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It is widely recognised that few antimicrobials remain effective in the treatment of Neisseria gonorrhoeae infection and that gonorrhoea could become untreatable in the future. Key factors in maintaining treatment options include timely diagnosis, adherence to treatment, effective follow-up, test of cure (TOC) and partner notification (PN), identification of potential treatment failures and surveillance for emerging resistance.1 The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) provides susceptibility data which has twice led to changes in national treatment guidelines, most recently in 2011 to recommend 500 mg ceftriaxone intramuscularly combined with 1 g azithromycin orally.2 GRASP provides invaluable data to inform treatment policy, but is a 3 month sentinel surveillance programme. Many laboratories additionally refer isolates showing potential resistance to front-line antimicrobials to the Sexually Transmitted Bacteria Reference Unit (STBRU). In early 2015, STBRU issued a letter requesting all microbiology laboratories in England to adopt this referral practice, to improve temporal and geographical monitoring for potential emerging resistance.
From November 2014 to March 2015, eight gonococcal isolates exhibiting high-level azithromycin resistance (HL-AziR) (MIC >256 mg/L) were identified in Leeds. To put this into context, just one other case was identified in Leeds in 2014, and one in 2012. There has been only a moderate increase in the number of cases of gonorrhoea in Leeds, so this would not account for the increase of HL-AziR. HL-AziR is a rare phenotype observed sporadically in the UK.3 This increased incidence suggested an outbreak, necessitating epidemiological and microbiological investigation.
Isolates resistant to azithromycin and/or ceftriaxone were referred from primary laboratories to STBRU for confirmatory testing by Etest.
Whole genome sequencing
Whole genome sequencing (WGS) was performed on seven of the HL-AziR ‘outbreak’ isolates by the Public Health England (PHE) Genome Sequencing Unit using Nextera library preparation and the Illumina HiSeq 2500. Additionally six strains with azithromycin MICs ranging from 0.125 mg/L to 0.75 mg/L and two HL-Azi R strains from Leeds and London sourced during January 2014 and March 2015 were included as controls.
Reads were mapped to NCCP11945 genome using bwa.4 Variants were called using Genome Analysis Tool Kit (GATK) 2.6.5 and parsed to retain high quality single nucleotide polymorphisms (SNPs) based on the following conditions: (depth ≥5, allelic depth ratio ≥0.8, mapping quality ≥30, MQ0 ≤0.05 and distance to nearest SNP >10). A maximum likelihood tree using RAxML5 was implemented on the CyberInfrastructure for Phylogenetic Research (CIPRES) portal.
NG multiantigen sequence type
To extract N. gonorrhoeae multi-antigen sequence type (NG-MAST), all WGS samples were de novo assembled using SPAdes V.3.5.0 with default settings and kmers 21,33,55,77 and 91. Reference por and tbpB alleles and Sequence Tags definitions were downloaded from http://www.ng-mast.net. Blast databases for por and tbpB were compiled using the NCBI Blast+ software suite 2.2.27, and scaffolds for each sample searched with blastn for matches to reference alleles.
Resistance mutation detection
Fastq files were aligned to a reference genome using the short read mapping tool Bowtie. Samtools & Mpileup software then generated positional information per base which could be interrogated for SNPs in relation to the reference. Resistance analysis was run using a reference sequence for the 23S rRNA gene.
Five patients were male, three were female, and all were heterosexual. Two had previous gonorrhoea, five had previous chlamydia. Geographical spread was throughout Leeds, four from areas of high deprivation. All had genital infection, but two women also had pharyngeal infection. Two men and one woman were coinfected with chlamydia. All men and one woman were treated with ceftriaxone 500 mg and azithromycin 1gm; TOCs were negative. One woman, treated for pelvic inflammatory disease (ceftriaxone 500 mg and doxycycline 100 mg twice daily for 14 days), declined to return for TOC. The other woman (who also had pharyngeal infection) was treated with spectinomycin 2 g. At TOC she was cervix-negative but pharyngeal-positive. She was successfully retreated with ciprofloxacin 500 mg. Spectinomycin has been associated with pharyngeal treatment failures and is not recommended to treat infection at this site.
