Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology

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pISSN 2288-0585 eISSN 2288-6850

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

Original article

Annals of Clinical Microbiology (Ann Clin Microbiol) 2009 March, Volume 12, Issue 1, pages 11-16.

https://doi.org/10.5145/ACM.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

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

Keywords

Clostridium difficile, Toxin, Polymerase chain reaction