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With an estimated 1.2 million cases annually worldwide, bacterial meningitis is a medical emergency and primary prevention as well as accurate diagnosis and treatment of this often fatal disease is paramount. The most common microorganisms associated with bacterial meningitis are Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. Several vaccines are available for the prevention of bacterial meningitis caused by these three microorganisms; however, these vaccines offer limited serotype coverage and as a consequence bacterial meningitis cases as a result of non-vaccine serotypes have emerged. Furthermore, limited vaccine availability in resource poor countries in addition to vaccine failures is a major concern in the prevention of bacterial meningitis worldwide. As such, patients with suspected meningitis require immediate medical assessment and accurate diagnosis of the disease in order to provide an optimal therapeutic regime. Moreover, as the incidence of bacterial meningitis varies significantly by age and geographic location, the accurate diagnosis of the causative microorganism would enable unambiguous epidemiological studies to be carried out. Presently, culture remains the gold standard for the diagnosis of bacterial meningitis, however, this is limited in its ability to rapidly and accurately diagnose and nucleic acid based diagnostic methods are more sensitive and specific for the diagnosis of this fatal disease. The overall aim of this study was to design, develop, optimise and validate robust, internally controlled nucleic acid based in-vitro amplification methods for the rapid and accurate identification of H. influenzae, N. meningitidis and S. pneumoniae. This was achieved using sequential experimental design consisting of three main studies. In the first study, RNA transcripts encoded for by the ssrA (tmRNA) and lepA (lepA mRNA) genes were evaluated using real-time Nucleic Acid Sequence Based Amplification (NASBA) as potential diagnostic targets for species specific identification of H. influenzae, N. meningitidis and S. pneumoniae. This established that tmRNA and lepA mRNA have potential as diagnostic targets for the species specific identification of N. meningitidis and S. pneumoniae respectively. However, neither the tmRNA transcript nor the lepA mRNA transcript can be used as a diagnostic target to unequivocally differentiate H. influenzae from its most closely related species Haemophilus haemolyticus. In the second study, whole genome comparative analysis of H. influenzae and H. haemolyticus identified two novel gene targets, phoB and pstA, present in H. influenzae and absent in all other closely related species including H. haemoylticus. Subsequently, two internally controlled real-time PCR diagnostic assays were developed targeting both phoB and pstA and validated against an extensive panel of H. influenzae isolates and non-H. influenzae closely related species for the specific identification of H. influenzae. Analytical sensitivities of the real-time PCR diagnostic assays developed were determined to be comparable to other published diagnostic targets and more superior in terms of specificity than a previously published diagnostics targets for the identification of H. influenzae. In the third study, phoB was further validated as a diagnostic target for the species specific detection of H. influenzae by targeting the phoB mRNA transcript in a duplex real-time NASBA diagnostic assay. In addition, two duplex real-time NASBA diagnostic assays were also developed targeting the RNA transcripts encoded for by the ssrA gene and lepA gene for the species specific identification of N. meningitidis and S. pneumoniae, respectively. All three duplex real-time NASBA diagnostic assays were determined to be 100% specific for the target species tested for and analytical sensitivities of less than 60 cell equivalents were determined for each of the diagnostic assays in duplex format. Each real-time NASBA diagnostic assay developed in this study includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify transcript 1 of the Homo Sapiens TBP gene from human total RNA. By incorporating an endogenous internal amplification control stably expressed at low levels in human blood, these duplex real-time NASBA diagnostic assays have the potential to be used in a clinical setting for the specific, sensitive and rapid (< 60 mins) detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. In summary, nucleic acid based in-vitro amplification diagnostic assays described in this study are the first description of internally controlled real-time PCR diagnostic assays and real-time NASBA diagnostic assay for the accurate, species specific identification of H. influenzae, N. meningitidis and S. pneumoniae.

Journal article We present a disc-based module for rotationally controlled solid-phase purification of RNA from cell lysate. To this end, multi-stage routing of a sequence of aqueous and organic liquids into designated waste and elution reservoirs is implemented by a network of strategically placed, solvent-selective composite valves . Using a bead-based stationary phase at the entrance of the router, we show that total RNA is purified with high integrity from cultured MCF7 and T47D cell lines, human leucocytes and Haemophilus influenzae cell lysates. Furthermore, we demonstrate the broad applicability of the device through the in vitro amplification of RNA purified on-disc using RT-PCR and NASBA. Our novel router will be at the pivot of a forthcoming, fully integrated and automated sample preparation system for RNA-based analysis Science Foundation Ireland - Grant 10/CE/B1821 peer-reviewed