Microbiological analysis and whole-genome sequencing of Neisseria gonorrhoeae from the microbiological failures in the international, zoliflodacin, phase 3, clinical trial for treatment of uncomplicated urogenital gonorrhoea: a retrospective, genomic, observational study.
BACKGROUND: Zoliflodacin, a first-in-class oral bacterial, DNA gyrase (GyrB) inhibitor, showed non-inferiority to ceftriaxone combined with azithromycin in a recent large international, phase 3, randomised controlled trial for treatment of uncomplicated urogenital gonorrhoea. The aim of this study was to describe the microbiological and whole-genome sequencing (WGS) analyses of paired baseline (pre-treatment) and test-of-cure (TOC) gonococcal isolates from the zoliflodacin phase 3, randomised controlled trial to further characterise and evaluate the protocol-specified microbiological failures with zoliflodacin (n=22) or ceftriaxone and azithromycin (n=1). METHODS: In this retrospective, genomic, observational study, results from antimicrobial susceptibility testing (agar dilution method) of isolates (n=960; 936 baseline isolates from 763 participants and 24 TOC isolates [23 with a paired baseline isolate in the same anatomical site] from 20 participants) collected during the zoliflodacin phase 3, randomised controlled trial done in 16 outpatient clinics in Belgium, the Netherlands, South Africa, Thailand, and the USA (Nov 6, 2019-March 16, 2023) are described. WGS analysis was performed on paired baseline and TOC isolates from participants with microbiological failures (zoliflodacin 44 isolates [19 participants]; ceftriaxone and azithromycin two isolates [one participant]), and the three baseline isolates with highest zoliflodacin minimum inhibitory concentration (MIC 0·5 mg/L). FINDINGS: All isolates were inhibited by the same zoliflodacin concentrations (MICs ≤0·008 to 0·5 mg/L) as wild-type strains cultured internationally in 2013-23. In participants with a microbiological failure after zoliflodacin treatment (n=22, 19 participants), zoliflodacin MIC values for baseline and TOC isolates were similar, and resistance selection was lacking. WGS showed that five (23%) of 22 infections (95% CI 10-43 [in four participants]) of zoliflodacin microbiological failures had different strains at TOC versus baseline. In 17 zoliflodacin microbiological failures (15 participants), isolates at baseline and TOC were indistinguishable. 13 of these 17 microbiological failures, corresponding to 59% (95% CI 39-77; 13 of 22) of all zoliflodacin microbiological failures, were in urogenital or rectal sites in 11 participants and the isolates had zoliflodacin MICs less than or equal to 0·008 to 0·25 mg/L. The single microbiological failure after ceftriaxone and azithromycin treatment had different strains at TOC versus at baseline. No sequenced isolates had mutations associated with elevated zoliflodacin MICs. INTERPRETATION: In the zoliflodacin phase 3, randomised controlled trial, 23% of the zoliflodacin microbiological failures and the single ceftriaxone and azithromycin microbiological failure had different gonococcal strains at TOC versus baseline, which suggests reinfections and not treatment failures. In addition, 59% of the zoliflodacin microbiological failures, all in anogenital sites, had no obvious microbiological explanation based on the low zoliflodacin MICs, previous pharmacodynamic studies, and no evidence of resistance selection after zoliflodacin therapy. A reinfection as the cause for these microbiological failures could not be excluded. We recommend that WGS is implemented in future randomised controlled trials for gonorrhoea treatment to further evaluate possible microbiological failures, exclude reinfections (to avoid underestimating the cure rates), and characterise antimicrobial resistance determinants. FUNDING: GARDP through grants from Germany BMFTR (03KA1831), UK DHSC as part of GAMRIF, Japan MHLW, the Netherlands' Ministry of Health, Welfare and Sport and Directorate-General for International Cooperation, the Federal Office of Public Health of Switzerland, the Canton of Geneva, Switzerland, and Örebro University Hospital, Sweden.