Evaluation of the SpeeDx ResistancePlus^® GC and SpeeDx GC 23S 2611 (beta) molecular assays for prediction of antimicrobial resistance/susceptibility to ciprofloxacin and azithromycin in Neisseria gonorrhoeae Free

Abstract

Background

Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance.

Objectives

We evaluated the new ResistancePlus^® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively.

Methods

Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays.

Results

The sensitivity and specificity of the ResistancePlus^® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus^® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions.

Conclusions

The new SpeeDx ResistancePlus^® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).

Introduction

Gonorrhoea remains a global public health concern due to increasing prevalence, particularly in many more-resourced settings and because Neisseria gonorrhoeae (Ng) has developed antimicrobial resistance (AMR) to all available therapeutic antimicrobials.^1,2 Capacity to culture and perform AMR testing of Ng is also declining worldwide because in more-resourced settings nucleic acid amplification tests (NAATs) are rapidly replacing culture for diagnosis, and in many less-well-resourced countries STI syndromic management is mainly practised without laboratory diagnostics. Sensitive and specific molecular assays for prediction of Ng AMR or antimicrobial susceptibility are imperative, to inform individualized treatment and for AMR surveillance, which is further highlighted by the WHO global action plan.^3–8

Ciprofloxacin resistance is primarily due to mutations in the gyrA gene, encoding the GyrA subunit of DNA gyrase. The main resistance mutation, which is highly predictive of ciprofloxacin resistance,^1,2,8–15 is the GyrA S91F mutation and mutations in GyrA D95 increase the ciprofloxacin resistance. Simultaneous mutation(s) in the parC gene (encoding the ParC subunit of topoisomerase IV) further increase the ciprofloxacin MIC.^1,3,9,11 Multiple molecular assays have been developed to detect ciprofloxacin resistance determinants, with high agreement between these assays and Ng phenotypic ciprofloxacin resistance testing.^3,5–8 However, many of these molecular assays have lacked an appropriate Ng internal control, only a few extragenital NAAT samples were evaluated and cross-reactivity with non-gonococcal Neisseria species, frequently colonizing the oropharynx in particular, was detected.^3,5,12–15 Regarding macrolides, target mutations in the 23S rRNA gene alleles, namely C2611T and A2059G mutations, are associated with moderate- and high-level azithromycin resistance, respectively. The level of azithromycin resistance is dependent on the number of the four 23S rRNA gene alleles that are mutated.^1,3,8,16,17 However, additional AMR determinants increase the MICs and can cause resistance to azithromycin, such as: mtrR mutations and mosaic mtr locus causing overexpression of the MtrCDE efflux pump; erm genes that encode rRNA methylases, which block the macrolide binding to the 23S rRNA; overexpression of the MacAB efflux pump; and the mef-encoded efflux pump.^1,18 To date, few molecular assays have been developed for detection of Ng azithromycin resistance in NAAT samples and cross-reactivity with non-gonococcal Neisseria species has also been observed in these assays.^3,8,19 Recently, the first commercially available Ng molecular resistance/susceptibility assay, ResistancePlus^® GC (SpeeDx Pty Ltd, Sydney, Australia), was developed for detection of Ng and ciprofloxacin resistance/susceptibility and an evaluation of the beta version of this assay showed promising results.²⁰ This assay includes five targets: opa genes and porA pseudogene for detection of Ng, GyrA S91F and S91 WT, for prediction of ciprofloxacin resistance/susceptibility, and an internal control. A molecular assay for prediction of azithromycin resistance/susceptibility in Ng, herein named ‘GC 23S 2611 (beta)’, is under development by SpeeDx for commercial use. This assay currently includes three targets: porA pseudogene for Ng detection and 23S rRNA C2611T and C2611 WT for prediction of resistance/susceptibility to azithromycin.

We evaluated the new SpeeDx ResistancePlus^® GC assay and the GC 23S 2611 (beta) assay for molecular prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively, by examining a large collection of previously whole-genome sequenced Ng isolates from 20 European countries (n = 967)⁹ and clinical Ng-positive (n = 143) and Ng-negative (n = 167) samples using the Aptima Combo 2 assay (AC2; Hologic, San Diego, CA, USA). Additionally, ciprofloxacin- and azithromycin-resistant and -susceptible isolates (n = 143) of more than eight non-gonococcal commensal Neisseria species (n = 135), Neisseria meningitidis (n = 6) and two closely related species (n = 2) were examined to substantially challenge the exclusivity of both assays.

