Novel multiplex isothermal nucleic acid amplification technologies for the detection of bacterial meningitis associated pathogens
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Infectious diseases, caused by pathogenic microorganisms such as bacteria, fungi or viruses, are a leading cause of significant global morbidity and mortality, with over 10 million associated deaths reported each year. Respiratory tract infections, such as bacterial meningitis, account for the highest rates of infectious disease related mortality. Bacterial meningitis infection is caused by human commensal bacteria that invade the respiratory tract and central nervous system leading to inflammation and rapid onset of symptoms, which can be fatal if untreated. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are the most common etiological agents of bacterial meningitis infection. S. pneumoniae and N. meningitidis are responsible for over 80% of all bacterial meningitis cases since the implementation of the H. influenzae type b (Hib) conjugate vaccine. However, H. influenzae related meningitis remains a serious threat in regions without immunisation and especially amongst children under five years of age. Global vaccination programmes have significantly reduced the occurrence of bacterial meningitis infection, however, approximately 1.2 million cases and over 100,000 related deaths are still reported annually, with increasing incidents of drug resistance leading to treatment failures. The global burden of bacterial meningitis infection is directly related to socioeconomic factors, with the highest rates of infection in low-resourced developing regions. The rate of infection in developed regions is approximately 1 in 100,000 persons per year, however, this can be as high as 20-40 in 100,000 persons per year in developing regions, with significantly higher mortality rates and severe impairments in survivors. The rapid and accurate detection of bacterial meningitis pathogens is essential for early and appropriate treatment which is directly linked with lower morbidity and mortality rates, and prevents broad-range antibiotic administration, a major contributing factor to antimicrobial-resistance dissemination. Isothermal nucleic acid amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) offer rapid, sensitive and specific diagnostic approaches for the identification of bacterial meningitis associated pathogens. Additionally, these methods are low-cost, easy-to-use and compatible with point-of-care (POC) diagnostic technologies, essential for near-patient testing in poorer disease-burdened regions. This thesis describes the development and evaluation of novel RPA and LAMP technologies for the detection of S. pneumoniae, N. meningitidis and H. influenzae. These technologies include: three internally controlled duplex RPA assays for the detection of each pathogen; a novel Tth endonuclease cleavage loop-mediated isothermal amplification (TEC-LAMP) assay for the internally controlled multiplex detection of these pathogens; and novel loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) technology for the singleplex or multiplex detection of each pathogen with flexible single-base specificity for effective single nucleotide polymorphism detection. All technologies demonstrated efficient analytical specificity when tested with extensive exclusivity and inclusivity bacterial reference strains, and low limits of detection using Probit regression analysis. Clinical testing of each technology, using samples from confirmed cases of bacterial meningitis infection, demonstrated efficient diagnostic specificity and sensitivity. This thesis advanced the current state-of-the-art in multiplex isothermal nucleic acid amplification detection of these major bacterial meningitis pathogens, providing novel transferable diagnostics technology for infectious disease POC testing.