Abstract
Legionnaires’ disease (LD) is a severe form of community-acquired pneumonia (CAP) caused by Legionella bacteria. Since its discovery in 1976, over 50 distinct species and more than 70 serogroups have been discovered, with at least half determined to be pathogenic to humans. In New Zealand, Legionella longbeachae and Legionella pneumophila are the main causative agents of LD. The clinical and radiological features of LD resemble those of CAP caused by non-Legionella pathogens. As such, the current gold standard for a definitive LD diagnosis requires either culture or quantitative polymerase chain reaction (qPCR) of respiratory samples such as sputum. Unfortunately, many patients are unable to produce sputum samples due to a dry cough or confusion; therefore, induced sputum samples are often required for diagnosis, which is unpleasant and invasive. Existing non-invasive urinary antigen tests (UAT) are limited to detecting L. pneumophila serogroup 1 leading to the underreporting of all other Legionella species. This underreporting is critical for countries like New Zealand, where L. longbeachae-associated pneumonia is more common than L. pneumophila, rendering commercial UATs unsuitable as the sole non-invasive diagnostic method. Currently, empiric antibiotics are often administered to patients admitted with pneumonia until a definitive LD diagnosis is made. If LD is diagnosed or suspected, then macrolide and fluoroquinolone antibiotics are initiated. If LD diagnosis is delayed the use of incorrect anti-microbial therapy can promote antimicrobial resistance, increase adverse side effects for patients and further burden the healthcare system. Therefore, the development of a sensitive and rapid test that is non-invasive and specific for all Legionella species is paramount to enhance diagnosis and improve patient outcomes.
This thesis investigates the development and efficiency of a novel, non-invasive diagnostic test for LD using magnetic hybridisation capture of Legionella cell-free DNA in urine coupled with qPCR. The primary aim of this work was proof of principle for the use of this hybridisation assay for the detection of Legionella cfDNA by evaluating its baseline sensitivity and specificity with non-fragmented Legionella DNA.
A total of nine previously published primer pairs were tested for Legionella to assess qPCR efficiency. Specificity against seven Legionella spp and four non-Legionella bacteria were tested and the L5S, Pan-Leg and LS/PQ primer pairs were found to be suitable to detect all Legionella spp. The Pan-Leg primer set reported the best specificity alongside well-defined melt points. While both L5S and LS/PQ primers reported poorer specificity, the LS/PQ primer set was tested further since the observed melt peaks could distinguish between bacterial species. Unfortunately, the L5S primer set reported the same melt peak for the negative template control (NTC), Legionella and non-Legionella species; hence, they were excluded from further use. Further optimisation of the LS/PQ and Pan-Leg primer sets indicated that the Pan-Leg primers were superior and would be used to design the probes for the hybridisation assay.
Sequence-specific probes (BP1 and BP2) for the Pan-Leg primers were designed with a poly-A tail and 3-carbon spacer (C3) modification. These biotinylated probes were successfully coupled to streptavidin-coated magnetic beads. Urine samples were spiked with a known number of theoretical unfragmented DNA copies, extracted through the hybridisation assay and subsequently amplified on the qPCR program. The results revealed that the hybridisation assay recovered less than 1% of spiked unfragmented DNA, which was significantly lower than the recovery rate observed by Oreskovic et al. However, the number of DNA copies extracted was still significantly higher than from the commercially available Quick-DNATM Urine Kit, which also recovered less than 1%.
These findings suggest that the hybridisation assay could be refined to enhance its limit of detection, sensitivity, and specificity. Future research should focus on testing and validating the assay using short DNA fragments, by optimising the Pan-Leg primers and qPCR protocol to improve detection limits and by standardising the hybridisation assay to minimise bead aggregation and variability through automation. In conclusion, this study provides a foundation for developing a sensitive and specific non-invasive diagnostic tool for LD using urine samples. While further optimisation and validation are required, the hybridisation capture assay coupled with qPCR holds promise as a rapid and efficient diagnostic method for detecting Legionella infections, potentially improving patient outcomes and public health surveillance.