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dc.contributor.advisorMurdoch, David
dc.contributor.advisorGeorge, Peter
dc.contributor.authorWerno, Anja Maria
dc.date.available2013-04-04T00:51:22Z
dc.date.copyright2013
dc.identifier.citationWerno, A. M. (2013). Development of improved diagnostic testing methods for invasive pneumococcal disease (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/3826en
dc.identifier.urihttp://hdl.handle.net/10523/3826
dc.description.abstractStreptococcus pneumoniae (the pneumococcus) is one of the most important bacterial pathogens and contributes extensively to global infectious disease morbidity and mortality, especially in children. To unequivocally identify S. pneumoniae as the causative pathogen of diseases like pneumonia, septicaemia or meningitis, has been a challenge ever since its first description in the late 19th century, reflecting, in part, short-comings of available diagnostic tests in terms of sensitivity and specificity. In recent history pneumococcal diagnostic advances have been slow and the urine antigen test and several molecular-based techniques appear to be the sole novel diagnostic approaches over the last three decades. The focus of this study was to explore new diagnostic avenues and evaluate their usefulness in the clinical setting. The thesis starts with a landscape analysis looking at the diagnostic methods and clinician’s ordering patterns over the last 11 years, followed by the evaluation of diagnostic assays. Three methods were included in the study, namely antigen detection, polymerase chain reaction (PCR) and mass spectrometry (MS). The targets chosen for the antigen detection assays, pneumolysin (Ply) and pneumococcal surface adhesin A (PsaA) were known to be immunogenic and present in most, if not all pneumococcal serotypes. The PCR target, autolysin (lytA), though not a novel PCR target was investigated in a quantitative assay as a potential marker for disease severity. Finally, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) MS, a technology that has been used in other areas, such as biochemistry for a few decades, has only in the last decade made advances into routine microbiological diagnostics, was explored as a tool to differentiate S. pneumoniae from related non-pneumococcal viridians streptococci. Both antigen detection assays were developed successfully. However, the Ply-assay revealed a problem with the non-specificity of the polyclonal antibody in serum specimens and was therefore not evaluated on clinical specimens. The PsaA-assay was applied to a selection of well characterised patient sera, and was showed to be specific, but needs to be of greater sensitivity to be clinically useful. The quantitative lytA PCR assay did demonstrate correlation of bacterial load and severity of disease and might be a useful adjunct in the clinical management of patients. MALDI-TOF MS is a novel tool applied to microbiological bacterial and fungal identification which needs further development in the area of typing and species level identification of certain bacteria, such as the viridians streptococci. This study demonstrated that species differentiation of S. pneumoniae from non-pneumococcal streptococci of the S. mitis group might be possible by this method. In summary, this study describes the investigation of novel diagnostic approaches which aim to provide solutions to three main ongoing challenges in the diagnosis of pneumococcal disease: (i) to increase the sensitivity of assays to detect S. pneumoniae, (ii) to differentiate between colonisation and invasive disease and (iii) to ensure more reliable identification of pneumococcal and non-pneumococcal viridians streptococci at the species level. The findings of this thesis have allowed an in depth assessment of the current state of pneumococcal diagnostics and have allowed the realistic assessment of the implementation of technologies such as antigen detection assays, quantitative PCR and mass spectrometry in this area; the latter two almost certainly will become more firmly established in the diagnosis of pneumococcal disease in future.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectStreptococcus pneumoniae
dc.subjectMALDI-TOF MS
dc.subjectQuantitative PCR
dc.subjectdiagnostic methods
dc.subjectantigen detection
dc.titleDevelopment of improved diagnostic testing methods for invasive pneumococcal disease
dc.typeThesis
dc.date.updated2013-04-03T21:20:43Z
dc.language.rfc3066en
thesis.degree.disciplineDepartment of Pathology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
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