A Point of Care Antimicrobial Resistance test for Neisseria gonorrhoeae and Mycoplasma genitalium infection. Ensuring accurate therapy and antibiotic stewardship in sexual health medicine.

Background. Antimicrobial resistance (AMR) is undermining treatment of sexually transmitted infections (STIs), by limiting capacity to give empirical therapy. Despite widespread AMR within N. gonorrhoeae and M. genitalium, most strains remain susceptible to cheap and easy to use oral antibiotics such as ciprofloxacin and azithromycin respectively, and genetic determinants of AMR for these drugs correlate closely with phenotypic susceptibility. Aims. We will develop the following rapid, point of care (PoC), AMR tests contingent on feedback from patients, the public, healthcare workers and the market: 1.a) Single disposable cartridge assays on Atlas io™ (Aio™) PoC system for detecting mutations for N. gonorrhoeae AMR to ciprofloxacin or for M. genitalium to azithromycin, within 30 minutes of unprocessed sample collection for use on genital or extra-genital swab samples, following a positive result for infection, such as Gram stain microscopy or laboratory tests; b) Combining PoC diagnostic assays with genotypic AMR detection, as above, with antimicrobial susceptibility reported if the organism is detected. We will explore the possibility of integrating Chlamydia trachomatis and Trichomonas vaginalis infections on to AMR tests, as the diagnostic assays for these infections have already been developed on the Aio™. AioTM currently have two PCR amplification chambers (AC), two detection chambers (DC) per AC and four electrochemical label channels per DC. Two mismatched probes (for wild-type and mutant), give capacity to include gene targets for diagnosis, internal controls and up to five SNPs of interest. Plan. Work will be divided into two workstreams, at SGUL. We will conduct a literature review confirming validity of chosen gyrA and 23sRNA mutations conferring AMR in N. gonorrhoeae and M. genitalium respectively, as well as market research and qualitative surveys with patients and the public to obtain data on acceptability and preferences for combined or single diagnostic & resistance assays in the context of clinical care pathways. Molecular assay design will be followed by N. gonorrhoeae & M. genitalium AMR gene targets transfer together with diagnostic targets for C. trachomatis and T. vaginalis, onto an AIo™ prototype, fully representative of the AIo™ system but allowing more rapid assay development. We will test the prototype, initially with samples spiked with DNA from drug resistant and susceptible N. gonorrhoeae and M. genitalium and then with clinical samples positive for N. gonorrhoea, M. genitalium, C. trachomatis and T. vaginalis, fully characterized for AMR by genotyping and sequencing. Most N. gonorrhoeae positive samples will have AMR status confirmed phenotypically. Being difficult to culture, it is estimated that 50% of M. genitalium samples will be phenotypically confirmed. Assays will then be transferred to an integrated and dedicated production AIo™ cartridge system (pre in-vitro diagnostic; IVD), tested on clinical samples to set signal thresholds and finally evaluated by comparing performance to laboratory assays of genotype, and phenotype. Data from this evaluation will be analysed to assess potential clinical impact of PoC AIo™ if used in clinic for resistance prediction and rational antibiotic prescribing. The entire work will be enabled by the prospective collection of 2000 clinical samples from urethritis/cervicitis and pelvic inflammatory disease patients and controls from sexual health clinics, in two phases in the project, under existing ethics and R&D protocols, specifically approved for undertaking novel diagnostic evaluations. These will yield the required numbers of positive samples for all four organisms to perform the work. Following this project, IVD(s) will be produced and evaluated by Atlas and SGUL for CE marking and further funding sought to determine the clinical and public health impacts of placing these PoC-AMR tests in clinical care p