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Journal of Clinical Microbiology, July 2005, p. 3390-3397, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3390-3397.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

Sven Poppert,1* Andreas Essig,1 Barbara Stoehr,1 Adelinde Steingruber,1 Beate Wirths,1 Stefan Juretschko,3,{dagger} Udo Reischl,2 and Nele Wellinghausen1

Department of Medical Microbiology and Hygiene, University of Ulm, Ulm,1 Department of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany,2 University of Washington, Seattle, Washington3

Received 14 July 2004/ Returned for modification 6 October 2004/ Accepted 14 February 2005

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

* Corresponding author. Mailing address: Abteilung für Medizinische Mikrobiologie und Hygiene, Universitaet Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. Phone: 731 500 24610. Fax: 731 500 24619. E-mail: sven.poppert{at}medizin.uni-ulm.de.

{dagger} Present address: Department of Pediatric Pathology, Arkansas Children’s Hospital, Little Rock, AR 72202-3591.

Journal of Clinical Microbiology, July 2005, p. 3390-3397, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3390-3397.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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