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Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology. (Jonas Blomberg)
Department of Infectious Diseases, Örebro University Hospital, Örebro.
Department of Chest Diseases, Aarhus University Hospital, Aalborg, Denmark.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
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2010 (English)In: BMC Microbiology, ISSN 1471-2180, Vol. 10, 310- p.Article in journal (Refereed) Published
Abstract [en]

Background. Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.

Results. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria.

Conclusions. The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.

Place, publisher, year, edition, pages
2010. Vol. 10, 310- p.
Keyword [en]
S. pneumoniae, H. influenzae, N. meningitidis, quantitative multiplex PCR, lower respiratory tract infection
National Category
Medical and Health Sciences
Research subject
Clinical Bacteriology
URN: urn:nbn:se:uu:diva-107967DOI: 10.1186/1471-2180-10-310ISI: 000285888900001PubMedID: 21129171OAI: oai:DiVA.org:uu-107967DiVA: diva2:233757
Available from: 2009-09-02 Created: 2009-09-02 Last updated: 2011-11-11Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 479
Lower respiratory tract infections, Pneumonia, Streptococcus pneumoniae, Haemophilus influenzae, real-time PCR
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)
Available from: 2009-09-25 Created: 2009-08-31 Last updated: 2011-02-07Bibliographically approved

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