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Journal of Clinical Microbiology, September 2001, p. 3129-3134, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3129-3134.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCR

Oliver Greiner,1 Philip J. R. Day,1,2 Philipp P. Bosshard,3 Fatime Imeri,3 Martin Altwegg,3 and David Nadal1,*

Division of Infectious Diseases1 and Division of Oncology,2 University Children's Hospital of Zurich, CH-8032 Zurich, and Department of Medical Microbiology, University of Zurich, CH-8028 Zurich,3 Switzerland

Received 14 March 2001/Returned for modification 13 May 2001/Accepted 28 June 2001

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 106 cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were >= 106 cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.

* Corresponding author. Mailing address: Division of Infectious Diseases, University Children`s Hospital of Zurich, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland. Phone: 41-1-266-7562. Fax: 41-1-266-7157. E-mail: david.nadal{at}kispi.unizh.ch.

Journal of Clinical Microbiology, September 2001, p. 3129-3134, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3129-3134.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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