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Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology. (Björn Herrmann)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology. (Björn Herrmann)
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2010 (English)In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 16, no 8, 1135-1141 p.Article in journal (Refereed) Published
Abstract [en]

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.

Place, publisher, year, edition, pages
2010. Vol. 16, no 8, 1135-1141 p.
Keyword [en]
lytA gene, plasma, ply gene, pneumococcal pneumonia, real-time PCR, Spn9802 fragment, Streptococcus pneumoniae
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-125332DOI: 10.1111/j.1469-0691.2009.03069.xISI: 000280359900015PubMedID: 19832718OAI: oai:DiVA.org:uu-125332DiVA: diva2:319400
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12Bibliographically 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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