Objectives: Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide. Methods:The 2016 WHO reference strains (n¼14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n¼ 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing. Results:The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n¼ 14) were presented. Conclusions:The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.

The 2008 WHO N. gonorrhoeae reference strain panel was extensively characterized, which is crucial for the expansion of gonococcal AMR surveillance nationally and internationally. The panel is available through WHO sources for quality assurance and quality control aspects of current phenotypic testing protocols, to allow valid comparison of AMR data derived by divergent methods, and also for the control of present and future molecular assays for AMR detection. Additional WHO reference strains can be included as required by the emergence of additional resistant phenotypes and/or genotypes.

Molecular detection ofNeisseria gonorrhoeae causes the second most prevalent bacterial sexually transmitted infection (STI) in men and women globally (42). As clinical signs of gonococcal infection may overlap with those of other STIs, laboratory testing is crucial for appropriate diagnosis and subsequent adequate treatment. The accurate diagnosis of gonorrhea relies on laboratory tests that are sensitive, specific, reproducible, and robust because of nonspecific clinical manifestations or lack of symptoms in women as well as men (43). Nucleic acid amplification tests (NAATs) have a number of recognized advantages over other diagnostic assays, which include increased sensitivity of detection and ease of sample collection (including use of self-collected samples) and transport, compared to culture-based methods (13,26,27,31,32). A number of commercial NAAT systems and assays developed "in-house" are currently in use for the detection of urogenital gonococcal infection, and their use has seen many of the anticipated benefits of NAATs. However, with increasing use of commercial and "in-house" NAAT systems over time and in different geographical settings, the need for both a considered approach to the application of NAATs and for an awareness of their limitations has arisen. These considerations include the sensitivity and specificity of the primary NAAT screening test, and where used, supplementary "confirmatory" tests as well as the prevalence of infection in various population groups (18). A number of assays have been shown to cross-react with other Neisseria species (10,20,23,29,36). The Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia) and the Australian Public Health Laboratory Network have proposed a number of testing algorithms for confirmation of STIs by NAATs, which require the use of additional or supplementary assays (12,28,43). There is increasing evidence for the rise in infection in extragenital sites, which include the rectum and oropharynx, particularly in population groups of men who have sex with men (38). Commercial NAAT systems currently on the market have not been cleared by the FDA for diagnosis of specimens from the rectum, pharynx, or conjunctiva; however, as detection of N. gonorrhoeae by NAAT is more sensitive than culture (43), laboratories continue to offer these tests to diagnose extragenital gonorrhea (4). Extragenital sites carry a number of commensal Neisseria species and commonly Neisseria meningitidis, which due to having a high nucleic acid homology to N. gonorrhoeae may cross-react in the NAAT assay utilized (9). As crossreactions occur in screening assays, steps to include at least one

Diagnostic, genotypic and antibiotic-resistance determinants of Neisseria gonorrhoeae were analysed by molecular methods to verify the failure of ceftriaxone treatment in two cases of pharyngeal gonorrhoea. Monoplex assays were needed to define competitive inhibition of a positive Chlamydia PCR in a duplex assay. Different penA changes were detected in the N. gonorrhoeae isolated from the two cases. These were associated with raised ceftriaxone MICs of 0.03 and 0.016 mg l−1, which may have contributed to the treatment failures in these cases.