Antimicrobial resistance in Neisseria gonorrhoeae isolates and gonorrhoea treatment in the Republic of Belarus, Eastern Europe, 2009–2019

Background Limited antimicrobial resistance (AMR) data for Neisseria gonorrhoeae are available in Eastern Europe. We investigated AMR in N. gonorrhoeae isolates in the Republic of Belarus from 2009 to 2019, antimicrobial treatment recommended nationally, and treatment given to patients with gonorrhoea. Methods N. gonorrhoeae isolates (n = 522) cultured in three regions of Belarus in 2009–2019 were examined. Determination of minimum inhibitory concentrations (MICs) of eight antimicrobials was performed using Etest. Resistance breakpoints from the European Committee on Antimicrobial Susceptibility Testing were applied where available. A Nitrocefin test identified β-lactamase production. Gonorrhoea treatment for 1652 patients was also analysed. Statistical significance was determined by the Z-test, Fisher’s exact test, or Mann-Whitney U test with p-values of < 0.05 indicating significance. Results In total, 27.8% of the N. gonorrhoeae isolates were resistant to tetracycline, 24.7% to ciprofloxacin, 7.0% to benzylpenicillin, 2.7% to cefixime, and 0.8% to azithromycin. No isolates were resistant to ceftriaxone, spectinomycin, or gentamicin. However, 14 (2.7%) isolates had a ceftriaxone MIC of 0.125 mg/L, exactly at the resistance breakpoint (MIC > 0.125 mg/L). Only one (0.2%) isolate, from 2013, produced β-lactamase. From 2009 to 2019, the levels of resistance to ciprofloxacin and tetracycline were relatively high and stable. Resistance to cefixime was not identified before 2013 but peaked at 22.2% in 2017. Only sporadic isolates with resistance to azithromycin were found in 2009 (n = 1), 2012 (n = 1), and 2018–2019 (n = 2). Overall, 862 (52.2%) patients received first-line treatment according to national guidelines (ceftriaxone 1 g). However, 154 (9.3%) patients received a nationally recommended alternative treatment (cefixime 400 mg or ofloxacin 400 mg), and 636 (38.5%) were given non-recommended treatment. Conclusions The gonococcal resistance to ciprofloxacin and tetracycline was high, however, the resistance to azithromycin was low and no resistance to ceftriaxone was identified. Ceftriaxone 1 g can continuously be recommended as empiric first-line gonorrhoea therapy in Belarus. Fluoroquinolones should not be prescribed for treatment if susceptibility has not been confirmed by testing. Timely updating and high compliance with national evidence-based gonorrhoea treatment guidelines based on quality-assured AMR data are imperative. The need for continued, improved and enhanced surveillance of gonococcal AMR in Belarus is evident.


