title: A quantitative LightCycler PCR to detect Streptococcus pneumoniae in blood and CSF.
abstract: A quantitative real-time PCR targeting the Pneumolysin (ply) gene of Streptococcus pneumoniae was developed for the LightCycler instrument using Fluorescence Resonance Energy Transfer (FRET) probes. All common S. pneumoniae serotypes were detected while other bacteria and viruses were not. The sensitivity was determined to be between one and ten target copies per reaction. The PCR was applied to six CSF and 16 whole blood specimens from 17 patients with laboratory proven invasive pneumococcal disease. One hundred percent of CSF specimens and 69% of whole blood specimens were PCR positive. The bacterial loads were determined to be 7.6 to 6.01 x 10(5) copies/microL for the six CSF specimens, and 0.08 to 5.4 x 10(2) copies/microL for the 16 whole blood specimens. Ninety-seven percent of 30 culture and Gram's stain negative CSF specimens and 100% of 50 normal whole blood specimens were PCR negative. This highly sensitive and specific PCR assay has the potential to provide sufficiently rapid results to improve antibiotic treatment of S.pneumoniae infections, while bacterial load quantitation has opened up exciting possibilities for patient management.
authors.affiliation: Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth, Australia. robert.vanhaeften@health.wa.gov.au
authors.forename: Robert
authors.initials: R
authors.lastname: van Haeften
authors.forename: Silvano
authors.initials: S
authors.lastname: Palladino
authors.forename: Ian
authors.initials: I
authors.lastname: Kay
authors.forename: Tony
authors.initials: T
authors.lastname: Keil
authors.forename: Chris
authors.initials: C
authors.lastname: Heath
authors.forename: Grant W
authors.initials: GW
authors.lastname: Waterer
mesh_terms: D016470:Bacteremia / Q000175:diagnosis / Q000382:microbiology; D001426:Bacterial Proteins; D001769:Blood / Q000382:microbiology*; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D008586:Meningitis, Pneumococcal / Q000175:diagnosis / Q000382:microbiology; D011008:Pneumococcal Infections / Q000175:diagnosis* / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D013301:Streptolysins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae