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Antibody response to the patient's own Haemophilus influenzae isolate can support the aetiology in lower respiratory tract infections
Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
2004 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 112, no 4-5, 299-303 p.Article in journal (Refereed) Published
Abstract [en]

In order to understand the clinical importance of Haemophilus influenzae isolated from sputum samples, an indirect immunofluorescence (IF) assay was developed, using the patient's own isolate as the antigen. The method was tested on samples from six patients with lower respiratory tract infection (LRTI) and H. influenzae isolated from blood (n=2), sputum (n=3) or both (n=1), and on two healthy adults with H. influenzae isolated from the nasopharynx. Between acute and convalescent sera, a four-fold IgG antibody increase was achieved in five of six LRTI patients, including the three blood culture-positive patients. One LRTI patient and the two asymptomatic carriers showed stable antibody levels against their own isolate. Although small, the study indicates that indirect IF can be a promising tool for determining whether a H. influenzae strain represents the probable cause of infection or just a strain colonising the airways. More extensive studies should be performed in order to establish the usefulness of the assay.

Place, publisher, year, edition, pages
2004. Vol. 112, no 4-5, 299-303 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-24245DOI: 10.1111/j.1600-0463.2004.apm11204-0511.xLocal ID: 3846OAI: diva2:244562
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-26Bibliographically approved
In thesis
1. Diagnostic methods for bacterial etiology in adult community-acquired pneumonia
Open this publication in new window or tab >>Diagnostic methods for bacterial etiology in adult community-acquired pneumonia
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The etiologic agent is often unidentified in patients with community-acquired pneumonia (CAP). Development of new diagnostic methods has been encouraged. We aimed to develop a multiplex PCR (mPCR) assay for common bacterial pathogens and evaluate the diagnostic usefulness of this assay and of respiratory culture in CAP.

An mPCR was constructed for simultaneous identification of Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. Applied to 257 bacterial strains, the analytic sensitivity was 100% and the analytic specificity 99%.

In order to create appropriate reference standards, an indirect immunofluorescence test for H. influenzae was developed and two rapid urinary antigen tests for S. pneumoniae were evaluated. The indirect immunofluorescence test measured antibodies in paired sera against a H. influenzae strain isolated from the patient. A significant antibody rise was noted in 5/6 patients with lower respiratory tract infection (LRTI) and in 0/2 controls. The Binax NOW test and a serotype-specific latex agglutination test for 23 pneumococcal serotypes applied to urine samples, were positive in 24% and 7.4%, respectively, of 215 CAP patients tested. The Binax NOW test was false positive in 2/108 adult controls.

In a prospective study, 235 adult CAP patients and 113 controls were enroled. S. pneumoniae, H. influenzae, M. pneumoniae, and C. pneumoniae were considered definite etiologic agents in 17%, 11%, 5.5%, and 1.3% of the patients, respectively. When applied to sputum, nasopharyngeal aspirate (NpA), and/or nasopharyngeal swab (NpS), culture identified S. pneumoniae in 34% and H. influenzae in 23%, while mPCR identified S. pneumoniae in 48%, H. influenzae in 28%, M. pneumoniae in 12%, and C. pneumoniae in 1.3%. NpA and sputum yielded similar sensitivities and specificities and were generally more sensitive than NpS. In samples collected during antibiotic treatment, S. pneumoniae was identified by culture in 4.3% and by mPCR in 14% (P=0.004). Among the controls, NpS and/or NpA identified S. pneumoniae in 4.4% with culture and 8.0% with mPCR, H. influenzae in 2.7% with culture and 4.4% with mPCR, and M. pneumoniae in 0.88% with mPCR.

mPCR was also tested on bronchoalveolar lavage (BAL) samples from 156 adult patients with LRTI and 36 controls. BAL mPCR showed sensitivities of 86% for S. pneumoniae, 88% for H. influenzae, and 100% for M. pneumoniae, and specificities of 81% for S. pneumoniae, 64% for H. influenzae, 100% for M. pneumoniae, and 99% for C. pneumoniae. Among the controls, BAL mPCR identified S. pneumoniae in 11% and H. influenzae in 39%.

