FDA, CDC, and NIH Co-sponsored Public Workshop Summary-Development Considerations of Antimicrobial Drugs for the Treatment of Gonorrhea.

There is an unmet need for developing drugs for the treatment of gonorrhea, due to rapidly evolving resistance of Neisseria gonorrhoeae against antimicrobial drugs used for empiric therapy, an increase in globally reported multidrug resistant cases, and the limited available therapeutic options. Furthermore, few drugs are under development. Development of antimicrobials is hampered by challenges in clinical trial design, limitations of available diagnostics, changes in and varying standards of care, lack of robust animal models, and clinically relevant pharmacodynamic targets. On April 23, 2021, the U.S. Food and Drug Administration; Centers for Disease Control and Prevention; and National Institute of Allergy and Infectious Diseases, National Institutes of Health co-sponsored a workshop with stakeholders from academia, industry, and regulatory agencies to discuss the challenges and strategies, including potential collaborations and incentives, to facilitate the development of drugs for the treatment of gonorrhea. This article provides a summary of the workshop.

Detection of antimicrobial resistance in <5 h in Neisseria gonorrhoeae isolates using flow cytometry-proof of concept for seven clinically relevant antimicrobials.

INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

Pharmacodynamic evaluation of ceftriaxone single-dose therapy (0.125-1 g) to eradicate ceftriaxone-susceptible and ceftriaxone-resistant Neisseria gonorrhoeae strains in a hollow fibre infection model for gonorrhoea.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is threatening the gonorrhoea treatment, and optimizations of the current ceftriaxone-treatment regimens are crucial. We evaluated the pharmacodynamics of ceftriaxone single-dose therapy (0.125-1 g) against ceftriaxone-susceptible and ceftriaxone-resistant gonococcal strains, based on EUCAST ceftriaxone-resistance breakpoint (MIC > 0.125 mg/L), in our hollow fibre infection model (HFIM) for gonorrhoea. METHODS: Gonococcal strains examined were WHO F (ceftriaxone-susceptible, MIC < 0.002 mg/L), R (ceftriaxone-resistant, MIC = 0.5 mg/L), Z (ceftriaxone-resistant, MIC = 0.5 mg/L) and X (ceftriaxone-resistant, MIC = 2 mg/L). Dose-range HFIM 7 day experiments simulating ceftriaxone 0.125-1 g single-dose intramuscular regimens were conducted. RESULTS: Ceftriaxone 0.125-1 g single-dose treatments rapidly eradicated WHO F (wild-type ceftriaxone MIC). Ceftriaxone 0.5 and 1 g treatments, based on ceftriaxone human plasma pharmacokinetic parameters, eradicated most ceftriaxone-resistant gonococcal strains (WHO R and Z), but ceftriaxone 0.5 g failed to eradicate WHO X (high-level ceftriaxone resistance). When simulating oropharyngeal gonorrhoea, ceftriaxone 0.5 g failed to eradicate all the ceftriaxone-resistant strains, while ceftriaxone 1 g eradicated WHO R and Z (low-level ceftriaxone resistance) but failed to eradicate WHO X (high-level ceftriaxone resistance). No ceftriaxone-resistant mutants were selected using any ceftriaxone treatments. CONCLUSIONS: Ceftriaxone 1 g single-dose intramuscularly cure most of the anogenital and oropharyngeal gonorrhoea cases caused by the currently internationally spreading ceftriaxone-resistant gonococcal strains, which should be further confirmed clinically. A ceftriaxone 1 g dose (±azithromycin 2 g) should be recommended for first-line empiric gonorrhoea treatment. This will buy countries some time until novel antimicrobials are licensed. Using ceftriaxone 1 g gonorrhoea treatment, the EUCAST ceftriaxone-resistance breakpoint is too low.

In silico gepotidacin target mining among 33 213 global Neisseria gonorrhoeae genomes from 1928 to 2023 combined with gepotidacin MIC testing of 22 gonococcal isolates with different GyrA and ParC substitutions.

OBJECTIVES: The novel dual-target triazaacenaphthylene, gepotidacin, recently showed promising results in its Phase III randomized controlled trial for the treatment of gonorrhoea. We investigated alterations in the gepotidacin GyrA and ParC targets in gonococci by in silico mining of publicly available global genomes (n = 33 213) and determined gepotidacin MICs in isolates with GyrA A92 alterations combined with other GyrA and/or ParC alterations. METHODS: We examined gonococcal gyrA and parC alleles available at the European Nucleotide Archive. MICs were determined using the agar dilution method (gepotidacin) or Etest (four antimicrobials). Models of DNA gyrase and topoisomerase IV were obtained from AlphaFold and used to model gepotidacin in the binding site. RESULTS: GyrA A92 alterations were identified in 0.24% of genomes: GyrA A92P/S/V + S91F + D95Y/A/N (0.208%), A92P + S91F (0.024%) and A92P (0.003%), but no A92T (previously associated with gepotidacin resistance) was found. ParC D86 alterations were found in 10.6% of genomes: ParC D86N/G (10.5%), D86N + S87I (0.051%), D86N + S88P (0.012%) and D86G + E91G (0.003%). One isolate had GyrA A92P + ParC D86N alterations, but remained susceptible to gepotidacin (MIC = 0.125 mg/L). No GyrA plus ParC alterations resulted in a gepotidacin MIC > 4 mg/L. Modelling of gepotidacin binding to GyrA A92/A92T/A92P suggested that gepotidacin resistance due to GyrA A92T might be linked to the formation of a new polar contact with DNA. CONCLUSIONS: In silico mining of 33 213 global gonococcal genomes (isolates from 1928 to 2023) showed that A92 is highly conserved in GyrA, while alterations in D86 of ParC are common. No GyrA plus ParC alterations caused gepotidacin resistance. MIC determination and genomic surveillance of potential antimicrobial resistance determinants are imperative.

