]
  20. Jensen M. B. F., Olsen K. E. P., Nielsen X. C., Hoegh A. M., Dessau R. B., Atlung T., Engberg J.. 2015; Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture. Eur J Clin Microbiol Infect Dis34:727–736 [CrossRef][PubMed]
    [Google Scholar]
  21. Kato H., Arakawa Y.. 2011; Use of the loop-mediated isothermal amplification method for identification of PCR ribotype 027 Clostridium difficile. J Med Microbiol60:1126–1130 [CrossRef][PubMed]
    [Google Scholar]
  22. Knetsch C. W., Lawley T. D., Hensgens M. P., Corver J., Wilcox M. W., Kuijper E. J.. 2013; Current application and future perspectives of molecular typing methods to study Clostridium difficile infections. Euro Surveill18:20381[PubMed]
    [Google Scholar]
  23. Lessa F. C., Mu Y., Bamberg W. M., Beldavs Z. G., Dumyati G. K., Dunn J. R., Farley M. M., Holzbauer S. M., Meek J. I., other authors. 2015; Burden of Clostridium difficile infection in the United States. N Engl J Med372:825–834 [CrossRef][PubMed]
    [Google Scholar]
  24. Loo V. G., Poirier L., Miller M. A., Oughton M., Libman M. D., Michaud S., Bourgault A.-M., Nguyen T., Frenette C., other authors. 2005; A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med353:2442–2449 [CrossRef][PubMed]
    [Google Scholar]
  25. Loo V. G., Bourgault A.-M., Poirier L., Lamothe F., Michaud S., Turgeon N., Toye B., Beaudoin A., Frost E. H., other authors. 2011; Host and pathogen factors for Clostridium difficile infection and colonization. N Engl J Med365:1693–1703 [CrossRef][PubMed]
    [Google Scholar]
  26. Lyerly D. M., Krivan H. C., Wilkins T. D.. 1988; Clostridium difficile: its disease and toxins. Clin Microbiol Rev1:1–18[PubMed]
    [Google Scholar]
  27. Lyon E.. 2001; Mutation detection using fluorescent hybridization probes and melting curve analysis. Expert Rev Mol Diagn1:92–101 [CrossRef][PubMed]
    [Google Scholar]
  28. 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. Nature458:1176–1179 [CrossRef][PubMed]
    [Google Scholar]
  29. Magill S. S., Edwards J. R., Bamberg W., Beldavs Z. G., Dumyati G., Kainer M. A., Lynfield R., Maloney M., McAllister-Hollod L., other authors. 2014; Multistate point-prevalence survey of health care-associated infections. N Engl J Med370:1198–1208 [CrossRef][PubMed]
    [Google Scholar]
  30. Markham N. R., Zuker M.. 2005; DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res33:W577–W581 [CrossRef][PubMed]
    [Google Scholar]
  31. McDonald L. C., Killgore G. E., Thompson A., Owens R. C. Jr, Kazakova S. V., Sambol S. P., Johnson S., Gerding D. N.. 2005; An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med353:2433–2441 [CrossRef][PubMed]
    [Google Scholar]
  32. O'Connor J. R., Johnson S., Gerding D. N.. 2009; Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. Gastroenterology136:1913–1924 [CrossRef][PubMed]
    [Google Scholar]
  33. O'Horo J. C., Jones A., Sternke M., Harper C., Safdar N.. 2012; Molecular techniques for diagnosis of Clostridium difficile infection: systematic review and meta-analysis. Mayo Clin Proc87:643–651 [CrossRef][PubMed]
    [Google Scholar]
  34. Pallis A., Jazayeri J., Ward P., Dimovski K., Svobodova S.. 2013; Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR. J Med Microbiol62:1350–1356 [CrossRef][PubMed]
    [Google Scholar]
  35. Pecavar V., Blaschitz M., Hufnagl P., Zeinzinger J., Fiedler A., Allerberger F., Maass M., Indra A.. 2012; High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile. J Med Microbiol61:780–785 [CrossRef][PubMed]
