Identification of pneumococcal serotypes from culture-negative clinical specimens by novel real-time PCR

Abstract

Pneumococcal parapneumonic empyema is an increasingly common complication in children. Conventional microbiological cultures indicate bacterial causes in as few as 8% of cases; therefore, there is a vital need for new molecular methods of detection and diagnosis. The development and clinical evaluation of real-time PCR-based assays to detect the pneumococcal capsular wzg gene of all serotypes tested are reported here, and 24 of them have been identified in clinical specimens. Using real-time PCR assays with highly specific TaqMan MGB probes that target DNA sequences within the capsular polysaccharide gene cluster, it was possible to differentiate serotypes 1, 3, 5, 4, 6A, 6B, 7F/A, 8, 9V/A/N/L, 14, 15B/C, 18C/B, 19A, 19F/B/C, 23F and 23A. These assays showed high sensitivity (five to ten pneumococcal DNA equivalents) and they were validated with 175 clinical isolates of known serotypes. The clinical value of this approach was demonstrated by analysis of 88 culture-negative pleural fluids from children diagnosed with parapneumonic empyema in three Spanish hospitals. Pneumococcal DNA was detected in 87.5% of pleural fluids, and serotypes 1, 7F and 3 were responsible for 34.3%, 16.4% and 11.9%, respectively, of cases of parapneumonic empyema in children. Such molecular methods are critical for the diagnosis of invasive pneumococcal disease and continued epidemiological surveillance in order to monitor serotype vaccine effectiveness.

Characterization of a New Mouse Model of Empyema and the Mechanisms of Pleural Invasion by Streptococcus pneumoniae

“…Further research is required to clarify whether similar mechanisms are relevant for translocation across mesothelial cells, characterization of which could offer potential targets for therapies that prevent empyema. Differences in the ability to translocate across mesothelial cells may explain why certain bacterial pathogens and strains are particularly associated with pleural infection (e.g., the S. milleri group and capsular serotype 1 S. pneumoniae) (30)(31)(32). During later stages of infection, the pleural layer showed evidence of severe disruption, which could allow significant bacterial movement into the pleural space from the lung and vice versa via a paracellular route.…”

DNA bacterial load in children and adolescents with pneumococcal pneumonia and empyema

“…Detection of pneumococcal serotypes in clinical samples with negative culture were performed in our laboratory, according to Multiplex Real-Time PCR [13].This sequential PCR approach distinguishes among 24 serotypes (1, 3 Selected samples were performed in parallel in order to compare the recovery of bacterial DNA using both DNA extraction methods.…”

Severe necrotizing pneumonia in a child with pandemic (H1N1) influenza

“…Pneumococcal DNA has been identified in up to 87.5% of culture-negative pleural fluid in children with parapneumonic empyema. 4 S. pneumoniae is a gram-positive, α-hemolytic coccus with 91 serotypes differentiated by their capsular polysaccharide. 3 The ability of S. pneumoniae to cause disease in humans is based on its ability to adhere to human cells via surface adhesions, avoid phagocytosis by the host immune response and activate a significant inflammatory response.…”