Development and Use of Loop-Mediated Isothermal Amplification (LAMP) Assays to Detect the Presence of Pathogens Which Cause Urinary Tract Infection and Genes Which Cause Resistance to 3rd Generation Cephalosporins

Urinary tract infection (UTI) is an area of infectious disease which has long been neglected, despite <br/>the fact it affects ~50% of the female population worldwide and if left untreated/mis-diagnosed can <br/>cause life threatening illness, such as sepsis. <br/><br/>Current methods of diagnosis, although known to be imperfect, have not been changed or improved<br/>in many years. However, with the emergence of antimicrobial resistance (AMR) and the focus being <br/>firmly placed on improving diagnostic pathways, this is set to change. There is a fundamental need <br/>to reduce the misuse and overuse of antibiotics, which is driving AMR and causing an increasing <br/>number of deaths worldwide.<br/><br/>In June 2023, the World Health Organisation published their Global Research Agenda for AMR in <br/>Human Health. Listing 40 research topics which should be prioritised in the fight against AMR, some <br/>of which feed directly into the aim of this research.<br/><br/>Aim - To develop and test a rapid molecular diagnostic test designed for near patient/point-of-care, <br/>for the detection of bacterial uropathogens direct from urine, using loop-mediated isothermal <br/>amplification (LAMP) technology. Then, to assess whether the same technology could be adapted to <br/>detect bacterial AMR genes, with the potential to direct treatment decisions and improve current <br/>methods of UTI diagnosis.<br/><br/>Using the Llusern Scientific Lodestar DX analyser and LAMP bacterial ID tests, initial assessment was<br/>carried out for assays designed to detect 6 common uropathogens, selected using pathogen <br/>prevalence data from Cardiff, Wales in 2022. These were combined into a UTI test panel with ~87% <br/>coverage of all UTIs. Throughout testing, improvements were made to the sampling methodology<br/>and testing algorithm, prior to design freeze. A clinical performance evaluation was then carried out, <br/>comparing the Lodestar DX UTI test with standard laboratory methods (flow cytometry and culture)<br/>for a total of 199 urine samples from symptomatic adult females, suspected of experiencing acute, <br/>uncomplicated UTI.<br/><br/>An overall sensitivity and specificity of 88.1% (95% CI: 77.8%– 94.7%) and 83.9% (95% CI: 72.3%–<br/>92.0%) respectively was achieved. Along with a positive predictive value (PPV) of 85.5% (95% CI: <br/>76.9%–91.3%), a negative predictive value (NPV) of 86.7% (95% CI: 77.1%–92.6%) and an overall <br/>accuracy of 86.1% (95% CI: 78.9%–91.5%). This data allowed the test to achieve UK CA accreditation <br/>and it is now commercially available in the UK as a result.<br/><br/>Following this, Llusern Scientific provided LAMP assays designed to detect antibiotic resistance <br/>genes. The Lodestar DX platform was used to demonstrate proof-of-concept in successfully detecting <br/>these targets, with minor changes to the sampling methodology.<br/><br/>The Lodestar DX platform and UTI test performs well in comparison with culture, for acute <br/>uncomplicated UTI in symptomatic adult females. However, there is much scope for future <br/>improvements, further development, and assessment of its true point-of-care compatibility. There is<br/>a need to assess its effectiveness in improving current prescribing methods and the question <br/>remains whether knowing the bacterial ID will have any effect on clinical decision making and<br/>whether a positive impact on patient outcomes is possible.

Name dc:type.qualificationname

Master's Thesis

Level dc:type.qualificationlevel

Student thesis

Year dc:date.issued

2025

Author dc:creator

• Diggle, Joanna Morfydd

Advisor dc:contributor.advisor

• Nieuwland, Jeroen

Language dc:language

eng

Identifier

oai:pure.atira.dk:studenttheses/15338b5a-4ccd-462e-ba09-e8d730be85e1

OAI identifier oai:identifier

oai:pure.atira.dk:studenttheses/15338b5a-4ccd-462e-ba09-e8d730be85e1