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

Abstract

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 10(6) 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 > or =10(6) 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.

FIG. 1

Sensitivity and detection range of the real-time PCR assay for S. pneumoniae. (A) Reproducibility of the assay when testing dilution series of S. pneumoniae followed by DNA extraction in a single assay with replicates from the same DNA extraction (●) and in independent assays with the same DNA extraction (□). The intra- and interassay variabilities were 2.0 and 3.3%, respectively. (B) Absence of inhibition when spiking genomic DNA from S. pneumoniae with DNA from NPS samples shown to be negative for S. pneumoniae. Threshold cycle is the cycle number when the threshold fluorescence is reached. The standard deviations from three measurements are shown in error bars. ●, S. pneumonia DNA alone; ▵, S. pneumonia DNA spiked with DNA from NPS sample 1; □, S. pneumoniae DNA spiked with DNA from NPS sample 2.

FIG. 2

Specificity of the real-time PCR assay for S. pneumoniae. Serial dilutions of liquid cultures from different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay targeting the pneumolysin gene. The values are the means of duplicate measurements. (A) Assay executed at an annealing temperature of 60°C. (B) Assay executed at an annealing temperature of 65°C.

FIG. 3

Sensitivity and specificity of the real-time PCR assay for S. pneumoniae in clinical samples (NPSs) compared with culture results. The dashed line indicates the arbitrary cutoff C_T value. C_T values below 25 were regarded as positive for S. pneumoniae. Horizontal bars indicate medians, which are also given as absolute values. (A) Assay executed at an annealing-extension temperature of 60°C. (B) Assay executed at an annealing-extension temperature of 65°C.

FIG. 4

Influence of temperature on C_T values and on specificity. The standard amplification parameters used were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles with each cycle comprising 95°C for 15 s and either 60, 62, or 65°C for 1 min.

FIG. 5

Comparison of quantification of S. pneumoniae by real-time PCR with semiquantitative cultures. The samples were inoculated onto sheep blood agar by fractionation with a calibrated wire loop (5 μl). Growth only in the first fraction was defined a low growth (+), growth also in the second fraction was defined as intermediate growth (++), and growth in all three fractions was defined as abundant growth (+++). The real-time PCR assay was executed at an annealing temperature of 65°C.