Assessing isothermal technology for detection of antimicrobial pathogens

Abstract

This thesis aims to provide solutions for antibiotic resistance by developing rapid point-of-care tests for key bacteria and associated resistances that have been flagged by the World Health Organization (WHO) and Centre for Disease prevention and Control (CDC). The studies initially focused on detection of New Delhi Metallo (NDM), and Verona Integrated Metallo (VIM) Beta Lactamase genes among carbapenemase-producing organisms Gram negative bacteria. The project then expanded to detect Neisseria gonorrhoeae (by targeting the gonococcal porA pseudogene) and associated resistances to ciprofloxacin (by the detection of a single nucleotide polymorphism [SNP] within the gonococcal gyrA gene) and ceftriaxone (by targeting the gonococcal penA 60.001 gene). The technology used for detection was iso-thermal recombinase polymerase amplification (RPA) and the detection was coupled with a lateral flow detection (LFD) system. Recognising the complexity for SNP detection by RPA (e.g., its ability to accommodate several mismatches in assay targets), this thesis explored several approaches to tackle this, including adding additional artificial mismatches in oligonucleotides, as well as using blocker primer and probe approaches. Assays performances compared well to traditional methods of detection (e.g., polymerase chain reaction; PCR), and time needed to perform/read results was less than thirty minutes compared to ninety minutes using PCR. Unlike PCR, RPA can be performed at a relatively low temperature, and assays can be successfully conducted by holding the reaction tubes in the palm of a hand. In addition to the wet-lab experiments, extensive in-silico sequence-based analyses were conducted to help further accelerate the development of point of care (POC) assays; these included improving our understanding of gonococcal gyrA diversity to inform target selection (using publicly available databases; National Centre for Biotechnology Information (NCBI), Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), Pathogen Watch) as well as to identify new potential N. gonorrhoeae diagnostic gene targets (noting that, for example, the gonococcal porA pseudogene can lead to false-negative results for certain N. gonorrhoeae strains). Overall, this thesis provides further potential POC solutions for antibiotic resistance; specifically, the ability to detect gram negative bacteria-harbouring blaNDM and blaVIM genes, and N. gonorrhoeae gyrA and penA 60.001 resistance targets. These advances will provide valuable information to help clinicians and healthcare teams to tailor antibiotic therapy in a timely manner, with limited skills and training needed to perform and read results.