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

  • Joanna Morfydd Diggle

    Student thesis: Master's Thesis

    Abstract

    Urinary tract infection (UTI) is an area of infectious disease which has long been neglected, despite
    the fact it affects ~50% of the female population worldwide and if left untreated/mis-diagnosed can
    cause life threatening illness, such as sepsis.

    Current methods of diagnosis, although known to be imperfect, have not been changed or improved
    in many years. However, with the emergence of antimicrobial resistance (AMR) and the focus being
    firmly placed on improving diagnostic pathways, this is set to change. There is a fundamental need
    to reduce the misuse and overuse of antibiotics, which is driving AMR and causing an increasing
    number of deaths worldwide.

    In June 2023, the World Health Organisation published their Global Research Agenda for AMR in
    Human Health. Listing 40 research topics which should be prioritised in the fight against AMR, some
    of which feed directly into the aim of this research.

    Aim - To develop and test a rapid molecular diagnostic test designed for near patient/point-of-care,
    for the detection of bacterial uropathogens direct from urine, using loop-mediated isothermal
    amplification (LAMP) technology. Then, to assess whether the same technology could be adapted to
    detect bacterial AMR genes, with the potential to direct treatment decisions and improve current
    methods of UTI diagnosis.

    Using the Llusern Scientific Lodestar DX analyser and LAMP bacterial ID tests, initial assessment was
    carried out for assays designed to detect 6 common uropathogens, selected using pathogen
    prevalence data from Cardiff, Wales in 2022. These were combined into a UTI test panel with ~87%
    coverage of all UTIs. Throughout testing, improvements were made to the sampling methodology
    and testing algorithm, prior to design freeze. A clinical performance evaluation was then carried out,
    comparing the Lodestar DX UTI test with standard laboratory methods (flow cytometry and culture)
    for a total of 199 urine samples from symptomatic adult females, suspected of experiencing acute,
    uncomplicated UTI.

    An overall sensitivity and specificity of 88.1% (95% CI: 77.8%– 94.7%) and 83.9% (95% CI: 72.3%–
    92.0%) respectively was achieved. Along with a positive predictive value (PPV) of 85.5% (95% CI:
    76.9%–91.3%), a negative predictive value (NPV) of 86.7% (95% CI: 77.1%–92.6%) and an overall
    accuracy of 86.1% (95% CI: 78.9%–91.5%). This data allowed the test to achieve UK CA accreditation
    and it is now commercially available in the UK as a result.

    Following this, Llusern Scientific provided LAMP assays designed to detect antibiotic resistance
    genes. The Lodestar DX platform was used to demonstrate proof-of-concept in successfully detecting
    these targets, with minor changes to the sampling methodology.

    The Lodestar DX platform and UTI test performs well in comparison with culture, for acute
    uncomplicated UTI in symptomatic adult females. However, there is much scope for future
    improvements, further development, and assessment of its true point-of-care compatibility. There is
    a need to assess its effectiveness in improving current prescribing methods and the question
    remains whether knowing the bacterial ID will have any effect on clinical decision making and
    whether a positive impact on patient outcomes is possible.
    Date of Award2025
    Original languageEnglish
    SponsorsPublic Health Wales & Llusern Scientific
    SupervisorJeroen Nieuwland (Supervisor) & Emma Hayhurst (Supervisor)

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