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Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk bakteriologi. (Björn Herrmann)
Department of Infectious Diseases, Örebro University Hospital.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. (Leif Kirsebom)
Department of Clinical Microbiology, Örebro University Hospital.
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2009 (Engelska)Ingår i: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, nr 4, s. 366-373Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Ort, förlag, år, upplaga, sidor
Elsevier , 2009. Vol. 64, nr 4, s. 366-373
Nyckelord [en]
H. influenzae, Pneumonia, Real-time PCR, Outer membrane protein P6, fucK, rnpB
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Forskningsämne
Mikrobiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-106189DOI: 10.1016/j.diagmicrobio.2009.03.030ISI: 000269337900002PubMedID: 19446978OAI: oai:DiVA.org:uu-106189DiVA, id: diva2:224210
Tillgänglig från: 2009-06-17 Skapad: 2009-06-17 Senast uppdaterad: 2018-01-13Bibliografiskt 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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