    [Google Scholar]
  36. Persson S., Jensen J. N., Olsen K. E. P.. 2011; Multiplex PCR method for detection of Clostridium difficile tcdA, tcdB, cdtA, and cdtB and internal in-frame deletion of tcdC. J Clin Microbiol49:4299–4300 [CrossRef][PubMed]
    [Google Scholar]
  37. Petrella L. A., Sambol S. P., Cheknis A., Nagaro K., Kean Y., Sears P. S., Babakhani F., Johnson S., Gerding D. N.. 2012; Decreased cure and increased recurrence rates for Clostridium difficile infection caused by the epidemic C. difficile BI strain. Clin Infect Dis55:351–357 [CrossRef][PubMed]
    [Google Scholar]
  38. Ririe K. M., Rasmussen R. P., Wittwer C. T.. 1997; Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem245:154–160 [CrossRef][PubMed]
    [Google Scholar]
  39. Rupnik M., Avesani V., Janc M., von Eichel-Streiber C., Delmée M.. 1998; A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol36:2240–2247[PubMed]
    [Google Scholar]
  40. Schwartz S., Zhang Z., Frazer K. A., Smit A., Riemer C., Bouck J., Gibbs R., Hardison R., Miller W.. 2000; PipMaker -a web server for aligning two genomic DNA sequences. Genome Res10:577–586 [CrossRef][PubMed]
    [Google Scholar]
  41. Skow A., Mangold K. A., Tajuddin M., Huntington A., Fritz B., Thomson R. B. Jr, Kaul K. L.. 2005; Species-level identification of staphylococcal isolates by real-time PCR and melt curve analysis. J Clin Microbiol43:2876–2880 [CrossRef][PubMed]
    [Google Scholar]
  42. 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 Microbiol48:3719–3724 [CrossRef][PubMed]
    [Google Scholar]
  43. Tsiatis A. C., Norris-Kirby A., Rich R. G., Hafez M. J., Gocke C. D., Eshleman J. R., Murphy K. M.. 2010; Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn12:425–432 [CrossRef][PubMed]
    [Google Scholar]
  44. Untergasser A., Nijveen H., Rao X., Bisseling T., Geurts R., Leunissen J. A. M.. 2007; Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res35:W71–W74 [CrossRef][PubMed]
    [Google Scholar]
  45. van der Stoep N., van Paridon C. D. M., Janssens T., Krenkova P., Stambergova A., Macek M., Matthijs G., Bakker E.. 2009; Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner. Hum Mutat30:899–909 [CrossRef][PubMed]
    [Google Scholar]
  46. Vonberg R. P., Kuijper E. J., Wilcox M. H., Barbut F., Tüll P., Gastmeier P., van den Broek P. J., Colville A., Coignard B., other authors. 2008; Infection control measures to limit the spread of Clostridium difficile. Clin Microbiol Infect14:(Suppl 5)2–20 [CrossRef][PubMed]
    [Google Scholar]
  47. White H. E., Hall V. J., Cross N. C. P.. 2007; Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader–Willi and Angelman syndromes. Clin Chem53:1960–1962 [CrossRef][PubMed]
    [Google Scholar]
  48. Wiegand P. N., Nathwani D., Wilcox M. H., Stephens J., Shelbaya A., Haider S.. 2012; Clinical and economic burden of Clostridium difficile infection in Europe: a systematic review of healthcare-facility-acquired infection. J Hosp Infect81:1–14 [CrossRef][PubMed]
    [Google Scholar]
  49. Wolff D., Brüning T., Gerritzen A.. 2009; Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis. Eur J Clin Microbiol Infect Dis28:959–962 [CrossRef][PubMed]
    [Google Scholar]
  50. Yeh S.-H., Tsai C.-Y., Kao J.-H., Liu C.-J., Kuo T.-J., Lin M.-W., Huang W.-L., Lu S.-F., Jih J., other authors. 2004; Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis. J Hepatol41:659–666 [CrossRef][PubMed]
    [Google Scholar]
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