DETECTION OF STREPTOCOCCUS-PNEUMONIAE IN WHOLE-BLOOD BY PCR

Streptococcus pneumoniae is a major cause of bacteremia in both childr en and adults. Currently, the diagnosis of pneumococcal bacteremia rel ies on the isolation and identification of the bacteria from blood cul tures. We have developed a sensitive assay for the detection of S. pne umoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-bindi ng protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, incl uding 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demon strated by its inability to support amplification from a series of hum an, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any p athogens in whole blood was developed. With this protocol it was possi ble to detect S. pneumoniae-specific DNA from whole blood specimens in oculated with as little as 4 CFU/ml. Copurified human blood DNA, rangi ng from 0 to 4.5 mu g per PCR, did not affect the sensitivity of S. pn eumoniae detection by PCR. A blinded clinical trial was used to compar e the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained fro m pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh. With culture as the ''gold standard,'' the PCR-based a ssay had a sensitivity of 80% (4 of 5 culture-positive specimens were PCR positive) and a specificity of 84% (26 of 31 culture-negative spec imens were PCR negative). However, three patients whose specimens were PCR positive and culture negative had histories suggestive of bactere mia, including recent positive blood cultures, treatment with antibiot ics, cellulitis, and multiple emergency room visits for fever within a 24-h period. These data suggest that PCR-based assays for S. pneumoni ae may prove useful to augment current methods of detection for S. pne umoniae bacteremia.