Molecular epidemiology and antimicrobial resistance determinants of Neisseria gonorrhoeae among males with urethritis in Kampala, Uganda

Abstract

Gonorrhoea is a highly prevalent bacterial sexually transmitted infection (STI) that negatively impacts sexual and reproductive health (SRH) and that is also greatly associated with serious clinical sequelae. Gonorrhoea is mostly associated with an acute urogenital infection in males and sub-acute or acute urogenital infection in females. Although primarily an infection of the genitals, extragenital infection is increasingly common affecting the rectum and pharynx, especially among men who have sex with men (MSM). Antibiotics are the mainstay for treatment of gonorrhoea, with good treatment outcomes. However, the emergence of antimicrobial resistance (AMR) is threatening our ability to cure gonorrhoea optimally. In addition to the rapidly increasing burden of gonorrhoea and associated AMR is the extremely low capacity for surveillance especially in low-and-middle income countries (LMICs). Lack of quality-assured data, especially on the circulating Neisseria gonorrhoeae (NG) strains in the population limits the ability to predict infection transmission, identify novel targets for antimicrobial therapy and precise information towards vaccines development. We conducted a literature review of the burden of gonococcal AMR in Africa and capacities of the surveillance programs to address objective one of this thesis. In objective two, we set up and evaluated an NG AMR surveillance program in Kampala, Uganda. In objectives three and four, we used isolates obtained from objective two to identify the molecular AMR determinants and other genomic characteristics of gonococcal strains circulating in Kampala, Uganda. We compared the identified NG strains to NG strains isolated in four other African countries. The narrative review conducted in objective one of this thesis showed gaps in NG AMR surveillance systems in Africa, including sub-optimal data collection and reporting systems, lack of use of standardised surveillance and microbiology laboratory protocols. In objective two we demonstrated the feasibility of rolling out a quality assured NG surveillance program in Kampala, Uganda, collecting an adequate number of isolates for reporting to World Health Organization (WHO) Global AMR and USE Surveillance System (GLASS). We also collected 433 NG isolates, which is notably higher than what has been reported in the literature from other NG surveillance programs in Africa. The antimicrobial susceptibility testing (AST) data generated from the surveillance program in Kampala is consistent with other African reports indicating very high susceptibility to extended spectrum cephalosporins (ESCs) particularly ceftriaxone and cefixime and very high resistance to ciprofloxacin. Of concern in our study findings was the emerging resistance to Azithromycin observed in one isolate. In objective three, no NG isolates had any mosaic penA allele and penicillin-binding protein 2 (PBP2) A501V/T substitutions were very scarce at only 0.2%. Furthermore, no gonococcal isolates harboured any other potential ESCs resistance associated mutations validating the susceptibility to ceftriaxone and cefixime. 23S rRNA gene mutations and mosaic MtrD were were found at a prevalence of 0.1% and 0% respectively, with only one isolate having a 23S rRNA gene C2611T mutation. All isolates analyzed were ciprofloxacin-resistant with presence of both the GyrA S91F and GyrA D95A/G/N substitutions. In objective four, we found two distinctive gonococcal lineages with different genotypic profiles circulating in Kampala, Uganda. The two identified lineages were still highly susceptible to ceftriaxone, cefixime and azithromycin, which is a significant contrast with findings reported from other continents. In addition, we observed a very high level of diversity with many novel sequence types (STs) in the Ugandan gonococcal population. Multi-Locus Sequence Types (MLSTs) ST 1893, 1903, 11366 and 1599 were the most prevalent among the 433 gonococcal isolates. In conclusion, findings of chapter four provides further evidence for the need of strengthening AMR surveillance programs on the continent to ensure quality-assured collection and analysis of NG AMR data. In the different results chapter, we exhibited the practicality of establishing a standardized NG AMR surveillance system including the generation of phenotypic data. The summary AST results were 100% susceptibility to ESCs and 100% resistance to ciprofloxacin. The genotypic profiles elucidated in the findings validated the reported phenotypic AST results reported in the study. Finally, the strain typing (MLST and NG-STAR) and phylogenetic analysis shown the evolutionary trends of NG isolates in Uganda including the AMR determinants they carry, lineages and associated characteristics (including meta-data).