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A prospective study of 154 consecutive high-risk hospitalized children with lower respiratory infections was conducted to determine the clinical utility of a pneumolysin-based polymerase chain reaction (PCR) assay compared with blood and pleural fluid cultures and serological and urinary antigen tests to determine the incidence of Streptococcus pneumoniae. Whole blood, buffy coat, or plasma samples from 67 children (44%) tested positive by PCR. Sensitivity was 100% among 11 promptly tested culture-confirmed children and specificity was 95% among control subjects. Age, prior oral antibiotic therapy, and pneumococcal nasopharyngeal colonization did not influence PCR results, whereas several surrogates of disease severity were associated with positive tests. Although serological and urinary antigen tests had comparable sensitivity, specificity varied among infected children, and statistical agreement among all assays was limited. These findings support the use of PCR tests to evaluate the protective efficacy of pneumococcal conjugate vaccines and to identify promptly children with pretreated or nonbacteremic pneumococcal lower respiratory infections.