LETTER TO THE EDITOR The laboratory diagnosis of Bordetella pertussis infection: a comparison of semi-nested PCR and real-time PCR with culture B. Abu Raya & E. Bamberger & R. Gershtein & M. Peterman & I. Srugo Received: 5 March 2011 /Accepted: 16 June 2011 /Published online: 9 July 2011 # Springer-Verlag 2011 Introduction Pertussis is an infection of the respiratory tract caused by Bordetella pertussis (B. pertussis)[1]. Despite high vacci- nation coverage, there has been a worldwide resurgence of B. pertussis infection [1–3]. Increasing atypical clinical presentations warrant a sensitive, specific and rapid labora- tory diagnostic tool [4]. While culture is still considered the "gold standard" for laboratory diagnosis, its isolation rates are variable and low [1, 5, 6]. Conventional-semi nested (CsnPCR) and real-time PCR (RtPCR) are two PCR tools employed for the detection of B. pertussis with the former detecting amplified target gene products visualized by ultra violet (UV) light following agarose gel electrophoresis and the latter monitoring the fluorescence emitted throughout PCR reaction phases indicating gene amplification. Little research has compared CsnPCR and RtPCR for the detection of B. pertussis [7, 8]. The aim of this study was to compare the performance characteristics and diagnostic yield (+PCR/+Culture ratio) of CsnPCR with RtPCR in the detection of B. pertussis and to examine how epidemiological features moderate these characteristics. Materials and methods CsnPCR were performed on nasopharyngeal samples from 1/2004 to 10/2007 (n =1,891) and compared with RtPCR samples from 11/2007 to 8/2009 (n =1,693). All samples were sent to the Clinical Microbiology Laboratory at Bnai Zion Medical Center, Haifa, Israel, where PCR analysis was performed by the same highly trained laboratory personnel. The specimens were obtained from both in- and outpatient patients ages 1 day to 85-years old. Prior to the introduction of the new RtPCR method to the routine laboratory work, a pilot study of 92 samples was performed whereby both CsnPCR and RtPCR were performed on all samples. The PCR detection of B. pertussis was based on the amplification of Insertion Sequence 481, and positive B. pertussis cultures were regarded as the "gold standard". CsnPCR detection Sample collection and DNA isolation Nasopharyngeal Dacron swabs and aspirates were used. Swabs were placed in sterile physiological solution and stored at -20°C. After defrosting, samples were vortexed and transferred into Eppendorf tubes. A 10 min 5000×g centrifugation was performed and DNA was extracted employing QIAamp DNA kit (Giagen, Hilden, Germany) and used directly for PCR. CsnPCR analysis Primers for IS481 as previously pub- lished by Lichtinghagen et al. [9] were used. Upstream primer 5′-GATTCAATAGGTTGTATGCATGGTT-3′ and downstream primer 5′ -GCTTCAGGCACACAAACTT GATGG -3′ were used in the first PCR round which included 4-μl sample DNA, 2x Reddy mix PCR Master B. Abu Raya (*) : I. Srugo Department of Pediatrics, Bnai Zion Medical Center, The Bruce Rappaport Faculty of Medicine, Haifa, Israel e-mail: baha_aboraya@yahoo.com E. Bamberger : R. Gershtein : M. Peterman : I. Srugo Clinical Microbiology Laboratory, Bnai Zion Medical Center, The Bruce Rappaport Faculty of Medicine, Haifa, Israel Eur J Clin Microbiol Infect Dis (2012) 31:619–622 DOI 10.1007/s10096-011-1327-6