Enhanced patient contact and PN (performed using texts, phone calls and letters as necessary until all identifiable contacts had been notified and TOCs performed) was commenced rapidly. One man named two of the women as contacts. The remaining five cases had traceable partners who were gonorrhea-negative as well as unidentifiable contacts. A control team convened to support the outbreak investigation informed all general practitioners in Leeds and all regional sexual health clinics and microbiology laboratories about the outbreak.
Characterisation of bacterial isolates exhibiting HL-AziR
All eight isolates were sensitive to ceftriaxone (MIC range 0.004–0.023 mg/L).
All seven strains characterised by WGS were NG-MAST ST, 9768 (por and tbpB alleles 5759 and 29, respectively). Comparison of WGS data to achieve greater discrimination than NG-MAST alone, confirmed that the genomes were identical with zero or just a single SNP difference among this cluster of strains, which was distinct from strains with lower azithromycin MICs and other HL-AziR strains from earlier time periods or different regions (figure 1).
All HL-AziR isolates showed mutation A2143G in all four alleles of the 23S rRNA gene.
We report an increased incidence of N. gonorrhoeae infections exhibiting HL-AziR in Leeds referred to STBRU from November 2014 to March 2015. All cases were identified in heterosexual patients, three of whom were linked. The use of WGS to define NG-MAST type and to compare whole genomes for seven of the HL-AziR strains showed that they were virtually identical. The rapid emergence of a cluster of identical strains in a relatively restricted patient group suggested an outbreak requiring urgent intervention to prevent further dissemination of a phenotype presenting a significant threat to the current front-line therapy for gonorrhoea.
A potential outbreak of HL-AziR, predominantly in Liverpool, was identified in 2007.6 However, this was identified retrospectively via GRASP at a time when azithromycin was not a recommended front-line therapy. In contrast this outbreak was identified quickly and interventions included treatment reviews and ensuring TOC were performed for seven of the cases. While PN identified some linked cases, five patients had unidentifiable contacts, highlighting the difficulties in curtailing an outbreak and the importance of culture and susceptibility testing to identify further cases requiring strict management and follow-up. Proactively managing an outbreak in this way is however reliant on the correct and timely referral of resistant isolates to STBRU in the first instance and the availability and inclusions of appropriate control strains. All suspected cases of HL-AziR N. gonorrhoeae in England should be referred to the STBRU, who routinely confirm this phenotype.
The HL-AziR phenotype first emerged in Scotland in 2004, where it peaked in 2007 and subsequently declined.3 Reports of HL-AziR have otherwise been sporadic, with strains reported in Europe, the Americas and Australia generally in the heterosexual population.6–8 NG-MAST typing of HL-AziR strains globally has demonstrated considerable strain diversity (analysis not shown), although within the UK most strains typed to date, have been part of a cluster of strains closely related to ST649 which was predominant in the UK in 2007.6 ,9 The current outbreak strain differs from ST649 by a single bp within por, and showed the same resistance mechanism (A2143G mutation in all 23S rRNA alleles), documented previously for ST649.10 While ST649 has been reported recently in Australia,8 it seems likely that the current outbreak strain was endemic in the gonococcal population in the UK, given the former predominance of this type.3 ,6 ,9
The dual therapy currently recommended for gonorrhoea was introduced to delay the emergence of resistance to the few remaining antimicrobials effective against N. gonorrhoeae. Emergence and further dissemination of the HL-AziR phenotype is of great concern as this renders the azithromycin component ineffective. To date most reported HL-AziR strains have been ceftriaxone sensitive, as were the strains in the current outbreak. However the diversity of strains exhibiting HL-AziR globally and the growing evidence of emergence of decreased susceptibility to the cephalosporins in the heterosexual population in the UK demonstrate that there is real potential for emergence of strains resistant to both components of dual therapy.
The continued vigilance by clinicians and microbiologists to prevent further dissemination of the HL-AziR strain is required. Future work should focus on rapid identification and effective management of new cases and any potential treatment failures, and on further characterisation by WGS of HL-AziR strains observed in the UK and elsewhere to date, to better understand their emergence and dissemination. This will be invaluable in considering the longevity of azithromycin as an effective component of dual therapy.
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