Materials and methods

Bacterial isolates

Whole-genome sequenced Ng isolates cultured in 20 European countries in 2013 were included. These isolates represented 921 WGS genotypes, 103 MLST STs and 377 NG-MAST STs (including 249 singletons) (https://pathogen.watch/collection/eurogasp2013).⁹ However, only 967 (91.7%; 967/1054) of these previously published isolates were viable and/or available for the present study [81 isolates from the UK and six isolates from Norway were not tested using the ResistancePlus^® GC assay; and three additional isolates from the Netherlands were not tested with the GC 23S 2611 (beta) assay]. Using MIC gradient strip tests or agar dilution for susceptibility testing⁹ and the EUCAST 2019 Ng breakpoints (http://www.eucast.org/clinical_breakpoints/), 446 of these isolates were susceptible, 1 was intermediate and 520 were resistant to ciprofloxacin; and 951 were susceptible and 13 resistant to azithromycin [23S rRNA C2611T (n = 8; 6 also had the characteristic mtrR promoter A-deletion^1,3), only mtrR promoter A-deletion (n = 3) and unknown azithromycin resistance mutations (n = 2)], i.e. using the azithromycin epidemiological cut-off of MIC > 1 mg/L to indicate isolates with azithromycin resistance determinants (referred to as resistant in this study). Furthermore, isolates of more than eight non-gonococcal commensal Neisseria species (n = 135) and two closely related species (n = 2; Moraxella catarrhalis, Moraxella osloensis) were tested. Using Etest (BioMérieux, Marcy l’Étoile, France) and EUCAST 2019 Ng breakpoints (http://www.eucast.org/clinical_breakpoints/), because EUCAST lacks breakpoints for non-gonococcal Neisseria species, 42 of these non-gonococcal isolates were susceptible, 39 intermediate, and 56 resistant to ciprofloxacin; and 7 were susceptible and 130 resistant to azithromycin. Six N. meningitidis reference strains, susceptible to both ciprofloxacin and azithromycin (using EUCAST 2019 Ng breakpoints because of lack of azithromycin breakpoints for N. meningitidis), were also examined: serogroup A (CCUG 3269), B (CCUG 3270), C (CCUG 3271), W (CCUG 27650), X (CCUG 27645) and Y (CCUG 38303).

Clinical NAAT samples

One hundred and forty-three Ng-positive AC2 samples (one sample per gonorrhoea patient/episode), confirmed in culture (n = 37) or Aptima GC assay (n = 106), from November 2014 to May 2018 were examined. Of the 37 paired Ng isolates, 17 were susceptible and 20 were resistant to ciprofloxacin, and 36 were susceptible and 1 was resistant to azithromycin, using Etest. The Ng-positive AC2 samples included urine (n = 54), pharyngeal (n = 43), rectal (n = 19), vaginal (n = 18) and cervical swabs (n = 9). Ng-negative AC2 samples (n = 167) were also examined: cervical (n = 65), pharyngeal (n = 48), urine (n = 20), vaginal (n = 20) and rectal (n = 14).

DNA extraction and ResistancePlus^® GC assay and GC 23S 2611 (beta) assay

Genomic DNA of all isolates was extracted using single colonies or cryobeads boiled in 100 μL of sterile distilled water for 10 min, followed by centrifugation at 2250 g for 10 min. The samples were diluted 1:400 for the ResistancePlus^® GC assay and 1:100 for the GC 23S 2611 (beta) assay prior to analysis. In the case of indeterminate results, isolates were re-extracted using the QIAsymphony platform with the Virus/Pathogen Midi Kit (QIAGEN, GmbH, Hilden, Germany). All AC2 samples were extracted using the MagNA Pure Compact Nucleic Acid Isolation Kit Large Volume (Roche Diagnostics, Mannheim, Germany) on MagNA Pure Compact (Roche), including an internal control for the ResistancePlus^® GC assay.

