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Detection of bacterial pneumonia by quantitative PCR, with special reference to Streptococcus pneumoniae and Haemophilus influenzae

Abstract number: P1984

Abdeldaim G., Strålin K., Korsgaard J., Olcén P., Blomberg J., Herrmann B.

Objectives: To develop sensitive and specific quantitative multiplex real-time PCR for detection of Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

Methods: A multiplex quantitative real-time PCR assay was developed for detection of S. pneumoniae (9802 gene fragment), H. influenzae (P6 gene) and N. meningitidis (ctrA gene). The analytical sensitivity of the assay was determined by serial dilutions of target DNAs. The method was evaluated on bronchoalveolar lavage from 156 immunocompetent adult patients with lower respiratory tract infection (LRTI) and 44 adult controls who underwent bronchoscopy for suspected malignancy. The PCR results were compared with the results of culture.

Results: The analytical sensitivity was 10–60 copies/reaction for S. pneumoniae and 3–10 copies/reaction for H. influenzae. The detection capacity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. By culture, S. pneumoniae and H. influenzae were aetiological agents in 21 and 33 of the LTRI patients, respectively.

These pathogens were identified by real time PCR in 45 and 67 of the cases, respectively, yielding sensitivities of 76% for S. pneumoniae and 90% for H. influenzae, and specificities of 78% for S. pneumoniae and 68% for H. influenzae. A cutoff for clinically significant positivity (104 DNA copies/ml for S. pneumoniae and 5×104 DNA copies/ml for H. influenzae) yielded sensitivities of 71% and 78%, and specificities of 85% and 86% for S. pneumoniae and H. influenzae, respectively. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 7% and 17% of the cases, respectively, and by the PCR in 28% and 52% of the cases, respectively.

Among the 44 controls, S. pneumoniae and H. influenzae were identified by culture in 3 and 5 cases, respectively, by PCR in 14 and 17 cases, respectively, and by PCR with the defined cutoff limits in 5 and 9 cases, respectively.

N. meningitidis was detected in 7 LRTI patients which indicated that the multiplex assay may be useful in the diagnosis of meningitis.

Conclusions: The multiplex format of the assay facilitates diagnosis of S. pneumoniae and H. influenzae and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of aetiology for pneumonia.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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