DEVELOPING RAPID MOLECULAR TECHNIQUES FOR DIAGNOSIS OF NEISSERIA GONORRHOEAE AND CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE

Abstract

Multidrug-resistant Neisseria gonorrhoeae (Ng) is a global health emergency. To stop the spread of untreatable gonorrhea, improved molecular diagnostics and characterization of antimicrobial resistance (AMR) determinants are crucial. For this research, I developed an RT-PCR based molecular diagnostic test to identify Ng as well as a second test, utilizing novel multiplex primers for gyrA, to simultaneously identify Ng and predict ciprofloxacin susceptibility. Although ciprofloxacin is no longer recommended for the treatment of gonorrhea, susceptible isolates can be prevalent in some regions. Thus, if the ciprofloxacin susceptibility of an isolate is known, this antibiotic could be used for treatment. Both the Ng diagnostic test and the test for ciprofloxacin susceptibility performed with high sensitivity and specificity with DNA from cultures and gave more variable results with clinical specimens (i.e. urines and remnant Aptima urine specimens). These tests were modified for the incorporation of the primers into a hydrogel-based diagnostic platform, for eventual point-of-care applications. The hydrogel platform performed with high sensitivity and specificity for Ng diagnosis, depending on the specimen type. Additionally, to identify emerging AMR genotypes, various genes implicated in Ng AMR resistance were amplified and sequenced from Ng positive remnant Aptima urine specimens (for which AMR testing is not possible). Phenotype predictions based on DNA sequence analysis indicated emerging azithromycin resistance in Saskatchewan and highlighted the importance of molecular AMR surveillance to prevent Ng outbreaks. Finally, the properties of a β-lactamase produced by an uncharacterized Ng plasmid was investigated. A novel 6bp deletion in the start codon of blaTEM was identified. Due to this deletion, the β-lactamase was truncated and resulted in slow ampicillin hydrolysis coupled with a low penicillin MIC (0.125 mg/L). Overall, my research produced molecular diagnostic tests for Ng and ciprofloxacin susceptibility prediction and characterized and predicted emerging AMR resistance by molecular analysis.