The ResistancePlus^® GC assay and GC 23S 2611 (beta) assay (SpeeDx Pty Ltd, Sydney, Australia) were performed, according to the manufacturer’s instructions, on a 7500 Fast Real-Time PCR System (Applied Biosystems). All data were analysed and reported using the ResistancePlus GC (7500) analysis software and GC 23S 2611 (beta) analysis software (still under development), respectively. Ng-positive AC2 samples and cross-reactive non-gonococcal Neisseria isolates in the ResistancePlus^® GC assay were further analysed using an in-house Ng GyrA S91 WT/S91F PCR.²¹ Indeterminate samples had an invalid internal control, similar amplification of both AMR and WT targets in gyrA or 23S rRNA gene, or lack of detection of both Ng and AMR target.

Results

ResistancePlus^® GC assay for ciprofloxacin resistance/susceptibility prediction

Detection of N. gonorrhoeae

The ResistancePlus^® GC assay detected all of the 967 European Ng isolates⁹ and 141 (98.6%) of the 143 Ng-positive AC2 samples. Furthermore, 135 (98.5%) of 137 non-gonococcal commensal isolates, all N. meningitidis isolates (n = 6) and all Ng-negative AC2 samples (n = 167) were negative for Ng; however, two Ng-negative AC2 samples were invalid and excluded from further calculations (Table 1). Compared with the AC2 assay, the overall sensitivity and specificity of ResistancePlus^® GC for detection of Ng in AC2 samples were 98.6% and 100%, respectively.

Detection of GyrA S91 WT/S91F

The GyrA S91 WT/S91F targets were correctly identified by the ResistancePlus^® GC assay in all of the 967 genome-sequenced European Ng isolates.⁹ Of the 141 (98.6%) AC2 samples reported as Ng-positive by ResistancePlus^® GC, indeterminate gyrA results were obtained for four (2.8%) samples. These four samples were detected with the in-house gyrA PCR.²¹ Of the GyrA S91F-positive AC2 samples (n = 57) using the ResistancePlus^® GC assay, the in-house gyrA PCR²¹ confirmed all but two (indeterminate). Of the GyrA S91 WT AC2 samples (n = 80), the in-house gyrA PCR²¹ confirmed all except four samples [GyrA S91F (n = 1) or indeterminate (n = 3)]. Accordingly, 92.9% (131/141) of the evaluable gyrA results were concordant between the ResistancePlus^® GC and the in-house gyrA PCR assay.²¹ The positive percentage agreement (PPA) and negative percentage agreement (NPA), after excluding samples with indeterminate results in any of the assays (n = 9), of ResistancePlus^® GC and the in-house gyrA PCR assay²¹ in the detection of GyrA S91F were 98.2% (55/56) and 100% (76/76), respectively. Regarding cross-reaction, GyrA S91 WT signal was visually observed in eight (4.8%) Ng-negative AC2 pharyngeal samples (all gyrA negative using the in-house gyrA PCR²¹) and GyrA S91 WT signal or amplification of both gyrA targets was observed in isolates of several non-gonococcal Neisseria species: Neisseria macacae (5/9 isolates), Neisseria sicca (2/4 isolates), Neisseria species (2/34 isolates) and Neisseria mucosa (1/6 isolates) (Table 1). However, all these samples were correctly reported as Ng negative using ResistancePlus GC analysis software. Two other non-gonococcal isolates (N. mucosa and Neisseria animalis) were reported as Ng positive but were only positive for the opa signal and showed no GyrA S91 call. All the cross-reactive non-pathogenic commensal Neisseria isolates were negative for the gyrA targets in the in-house Ng gyrA PCR.²¹ Notably, many (81%) of the Ng-positive AC2 samples visually showed some level of amplification of both GyrA S91 WT and S91F, although the amplification of one of the targets was considerably stronger (lower cycle threshold value) than the other, illustrating the necessity of using the ResistancePlus GC analysis software. Almost all cultured Ng isolates showed some level of amplification of both gyrA targets, likely a result of the high bacterial load that was tested and possibly due to multiple Ng isolates in some samples.

For the 967 European Ng isolates⁹ and the 37 Ng isolates paired with Ng-positive AC2 samples, both the sensitivity and specificity of the ResistancePlus^® GC assay to predict phenotypic ciprofloxacin resistance by detection of GyrA S91F was 99.8%.

In total, 2.6% (n = 8) of all Ng-positive or Ng-negative AC2 samples were indeterminate in the ResistancePlus^® GC assay due to invalid internal control (n = 2), indeterminate gyrA detection but with a valid Ng detection (n = 4), or the assay could not detect any of the Ng or gyrA targets (n = 2).

