Associations between antimicrobial susceptibility/resistance of Neisseria gonorrhoeae isolates in European Union/European Economic Area and patients’ gender, sexual orientation and anatomical site of infection, 2009–2016

Euro-GASP has been previously described in detail [6, 7, 27‐29, 47‐51]. Briefly, participating EU/EEA countries submit a sample of consecutive N. gonorrhoeae isolates (ideally 100–200 isolates dependent on the number of gonorrhoea cases reported annually in the country) from their routine diagnostics for centralised antimicrobial susceptibility testing (AST) or, if countries have fulfilled set quality assurance criteria, they perform decentralised AST in their own countries. Countries include only one isolate per patient from those who were infected multiple times within a 4-week period or at several anatomical sites, to represent different gonorrhoea episodes. When multiple isolates per patient and gonorrhoea episode, priority is given to test extragenital isolates, particularly pharyngeal ones [48]. For decentralised AST, countries should have performed consistently well in the annual mandatory Euro-GASP external quality assessment (EQA) [50] and have had a good comparability between the laboratories own national or regional AST data and AST data generated by centralised testing [48]. During 2009–2016, the number of Euro-GASP countries increased from 17 to 23 and the number of decentralised Euro-GASP countries increased from 12 to 17. Centralised AST is currently conducted using minimum inhibitory concentration (MIC; mg/L) gradient strip tests for ceftriaxone, cefixime, and azithromycin, and MIC gradient strip test or agar dilution breakpoint method, which does not provide MIC and only susceptibility category (using agar plates with concentrations of 0.03 mg/L and 0.06 mg/L), for ciprofloxacin. For quality control of the AST in Euro-GASP [48], the 2016 WHO N. gonorrhoeae reference strains G, K, M, P and O (only when spectinomycin is tested) [51] are used. Furthermore, all centralised and decentralised Euro-GASP countries participate in the mandatory annual Euro-GASP EQA [50]. The MICs of each antimicrobial are interpreted into resistant (R); susceptible, increased exposure (I; formerly intermediate susceptibility); or susceptible (S) using current clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.​eucast.​org/​clinical_​breakpoints/​), i.e. ceftriaxone and cefixime (S ≤ 0.125 mg/L, R > 0.125 mg/L), and ciprofloxacin (S ≤ 0.032 mg/L, R > 0.064 mg/L). In 2019, EUCAST excluded their clinical breakpoints for azithromycin. However, because agar dilution breakpoint method was previously used by many Euro-GASP countries also for azithromycin and accordingly no MICs of azithromycin are available, in the present study, we had to use the previous clinical azithromycin breakpoints (S ≤ 0.25 mg/L; R > 0.5 mg/L). Due to the low number of isolates with ceftriaxone resistance, analysis was performed using isolates with resistance (MIC> 0.125 mg/L) combined with decreased susceptibility (DS; MIC> 0.032–0.125 mg/L) to ceftriaxone. In Euro-GASP, the AST and N. gonorrhoeae species identification should be repeated for all isolates that are resistant to ceftriaxone, have elevated resistance to cefixime (MIC> 0.25 mg/L), and all isolates showing high-level resistance to azithromycin (MIC≥256 mg/L). Those isolates are also recommended to be sent to the Euro-GASP Reference Laboratory Hub (Public Health England/Örebro University Hospital) for further verification and whole genome sequencing [48].

All countries participating in Euro-GASP report all results through The European Surveillance System (TESSy), a web-based reporting system managed by the ECDC, where in addition to antimicrobial susceptibility/resistance data also epidemiological and clinical data of the corresponding gonorrhoea patients are included. The collected epidemiological and clinical data include gender, age, country of origin, mode of transmission (referred to as sexual orientation below: females, heterosexual males and MSM), anatomical site of infection (urogenital, pharyngeal and anorectal), HIV status, previous gonorrhoea diagnosis, and probable country of infection [6, 7, 27‐29, 47‐50].