Conclusions:
The gonococcal resistance to ciprofloxacin and tetracycline was high, however, the resistance to azithromycin was low and no resistance to ceftriaxone was identified. Ceftriaxone 1 g can continuously be recommended as empiric first-line gonorrhoea therapy in Belarus. Fluoroquinolones should not be prescribed for treatment if susceptibility has not been confirmed by testing. Timely updating and high compliance with national evidence-based gonorrhoea treatment guidelines based on quality-assured AMR data are imperative. The need for continued, improved and enhanced surveillance of gonococcal AMR in Belarus is evident.
Keywords: Neisseria gonorrhoeae, Antimicrobial resistance, Surveillance, Treatment, Ceftriaxone, Azithromycin, Belarus, Eastern Europe Background Gonorrhoea, etiological agent: Neisseria gonorrhoeae, is one of the most common sexually transmitted infections (STIs) worldwide [1][2][3]. According to the World Health Organization (WHO), in 2016 86.9 million new adult cases of gonorrhoea were estimated [2]. In the Republic of Belarus the incidence of reported gonorrhoea (cases per 100,000 inhabitants) has substantially decreased, i.e., from 63 in 2005 to 44.4 in 2009, and finally, to 10.5 in 2018 [4]. The incidence of gonorrhoea in Belarus and many other East European countries is most likely underestimated because of limited testing (especially pharyngeal and rectal samples), insufficient use of nucleic acid amplification tests, incomplete reporting using multiple methods and epidemiological surveillance [5][6][7].
Alarmingly, the level of antimicrobial resistance (AMR) in Neisseria gonorrhoeae to a wide range of drugs has increased dramatically worldwide over the past decades, including first-line treatment [3,5,[8][9][10][11]. Ceftriaxone, an extended-spectrum cephalosporin (ESC), is the last option for first-line empiric monotherapy internationally. It is a concern that in vitro and clinically decreased susceptibility or resistance to ceftriaxone has been reported from many countries [3,5,. This has resulted in that the WHO global gonorrhoea treatment guidelines and international and national guidelines in Europe, Australia, and Canada recommend antimicrobial combination therapies, mostly ceftriaxone 250-1000 mg × 1 intramuscularly (IM) combined with azithromycin 1-2 g × 1 orally [8,11,29,30]. However, some countries, such as Japan, Ukraine and, since 2019 and 2020, the United Kingdom and USA, respectively, recommend high-dose (1 g except in the USA where 500 mg is recommended) ceftriaxone monotherapy, i.e., when chlamydial infection has been excluded [31][32][33][34]. Nevertheless, the first treatment failure with a recommended dual therapy for gonorrhoea was reported in 2016 [27]. In 2018, the first gonococcal strain with ceftriaxone resistance combined with high-level resistance to azithromycin was identified in England [25] and Australia [35]. WHO has developed a global action plan to control the spread and minimise the impact of AMR in N. gonorrhoeae [10]. One of its key strategies is to globally enhance quality-assured gonococcal AMR surveillance; to identify emerging AMR, monitor AMR trends, and ensure effective patient management by timely and evidence-based refinements of treatment guidelines. In the European Union/European Economic Area (EU/ EEA) the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) has been monitoring the patterns of AMR since 2004 [12,[36][37][38]. It is a grave concern that gonococcal AMR surveillance is dreadfully limited [5,6] in the non-EU/EEA countries of the WHO European Region, and only available in Ukraine, Belarus, and Russia [33,[39][40][41][42][43][44].
The empirical treatment of gonococcal infections in Belarus is guided by the Clinical protocol for the diagnosis and treatment of patients with STIs and approved by the order of the Ministry of Health of the Republic of Belarus 10/29/2009, No. 1020 (below referred to as '2009 Belarusian national gonorrhoea guideline') [45]. For treatment of uncomplicated gonorrhoea, recommended first-line therapy is ceftriaxone 1 g × 1 IM. Recommended alternative regimens are cefixime 400 mg × 1 orally or spectinomycin 2 g × 1 IM for men and 4 g × 1 IM for women or ofloxacin 400 mg × 1 orally, or lomefloxacin 800 mg × 1 orally [45]. To treat complicated gonorrhoea ceftriaxone 1 g IM or IV every 24 h for 7 days is the recommended first-line therapy. Recommended alternative regimens are spectinomycin 2 g IM every 12 h for 7 days or ofloxacin 200 mg orally every 12 h for 7-14 days, or lomefloxacin 400 mg orally every 24 h for 7-14 days [45]. Notable, spectinomycin is currently not available throughout Belarus [46].
The aims of this study were to 1) analyse the trends in N. gonorrhoeae AMR levels in Belarus (2009-2019), 2) review the antimicrobial treatments recommended nationally as well as compliance with these recommendations in Belarus, and 3) provide quality-assured gonococcal AMR data for informing the 2009 Belarusian national gonorrhoea guideline [45].