GC 23S 2611 (beta) assay for azithromycin resistance prediction

Detection of N. gonorrhoeae

The GC 23S 2611 (beta) assay detected Ng in all except one of the 964 European Ng isolates (99.9%).⁹ This sample was repeatedly reported as negative by the analysis software, although a 23S rRNA gene signal was detected. For Ng-positive AC2 samples (n = 143), the assay detected Ng in 137 (95.8%) samples, missing Ng in three rectal samples, two urine samples and one pharyngeal sample. All Ng-negative AC2 samples and isolates of non-gonococcal Neisseria species were correctly identified as Ng negative (Table 2). The sensitivity and specificity of the assay for Ng detection in AC2 samples were 95.8% and 100%, respectively.

Detection of 23S rRNA C2611 WT/C2611T

Examining the 964 European Ng isolates,⁹ the 23S rRNA gene C2611 WT target was detected by the GC 23S 2611 (beta) assay in 955 (99.1%) isolates, with all (100%) isolates in agreement with the WGS results, and the C2611T mutation in 9 (0.9%) isolates (Table 2), with 8 (88.9%) isolates in concordance with the WGS results. Of the 955 isolates with 23S rRNA C2611 WT, 647 had azithromycin MIC values of 0.016–1.0 mg/L, 5 had azithromycin MICs of 2 mg/L (likely due to their overexpressed MtrCDE efflux pump) and 303 with exact MIC lacking due to the use of agar dilution breakpoint technique in some European countries in 2013 (all susceptible, i.e. MICs ≤ 0.25 mg/L). The eight isolates with 23S rRNA gene C2611T had azithromycin MICs ranging from 2 to 12 mg/L and all had 3–4 mutated 23S rRNA gene alleles according to the previous WGS.⁹ Among the Ng-positive AC2 samples detected as Ng positive (n = 137), the GC 23S 2611 (beta) assay reported 23S rRNA C2611 WT in 128 samples (89.5%, n = 34 with known azithromycin MICs of 0.016–1 mg/L), C2611T in 2 samples (1.4%, one with known azithromycin MIC of 8 mg/L) and 7 (4.9%) samples were indeterminate. Examining the Ng-negative AC2 samples, 23S rRNA gene C2611 WT signals were visually observed in 48 (28.7%) samples, i.e. pharyngeal (n = 43), cervical (n = 3) and vaginal (n = 2) samples; however, all these samples were reported as Ng negative by the analysis software of the GC 23S 2611 (beta) assay. Furthermore, 62 (45.3%) of the non-pathogenic commensal Neisseria isolates (azithromycin MIC: 1–24 mg/L) and all six N. meningitidis reference strains visually had C2611 WT signals but all these samples were reported as Ng negative by the analysis software of the GC 23S 2611 (beta) assay (Table 2). Notably, the GC 23S 2611 (beta) assay showed visual amplification of both the 23S rRNA gene C2611 WT and C2611T targets in 23.8% (n = 34) of the Ng-positive AC2 samples and 1.9% (n = 18) of the non-gonococcal Neisseria isolates including all N. meningitidis reference strains, emphasizing the requirement for the GC 23S 2611 (beta) analysis software in the analysis of raw results.

For the 964 European Ng isolates and the 37 Ng isolates paired with Ng-positive AC2 samples, the overall sensitivity and specificity of the GC 23S 2611 (beta) assay to predict azithromycin resistance by detection of 23S rRNA C2611T mutation compared with the phenotypic azithromycin resistance testing were 64.3% and 99.9%, respectively. However, the sensitivity and specificity of the GC 23S 2611 (beta) assay to detect the 23S rRNA C2611T mutation, compared with the WGS data, were 100% and 99.9%, respectively.