Associations between resistance/decreased susceptibility and patients’ gender, sexual orientation and anatomical site of infection were investigated using univariate and multivariate logistic regression analysis with MSM as the reference for sexual orientation and urogenital for site of infection. To control for the different years, year of isolate collection, with 2009 as the base, was included in the multivariate analysis. The odds ratios (OR) and 95% confidence intervals (CI) were calculated and Pearson’s χ2 test was used to measure if these odds ratios differed significantly from 1. Using a forward step-wise approach, the most significant and strongest associations from the univariate analysis were added to a multivariable logistic regression model sequentially. The statistical significance was determined by Pearson χ²-test or by Fisher’s exact test if cell numbers were less than 5, considering two-sided p-values of < 0.05 indicating significance. Statistical analysis was performed in Stata 15.1 (StataCorp, Texas, USA).

The number of Euro-GASP isolates reported in 2009–2016, which significantly increased from 1366 isolates in 2009 to 2663 isolates in 2016, gender and sexual orientation of the corresponding patients, and anatomical site of infection have been summarised in Table 1.

Briefly, the overall coverage of reporting gender, sexual orientation, and anatomical site of infection was 98.8% (range: 96.1–99.7%), 56.2% (range: 49.9–63.9%), and 96.1% (range: 93.8–98.5%), respectively. The isolates were mainly collected from males (n = 13,083, 83.8%; females n = 2536, 16.2%; gender not reported, n = 184). Of these patients, 3939 (25.2%), 3464 (22.2%), and 2536 (16.2%) reported sexual orientation as MSM, heterosexual males, and females, respectively. The isolates were cultured from urogenital samples (n = 12,113; 79.7%), anorectal samples (n = 1822; 12.0%), pharyngeal samples (n = 883; 5.8%), and samples from other anatomical sites (n = 372; 2.5%). The site of infection was not reported for 613 isolates (Table 1). Males who did not report as MSM and females were most likely to have urogenital infections, whereas MSM had the highest proportion of anorectal and pharyngeal infections (Table 2).

During 2009–2016, the overall resistance to ciprofloxacin, azithromycin, cefixime, and ceftriaxone was 51.7% (range during the years: 46.5–63.5%), 7.1% (4.5–13.2%), 4.3% (1.8–8.7%), and 0.2% (0.0–0.5%), respectively. The level of resistance combined with decreased susceptibility to ceftriaxone was 10.2% (5.7–15.5%). The proportion of isolates with resistance combined with decreased susceptibility to ceftriaxone, and resistance to cefixime, azithromycin and ciprofloxacin over time by gender and sexual orientation are shown in Fig. 1.

Despite that the overall level of resistance combined with decreased susceptibility to ceftriaxone was relatively high (10.2%), it decreased from 2009 to 2016 (Fig. 1a). Furthermore, the overall number of isolates with ceftriaxone resistance was low (n = 26, 0.2%) and none were collected from females. Rare isolates with ceftriaxone resistance initially emerged in 2011 (three isolates in Austria and seven in Germany) among five patients without gender or sexual orientation reported, four male patients with sexual orientation not reported, and one heterosexual male. Ceftriaxone resistance was subsequently identified in two MSM (Ireland and Slovenia) and one patient without gender or sexual orientation reported (Germany) in 2012, in six males with sexual orientation not reported (Spain) and one heterosexual male (Germany) in 2013, in three males with sexual orientation not reported (Germany, Greece, and Norway) and two heterosexual males (Greece) in 2014, and finally in one heterosexual male (Greece) in 2015 (no ceftriaxone resistance was found in 2016). The overall level of resistance combined with decreased susceptibility to ceftriaxone was 7.6% in MSM, 12.6% in heterosexual males, 10.6% in males with sexual orientation not reported, and 9.1% in females. Resistance combined with decreased susceptibility to ceftriaxone was significantly positively associated with isolates from heterosexual males, males with sexual orientation not reported, and females (p < 0.001, p < 0.001, and p = 0.025; ORs 1.76, 1.44, and 1.23, respectively). The level of gonococcal resistance to cefixime was 2.1, 6.0, 4.4, and 4.6% in MSM, heterosexual males, males with sexual orientation not reported, and females, respectively. Also cefixime resistance was significantly associated with isolates from heterosexual males, males with sexual orientation not reported, and females (all p < 0.001; ORs 3.0, 2.14, and 2.26). Regarding azithromycin, 6.5% of the isolates from MSM, 8.5% from heterosexual males, 7.3% from males with sexual orientation not reported, and 5.6% from females were resistant. Azithromycin resistance was significantly associated with heterosexual males (p < 0.001; OR 1.33). Finally, 43.2% of the isolates from MSM, 57.4% from heterosexual males, 56.0% from males with sexual orientation not reported, and 46.6% from females were resistant to ciprofloxacin. Ciprofloxacin resistance was significantly associated with heterosexual males, males with sexual orientation not reported, and females (p < 0.001, p < 0.001, and p = 0.007; ORs 1.78, 1.67, and 1.15). See supplementary tables S1, S2, S3 and S4 for all p-values, ORs and CIs.

