Epidemiology and antimicrobial resistance profile of Neisseria gonorrhoeae in Catalonia, Spain, 2016–2019

Antimicrobial resistance data for Neisseria gonorrhoeae is globally sparse and resistant strains are emerging in Catalonia. We aim to describe epidemiological and antimicrobial resistance in all patients infected with N. gonorrhoeae during the period from 2016 to 2019, using available antimicrobial susceptibility data. We retrospectively analysed confirmed N. gonorrhoeae cases notified to Catalonia’s microbiological reporting system. Antibiotic susceptibility testing (azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, spectinomycin, and tetracycline) was assessed using clinical breakpoints published by the European Committee on Antimicrobial Susceptibility Testing. Incidence rates were calculated and proportions were compared using the χ2 test or Fisher’s exact test, and analysed using the Statistical Package for Social Sciences (SPSS 18.0). A total of 14,251 confirmed cases of N. gonorrhoeae were notified. Incidence increased from 30.7 cases/100,000 person-years (p < 0.001) in 2016 to 64.7 in 2019. Culture was available in 6,292 isolates (44.2%), of which 5,377 (85.5%) were resistant to at least one of the antibiotics tested. Azithromycin resistance rose from 6.1% in 2016 to 16% in 2019 (p < 0.001). Only 1.0% (45 cases) were resistant to ceftriaxone. Multidrug-resistant N. gonorrhoeae increased from 0.25% in 2016 to 0.42% in 2019 (p = 0.521). One case presented extensively drug-resistant N. gonorrhoeae. In Catalonia, 10% of the N. gonorrhoeae isolates were resistant to azithromycin in the 2016–2019 period. According to World Health Organization guidelines, resistance above 5% indicates an alert to review treatment guidelines. Antimicrobial susceptibility testing in clinical practice followed by surveillance and interventions are essential to monitor trends and prevent the spread of antimicrobial resistance.


Introduction
Gonorrhoea is one of the most common sexually transmitted bacterial infections (STI) and an important cause of morbidity worldwide. Without treatment, the disease can cause severe complications such as pelvic inflammatory disease and ectopic pregnancy in women, and infertility in both genders. In addition, neonatal gonococcal conjunctivitis may lead to scarring and blindness [1].
Laboratory diagnosis is determined by gram staining, conventional agar-based culture method, and/or nucleic acid amplification test (NAAT). In the case of gonococcus, it is especially important to obtain a sample of urethral/cervical exudate for culture before starting treatment; this sample will be used to perform an antibiogram, confirm the appropriate treatment and track changes in antimicrobial resistance (AMR) [2].
Antimicrobial resistance is a major public health problem. Systematic epidemiological surveillance of AMR to N. gonorrhoeae is essential for several reasons. First, to detect emerging resistance to antimicrobials in strains circulating among the population; second, to contribute to the design of action plans for the prevention and control of gonorrhoea infection, and third, to allow professionals to evaluate its extension worldwide and report on local and global strategies to control AMR to gonococcal disease. Current surveillance is often suboptimal and presents many challenges, especially in countries with the highest burden of resistance [3].
Treatment of gonococcal infection is currently affected by the appearance of strains resistant to almost all groups of antibiotics used, as well as by the recent reduced susceptibility appearing worldwide in third-generation cephalosporins [4].
In order to reduce the risk of resistance to the use of antibiotic monotherapy, the World Health Organization (WHO) recommends that local resistance data determine the choice of therapy (both dual therapy and monotherapy). In territories where local resistance data are unavailable, the guidelines suggest combining azithromycin and ceftriaxonebased therapy or, alternatively, treating with cefixime prior to monotherapy [5].
Since 2009, the European Centre for Disease Prevention and Control (ECDC) has been coordinating the Gonococcal Antimicrobial Surveillance Programme (Euro-GASP), a specific epidemiological surveillance programme for antibiotic resistance in Neisseria gonorrhoeae. Funded by an international network, the programme is led by Public Health England in the UK and includes the Örebro University Hospital in Sweden [6].
According to the ECDC, incidence rates (IR) of gonococcal infection have increased significantly in Europe over the years, rising from 8.7 cases/100,000 persons in 2010 to 18.9/100,000 in 2016; 46% of these cases were registered as men who have sex with men [6]. In Spain, the gonococcal infection incidence has increased from 2.02 cases/100,000 persons in 2001 to 13.89/100,000 in 2016. Of 6,456 notified cases, 83.7% occurred in males, indicating a male/female ratio of 5:1.
With respect to age groups, the highest incidence occurs in 20 to 24-years-old (89.2/100,000 in males and 21.5/100,000 in women) in Europe and worldwide. In 2016, the highest IR in Spain was in Catalonia, followed by Asturias and Madrid [7]. In Barcelona, between 2013 and 2017, the observed proportions of cases resistant to first-line treatments were 4.9% for cefixime, 0.9% for ceftriaxone, and 3.0% for azithromycin. However, the proportion of isolates showing resistance to all antibiotics was approximately 1% of cases [8].
In Catalonia, the Epidemiological Surveillance Network (Decree 203/2015 [9], of 15 September) was created in 2015 to regulate reporting systems for notifiable diseases and epidemic outbreaks, and establish a microbiological reporting system for Catalonia (MRSC) as a notifiable system. Through the MRSC, microbiologists notify confirmed cases of acute illnesses, enabling the study of microorganisms with antibiotic susceptibility and notifiable resistance profiles.
In this study, we aim to describe the epidemiological characteristics of confirmed cases of N. gonorrhoeae and antimicrobial susceptibility notified to the MRSC between 2016 and 2019.

