Azithromycin-resistant Neisseria gonorrhoeae spreading amongst men who have sex with men (MSM) and heterosexuals in New South Wales, Australia, 2017.

Objectives: To identify the genetic basis of resistance as well as to better understand the epidemiology of a recent surge in azithromycin-resistant Neisseria gonorrhoeae in New South Wales, Australia. Methods: Azithromycin-resistant N. gonorrhoeae isolates (n = 118) collected from 107 males, 10 females and 1 transsexual between January and July 2017 were genotyped using a previously described iPLEX method. The results were compared with phenotypic resistance profiles and available patient data. Results: The iPLEX results revealed 10 different N. gonorrhoeae genotypes (designated AZI-G1 to AZI-G10) of which three were responsible for the majority of infections; AZI-G10 (74.6%, 88 isolates; 87 males and 1 transsexual), AZI-G4 (11.0%, 13 isolates; 7 males and 6 females) and AZI-G7 (6.8%, 8 isolates; 7 males and 1 female). The observed resistance was attributable to one of two different azithromycin resistance mechanisms; the 23S rRNA C2611T mutation was identified in 24% of isolates, whereas the majority of resistance (76%) was associated with a meningococcal-type mtrR variant. Additionally, one isolate was found to harbour both the 23S rRNA C2611T mutation and a type XXXIV mosaic penA sequence associated with cephalosporin resistance. Conclusions: These data indicate outbreaks of azithromycin-resistant gonococci amongst networks of MSM and heterosexuals in New South Wales. The results also provide further evidence that azithromycin may soon be an ineffective treatment option for gonococcal infection and highlight the urgent need to explore alternative therapies.

Molecular Antimicrobial Resistance Surveillance for Neisseria gonorrhoeae, Northern Territory, Australia.

Neisseria gonorrhoeae antimicrobial resistance (AMR) is a globally recognized health threat; new strategies are needed to enhance AMR surveillance. The Northern Territory of Australia is unique in that 2 different first-line therapies, based primarily on geographic location, are used for gonorrhea treatment. We tested 1,629 N. gonorrhoeae nucleic acid amplification test-positive clinical samples, collected from regions where ceftriaxone plus azithromycin or amoxicillin plus azithromycin are recommended first-line treatments, by using 8 N. gonorrhoeae AMR PCR assays. We compared results with those from routine culture-based surveillance data. PCR data confirmed an absence of ceftriaxone resistance and a low level of azithromycin resistance (0.2%), and that penicillin resistance was <5% in amoxicillin plus azithromycin regions. Rates of ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae were lower when molecular methods were used. Molecular methods to detect N. gonorrhoeae AMR can increase the evidence base for treatment guidelines, particularly in settings where culture-based surveillance is limited.

Changes in the rates of Neisseria gonorrhoeae antimicrobial resistance are primarily driven by dynamic fluctuations in common gonococcal genotypes.

Objectives: To examine how gonococcal genotypes and associated changes over time influence rates of Neisseria gonorrhoeae antimicrobial resistance. Methods: All available N. gonorrhoeae isolates collected in New South Wales, Australia in the first half of both 2012 and 2014 were genotyped using the Agena MassARRAY iPLEX platform. Genotypic data were compared with phenotypic antimicrobial resistance profiles over time. We focused on penicillin and ciprofloxacin as significant increases in resistance to both antibiotics were observed over this time period. Results: Genotyping data were obtained for 760 and 782 isolates in 2012 and 2014, respectively. A total of 162 distinct genotypes were identified in the study, including 36 (22.2%) genotypes present in both years ( persisting genotypes), 54 (33.3%) observed in 2012 only and 72 (44.4%) observed in 2014 only (s ingle-year genotypes). Overall, persisting genotypes comprised 15 of the 20 most common genotypes, 8 of which showed a significant change in proportion from 2012 to 2014. Persisting genotypes also comprised the majority (>70%) of ciprofloxacin- and penicillin-resistant isolates in both years. Significant fluctuations in the most common persisting genotypes accounted for the majority of observed increases in both ciprofloxacin and penicillin resistance. Single-year genotypes contributed to ∼20% of ciprofloxacin and penicillin resistance in each year. Conclusions: The results show that the gonococcal genotypes persisting in the study population fluctuated significantly within a 3 year period, with numerous other genotypes appearing or disappearing. It is the net effect of these changes that determines N. gonorrhoeae antimicrobial resistance levels within the population.