The number of Euro-GASP isolates collected from different anatomical sites and the number and proportion of isolates with resistance combined with decreased susceptibility to ceftriaxone, and resistance to cefixime, azithromycin and ciprofloxacin, by anatomical site is summarised in Table 3.

The majority (79.3%) of AMR isolates (resistant to at least one antimicrobial) were cultured from urogenital samples, reflecting that most (76.7%) Euro-GASP isolates during 2009 to 2016 were obtained from urogenital sites.

Where site of infection was known for ceftriaxone-resistant isolates (20/26), all were from urogenital site (n = 17), with exception of two pharyngeal isolates (2012, Germany and Slovenia (MSM)) and one rectal isolate from Ireland (2012, MSM). Overall, resistance combined with decreased susceptibility to ceftriaxone was found in 10.6% of the isolates from urogenital site, 8.2% from anorectal, and 8.4% from pharyngeal. Resistance combined with decreased susceptibility to ceftriaxone was significantly associated with urogenital samples compared to anorectal and pharyngeal samples (p = 0.002, p = 0.039; ORs 0.76 and 0.77). The level of resistance to cefixime was 4.8, 1.9, and 2.5% in urogenital, anorectal, and pharyngeal isolates, respectively, and also significantly associated with urogenital samples compared to anorectal and pharyngeal samples (p < 0.001, p = 0.002; ORs 0.39 and 0.51). For azithromycin 7.2% of the isolates from urogenital, 6.5% from anorectal, and 8.3% from pharyngeal sites were resistant; with no significant association to any anatomical site of infection. Finally, 53.8% of the isolates from urogenital, 42.5% from anorectal, and 41.3% from pharyngeal sites were resistant to ciprofloxacin (Table 3). Ciprofloxacin resistance was significantly associated with isolates from urogenital site when compared to anorectal and pharyngeal samples (both p < 0.001; ORs 0.63 and 0.61, respectively). See supplementary tables S1, S2, S3 and S4 for all p-values, ORs and CIs.

When combining gender, sexual orientation and anatomical site of infection over time, cefixime resistant isolates were mostly from the urogenital site. However, during the overall increase in 2010 and 2011 the proportion of anorectal and pharyngeal isolates increased by primarily anorectal isolates among MSM in 2010 and subsequently pharyngeal isolates among females in 2011 (Fig. 2).

Also azithromycin-resistant isolates were most frequently from urogenital site, however, the proportion of anorectal and pharyngeal isolates increased in MSM in 2014, and a small increase of anorectal isolates was seen in females in 2015 (Fig. 3).

Ciprofloxacin resistance was relatively high and stable from 2009 to 2016 and mostly identified in urogenital isolates, but also in anorectal isolates from MSM (Fig. 4).

Finally, most of the statistical associations reported above remained valid in the multivariate analysis in respect to cefixime and ciprofloxacin resistance/susceptibility and gender, sexual orientation, and site of infection with the exception of the association between ciprofloxacin resistance and females (supplementary tables S1, S2, S3 and S4). Ceftriaxone resistance combined with decreased susceptibility remained associated with non-MSM males, as well as unknown site of infection. When controlling for year, there was no longer an association between cefixime susceptibility and pharyngeal infections. All other azithromycin, ceftriaxone and ciprofloxacin associations remained when controlling for year in a multivariate analysis.