Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture

Yun Ha Jang; Jaewoo Chung; Seungmi Baek; Sookja Park; Heungsup Sung; Mi-Na Kim

doi:10.5145/KJCM.2009.12.1.11

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Jang, Chung, Baek, Park, Sung, and Kim: Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture

Original Article

Korean J Clin Microbiol 2009;12(1):11-16.

Published online: 22 March 2009

DOI: https://doi.org/10.5145/KJCM.2009.12.1.11

Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture

Yun Ha Jang, Jaewoo Chung, Seungmi Baek, Sookja Park, Heungsup Sung, Mi-Na Kim

Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

Correspondence: Mi-Na Kim, Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 388-1, Pungnap-dong, Songpa-gu, Seoul 138-736, Korea. (Tel) 82-2-3010-4511, (Fax) 82-2-478-0884, (E-mail) mnkim@amc.seoul.kr

Received 25 August 20083 December 2008 Accepted 20 January 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

We evaluated multiplex PCR for species identification and toxin typing to improve the sensitivity and turnaround time of toxigenic Clostridium difficile culture (TCDC).

Methods

We performed multiplex PCR using primers targeting the species-specific gene, tpi, and the toxin genes, tcdA and tcdB. From January to March 2008, 528 stool specimens were tested with direct toxin assay (DT) using C. difficile Tox A/B II (Techlab, Blacksburg, USA) and TCDC. For 288 specimens from early study period, toxin production by C. difficile isolates of TCDC was measured by enzyme immunoassay with culture supernatants using VIDAS C. difficile Toxin A&B (CDAB; bioMérieux, Marcy-l'Etoile, France) and multiplex PCR with isolated colonies. For 240 specimens from late period, only multiplex PCR was used to test toxin production by the isolates.

Results

During the early period, 29 C. difficile were isolated and their toxin-positive rates were 65.5% by PCR and 44.8% by CDAB (P＜0.05). Among 528 stool specimens, the results of DT+/TCDC+, DT+/ TCDC-, and DT-/TCDC+ were 32 (6.1%), 33 (6.3%), and 10 (1.9%), respectively, when tested with PCR. 13.3% of total 75 positive specimens was detected only by TCDC. Of the 42 toxigenic C. difficile isolates, all were positive for tpi, 30 (71.4%) were tcdA+/tcdB+, and 12 (28.6%) were tcdA-/tcdB+.

Conclusion

TCDC using multiplex PCR for species identification and toxin typing is sensitive and rapid to be used as a routine diagnostic test.

Keywords: Clostridium difficile, Toxin, Polymerase chain reaction

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Fig. 1.

Electrophoresis of the PCR products of nine isolates of C. difficile using DNA templates extracted with GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, USA) (A) and boiling method (B) in parallel. The bands on all lanes of A are consistently denser than those of B except lane 3. Lane M; 100 bp DNA ladder, Lane 1; negative control, Lane 2 and 3; positive for species specific gene (tpi) but negative for toxin genes (tcdA and tcdB), indicating nontoxigenic strains, Lane 4-7; positive for tpi, tcdA, and tcdB genes, indicating A+B+ toxigenic strains, Land 8-10; positive for tpi, and tcdB, but negative (deleted) for tcdA gene, indicating A-B+ toxigenic strains.

Table 1.

Concordance between multiplex PCR and enzyme immunoassay using VIDAS C. difficile Toxin A&B (CDAB) for toxin production among 29 C. difficile isolates

CDAB	No. of isolates with toxin profile by multiplex PCR
CDAB	A+B+	A-B+	A-B-	Total
Positive Equivocal Negative Total	5 2 5 12	4 2 1 7	0 0 10 10	9 4 16 29

Abbreviations: A+B+, tcdA+/tcdB+; A-B+, tcdA-/tcdB+; A-B-, tcdA-/tcdB-.

Table 2.

Concordance between direct toxin assay and toxigenic C. difficile culture

	Number of isolates
	Total	No growth	A+B+	A-B+	A-B-
DT+/TCDC+ DT-/TCDC+ DT+/TCDC DT-/TCDC- Total	32 10 33 453 528	- - 28 443 471	22 8 - - 30	10 2 - - 12	- - 5 10 15

Abbreviations: A+B+, tcdA+/tcdB+; A-B+, tcdA-/tcdB+; A-B-, tcdA-/tcdB-; DT, direct toxin assay; TCDC, toxigenic C. difficile culture.

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