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Toward a quantitative DNA-based definition of pneumococcal pneumonia: a comparison of Streptococcus pneumoniae target genes, with special reference to the Spn9802 fragment
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk bakteriologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk virologi.
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2008 (Engelska)Ingår i: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 60, nr 2, s. 143-150Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The current shift from phenotypically toward genotypically based microbial diagnosis is not unproblematic. A novel quantitative real-time polymerase chain reaction (PCR) assay based on the Spn9802 DNA fragment was therefore developed for detection of Streptococcus pneumoniae. Out of 44 bacterial species, only S. pneumoniae and Streptococcus pseudopneumoniae were positive in Spn9802 PCR. In an evaluation on nasopharyngeal aspirates from 166 patients with community-acquired pneumonia, the assay was positive in 49 of 50 culture-positive cases. Of 19 culture-negative but Spn9802 PCR-positive cases, 12 were confirmed as S. pneumoniae by rnpB sequence analysis. With an expanded reference standard, including culture and rnpB sequencing, Spn9802 had a sensitivity of 94% and a specificity of 98%. A cutoff for clinically significant positivity was 10(4) DNA copies/mL, giving 71% sensitivity and 100% specificity. In conclusion, Spn9802 real-time PCR is highly sensitive and specific. The quantification it provides enables differentiation between pneumococcal pathogenicity and commensalism.

Ort, förlag, år, upplaga, sidor
2008. Vol. 60, nr 2, s. 143-150
Nyckelord [en]
S. pneumoniae, Pneumonia, Real-time PCR, Spn9802
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-11875DOI: 10.1016/j.diagmicrobio.2007.08.010ISI: 000252915100002PubMedID: 17916422OAI: oai:DiVA.org:uu-11875DiVA, id: diva2:39644
Tillgänglig från: 2008-12-02 Skapad: 2008-12-02 Senast uppdaterad: 2017-12-11Bibliografiskt granskad
Ingår i avhandling
1. PCR detection of Streptococcus pneumoniae and Haemophilus influenzae in pneumonia patients
Öppna denna publikation i ny flik eller fönster >>PCR detection of Streptococcus pneumoniae and Haemophilus influenzae in pneumonia patients
2009 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2009. s. 62
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 479
Nyckelord
Lower respiratory tract infections, Pneumonia, Streptococcus pneumoniae, Haemophilus influenzae, real-time PCR
Identifikatorer
urn:nbn:se:uu:diva-107931 (URN)978-91-554-7598-7 (ISBN)
Disputation
2009-10-16, Hörsalen, Uppsala University Hospital, Department of Clinical Microbiology, Dag Hammarskjölds Väg 17, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2009-09-25 Skapad: 2009-08-31 Senast uppdaterad: 2011-02-07Bibliografiskt granskad

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