Study population
The study population considered all patients notified to the MRSC who received treatment in hospitals and primary health care centres in Catalonia or presented with a new confirmed episode of acute infection by N. gonorrhoeae between 2016 and 2019.
Data collection All data was processed by the Subdirectorate General for Public Health Surveillance and Emergency Response at the Catalonian Public Health Agency, including the introduction, validation, cleaning and analysis of data records according to the diagnostic criteria, ensuring that cases met the confirmed case definition in line with established criteria. The epidemiological, laboratory and clinical data collected included the age and sex of the patient, anatomical site of infection, previous gonorrhoea diagnosis, diagnostic technique, type of centre, coinfection with other STIs, and antibiotic sensitivity in isolated strains. The site of infection was classified as urethral/balanopreputial, vaginal/endocervix, pharynx, anorectal, urine and others (broadly categorized under genital/anorectal area and non-genital area). The age groups were split as follows: 0-11 years, 12-19 years, 20-29 years, 30-39 years, 40-49 years, and ≥ 50 years.
A case was considered laboratory-confirmed when at least one of the following diagnostic criteria was met [10]: • isolation of N. gonorrhoeae in a clinical sample • detection of nucleic acids from N. gonorrhoeae in a clinical sample • observation of intracellular gram-negative diplococcus obtained in a smear of male's urethral material (direct microscopy) Data analysis A descriptive analysis of the study variables was conducted. With regard to statistical analysis, IR per 100,000 person-years were calculated according to demographic data delivered by the Institute of Statistics of Catalonia [11] for the studied age groups and sex. Proportions were compared using the χ 2 test or Fisher's exact test, as appropriate. Incidence rates were calculated and changes were assessed according to IR ratios and expressed as the percentage change in incidence. Two-sided p-values < 0.05 were considered statistically significant. Analyses were conducted using the Statistical Package for Social Sciences (SPSS 18.0).

Antimicrobial susceptibility
The determination of antimicrobial susceptibility testing was studied for seven antibiotics: azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, spectinomycin, and tetracycline. The laboratories used an "antibiogram kit"; where not available, manual susceptibility testing was performed for each antibiotic. For the study of resistance for each antibiotic, the breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (version 6.0) were used, with the consensus of the MRSC working group included in the Protocol for the Surveillance of Antimicrobial Resistance in Catalonia [12].  [13].
Multi-drug resistant gonococci are strains resistant to one category I antibiotic -extended spectrum cephalosporins (ESC) or spectinomycin -plus at least two other antibiotics in category II (penicillin, ciprofloxacin and azithromycin). Extensively drug-resistant gonococci strains are resistant to two or more category I antibiotics (ESC and spectinomycin); and to three or more in category II (penicillin, ciprofloxacin, azithromycin, aminoglycosides and carbapenem).
In our definition of MDR-NG, we used cefixime and ceftriaxone as ESC or XDR-NG and did not consider aminoglycosides and carbapenem.
We reviewed patient files for studied cases presenting multi-drug resistant and extensively drug-resistant gonococci.
Co-infections with other STIs were studied, considering a three-week window period between the gonorrhoea infection and other STIs. Analyses of all cases of N. gonorrhoeae (14,251) were conducted to determine whether other STIs (chlamydia, trichomonas, herpes or syphilis) were present during the three-week period from the initial diagnosis of N. gonorrhoeae.
Reinfection episodes were studied for each confirmed case of N. gonorrhoeae subsequent to the first infection. N. gonorrhoeae positive tests within 60 days of a previous positive confirmed case were considered a single episode.