Mixed gonococcal infections in a high-risk population, Sydney, Australia 2015: implications for antimicrobial resistance surveillance?

OBJECTIVES: Previous studies have shown that mixed-strain gonococcal infections can occur. However, it remains unclear whether such infections impact upon the reliability of Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance. In this study, we aimed to resolve this question by intensively sampling isolates from gonorrhoea-positive specimens in a high-risk population in Sydney, Australia. METHODS: A total of 615 N. gonorrhoeae isolates, originating from 63 clinical samples (31 rectal swabs and 32 throat swabs), were characterized. All isolates were subject to N. gonorrhoeae identification, antimicrobial susceptibility testing and genotyping by SNP-based MLST. RESULTS: Only 2 of the 63 (3.2%) samples provided evidence of mixed-strain infections. These comprised two rectal swabs that harboured isolates of different SNP-based MLST genotypes; however, the AMR susceptibility profiles of the different genotypes from these samples were indistinguishable. Within-sample differences in the AMR susceptibility profiles were observed for a further seven samples; however, the differences were not considered significant; MIC values were typically within a 2-fold difference or were close to test breakpoints. CONCLUSIONS: Results of this study provide further evidence that mixed-strain gonococcal infections do occur, although at low prevalence. Our data indicate that at a population level such infections are unlikely to impact significantly upon N. gonorrhoeae AMR surveillance.

A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility.

OBJECTIVES: The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. METHODS: The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. RESULTS: The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. CONCLUSIONS: The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

Real-time PCR detection of Neisseria gonorrhoeae susceptibility to penicillin.

OBJECTIVES: The objective of this study was to develop a real-time PCR assay targeting the gonococcal porB gene (PorB-PCR) for predicting susceptibility of Neisseria gonorrhoeae to penicillin. This complements a previously described PCR assay for detecting penicillinase-producing N. gonorrhoeae (PPNG) developed by our laboratory (PPNG-PCR). METHODS: The PorB-PCR assay was designed using six probes to characterize various combinations of amino acids at positions 101 and 102 of the PorB1b class protein, including the WT G101/A102 and mutant G101K/A102D, G101K/A102N and G101K/A102G sequences, as well as the PorB1a sequence. The ability of these sequences to predict penicillin susceptibility was initially assessed using 2307 N. gonorrhoeae isolates from throughout Australia for which phenotypic susceptibility data were available. The assay was then applied to N. gonorrhoeae-positive clinical specimens (n = 70). Specificity was assessed by testing commensal Neisseria strains (n = 75) and N. gonorrhoeae-negative clinical specimens (n = 171). RESULTS: Testing of the 2307 N. gonorrhoeae isolates using PorB-PCR to detect G101/A102 and PorB1a sequences identified a total of 78.4% (61.2% and 17.2%, respectively) of penicillin-susceptible isolates with specificities of 97.4% and 99.3% and positive predictive values of 98.8% and 98.9%, where PPNG strains were simultaneously identified and excluded. Similar performance data were obtained when the PorB-PCR assay was applied to the N. gonorrhoeae-positive clinical specimens. No false-positive results were observed for the N. gonorrhoeae-negative samples and no cross-reactions were observed with the non-gonococcal species. CONCLUSIONS: When used in parallel with the previously described PPNG-PCR, the PorB-PCR approach has the potential to facilitate individualized treatment of gonorrhoea using penicillin.

Is gentamicin a viable therapeutic option for treating resistant Neisseria gonorrhoeae in New South Wales?

