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PCR detection of Streptococcus pneumoniae and Haemophilus influenzae in pneumonia patients
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Jonas Blomberg)
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. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 479
Keywords [en]
Lower respiratory tract infections, Pneumonia, Streptococcus pneumoniae, Haemophilus influenzae, real-time PCR
Identifiers
URN: urn:nbn:se:uu:diva-107931ISBN: 978-91-554-7598-7 (print)OAI: oai:DiVA.org:uu-107931DiVA, id: diva2:233546
Public defence
2009-10-16, Hörsalen, Uppsala University Hospital, Department of Clinical Microbiology, Dag Hammarskjölds Väg 17, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-09-25 Created: 2009-08-31 Last updated: 2011-02-07Bibliographically approved
List of papers
1. Toward a quantitative DNA-based definition of pneumococcal pneumonia: a comparison of Streptococcus pneumoniae target genes, with special reference to the Spn9802 fragment
Open this publication in new window or tab >>Toward a quantitative DNA-based definition of pneumococcal pneumonia: a comparison of Streptococcus pneumoniae target genes, with special reference to the Spn9802 fragment
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2008 (English)In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 60, no 2, p. 143-150Article in journal (Refereed) 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.

Keywords
S. pneumoniae, Pneumonia, Real-time PCR, Spn9802
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-11875 (URN)10.1016/j.diagmicrobio.2007.08.010 (DOI)000252915100002 ()17916422 (PubMedID)
Available from: 2008-12-02 Created: 2008-12-02 Last updated: 2017-12-11Bibliographically approved
2. Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?
Open this publication in new window or tab >>Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?
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2009 (English)In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 15, no 6, p. 565-570Article 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.

Keywords
Bronchoalveolar lavage, PCR, pneumolysin, pneumonia, Streptococcus pneumoniae
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102538 (URN)10.1111/j.1469-0691.2009.02714.x (DOI)000268261300011 ()19416297 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
3. Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia
Open this publication in new window or tab >>Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia
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2010 (English)In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 16, no 8, p. 1135-1141Article 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.

Keywords
lytA gene, plasma, ply gene, pneumococcal pneumonia, real-time PCR, Spn9802 fragment, Streptococcus pneumoniae
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-125332 (URN)10.1111/j.1469-0691.2009.03069.x (DOI)000280359900015 ()19832718 (PubMedID)
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12Bibliographically approved
4. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction
Open this publication in new window or tab >>Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction
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2009 (English)In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, no 4, p. 366-373Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Elsevier, 2009
Keywords
H. influenzae, Pneumonia, Real-time PCR, Outer membrane protein P6, fucK, rnpB
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-106189 (URN)10.1016/j.diagmicrobio.2009.03.030 (DOI)000269337900002 ()19446978 (PubMedID)
Available from: 2009-06-17 Created: 2009-06-17 Last updated: 2018-01-13Bibliographically approved
5. Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis
Open this publication in new window or tab >>Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis
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2010 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, p. 310-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.

Keywords
S. pneumoniae, H. influenzae, N. meningitidis, quantitative multiplex PCR, lower respiratory tract infection
National Category
Medical and Health Sciences
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:uu:diva-107967 (URN)10.1186/1471-2180-10-310 (DOI)000285888900001 ()21129171 (PubMedID)
Available from: 2009-09-02 Created: 2009-09-02 Last updated: 2017-12-13Bibliographically approved

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