Results
Between 2016 and 2019, a total of 14,251 confirmed cases of N. gonorrhoeae were reported in Catalonia. In Table 1, we summarize the case incidence according to year, age group and sex; and diagnosis technique, anatomical site of infection and origin of the sample (clinical setting).
Age and sex were available for almost all cases (age was missing in only six cases). Over 80% of cases were males. Overall ratio of male cases to female cases was 4:1, with this distribution exhibiting a diminishing trend over time, from 4.7:1 in 2016 to 3.4:1 in 2019 (p < 0.001). The incidence rose by 98% in males and by 172% in females between 2016 and 2019. The median age was 30 years (with a range of 0 to 89 years), with 31 years for males and 25 years for females. Sex and age distributions were similar throughout the period, with a high increase in female incidence in 2019 compared to 2016 (10.5 cases per 100,000 persons in 2016; and 28.6 cases/100,000 persons in 2019; p < 0.001). The highest incidence occurred in the 20-29 age group (185.6 cases/100,000 persons/year), followed by the 30-39 age group (98.3 cases/100,000 persons/year).
Regarding diagnostic technique, culture was available in 6,292 cases (44.2%); 7,955 (55.8%) cases were analysed only by PCR and 4 of these (0.03%) were analysed by direct microscopy (Table 1 and Fig. 1). Laboratory culture decreased slightly, from 47.7% in 2016 to 42.9% in 2019, but not significantly, due to the shift from use of bacterial culture to NAAT for diagnosis of gonorrhoea. Of 16,202 samples, 12,322 (76.1%) were of genitalanorectal origin, 2,368 samples (14.6%) were pharyngeal, 1,451 samples (9.0%) were from urine, and 61 samples (0.4%) were from other anatomical site. In one case, site of infection was not reported. Confirmed cases of gonococcal infection came both from primary care (48.3%) and hospital care (51.7%).

Extensively drug-resistant N. gonorrhoeae (XDR-NG).
Only one case presented XDR-GC in 2019. The case was resistant to ceftriaxone, cefixime, azithromycin, penicillin and tetracycline and was only sensitive to spectinomycin (Figs. 1 and 2).
When the characteristics of the 20 patients with MDR-NG and XDR-NG were further analysed, twelve of the patients were of Spanish origin, three were from Morocco, two from South America and three patients had no recorded origin. The median age of these patients was 31 years old (with a range from 22 to 60 years) (Figs. 1 and 2).   Table 3). The boxplot in Fig. 3 represents the number of patients that had between one and seven reinfection episodes subsequent to the first primary infection. For each episode, the median time elapsed in months is shown. In females, the median  time between one infection and the next was shorter throughout the period. The distribution of reinfections shows 8.9% (n = 1,273) cases with one reinfection, and 2.2% (n = 316) with two. In 149 cases (1.1%) there were three or more reinfections, including one patient with seven reinfections.

