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The widespread uptake of pneumococcal vaccines has substantially reduced the incidence of invasive pneumococcal disease, such that pneumococcal bacteremia in children is now considered a relatively rare event. The objective of this study was to ascertain the clinical utility of a Streptococcus pneumoniae real-time polymerase chain reaction (PCR) assay compared with standard blood culture for the diagnosis of pneumococcal bacteremia in children in the post-vaccine era.A systematic retrospective review of laboratory and patient records from Children’s University Hospital, Temple Street, during a 6-year period was performed. Paired blood PCR and blood culture specimens from children younger than 16 years of age were investigated. Statistical analysis was performed to measure the diagnostic accuracy of PCR versus routine bacterial culture techniques.More than 1900 PCR test requests were examined from 2010 to 2015, of which 1561 paired PCR and blood culture specimens met criteria for inclusion in the statistical analysis. The PCR assay demonstrated high specificity (99%, confidence interval 95%: 98.81%–99.69%); however, the sensitivity was low compared with that of blood culture (47%, confidence interval 95%: 21.27%–73.41%). Investigation of 10 PCR-positive/culture-negative cases revealed that these cases ranged from definite, probable, and possible significance, indicating a low false positivity rate associated with the assay.This study demonstrates the limited utility of blood PCR testing for S. pneumoniae in pediatric patients without radiographic evidence pneumonia or empyema. Moreover, we report that PCR may be a useful diagnostic tool when blood cultures are negative because of antimicrobial therapy before sampling. Given that the incidence of pneumococcal disease has decreased considerably in recent years, justification of S. pneumoniae PCR requisition is necessary. Hence, new guidelines for pediatric pneumococcal blood PCR testing have been introduced at the Irish Meningitis and Sepsis Reference Laboratory.