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Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology. (Herrmann)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology. (Herrmann)
Köpenhamns Universitet.
Örebro Universitet, klinisk mikrobiologi.
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2009 (English)In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 15, no 6, 565-570 p.Article in journal (Refereed) Published
Abstract [en]

The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply-based PCR have been reported. The aim here was to study the performance of a quantitative ply-based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply-based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at >/=10(3) genome copies/mL in 61% and 71% of the subjects, at >/=10(5) genome copies/mL in 40% and 58% of the subjects, and at >/=10(7) genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply-based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 10(3) genome copies/mL, of 84% and 66% at a cut-off of 10(5) genome copies/mL, and of 53% and 90% at a cut-off of 10(7) genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply-based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply-based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated.

Place, publisher, year, edition, pages
2009. Vol. 15, no 6, 565-570 p.
Keyword [en]
Bronchoalveolar lavage, PCR, pneumolysin, pneumonia, Streptococcus pneumoniae
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-102538DOI: 10.1111/j.1469-0691.2009.02714.xISI: 000268261300011PubMedID: 19416297OAI: oai:DiVA.org:uu-102538DiVA: diva2:216325
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2011-04-13Bibliographically approved
In thesis
1. PCR detection of Streptococcus pneumoniae and Haemophilus influenzae in pneumonia patients
Open this publication in new window or tab >>PCR detection of Streptococcus pneumoniae and Haemophilus influenzae in pneumonia patients
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs.

Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%.

In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved.

Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%.

Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%.

In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively.

In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 479
Keyword
Lower respiratory tract infections, Pneumonia, Streptococcus pneumoniae, Haemophilus influenzae, real-time PCR
Identifiers
urn:nbn:se:uu:diva-107931 (URN)978-91-554-7598-7 (ISBN)
Public defence
2009-10-16, Hörsalen, Uppsala University Hospital, Department of Clinical Microbiology, Dag Hammarskjölds Väg 17, Uppsala, 09:15 (English)
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Available from: 2009-09-25 Created: 2009-08-31 Last updated: 2011-02-07Bibliographically approved

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