Discussion

Sensitive and specific molecular assays for prediction of Ng AMR or antimicrobial susceptibility are imperative, both to inform individualized treatment and for AMR surveillance, which is further highlighted by the WHO global action plan.^3–8 In the present study, the new commercially available ResistancePlus^® GC assay showed high sensitivity and specificity in the detection of Ng, compared with the AC2 assay, and ability to detect and distinguish GyrA S91 WT and S91F. Both the sensitivity and specificity of the ResistancePlus^® GC assay to predict ciprofloxacin resistance compared with phenotypic ciprofloxacin resistance testing were 99.8%, supporting the proposition that the assay can be effectively used for AMR surveillance and individualized treatment with ciprofloxacin, which is easily accessible and administered as a single 500 mg oral dose. Although signals for both the GyrA S91F resistance mutation (3.6%) and GyrA WT (7.3%) were visually observed in non-gonococcal commensal Neisseria isolates, all these samples were reported as Ng negative by the analysis software. Nevertheless, the high bacterial load of the non-gonococcal Neisseria isolates substantially challenging the assay likely seriously affected the probability of cross-reaction and, in future similar analytical evaluations, the bacterial load should ideally be standardized at more clinically relevant concentrations. These results regarding cross-reaction are comparable to those from previous studies, including one evaluating the beta version of the ResistancePlus^® GC assay that reported cross-reactions in non-pathogenic isolates and pharyngeal samples, as well as indeterminate gyrA calls.^13,20 Another study²² showed that the gyrA genotype determination was significantly improved using the ResistancePlus^® GC assay compared with an in-house PCR assay using high-resolution melt gyrA analysis. However, a proportion of samples remained indeterminate for gyrA, which is likely due to the low Ng load in some clinical NAAT samples and/or inhibition and cross-reactions with other Neisseria species.²² In particular, extragenital sites, such as the pharynx, are challenging for molecular Ng AMR prediction, because these sites frequently harbour non-gonococcal Neisseria species as commensals and many DNA sequences, including AMR determinants, are identical or very similar in several Neisseria species.^1–3,18 Accordingly, the cross-reactive non-gonococcal Neisseria isolates and AC2 pharyngeal samples in the present study illustrate some of the main problems faced by the field of molecular prediction of AMR in Ng. Previous studies of molecular assays for prediction of ciprofloxacin AMR or susceptibility have reported an overall sensitivity of 95.8%–100% and a specificity of 97.9%–100%. However, several of these assays did not use any internal control in their molecular assay,^12,15 included a low number or no extragenital samples^13,21,23 or Ng-negative clinical NAAT samples,^15,24 which are essential for assessing cross-reactivity. Finally, the cost-effectiveness of using the ResistancePlus^® GC assay or similar assays to predict ciprofloxacin resistance and inform individualized treatment in a setting depends on the local prevalence of ciprofloxacin resistance, the cost of the assay used, the proportion of NAAT samples that can be typed in regard to ciprofloxacin resistance/susceptibility, the testing frequency, the cost of ciprofloxacin locally and the cost of the empirical treatment used for ciprofloxacin-resistant or indeterminate cases.²⁵

The GC 23S 2611 (beta) assay for detection of the 23S rRNA gene C2611 WT or C2611T resistance mutation and prediction of azithromycin resistance/susceptibility is still under development and not yet commercially available. This assay does not yet include any internal/inhibition control and contains only a single porA pseudogene target for detection of Ng. The proportion of indeterminate 23S rRNA C2611 results was 4.9% in Ng-positive AC2 samples, and many Ng-negative AC2 samples (28.7%), predominantly pharyngeal samples, and non-gonococcal Neisseria isolates (47.6%) visually showed some cross-reaction producing a C2611 WT signal; however, all these samples were reported as Ng negative by the analysis software. It was visually observed that the GC 23S 2611 (beta) assay frequently amplified both of the 23S rRNA gene targets, which also can be correct because it could be a mixed infection or only some of the four 23S rRNA gene alleles in the same gonococcal strain may be mutated,^9,16,17 and the GC 23S 2611 (beta) analysis software is imperative for analysis. The detection of 23S rRNA targets in non-gonococcal Neisseria isolates may be mostly reflecting the high bacterial load tested. Similarly, a study by Donà et al.²⁶ also showed cross-reaction to C2611 WT with difficulty in the accurate prediction of C2611T AMR in extragenital samples. To reduce this type of cross-reaction, Trembizki et al.¹⁹ used non-template bases in primers to distinguish the 23S rRNA gene amplification of Ng from commensals, with delayed amplification of the latter, in addition to melting curve analysis of the target. Similar to our findings, their 23S rRNA gene C2611 PCR assay mainly showed cross-reactivity with Ng-negative clinical pharyngeal NAAT samples and all had C2611 WT alleles.¹⁹

