Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Aetiological molecular identification of sexually transmitted infections that cause urethral discharge syndrome and genital ulcer disease in Brazilian men: a nationwide study.

BACKGROUND: Little is known about the aetiology of urethral discharge syndrome (UDS) and genital ulcer disease (GUD) in Brazil due to limited access to laboratory tests and treatment based mainly on the syndromic approach. OBJECTIVES: To update Brazilian treatment guidelines according to the current scenario, the first nationwide aetiological study for UDS and GUD was performed. METHODS: Male participants with urethral discharge (UD) and/or genital ulcer (GU) reports were enrolled. Sample collection was performed by 12 sentinel sites located in the five Brazilian regions. Between 2018 and 2020, 1141 UD and 208 GU samples were collected in a Universal Transport Medium-RT (Copan). A multiplex quantitative PCR kit (Seegene) was used to detect UD: Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), M. hominis (MH), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Ureaplasma parvum (UP), U. urealyticum (UU) and another kit to detect GU: cytomegalovirus (CMV), Haemophilus ducreyi (HD), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), lymphogranuloma venereum (LGV), Treponema pallidum (TP) and varicella-zoster virus (VZV). RESULTS: In UD samples, the frequency of pathogen detection was NG: 78.38%, CT: 25.6%, MG: 8.3%, UU: 10.4%, UP: 3.5%, MH: 3.5% and TV: 0.9%. Coinfection was assessed in 30.9% of samples, with 14.3% of NG/CT coinfection. The most frequent pathogen identified in GU was HSV2, present in 40.8% of the samples, followed by TP at 24.8%, LGV and CMV at 1%, and HSV1 at 0.4%. Coinfection of TP/HSV2 was detected in 4.4% of samples. VZV and HD were not detected. In 27.7% of the GU samples, no pathogen was detected. CONCLUSION: This study provided the acquisition of unprecedented data on the aetiology of UDS and GUD in Brazil, demonstrated the presence of a variety of pathogens in both sample types and reaffirmed the aetiologies known to be most prevalent globally.

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance.

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC.

National surveillance of Neisseria gonorrhoeae antimicrobial susceptibility and epidemiological data of gonorrhoea patients across Brazil, 2018-20.

Objectives: To (i) describe the nationwide antimicrobial susceptibility of Neisseria gonorrhoeae (NG) isolates cultured across Brazil in 2018-20 and compare it with NG antimicrobial resistance data from 2015-16, and (ii) present epidemiological data of the corresponding gonorrhoea patients in 2018-20. Methods: Twelve representative sentinel sites cultured NG isolates from men with urethral discharge. Susceptibility to eight antimicrobials was examined using agar dilution method, according to WHO standards. The consenting participants were invited to provide epidemiological data. Results: In total, 633 NG isolates (one isolate per participant) were analysed, and 449 (70.9%) questionnaires were answered. Heterosexual (68.2%) and homosexual (23.1%) sexual orientations were common, and most prevalent types of unprotected sexual intercourse were vaginal insertive (69.9%), oral giving (56.6%) and anal insertive (47.4%). The levels of in vitro NG resistance to ciprofloxacin, tetracycline, benzylpenicillin, azithromycin, cefixime, gentamicin, spectinomycin and ceftriaxone were 67.3%, 40.0%, 25.7%, 10.6%, 0.3%, 0%, 0% and 0%, respectively. Compliance with the recommended first-line ceftriaxone 500 mg plus azithromycin 1 g therapy was high (90.9%). Conclusions: Compared with 2015-16, ciprofloxacin resistance has remained high and azithromycin and cefixime resistance rates have increased in Brazil. Resistance remained lacking to ceftriaxone, gentamicin and spectinomycin, which all are gonorrhoea treatment options. The increasing azithromycin resistance in Brazil and internationally may threaten the future use of azithromycin in dual regimens for treatment of gonorrhoea. Consequently, continued and enhanced quality-assured surveillance of gonococcal AMR, and ideally also treatment failures and including WGS, is imperative in Brazil and worldwide.

Spectinomycin, gentamicin, and routine disc diffusion testing: An alternative for the treatment and monitoring of multidrug-resistant Neisseria gonorrhoeae?

INTRODUCTION: Neisseria gonorrhoeae is a major concern of public health due to its extraordinary capacity to develop and acquire resistance to different antimicrobials used to treat gonorrhoea. Limited treatment options and uncontrolled transmission have raised the need to assess the antimicrobial susceptibility profile of the isolates and to establish affordable alternatives for laboratory diagnosis. OBJECTIVES: This study aimed to (i) determine the susceptibility profile of 336 clinical isolates of N. gonorrhoeae to ceftriaxone, azithromycin, ciprofloxacin, spectinomycin and gentamicin by the gold standard agar dilution method; (ii) assess the agreement among agar dilution and disc diffusion results for ciprofloxacin, azithromycin, ceftriaxone, spectinomycin and gentamicin. RESULTS: All isolates were susceptible to ceftriaxone and spectinomycin. The levels of resistance to azithromycin and ciprofloxacin were 3.9% and 35.1%, respectively. Intermediate susceptibility to gentamicin was observed in 19.4% of isolates. There was 100% agreement between methods for spectinomycin and ceftriaxone, 99.7% for ciprofloxacin, and 85.7% for azithromycin. For gentamicin, there was 86.3% agreement between agar dilution and disc diffusion, resulting in intermediate susceptible by one method and susceptible by the other method, defined as minor errors. The discordance among agar dilution and disc diffusion results is acceptable for ciprofloxacin, ceftriaxone and spectinomycin as per CLSI M23-Ed4. CONCLUSIONS: Spectinomycin and gentamicin can be considered in some cases as options for the treatment of gonorrhoea in Brazil. Disc diffusion can be an alternative method in routine testing with comparable accuracy to agar dilution.