Objectives: XDR Neisseria gonorrhoeae imposes the threat of untreatable gonorrhoea. Gentamicin is considered for future treatment; however, no interpretation criteria for the CLSI and calibrated dichotomous sensitivity (CDS) disc diffusion (DD) techniques are available for N. gonorrhoeae. We investigated the in vitro gentamicin activity by MIC and DD methods, proposed DD breakpoints and determined DD ranges for 10 international quality control (QC) strains. Methods: Gentamicin susceptibility of

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... 333 N. gonorrhoeae isolates, including 323 clinical isolates and 10 QC strains, was determined. MIC determination (Etest) and DD methods (CLSI and CDS) were performed. The relationship between MIC, inhibition zone diameter and annular radius was determined by linear regression analysis and the correlation coefficient (r) was calculated. Results: Gentamicin MICs for the QC strains were within published ranges. Of the 323 clinical isolates, according to published breakpoints 75.9%, 23.5% and 0.6% were susceptible, intermediately susceptible and resistant, respectively. Based on error minimization with MICs of ≤4, 8 -16 and ≥32 mg/L, breakpoints proposed are susceptible ≥16 mm, intermediately susceptible 13 -15 mm and resistant ≤12 mm for the CLSI method and susceptible ≥6 mm, less susceptible 3 -5 mm and resistant ≤2 mm for the CDS technique. Conclusions: Low resistance to gentamicin was identified and gentamicin might be a future treatment option for gonorrhoea. Tentative gentamicin zone breakpoints were defined for two DD methods and QC ranges for 10 international reference strains were established. Our findings suggest that in resource-poor settings where MIC testing is not a feasible option, the DD methods can be used to indicate gentamicin resistance.