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Journal of Clinical Microbiology, September 2003, p. 4121-4126, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4121-4126.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii for Clinical Diagnosis

Kate E. Templeton,1 Sitha A. Scheltinga,1 Anneke van der Zee,2 Bram M. W. Diederen,1 Anna M. Kruijssen,3 Herman Goossens,1,4 Ed Kuijper,1 and Eric C. J. Claas1*

Department of Medical Microbiology, Center of Infectious Diseases,1 Department of Pediatrics, Leiden University Medical Center, Leiden,3 Laboratory of Molecular Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands,2 Department of Microbiology, University of Antwerp, Antwerp, Belgium4

Received 31 January 2003/ Returned for modification 29 April 2003/ Accepted 24 June 2003

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

* Corresponding author. Mailing address: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 526 3650. Fax: 31 71 524 8148. E-mail: E.Claas{at}LUMC.NL.

Journal of Clinical Microbiology, September 2003, p. 4121-4126, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4121-4126.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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