In conclusion, an mPCR for detection off our bacteria was developed. Both culture and mPCR applied to sputum, NpA, and NpS were useful for detection of etiologic agents in CAP patients. mPCR appears particularly useful in patients treated with antibiotics. It can also be useful in BAL samples. The urinary antigen tests can reliably establish pneumococcal pneumonia.

Abstract [sv]

Den mikrobiologiska orsaken till samhällsförvärvad lunginflammation kan ofta inte påvisas, varför utveckling av nya diagnostiska metoder har uppmuntrats. Vi avsåg att utveckla en multiplex PCR (mPCR) för vanliga luftvägsbakterier samt att utvärdera användningen av denna och luftvägsodling vid lunginflammation.

En mPCR metod konstruerades för samtidig identifiering av Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae och Chlamydophila pneumoniae. När metoden testades på 257 bakteriestammar var den analytiska sensitiviteten 100% och den analytiska specificiteten 99%.

För att få tillgång tilllämpliga referensmetoder utvecklade vi en indirekt immunafluorescens-metod för H. influenzae. Vi utvärderade också två snabbtest för påvisning av pneu-mokockantigen i urin. Med den indirekta immunafluorescensmetoden uppskattades antikroppslitrarna i parade sera mot en H. influenzae stam som isolerats från patienten. En signifikant antikroppsstegring påvisades hos 5/6 patienter med nedre luftvägsinfektion, men inte hos någon av två kontroller. Urintesterna Binax NOW och en seratyp-specifik latex agglutination test för 23 pneumokockserotyper var positiva hos 24% respektive 7.4% av 215 lunginflammationspatienter. Binax NOW var falskt positiv hos 2/108 kontrollpatienter.

I en prospektiv studie inkluderades 235 vuxna patienter med lunginflammation och 113 kontrollpatienter. S. pneumoniae, H. influenzae, M. pneumoniae och C. pneumoniae var säkra etiologiska agens hos 17%, 11%, 5.5% respektive 1.3%, av patienterna. Sputum, nasopharyngxaspirat (NpA), och nasopharynxpinne (NpS) analyserades med odling och mPCR. Odling påvisade S. pneumoniae hos 34% och H influenzae hos 23% samtidigt som mPCR påvisade S. pneumoniae hos 48%, H. influenzae hos 28%, M. pneumoniae hos 12% och C. pneumoniae hos 1.3%. NpA och sputum hade likvärdiga sensitivileter och specificiteter och var generellt känsligare än NpS. Bland prover tagna under antibiotikabehandling påvisades S. pneumoniae hos 4.3% med odling och hos 14% med mPCR (P=0.004). I kontrollgruppen identifierade NpA och/eller NpS S. pneumoniae hos 4.4% med odling och 8.0% med mPCR, H. influenzae hos 2.7% med odling och 4.4% med mPCR och M. pneumoniae hos 0.88% med mPCR.

mPCR testades också på bronkoalveolärt lavage (BAL) från 156 vuxna patienter med nedre luftvägsinfektion och 36 kontrollpatienter. Sensitiviteten av BAL mPCR var 86% för S. pneumoniae, 88% för H. injluenzae och 100% för M pneumoniae. Specificiteten var 81% för S. pneumoniae, 64% för H. influenzae, 100% för M. pneumoniae och 99% för C. pneumoniae. Bland kontrollpatienterna identifierade BAL mPCR S. pneumoniae hos 11% och H. influenzae hos 39%.

Sammanfattningsvis utvecklade vi en mPCR för detektion av fyra bakterier. Odling och mPCR av sputum, NpA och NpS är användbara för påvisning av etiologiska agens vid lunginflammation. mPCR verkar vara särskilt användbar hos patienter som behandlats med antibiotika före provtagning. Metoden kan också användas på BAL-prover. Urinantigentesterna kan användas för att diagnostisera pneumokockpneumoni.

Place, publisher, year, edition, pages
Linköping: Linköpings Universitet, 2005. 98 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 918
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-30047 (URN)15507 (Local ID)91-85299-32-4 (ISBN)15507 (Archive number)15507 (OAI)
Public defence
2005-11-18, Wilandersalen, Universitetssjukhuset, Örebro, 13:00 (Swedish)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-09-26Bibliographically approved

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