Antimicrobial resistance and molecular epidemiology using whole-genome sequencing of Neisseria gonorrhoeae in Ireland, 2014-2016: focus on extended-spectrum cephalosporins and azithromycin.

High-level resistance and treatment failures with ceftriaxone and azithromycin, the first-line agents for gonorrhoea treatment are reported and antimicrobial-resistant Neisseria gonorrhoeae is an urgent public health threat. Our aims were to determine antimicrobial resistance rates, resistance determinants and phylogeny of N. gonorrhoeae in Ireland, 2014-2016. Overall, 609 isolates from four University Hospitals were tested for susceptibility to extended-spectrum cephalosporins (ESCs) and azithromycin by the MIC Test Strips. Forty-three isolates were whole-genome sequenced based on elevated MICs. The resistance rate to ceftriaxone, cefixime, cefotaxime and azithromycin was 0, 1, 2.1 and 19%, respectively. Seven high-level azithromycin-resistant (HLAzi-R) isolates were identified, all susceptible to ceftriaxone. Mosaic penA alleles XXXIV, X and non-mosaic XIII, and G120K plus A121N/D/G (PorB1b), H105Y (MtrR) and A deletion (mtrR promoter) mutations, were associated with elevated ESC MICs. A2059G and C2611T mutations in 23S rRNA were associated with HLAzi-R and azithromycin MICs of 4-32 mg/L, respectively. The 43 whole-genome sequenced isolates belonged to 31 NG-MAST STs. All HLAzi-R isolates belonged to MLST ST1580 and some clonal clustering was observed; however, the isolates differed significantly from the published HLAzi-R isolates from the ongoing UK outbreak. There is good correlation between previously described genetic antimicrobial resistance determinants and phenotypic susceptibility categories for ESCs and azithromycin in N. gonorrhoeae. This work highlights the advantages and potential of whole-genome sequencing to be applied at scale in the surveillance of antibiotic resistant strains of N. gonorrhoeae, both locally and internationally.

Clinical and analytical evaluation of the new Aptima Mycoplasma genitalium assay, with data on M. genitalium prevalence and antimicrobial resistance in M. genitalium in Denmark, Norway and Sweden in 2016.

OBJECTIVES: Mycoplasma genitalium (MG) causes urethritis and cervicitis, potentially causing reproductive complications. Resistance in MG to first-line (azithromycin) and second-line (moxifloxacin) treatment has increased. We examined the clinical and analytical performance of the new Conformité Européene (CE)/in vitro diagnostics (IVD) Aptima Mycoplasma genitalium assay (CE/IVD AMG; Hologic); the prevalence of MG, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG); and MG resistance to azithromycin and moxifloxacin in Denmark, Norway and Sweden in 2016. METHODS: From February 2016 to February 2017, urogenital and extragenital (only in Denmark) specimens from consecutive attendees at three sexually transmitted disease clinics were tested with the CE/IVD AMG, the research-use-only MG Alt TMA-1 assay (Hologic), Aptima Combo 2 (CT/NG) assay and a laboratory-developed TaqMan real-time mgpB quantitative real-time PCR (qPCR). Resistance-associated mutations were determined by sequencing. Strains of MG and other mycoplasma species in different concentrations were also tested. RESULTS: In total 5269 patients were included. The prevalence of MG was 7.2% (382/5269; 4.9-9.8% in the countries). The sensitivity of the CE/IVD AMG, MG Alt TMA-1 and mgpB qPCR ranged 99.13-100%, 99.13-100% and 73.24-81.60%, respectively, in the countries. The specificity ranged 99.57-99.96%, 100% and 99.69-100%, respectively. The prevalence of resistance-associated mutations for azithromycin and moxifloxacin was 41.4% (120/290; 17.7-56.6%) and 6.6% (18/274; 4.1-10.2%), respectively. Multidrug resistance was found in all countries (2.7%; 1.1-4.2%). CONCLUSIONS: Both transcription-mediated amplification (TMA)-based MG assays had a highly superior sensitivity compared to the mgpB qPCR. The prevalence of MG and azithromycin resistance was high. Validated and quality-assured molecular tests for MG, routine resistance testing of MG-positive samples and antimicrobial resistance surveillance are crucial.

First antimicrobial resistance data and genetic characteristics of Neisseria gonorrhoeae isolates from Estonia, 2009-2013.

Gonorrhoea is a sexually transmitted infection with major public health implications and Neisseria gonorrhoeae has developed resistance to all antimicrobials introduced for treatment. Enhanced surveillance of antimicrobial resistance in N. gonorrhoeae is crucial globally. This is the first internationally reported antimicrobial resistance data for N. gonorrhoeae from Estonia (44 isolates cultured in 2009-2013). A high prevalence of resistance was observed for azithromycin, ciprofloxacin and tetracycline. One and two isolates with resistance and decreased susceptibility to the last remaining first-line treatment option ceftriaxone, respectively, were identified. It is crucial to implement surveillance of gonococcal antimicrobial resistance (ideally also treatment failures) in Estonia.

Four treatment failures of pharyngeal gonorrhoea with ceftriaxone (500 mg) or cefotaxime (500 mg), Sweden, 2013 and 2014.