Importantly, azithromycin resistance can also be caused by other 23S rRNA gene mutations such as the A2059G mutation,^1,17,18,27 which results in high-level azithromycin resistance.^8,16,17 Furthermore, four copies of the 23S rRNA gene are present in the gonococcal genome and a higher number of mutated alleles causes higher MICs of azithromycin.^17,28 In addition, many other molecular AMR determinants such as erm genes and overexpressed MtrCDE efflux pump can also cause decreased susceptibility and resistance to azithromycin,^{1,16–18,29,30} which is an inherent limitation of all Ng 23S rRNA-based molecular AMR assays. The GC 23S 2611 (beta) assay evaluated in the present study does not distinguish the number of mutated 23S rRNA gene alleles; however, this is likely not a problem because it has been shown that any mutated alleles will likely be shared, through recombination, as soon as the gonococcal strain is exposed to azithromycin or other similar macrolides.^1,16,17 Accordingly, independent of the azithromycin MIC of such gonococcal strains, 23S rRNA gene mutations should be detected and azithromycin should not be used for treatment of strains harbouring any mutated alleles.

In conclusion, the new SpeeDx ResistancePlus^® GC assay performed well in the detection of Ng and AMR determinants. Our results support the proposition that the SpeeDx ResistancePlus^® GC assay can be effectively used for individualized treatment, where patients with ciprofloxacin-resistant and indeterminate samples receive empirical treatment (for example, ceftriaxone plus azithromycin),^2,18 and AMR surveillance, particularly in urogenital samples. This type of individualized treatment will also reduce the use of empirical treatment (ceftriaxone and azithromycin), which is of importance to spare the last-line ceftriaxone, reduce azithromycin use and in general antimicrobial use, in accordance with antimicrobial stewardship strategies. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity for predicting phenotypic azithromycin resistance was suboptimal and further evaluation and optimizations are required, including detection of the 23S rRNA A2059G mutation as well as additional macrolide resistance determinant(s). Furthermore, adding detection of a multicopy gene, e.g. the opa genes, would increase the sensitivity of Ng detection. Cross-reactivity was visually observed with non-gonococcal commensal Neisseria isolates and foremost in clinical AC2 pharyngeal samples, particularly for the GC 23S 2611 (beta) assay, emphasizing the necessity to use the SpeeDx analysis software. It would be valuable to examine a larger collection of Ng-positive and Ng-negative clinical NAAT samples, with paired cultured Ng isolates and using both the ResistancePlus^® GC assay and the GC 23S 2611 (beta) assay. Clearly, the high sensitivity and specificity of the ResistancePlus^® GC assay and promising potential of the GC 23S 2611 (beta) assay encourage the further development of molecular AMR or antimicrobial susceptibility assays for Ng to complement culture-based methods in AMR surveillance and, ultimately, to inform individualized treatment of gonorrhoea. Nevertheless, it remains important to continuously strengthen culture-based phenotypic AMR surveillance for Ng, i.e. for national and international gonococcal antimicrobial surveillance programmes and to detect new AMR determinants.

Acknowledgements

We are grateful to SpeeDx Pty Ltd for providing the reagents for the ResistancePlus^® GC assay and the GC 23S 2611 (beta) assay for this study.

Members of the European collaborative group

Raquel Abad Torreblanca, Lena Rós Ásmundsdóttir, Eszter Balla, Irith De Baetselier, Beatrice Bercot, Thea Bergheim, Maria José Borrego, Susanne Buder, Robert Cassar, Michelle Cole, Alje van Dam, Claudia Eder, Steen Hoffmann, Blazenka Hunjak, Samo Jeverica, Vesa Kirjavainen, Panayiota Maikanti-Charalambous, Vivi Miriagou, Beata Młynarczyk-Bonikowska, Gatis Pakarna, Peter Pavlik, Monique Perrin, Joseph Pett, Paola Stefanelli, Kate Templeton, Magnus Unemo, Jelena Viktorova and Hana Zákoucká.

Funding

This work was supported by the Örebro County Council Research Committee and the Foundation for Medical Research at Örebro University Hospital, Örebro, Sweden.

Transparency declarations

M.J.C. and M.D. have no personal conflicts, however PHE’s AMRHAI Reference Unit has received financial support from SpeeDx for conference attendance and an evaluation of their Mycoplasma genitalium ResistancePlus kit. S. Ebeyan, L.Y. Tan and S. Erskine are employees of SpeeDx Pty Ltd. The remaining authors have none to declare.

References

Author notes