Study population
Gonorrhoea patients (n = 522) diagnosed in accordance with standard care at the following STI Healthcare Institutions were included in the study: Mogilev Regional  [40]. Cervical and urethral specimens from females and males, respectively, were collected in accordance with standard care and delivered to the bacteriological laboratory in Amies Transport Medium with Charcoal (Research and production center Chemmedsynthsis, Minsk, Belarus). Sex and age of the patients were recorded but no patient identification data. Exclusion criterium was: not confirmed gonococcal infection. All patients and their sexual partners were to be treated in accordance with the 2009 Belarusian national gonorrhoea guideline [45].
The isolates were stored in accordance with standard care in a liquid preservation medium containing trypticase-soy broth, yeast extract, agar, and horse serum in liquid nitrogen (− 196°С) or a low-temperature freezer (− 80°С).

Antimicrobial susceptibility testing
At the WHO Collaborating Centre for Gonorrhoea and other STIs, Sweden, the minimum inhibitory concentrations (MICs; mg/L) of ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline, benzylpenicillin, and gentamicin were determined by Etest (bioMerieux, Marcy-l'Etoile, France), following the manufacturer's instructions and as described previously [33,39,40]. Whole MIC dilutions were used for interpretation and clinical susceptibility (S) and resistance (R) breakpoints stated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [47] were applied, where available. For azithromycin, no clinical breakpoints exist, and the EUCAST azithromycin epidemiological cut-off value (ECOFF) of MIC > 1 mg/L [47] was applied to indicate isolates with azithromycin resistance determinants (considered as azithromycin resistant below). For gentamicin, breakpoints from a previous publication were used [48]. The Nitrocefin test (Oxoid, Basingstoke, England) was used to identify β-lactamase producing gonococcal strains. For quality controls of the antimicrobial susceptibility testing, the 2016 WHO N. gonorrhoeae reference strains [49] were used.

Treatment of gonorrhoea
The treatment of patients with gonorrhoea (n = 1652) diagnosed in accordance with standard care at the Minsk City Clinical Dermatovenerologic Dispensary in 2013-2018 (n = 749) and Mogilev Regional Dermatovenerologic Dispensary in 2010-2019 (n = 903) were analysed. Uncomplicated and complicated gonorrhoea cases were defined according to the international statistical classification of diseases and related health problems [50]. Gonorrhoea treatment compliance with the 2009 Belarusian national gonorrhoea guideline [45] was evaluated.

Statistical analysis
Statistical analysis was performed using MedCalc Statistical Software v18.11.3 (MedCalc Software bvba, Ostend, Belgium). The 95% confidence interval (95% CI) was calculated using the exact binomial distribution. The Ztest, Fisher's exact test, and Mann-Whitney U test were used to compare groups, as appropriate. The level of significance was set at P<0.05.

Results
Patients and Neisseria gonorrhoeae isolates Demographic data (sex and age) were available from 514/522 (98.5%) patients. Briefly, the median age for men was 25 years (range 16-61 years) and for women, also 25 years (range 16-74 years). The sex and age distributions were similar in 2009-2019. The median malefemale ratio was 5.9 (range 1.8-7.6), with decreasing trends over time.
The MIC distributions for ceftriaxone and azithromycin, included in the internationally recommended firstline dual antimicrobial therapy [8,11,29], and cefixime are presented in Fig. 1.
In total, 2.7% (14/522) of the isolates had a ceftriaxone MIC of 0.125 mg/L, which is on the ceftriaxone resistance breakpoint [47]. The proportion of isolates with a ceftriaxone MIC of ≤0.016 mg/L was 85.2%, and in general, the annual MIC distribution for ceftriaxone appeared to shift to lower MICs during 2009-2019 (data not shown). For cefixime, 78.7% (411/522) of the isolates had a MIC of ≤0.016 mg/L; however, 2.7% (14/522) of the isolates were resistant to cefixime, highest MIC = 2 mg/L (one isolate in 2013), and additionally, 1.7% (9/ 522) of the isolates had a cefixime MIC of 0.125 mg/L, i.e., exactly on the cefixime resistance breakpoint [47]. Except for four (0.8%) azithromycin-resistant isolates (MICs 2-12 mg/L), the azithromycin MIC distribution appeared to represent a wild-type distribution.