ABSTRACT: The key issues with Neisseria gonorrhoeae infections, in Australia and elsewhere, are coincident increases in disease rates and in antimicrobial resistance (AMR), although these factors have not been shown to be correlated. Despite advances in diagnosis, control of this disease remains elusive, and incidence in Australia continues to increase. Of the Australian jurisdictions, New South Wales (NSW) has the highest N. gonorrhoeae notifications, and over the five-year period 2015-2019, notifications in NSW have increased above the national average (by 116% versus 85%, respectively). Gonococcal disease control is reliant on effective antibiotic regimens. However, escalating AMR in N. gonorrhoeae is a global health priority, as the collateral injury of untreated infections has substantive impacts on sexual and newborn health. Currently, our first-line therapy for gonorrhoea is also our last line, with no ideal alternative identified. Despite some limitations, gentamicin is licensed and readily available in Australia, and is proposed for treatment of resistant N. gonorrhoeae in national guidelines; however, supportive published microbiological data are lacking. Analysis of gonococcal resistance patterns within Australia for the period 1991-2019, including 35,000 clinical isolates from NSW, illustrates the establishment and spread of population-level resistance to all contemporaneous therapies. An analysis of gentamicin susceptibility on 2,768 N. gonorrhoeae clinical isolates from NSW, for the period 2015-2020, demonstrates that the median minimum inhibitory concentration (MIC) for gentamicin in NSW has remained low, at 4.0 mg/L, and resistance was not detected in any isolate. There has been no demonstration of MIC drift over time (p = 0.91, Kruskal-Wallis test), nor differences in MIC distributions according to patients' sex or site of specimen collection. This is the first large-scale evaluation of gentamicin susceptibility in N. gonorrhoeae in Australia. No gentamicin resistance was detected in clinical isolates, 2015-2020, hence this is likely to be an available treatment option for resistant gonococcal infections in NSW.

A comparison of agar dilution with the Calibrated Dichotomous Sensitivity (CDS) and Etest methods for determining the minimum inhibitory concentration of ceftriaxone against Neisseria gonorrhoeae.

OBJECTIVES: The objective of this study was to compare the Calibrated Dichotomous Sensitivity (CDS) based agar dilution (CDS AD) method with the Etest (bioMérieux SA) methods using 2 method protocols for determining the minimum inhibitory concentration (MIC) of ceftriaxone against Neisseria gonorrhoeae. The two method protocols were the manufacturer's protocol for which the Clinical and Laboratory Standards Institute (CLSI) interpretative criteria for Neisseria gonorrhoeae could be applied, and the CDS-adapted protocol. Comparability of MIC data is critical for situation analysis and monitoring trends in global antimicrobial analysis. METHODS: Two hundred and forty eight clinical isolates of N. gonorrhoeae and the World Health Organisation (WHO) N. gonorrhoeae reference strains were tested using the three methods. RESULTS: When compared, CDS AD and CDS Etest gave a regression R(2) value of 94%, the Pearson's correlation coefficient was 97% and a paired comparison within one log2 dilution was 98%. The CDS AD and the Etest (CLSI) comparison gave a regression R(2) value of 90%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution was 98%. The comparison of the CDS Etest and CLSI Etest gave a regression R(2) value of 91%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution of 99%. Importantly, there was robust agreement between all three methods for the categorization of susceptibility of Neisseria gonorrhoeae isolates using the WHO nominated breakpoint for decreased susceptibility to ceftriaxone (≥0.125 µg/mL). CONCLUSIONS: The CDS Etest method is comparable to agar dilution and the Etest methods for determining the MIC of ceftriaxone against N. gonorrhoeae.

Pharyngeal Gonorrhoea in Women: An Important Reservoir for Increasing Neisseria gonorrhoea Prevalence in Urban Australian Heterosexuals?

We aim to characterize sexual behavioral aspects of heterosexual Neisseria gonorrhoea (NG) acquisition in two Sexually Transmitted Diseases clinics in Sydney, Australia, in 2008-2012. Of 167 NG cases, 102 were heterosexually acquired with a trend of increasing NG prevalence in heterosexuals from 1.1% (95% CI 0.6-2.1) in 2008 to 3.0% (95% CI 2.0-4.0) in 2012 (P = 0.027). Of heterosexual male cases, unprotected fellatio was the likely sexual activity for NG acquisition in 21/69 (30.4%) and commercial sex work (CSW) contact the likely source in 28/69 (40.6%). NG prevalence overall in CSW (2.2%) was not significantly higher than in non-CSW (1.2%) (P = 0.15), but in 2012 there was a significant increase in NG prevalence in CSW (8.6%) compared to non-CSW (1.6%) (P < 0.001). Pharyngeal NG was found in 9/33 (27.3%) female cases. Decreased susceptibility to ceftriaxone (MIC ≥ 0.03 mg/L) occurred in 2.5% NG isolates, none heterosexually acquired. All were azithromycin susceptible. A significant trend of increasing prevalence of heterosexual gonorrhoea in an urban Australian STD clinic setting is reported. We advocate maintenance of NG screening in women, including pharyngeal screening in all women with partner change who report fellatio, as pharyngeal NG may be an important reservoir for heterosexual transmission. Outreach to CSW should be enhanced.