Discussion
This study describes microbiological surveillance data, coinfections and reinfections of 14.251 confirmed N. gonorrhoeae cases in Catalonia over a four-year period. Antimicrobial susceptibility was performed on 5,377 strains. To our knowledge, this is the first study conducted in Spain to describe AMR of such a considerable amount of N. gonorrhoeae isolates, previously published works [8,14] having studied far fewer strains.
The results indicate a rise in the incidence of gonococcal infection in our region in 2019 (IR = 64.7 cases/100,000 persons) compared to 2016 (IR = 30.7 cases/100,000 persons). This increase can be explained in part by improved STIs diagnosis, in addition to public health campaigns and an increase in access to rapid point-of-care testing. Furthermore, Catalonia is a reception site for sex tourism [15]. Recent studies conducted in Europe, the Netherlands and the United Kingdom [16,17] describe that the use of substances that facilitate chemsex and other high-risk group sex activities has contributed to an increased incidence of STIs. Pre-exposure prophylaxis (PrEP) for HIV does not protect against other infections, and the use of condoms is frequently abandoned during these practices [18].
Among our study population, roughly 48.7% of cases were aged 29 or younger, and 30.5% were aged 30 to 39 at the time of diagnosis (similar to the Spanish national data profile) [19]. In our dataset, the overall ratio of male cases to female cases was 4:1, higher than the overall European ratio of 3.2:1 [6].
It is not surprising that a large proportion of our population with N. gonorrhoeae was coinfected with other STDs (gonorrhoea and chlamydia being the most frequent association), especially among young women [20]. This supports the findings of previous Spanish studies [21] and Spanish national guidelines that gonorrhoea infections should be treated with combination therapy (ceftriaxone or cefixime with azithromycin) to target both infections, in accordance with ECDC guidance [22]. Cervical ectopy of young women (< 25 years old) has been identified as a risk factor for chlamydia and gonorrhoea co-infection [23].
Antimicrobial surveillance data was available from the 6,292 (44.2%) culture isolates studied. Laboratory culture slightly decreased from 47.7% in 2016 to 42.9% in 2019, due to the shift from the use of bacterial culture to NAAT for gonorrhoea diagnosis. This trend has also been attested to in Canada, where 21.8% of gonococcal infections are diagnosed through culture [24]. This is a concern, as cultures are required for antimicrobial susceptibility testing and to monitor AMR. In 2015-16, the WHO's Gonococcal Antimicrobial Surveillance Programme (WHO-GASP) documented sustained high levels of ciprofloxacin resistance [25]. And although the resistance rate to ciprofloxacin observed (57.1%) is similar to that of neighbouring European countries (53%), it is notably higher than that reported in Portugal (46%) or the Netherlands (36%) [26].
Combination therapy is now recommended for treating noncomplicated gonorrhoea, given the growing resistance to cephalosporins described for N. gonorrhoeae in recent years [27] and treatment failures detected. Health professionals therefore need to be made aware of the importance of performing a cure test in cases where symptoms do not improve [5].
During the period of our study, we observed a decrease in the resistance rate for cefixime, especially during the later years (2018-2019). One probable explanation could be the low number of strains studied for this antibiotic, affecting the relevance of continued monitoring. It has been suggested [28] that cefixime could be reconsidered as an alternative to ceftriaxone in first-line dual therapy if this decrease is sustained over time.
The increase in resistance to azithromycin observed in Catalonia, from 6.1% in 2016 to 16% in 2019, is comparable to a similar increase in Germany (from 1.3% in 2014 to 12.2% in 2020) [29], but higher than that observed in Canada (from 0.8% in 2012 to 7.2% in 2016) [24]. Azithromycin resistance in N. gonorrhoeae is established and spreading in Catalonia, exceeding the 5% threshold at which WHO guidelines recommend a review to determine whether a particular antimicrobial is the appropriate treatment.
In our study, however, the rate of resistance to ceftriaxone remains low and stable, and AMR has led a number of European countries to adopt high-dose ceftriaxone monotherapy in recent years (ECDC, 2019) [30]. Therefore, close AMR surveillance following gonococcal culture is urgently needed.
Diminishing azithromycin susceptibility combined with ceftriaxone resistance is a major concern and threatens the effectiveness of the currently highly effective dual therapy regimen of ceftriaxone in conjunction with azithromycin. Even though resistance to cefixime was decreasing in our region in 2019, cefixime resistance needs to be monitored closely, particularly because gonococcal strains resistant to both cefixime and ceftriaxone continue to spread internationally [30].
We have also detected four multidrug-resistant strains that are resistant to azithromycin and ceftriaxone and cefixime, and similar evidence has emerged in other countries where the first cases of MDR-NG appeared in February 2018 (1 case in England and 2 cases in Australia) [31]. These strains are a sign of the growing threat of multidrug-resistant gonococcal strains in the context of limited therapeutic alternatives, lack of a vaccine and limited surveillance capacity in many regions of the world.
Most reinfections occurred between 3 to 6 months after the first episode and were more common in males. A recent study of 9,241 cases conducted in New Zealand showed that, of the 34% (3,151) retested at 6 weeks and 6 months, 21% tested positive [32]. Given that reinfection with chlamydia or gonorrhea is common and can lead to significant reproductive health complications, testing for reinfection after treatment might be recommended despite individual and social characteristics associated with gonorrhea patients limiting the success of this strategy [33].
The present study has a number of limitations. First, we are aware of a possible under-notification of cases, as notification is reliant on a passive system. However, the MRSC conducts a search for suspected cases reported by health professionals, which are then validated by epidemiologists and confirmed by microbiology laboratories. Second, cultures were carried out for less than half of the samples analysed, probably because of the comparative speed of diagnosis afforded by the molecular test.
The low percentage of cultures, as shown in the study, is hampering the monitoring and evaluation of AMR and facilitating the spread of infection. Lastly, antibiotic sensitivity detection rates are often low (samples arriving late to the laboratory are no longer viable, rendering antibiotic susceptibility testing impossible), indicating that a far larger number of samples is needed to build cultures and tests for antibiotic sensitivity.

Conclusion
In Catalonia, 10% of the N. gonorrhoeae isolates studied between 2016 and 2019 were resistant to azithromycin. According to the WHO, resistance above 5% indicates an alert to review treatment guidelines. Resistance to ciprofloxacin, tetracycline and penicillin was relatively high and stable, while resistance to ceftriaxone and cefixime remained low; and multidrug-resistant strains of N. gonorrhoeae increased throughout the period. Epidemiological surveillance of N. gonorrhoeae must be enhanced, paying particular attention to improving the culture of strains and AMR data.
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