Quantitative detection of streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR

Streptococcus pneumoniae is an important cause of community-acquired pneumo nia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pne umoniae may also occur in the upper respiratory tract, quantification of th ese bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagn ostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent pr obe specific for the pneumolysin gene, we were able to detect DNA from seri al dilutions of S. pneumoniae cells in which the quantities of DNA ranged f rom the amounts extracted from 1 to 10(6) cells. No difference was noted wh en the same DNA was mixed with DNA extracted from NPSs shown to be deficien t of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemoph ilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci oth er than S. pneumoniae were not amplified or were only weakly amplified when there were greater than or equal to 10(6) cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infecti ons, 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 organ isms detected by real-time PCR correlated with the numbers detected by semi quantitative cultures. A real-time PCR that targeted the pneumolysin gene p rovided a sensitive and reliable means for routine rapid detection and quan tification 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.