Antimicrobial treatment of gonorrhoea patients in Belarus, 2010-2019
Compliance with the treatment of gonococcal infections to the 2009 Belarusian national gonorrhoea guideline [45] at the Minsk City Clinical Dermatovenerologic Dispensary (2013-2018) and the Mogilev Regional Clinical Dermatovenerologic Dispensary (2010-2019) for 1652 patients is described in Table 2.
Many antimicrobial treatment regimens were administered (Table 2). Overall, only 862 (52.2%) patients received the recommended first-line treatment (ceftriaxone 1 g (uncomplicated gonorrhoea) or > 1 g (complicated gonorrhoea) as monotherapy or in combination with other antimicrobials) in accordance with the 2009 Belarusian national gonorrhoea guideline [45]. Some 154 (9.3%) patients were given a recommended alternative treatment (cefixime 400 mg or ofloxacin 400 mg as a single oral dose) [45]. Consequently, 38.5% of the patients received treatment not compliant with the 2009 Belarusian national gonorrhoea guideline [45]. Worryingly, notwithstanding the high level of ciprofloxacin resistance, ofloxacin 400 mg × 1 was given to 149 (9.0%) patients.      Frequently, additional antimicrobials were given to treat other non-viral STIs, which had been confirmed or not excluded by appropriate laboratory diagnostics. These included doxycycline, other tetracyclines, different macrolides, fluoroquinolones, oral cephalosporins, penicillins, and nitroimidazoles b More than 1 g of ceftriaxone was quite often given; approximately 10% of the patients received benzylpenicillin, about 5% a tetracycline/macrolide regimen, and one (0.1%) patient was given rifampicin c More than 1 g of ceftriaxone was somewhat frequently given. A benzylpenicillin regimen was rarely given, and for a few patients, > 400 mg ofloxacin or a tetracycline/macrolide regimen was administered