We describe four cases in Sweden of verified treatment failures of pharyngeal gonorrhoea with ceftriaxone (500 mg; n=3) or cefotaxime (500 mg; n=1) monotherapy. All the ceftriaxone treatment failures were caused by the internationally spreading multidrug-resistant gonococcal NG-MAST genogroup 1407 clone. Increased awareness of treatment failures is crucial particularly when antimicrobial monotherapy is used. Frequent test of cure and appropriate verification/falsification of suspected treatment failures, as well as implementation of recommended dual antimicrobial therapy are imperative.

Treatment failure of pharyngeal gonorrhoea with internationally recommended first-line ceftriaxone verified in Slovenia, September 2011.

We describe the second case in Europe of verified treatment failure of pharyngeal gonorrhoea, caused by an internationally occurring multidrug-resistant gonococcal clone, with recommended first-line ceftriaxone 250 mg in Slovenia. This is of grave concern since ceftriaxone is last remaining option for empirical treatment. Increased awareness of ceftriaxone failures, more frequent test-of-cure, strict adherence to regularly updated treatment guidelines, and thorough verification/falsification of suspected treatment failures are essential globally. New effective treatment options are imperative.

Clinical Neisseria gonorrhoeae isolate with a N. meningitidis porA gene and no prolyliminopeptidase activity, Sweden, 2011: danger of false-negative genetic and culture diagnostic results.

We describe a Neisseria gonorrhoeae strain, found in Sweden in 2011, that harbours a N. meningitidis porA gene causing false-negative results in PCRs targeting the gonococcal porA pseudogene. Furthermore, the strain had no prolyliminopeptidase (PIP) activity that many commercial biochemical kits for species verification in culture rely on. Enhanced awareness of the spread of such strains and screening for them can be crucial.

Phenotypic and genotypic properties of Neisseria gonorrhoeae isolates in Norway in 2009: antimicrobial resistance warrants an immediate change in national management guidelines.

Despite rapidly diminishing treatment options for Neisseria gonorrhoeae and high levels of ciprofloxacin resistance worldwide, Norwegian guidelines still recommend ciprofloxacin as empirical treatment for gonorrhea. The present study aimed to characterize phenotypical and genotypical properties of N. gonorrhoeae isolates in Norway in 2009. All viable N. gonorrhoeae isolates (n = 114) from six university hospitals in Norway (2009) were collected, representing 42% of all notified gonorrhea cases. Epidemiological data were collected from the Norwegian Surveillance System for Communicable Diseases and linked to phenotypical and genotypical characteristics for each N. gonorrhoeae isolate. Resistance levels to the antimicrobials examined were: ciprofloxacin 78%, azithromycin 11%, cefixime 3.5%, ceftriaxone 1.8%, and spectinomycin 0%. The minimum inhibitory concentrations of gentamicin varied from 1.5 to 8 mg/L. Forty-one (36%) of the isolates were ß-lactamase-producing, 17 displayed penA mosaic alleles, and 72 different N. gonorrhoeae multiantigen sequence types (ST; 37 novel) were identified. The most common ST was ST1407 (n = 11), containing penA mosaic allele. Four of these isolates displayed intermediate susceptibility/resistance to cefixime. The N. gonorrhoeae strains circulating in Norway were highly diverse. The level of ciprofloxacin resistance was high and the Norwegian management guidelines should promptly exclude ciprofloxacin as an empirical treatment option for gonorrhea.

First Neisseria gonorrhoeae strain with resistance to cefixime causing gonorrhoea treatment failure in Austria, 2011.

We describe the first cefixime-resistant Neisseria gonorrhoeae strain in Austria that caused treatment failure.It follows the first five cases in Europe of cefixime treatment failure, reported in Norway in 2010 and the United Kingdom in 2011. Effective treatment of gonorrhoea is crucial for public health control and, at present, requires substantially enhanced awareness, more frequent test-of-cure, interaction with experts after therapeutic failure, tracing and therapy of contacts, and surveillance of gonococcal antimicrobial resistance and treatment failures worldwide.

Ceftriaxone treatment failure of pharyngeal gonorrhoea verified by international recommendations, Sweden, July 2010.

This report describes one case of verified treatment failure of pharyngeal gonorrhoea using ceftriaxone in Sweden. Previous reports described verified treatment failure of urogenital gonorrhoea using the internationally recommended first-line drug cefixime, but not with ceftriaxone, the last remaining option for empirical treatment of gonorrhoea. Enhanced awareness of clinical failures, pharmacodynamic considerations, follow-up and test of cure, adherence to appropriate case management and treatment guidelines as well as verification/falsification of presumed clinical treatment failures should be emphasised worldwide.

Two cases of verified clinical failures using internationally recommended first-line cefixime for gonorrhoea treatment, Norway, 2010.

Neisseria gonorrhoeae has developed resistance to most of the available therapeutic antimicrobials. The susceptibility to extended-spectrum cephalosporins, the last remaining first-line treatment option, is decreasing globally. This report describes the first two cases outside Japan of verified gonorrhoea clinical failures using internationally recommended first-line cefixime treatment. Enhanced awareness and more frequent follow-up examination, test-of-cure and appropriate verification/falsification of presumed clinical treatment failures, involving several clinical and laboratory parameters should be strongly emphasised worldwide.