Exploring diagnostic approaches and quality assurance data for improved management of gonorrhoea antimicrobial resistance

Abstract

Neisseria gonorrhoeae (NG), the etiological agent of gonorrhoea, accounts for 82 million cases among the 374 million new sexually transmitted infections recorded in 2020. NG is associated with high rates of antimicrobial resistance (AMR), with the World Health Organization designating it as an urgent AMR threat. There are several important factors when it comes to the ideal management of NG. One important approach to NG management entails enhancing AMR surveillance capabilities. Another involves applying accurate and specific diagnostics to detect both NG and related AMR genes/mutations directly from clinical samples, which can then guide treatment and/or inform surveillance. To effectively enhance surveillance of NG and associated AMR, it is crucial to comprehensively understand the factors that influence NG’s infection rates, distribution, and prevalence throughout geographic regions. Such knowledge contributes to effective public health interventions. Among the various public health concerns relevant to NG, COVID-19 has been a significant recent event to indirectly impact NG prevalence and transmission. Consequently, this thesis included an examination of the impact of COVID-19-related public health measures on the prevalence of NG and the distribution of NG genotypes among the population of Queensland (QLD) during the first half of 2020 (Chapter 2). The results of this chapter showed that there is a decrease in NG genotypic diversity post COVID-19 in 2020 in QLD, and the proportion of the isolates carrying an azithromycin AMR specific determinant have almost doubled post COVID-19. In Australia, NG treatment guidelines in most settings indicate 500 mg of ceftriaxone intramuscularly and 1 g of azithromycin orally to be the standard of care. However, the increase in azithromycin resistance has prompted a re-evaluation of this therapy regimen elsewhere, with 1 g of azithromycin no longer being recommended as a first-line therapy in many countries worldwide. This shift emphasises the importance of further understanding the spread of azithromycin resistance in Australia to inform its use as a first-line therapy. To do so, complementing culture-based surveillance by directly applying molecular AMR tests on clinical samples is required. As NG is known to constantly evolve, target testing variations may arise. It is therefore important to ascertain which genes and mutations are most prevalent and informative of azithromycin AMR. Accordingly, as part of this study (Chapter 3), the Pathogen-Watch online genomic database was explored to determine the prevalence of mutations known to be associated with azithromycin resistance within the global collection of NG isolate sequences. These outcomes then informed which diagnostic targets are most relevant when it comes to molecular assay targets; the meningococcal-mtrR and 23S-rRNA were found most significant. Following the above, real-time polymerase chain reaction (PCR) assays were developed and applied on NG-nucleic acid amplification test (NAAT)-positive clinical samples to enhance culture-based surveillance and better elucidate the distribution and prevalence of azithromycin resistance in QLD (Chapter 4). The results of this chapter indicated that there is a reduction in 23S rRNA determinants, while an increase the prevalence of meningococcal-mtrR harbouring strains (reinforcing Chapter 2 isolate data post COVID-19) which could put azithromycin treatment at risk in QLD. Molecular assay design complexities arose due to cross-reactivity with commensal Neisseria species, particularly for pharyngeal clinical samples. As routine molecular NG-AMR testing is on the horizon, I explored potential testing bias that may arise if patient pharyngeal clinical samples are excluded from the testing algorithm (Chapter 5). The results of this chapter suggested that excluding pharyngeal samples from N. gonorrhoeae AMR molecular testing in QLD will be of minimal impact with a loss of 13.17% of samples with the majority of infections appearing in two or more anatomical sites, therefore mostly accounted for. Finally, rapid and cost-effective diagnosis of both NG and AMR represents an important measure for controlling and managing gonococcal infection. Diagnostic tools such as PCR and culture require access to laboratory facilities and are associated with high costs and expertise, unfeasible in resource-poor settings with syndromic patient management. Therefore, alternative cost-effective diagnostic tools are urgently required. To further explore the latter as well as attempt to resolve commensal Neisseria cross-reactivity, I investigated the utility and feasibility of near-infrared spectroscopy (NIRS), for the identification and differentiation of NG from commensal Neisseria and detection of AMR (Chapter 6). This proof-of-concept study demonstrated the capability of the NIRS to distinguish N. gonorrhoeae from Neisseria commensals with accuracy of 98% for N. gonorrhoeae and 96% for commensals. Further, N. gonorrhoeae fully-susceptible strains were distinguished from resistant strains with an accuracy of 86% and 90%, respectively.

The data from this work reinforces how molecular methods enable to enhance culture-based surveillance in QLD to capture less represented regions and populations. Importantly, this data demonstrated that azithromycin use as a blanket therapy for NG in QLD may be undermined due to a significant increase of AMR determinants in local NG. The exclusion of pharyngeal samples from molecular AMR testing is unlikely to affect the representativeness of AMR in our population, yet this needs to be carefully monitored. The exploration of NIRS technology as an alternative test for NG and AMR detection is feasible, yet further validations are required. This body of work represents better understanding of the spread of NG-AMR in QLD and further informing potential gaps when rolling out molecular AMR testing in our population. Finally, paving the way for the development of alternative, rapid and cost-effective diagnostics to be further explored.