First characterisation of antimicrobial susceptibility and resistance of Neisseria gonorrhoeae isolates in Qatar, 2017–2020

Limited data are available regarding antimicrobial resistance in Neisseria gonorrhoeae strains circulating in WHO Eastern Mediterranean Region (EMR). We investigated the antimicrobial susceptibility/resistance of N. gonorrhoeae isolates to five antimicrobials (ceftriaxone, azithromycin, ciprofloxacin, tetracycline, and benzylpenicillin) currently or previously used for gonorrhoea treatment in Qatar, 2017–2020. Minimum inhibitory concentrations (MICs; mg/L) of antimicrobials were determined using Etest on gonococcal isolates collected during January 1, 2017-August 30, 2020 at Hamad Medical Corporation, a national public healthcare provider. During 2017–2020, resistance in isolates from urogenital sites of 433 patients was 64.7% (95% CI: 59.5–69.6%; range: 43.9–78.7%) for ciprofloxacin, 50.7% (95% CI: 45.3–56.1%; range: 41.3–70.4%) for tetracycline, and 30.8% (95% CI: 26.3–35.6%; range: 26.7–35.8%) for benzylpenicillin. Percentage of isolates non-susceptible to azithromycin was 4.1% (95% CI: 2.0–7.4%; range: 2.7–4.8%) and all (100%) isolates were susceptible to ceftriaxone. Two (1.6%) isolates from 2019 and one (2.2%) isolate from 2020 had high-level resistance to azithromycin (MIC≥256 mg/L). Overall, 1.0% (4/418) of isolates had a ceftriaxone MIC of 0.25 mg/L, which is at the ceftriaxone susceptibility breakpoint (MIC≤0.25 mg/L). Treatment with ceftriaxone 250 mg plus azithromycin 1 g can continuously be recommended for gonorrhoea therapy in Qatar. Continued quality-assured gonococcal AMR surveillance is warranted in EMR.

One key priority of the WHO global action plan is to enhance gonococcal AMR surveillance globally.Consequently, the WHO's Global Gonococcal Antimicrobial Surveillance Programme (GASP), initially stabled in 1990 [18], was revitalized and strengthened [11,13].WHO GASP is identifying emerging AMR, monitoring AMR trends, and informing refinements of global, regional, and national gonorrhoea treatment guidelines, as well as public health strategies and policies developed by WHO and other organisations [11].WHO recommends that treatment guidelines be refined based on data from recent and quality-assured gonococcal AMR surveillance and that use of an antimicrobial in empiric treatment is be discontinued when the rate of therapeutic failures and/or AMR reach 5% [11,13,16,19].However, exceedingly limited data regarding gonococcal AMR have been available in several WHO regions, especially in the WHO African and Eastern Mediterranean Regions ('Arab World', including the Middle East and North Africa) [11,13].
Qatar is an Arab country located in the Middle East with a resident population of 2.8 million people [20].However, 89% of the population are expatriates from over 150 countries [21,22].About 60% of the population consists of expatriate craft and manual workers, typically working in mega-development projects [23], who are predominantly young (20-49 years of age), male, and single [24].The extensive multi-national diversity of this population and its frequent travel between country of origin and Qatar may yield an unusual epidemiological situation for transmission of N. gonorrhoeae strains originating from different parts of the world.Thus Qatar provides a unique setting for gonococcal AMR surveillance.
The objectives of this study were to initiate a gonococcal AMR surveillance programme, quality assured in accordance with WHO standards in Qatar, and to investigate the antimicrobial susceptibility of N. gonorrhoeae isolates in this country, thereby informing the national STI treatment guidelines in Qatar, as well as contributing to the WHO GASP [11,13] at a critical time for gonococcal AMR surveillance.With the very limited gonococcal AMR surveillance to date in the WHO Eastern Mediterranean Region [11,13], this study makes the largest contribution to this surveillance effort from a region that constitutes about 10% of the global population [25].

Study population
The Microbiology Laboratory at Hamad Medical Corporation (HMC), the main national public healthcare provider in Qatar, cultures urogenital specimens collected from patients with gonorrhoea attending HMC and other public healthcare facilities in Qatar.All consecutive viable gonococcal isolates (n = 433, one per patient or episode of infection) cultured from January 1, 2017 to August 30, 2020 were included in the study.Demographic data (nationality, gender, and age) were collected, but no other socio-demographic information or sexual behaviour data were collected in the study.All patients were aimed to be treated in accordance with the national STI Screening and Prevention Guidelines that implement the United States Centers for Disease Control and Prevention (CDC) 2015 Sexually Transmitted Diseases Treatment Guidelines [26].
Urethral swab specimens from males and cervical swab specimens from females were collected in Amies transportation medium (International for Medical Equipment and Supplies, Doha, Qatar).

Culture of Neisseria gonorrhoeae
All swabs were transported in �12 h to the laboratory and inoculated on selective chocolate culture medium (International for Medical Equipment and Supplies, Doha, Qatar), followed by incubation in 5±1% CO 2 -enriched humid atmosphere at 36±1˚C for 24 h, and if negative, for an additional 24 h.Suspected colonies were verified as N. gonorrhoeae using an oxidase test, Bruker Maldi Biotyper (Bruker Daltonik, Bremen, Germany), and Phadebact Monoclonal GC Test (MKL Diagnostic AB, Sollentuna, Sweden).Isolates were stored in a Microbank cryovial system that incorporates 25 treated beads and a proprietary cryopreservative (Pro-Lab Diagnostics, Cheshire, UK) at -80˚C.

Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MICs; mg/L) of five antimicrobials (ceftriaxone, azithromycin, ciprofloxacin, tetracycline, and benzylpenicillin) were determined using Etest on GC agar base with 1% growth supplements (International for Medical Equipment and Supplies, Doha, Qatar), in accordance with the manufacturer's instructions (bioMe ´rieux, Marcy-l'Etoile, France).Azithromycin was only tested in 2018-2020 and some isolates were not tested against all antimicrobials, i.e. because they were not requested in routine diagnostics, because of a temporary lack of reagents, such as Etest strips, and/or because the isolates were not viable for subsequent antimicrobial susceptibility testing.MIC values were reported and interpreted using whole MIC doubling dilutions, and, where available, clinical breakpoints for susceptibility (S) and resistance (R), according to the United States Clinical Laboratory and Standards Institute (CLSI) [27].For ceftriaxone and azithromycin, no clinical resistance breakpoints exist and only the recommended susceptibility breakpoints could be used [27].β-lactamase production was identified using BD BBL Cefinase paper discs (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).The N. gonorrhoeae ATCC 49226 international reference strain was used for quality control.

Oversight
The study was approved by the Weill Cornell Medicine-Qatar and Hamad Medical Corporation Institutional Review Boards.The study was conducted following the ethics review boards guidelines and regulations.Waiver of consenting was granted as per the study design, retrospective collection of deidentified gonococcal isolates.The study was reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.The STROBE checklist can be found in S1

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates
Results of antimicrobial susceptibility testing of all N. gonorrhoeae isolates (n = 433) are summarised in Table 1 and listed in S2 Table of Supplementary Information.
In the present study, we substantially increased the available gonococcal AMR data from this WHO region, and provided AMR data for 433 isolates (five antimicrobials) cultured in Qatar in 2017-2020, which is available for the WHO GASP.All antimicrobial susceptibility testing was also based on MIC determination, i.e., Etest was used, which is recommended in the WHO GASP [11,13].
High levels of resistance to ciprofloxacin, tetracycline, and benzylpenicillin were found.The annual levels of resistance to ciprofloxacin and tetracycline appeared to decline from 2017 to 2020.However, the representativeness of the material during the different years was unclear and the number of isolates in 2020 was low (n = 46).Accordingly, enhanced, representative and quality-assured AMR surveillance will be required to provide appropriate evidence regarding these potential decreases in resistance levels.It is notable that resistance to ciprofloxacin in 2017-2020 was very high at 64.7%, which is similar to the high levels reported in several other WHO regions [11,13].Despite high levels of ciprofloxacin resistance, this antimicrobial or other fluoroquinolones remain in use for empirical treatment of gonorrhoea in many countries.This use needs to be abandoned and ciprofloxacin should only be used for treatment of gonorrhoea when the infection has been proven ciprofloxacin-susceptible as a result of laboratory testing [28].
No resistance to ceftriaxone was found among isolates in Qatar in 2017-2020 and only a small proportion (4.1%) of isolates demonstrated resistance to azithromycin.Accordingly, current first-line empiric treatment in Qatar, using ceftriaxone and azithromycin based on the United States CDC 2015 Sexually Transmitted Diseases Treatment Guidelines [26], remains an appropriate treatment, which additionally eradicates concomitant Chlamydia trachomatis infection and many Mycoplasma genitalium infections.Nevertheless, it is concerning that two isolates from 2019 and one isolate from 2020 in Qatar had high-level resistance to azithromycin (MIC�256 mg/L).Furthermore, recently published new recommendations for gonorrhoea treatment (ceftriaxone 500 mg monotherapy) in the United States CDC Sexually Transmitted Diseases Treatment Guidelines [29] will be considered.
The limitations of the present study included the lack of detailed epidemiological information of all gonorrhoea cases and uncertainties regarding the representativeness of the examined gonorrhoea cases.Individuals diagnosed with gonorrhoea at HMC, a national public healthcare provider, were examined, but we did not access any cases being managed in the private sector or at other public healthcare facilities, such as the Qatar Red Crescent Society, the main healthcare provider for craft and manual workers.Nevertheless, 28 nationalities were represented, which suggests that some expatriate craft and manual workers were represented.Only urogenital samples were collected.Azithromycin susceptibility testing was not conducted prior to 2018 and some of the isolates were not examined for susceptibility to all antimicrobials, because they were not requested in routine diagnostics, because of temporary lack of reagents, such as Etest strips, and/or because the isolates were not viable for subsequent antimicrobial susceptibility testing.Finally, only the recommended N. gonorrhoeae strain ATCC 49226 was used for quality control of the AMR testing, however, the 2016 WHO N. gonorrhoeae reference strains [30] are now available for use in quality assurance and control of the AMR surveillance in Qatar.
In conclusion, high levels of resistance to ciprofloxacin, tetracycline, and benzylpenicillin were found in Qatar, but less resistance to azithromycin and none to ceftriaxone.Treatment with ceftriaxone 250 mg plus azithromycin 1 g can be continuously recommended for gonorrhoea therapy in Qatar, but the recently recommended ceftriaxone 500 mg monotherapy [29] will also be considered.Continued and enhanced quality-assured gonococcal AMR surveillance is essential in Qatar and other countries in the WHO Eastern Mediterranean Region.Wholegenome sequencing of N. gonorrhoeae isolates is also in progress to enhance understanding of N. gonorrhoeae AMR determinants and transmission of N. gonorrhoeae, as well as its resistance in different subpopulations in Qatar, compared with the international population.