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. 1993 Mar;31(3):507–511. doi: 10.1128/jcm.31.3.507-511.1993

Specific detection of toxigenic strains of Clostridium difficile in stool specimens.

P H Gumerlock 1, Y J Tang 1, J B Weiss 1, J Silva Jr 1
PMCID: PMC262810  PMID: 8458943

Abstract

Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barroso L. A., Wang S. Z., Phelps C. J., Johnson J. L., Wilkins T. D. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 1990 Jul 11;18(13):4004–4004. doi: 10.1093/nar/18.13.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borriello S. P., Honour P. Concomitance of cytotoxigenic and non-cytotoxigenic Clostridium difficile in stool specimens. J Clin Microbiol. 1983 Oct;18(4):1006–1007. doi: 10.1128/jcm.18.4.1006-1007.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burg J. L., Grover C. M., Pouletty P., Boothroyd J. C. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol. 1989 Aug;27(8):1787–1792. doi: 10.1128/jcm.27.8.1787-1792.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Girolami P. C., Hanff P. A., Eichelberger K., Longhi L., Teresa H., Pratt J., Cheng A., Letourneau J. M., Thorne G. M. Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A. J Clin Microbiol. 1992 May;30(5):1085–1088. doi: 10.1128/jcm.30.5.1085-1088.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. George W. L., Sutter V. L., Citron D., Finegold S. M. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979 Feb;9(2):214–219. doi: 10.1128/jcm.9.2.214-219.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gumerlock P. H., Tang Y. J., Meyers F. J., 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 Nov-Dec;13(6):1053–1060. doi: 10.1093/clinids/13.6.1053. [DOI] [PubMed] [Google Scholar]
  7. Hatheway C. L. Toxigenic clostridia. Clin Microbiol Rev. 1990 Jan;3(1):66–98. doi: 10.1128/cmr.3.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kato N., Ou C. Y., Kato H., Bartley S. L., Brown V. K., Dowell V. R., Jr, Ueno K. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol. 1991 Jan;29(1):33–37. doi: 10.1128/jcm.29.1.33-37.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lyerly D. M., Krivan H. C., Wilkins T. D. Clostridium difficile: its disease and toxins. Clin Microbiol Rev. 1988 Jan;1(1):1–18. doi: 10.1128/cmr.1.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Martinez R. D., Wilkins T. D. Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin). Infect Immun. 1988 May;56(5):1215–1221. doi: 10.1128/iai.56.5.1215-1221.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McMillin D. E., Muldrow L. L., Laggette S. J. Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction. Can J Microbiol. 1992 Jan;38(1):81–83. doi: 10.1139/m92-013. [DOI] [PubMed] [Google Scholar]
  12. McMillin D. E., Muldrow L. L., Leggette S. J., Abdulahi Y., Ekanemesang U. M. Molecular screening of Clostridium difficile toxins A and B genetic determinants and identification of mutant strains. FEMS Microbiol Lett. 1991 Feb;62(1):75–80. doi: 10.1016/0378-1097(91)90258-c. [DOI] [PubMed] [Google Scholar]
  13. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  14. Patel R. J., Fries J. W., Piessens W. F., Wirth D. F. Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Mar;28(3):513–518. doi: 10.1128/jcm.28.3.513-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wilde J., Eiden J., Yolken R. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol. 1990 Jun;28(6):1300–1307. doi: 10.1128/jcm.28.6.1300-1307.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wren B. W., Clayton C. L., Tabaqchali S. Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction. FEMS Microbiol Lett. 1990 Jun 15;58(1):1–6. doi: 10.1016/0378-1097(90)90092-5. [DOI] [PubMed] [Google Scholar]
  17. Wren B., Clayton C., Tabaqchali S. Rapid identification of toxigenic Clostridium difficile by polymerase chain reaction. Lancet. 1990 Feb 17;335(8686):423–423. doi: 10.1016/0140-6736(90)90267-9. [DOI] [PubMed] [Google Scholar]
  18. von Eichel-Streiber C., Laufenberg-Feldmann R., Sartingen S., Schulze J., Sauerborn M. Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B. Med Microbiol Immunol. 1990;179(5):271–279. doi: 10.1007/BF00192465. [DOI] [PubMed] [Google Scholar]

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