Discussion
We report the first N. gonorrhoeae AMR surveillance data, quality-assured according to WHO standards [5,49,51,52], for isolates cultured in Belarus during an extended period (from 2009 to 2019 in Minsk, Vitebsk, and Mogilev). AMR levels were significant and comparable to those in neighbouring Russia [41][42][43][44], EU/EEA countries [12,36,37], and many other countries internationally [5,52]. However, the AMR levels were clearly higher than in neighbouring Ukraine [33]. In Belarus, the resistance levels to the earlier recommended gonorrhoea therapeutic antimicrobials tetracycline, ciprofloxacin, and benzylpenicillin were relatively high, i.e., at 27.8, 24.7, and 7%, respectively. These antimicrobials should not be recommended or used for empirical gonorrhoea treatment in Belarus, which is in concordance with most other countries [3, 5, 8-12, 28-34, 36, 37, 39-44]. It is a grave concern that fluoroquinolones (ofloxacin and lomefloxacin) remain to be recommended in the 2009 Belarusian national gonorrhoea guideline [45], i.e., despite the high resistance to ciprofloxacin. Furthermore, particularly ofloxacin was also frequently used in gonorrhoea monotherapy or in combination with other antimicrobials. When susceptibility has not been confirmed by laboratory testing, fluoroquinolones should not be used for treatment [8]. Accordingly, ofloxacin and lomefloxacin should be excluded from the empiric treatment recommended in the Belarusian national gonorrhoea guideline [45]. Of note, gonococcal strains producing β-lactamase were very rare (0.2%, 1/522 isolates) in Belarus, as earlier observed also in the neighbouring East European countries Russia and Ukraine [33,[39][40][41][42][43][44]. Accordingly, few β-lactamase producing strains appear to have emerged in Belarus or, if imported, managed to spread in the country. However, 7% of the 522 isolates had a chromosomally-mediated resistance to benzylpenicillin, and additionally, 36.7% displayed a decreased susceptibility. Azithromycin resistance (0.8%) was rare compared to most EU/EEA countries [12,36,37] and only sporadic isolates (n = 4) resistant to azithromycin were found. The overall resistance to cefixime was 2.7%, fluctuating from 0 to 22.2% in 2017. Only 36 gonococcal isolates, however, were examined in 2017 and the high cefixime resistance this year was suspected to mainly be caused by a single gonococcal clone. No gonococcal resistance to ceftriaxone, spectinomycin, and gentamicin was identified. However, spectinomycin is currently not available throughout Belarus [46]. The lack of ceftriaxone resistance and, in general, relatively low ceftriaxone MICs may be because of the long tradition to use ceftriaxone in high dose (1 g in monotherapy or dual antimicrobial therapy) for treatment of gonorrhoea [45] and that the less potent oral ESC cefixime has been rarely used for treatment of gonorrhoea in Belarus. Alarmingly, the compliance with the 2009 Belarusian national gonorrhoea guideline [45] was relatively low and many noncompliant suboptimal antimicrobials were prescribed to a large proportion of patients. This lack of compliance may select AMR in N. gonorrhoeae, etiological agents of other STIs, and bystander organisms [3,53]. Moreover, in Belarus antimicrobials, such as several penicillins and tetracyclines, are readily available over-the-counter in pharmacies without prescription, which is crucial to abandon to decrease the high level of self-medication and a further selection of AMR.
Our study has some limitations. First, gonococcal isolates were collected in only three of the six regions of Belarus. However, considering that these three regions represent 60% of the Belarusian population, including the capital city Minsk (https://en.wikipedia.org/wiki/ Regions_of_Belarus), this geographical bias should be limited and the results should be generalisable to the wider population. Second, the number of isolates per year was low. Third, no data were collected on pharyngeal or rectal specimens. Finally, no data on epidemiological or clinical characteristics (e.g., sexual behaviour and treatment outcomes) were available. Consequently, the gonococcal AMR surveillance in Belarus should be further improved and enhanced by increasing the number of isolates and representativeness of isolates collected each year, ideally including additional regions and collection of extragenital specimens such as pharyngeal and rectal specimens. Finally, gonococcal AMR surveillance in Belarus should also include more detailed patient epidemiological and clinical data.

Conclusions
The present study reports the first gonococcal AMR surveillance data for isolates cultured in Belarus over an extended period (i.e., from 2009 to 2019) in the Minsk, Mogilev, and Vitebsk regions, quality-assured according to WHO standards [5,49,51,52]. Based on the gonococcal AMR data presented in this paper, Belarus has also participated in the WHO Global GASP [5]. Briefly, in 2009-2019 the gonococcal population circulating in Belarus showed stable and high resistance to tetracycline, ciprofloxacin, and benzylpenicillin. More sporadic resistance to azithromycin and fluctuating resistance to cefixime were also found. However, no resistance to ceftriaxone, spectinomycin, or gentamicin was identified. Consequently, ceftriaxone 1 g can continuously be recommended as empiric first-line gonorrhoea therapy in Belarus. When susceptibility has not been confirmed by laboratory testing, fluoroquinolones should not be used for gonorrhoea treatment. Continued, improved and enhanced N. gonorrhoeae AMR surveillance of gonococcal AMR in Belarus is imperative to timely inform revisions of the national gonorrhoea guideline [45] in Belarus. Finally, a comparison of the Belarusian gonococcal population with the N. gonorrhoeae strains spreading internationally, i.e., using whole-genome sequencing, which is used in the Euro-GASP surveillance [37,54] and has also been recently applied in gonococcal AMR surveillance in Ukraine [55] and many other countries across the world [56][57][58][59][60][61][62], would be valuable.