The novel 2024 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations and superseded WHO N. gonorrhoeae reference strains-phenotypic, genetic and reference genome characterization.

OBJECTIVES: MDR and XDR Neisseria gonorrhoeae strains remain major public health concerns internationally, and quality-assured global gonococcal antimicrobial resistance (AMR) surveillance is imperative. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) and WHO Enhanced GASP (EGASP), including metadata and WGS, are expanding internationally. We present the phenotypic, genetic and reference genome characteristics of the 2024 WHO gonococcal reference strains (n = 15) for quality assurance worldwide. All superseded WHO gonococcal reference strains (n = 14) were identically characterized. MATERIAL AND METHODS: The 2024 WHO reference strains include 11 of the 2016 WHO reference strains, which were further characterized, and four novel strains. The superseded WHO reference strains include 11 WHO reference strains previously unpublished. All strains were characterized phenotypically and genomically (single-molecule PacBio or Oxford Nanopore and Illumina sequencing). RESULTS: The 2024 WHO reference strains represent all available susceptible and resistant phenotypes and genotypes for antimicrobials currently and previously used (n = 22), or considered for future use (n = 3) in gonorrhoea treatment. The novel WHO strains include internationally spreading ceftriaxone resistance, ceftriaxone resistance due to new penA mutations, ceftriaxone plus high-level azithromycin resistance and azithromycin resistance due to mosaic MtrRCDE efflux pump. AMR, serogroup, prolyliminopeptidase, genetic AMR determinants, plasmid types, molecular epidemiological types and reference genome characteristics are presented for all strains. CONCLUSIONS: The 2024 WHO gonococcal reference strains are recommended for internal and external quality assurance in laboratory examinations, especially in the WHO GASP, EGASP and other GASPs, but also in phenotypic and molecular diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and as complete reference genomes in WGS analysis.

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.

Prevalence of sexually transmitted infections and associated risk factors among female sex workers in Guinea-Bissau.

OBJECTIVE: To estimate the prevalence of the curable sexually transmitted infections (STIs) Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis and Treponema pallidum, to identify associated risk factors and to assess ciprofloxacin resistance in N. gonorrhoeae-positive specimens among female sex workers (FSWs) in Guinea-Bissau. METHODS: For this cross-sectional study, FSWs were recruited from October 2014 to May 2019. A questionnaire on STI risk factors was completed by the study participants, and the women were asked to provide a vaginal swab for nucleic acid amplification tests for C. trachomatis, N. gonorrhoeae, M. genitalium, T. vaginalis (Aptima, Hologica), as well as a blood sample for T. pallidum serological testing and discriminatory HIV-testing. The prevalence of STIs was determined, and multivariate logistic regression was used to identify STI risk factors. RESULTS: The study included 467 women. The prevalence of current infection with any curable STI was 46.7%, and the most common pathogen was T. vaginalis (26.3%), followed by M. genitalium (21.9%), C. trachomatis (11.8%), N. gonorrhoeae (10.1%) and T. pallidum (2.8%). The proportion of asymptomatic infections among the diagnosed STIs was 61.8%, 61.5%, 55.3%, 55.3% and 52.2% for C. trachomatis, T. pallidum, N. gonorrhoeae, T. vaginalis and M. genitalium, respectively. The prevalence of the gyrA S91F mutation conferring ciprofloxacin resistance in N. gonorrhoeae-positive specimens was 84.0%. Significant risk factors for having a curable STI were age and HIV-1 infection, while use of female condoms was a protective factor. CONCLUSION: This study demonstrated that the prevalence of curable STIs was high among FSWs in Guinea-Bissau during the study period, indicating an unmet need for STI services. Moreover, the results indicated that symptomatic treatment might be insufficient, highlighting a need for periodic aetiological testing to facilitate detection of asymptomatic as well as symptomatic STIs to stop ongoing transmission.

Clinic-based evaluation of the dual Xpert CT/NG assay on the GeneXpert System for screening for extragenital chlamydial and gonococcal infections amongst men who have sex with men.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections have increased globally. Asymptomatic infections represent a significant risk of long-term complications. Men who have sex with men (MSM) are disproportionally affected, underscoring the need to offer screening programmes to this population. CT/NG Point of Care Testing (POCT) constitutes a strategic tool to improve the continuum of STI care, however extensive real-life evaluations amongst at risk populations are lacking. The aim of this study is to estimate the GeneXpert CT/NG assay performance and usability for CT and NG at genital and extragenital sites for screening amongst MSM. METHODS: This study was a multi-site sexual health clinic-based evaluation (Italy, Malta and Peru) with consecutive enrolment. A first void urine sample (divided in two aliquots), two oropharyngeal and two anorectal swabs were collected for each study participant. One specimen set (one for each anatomical site) was tested with the dual index test (Cepheid) at the clinics by the healthcare staff, the other set with FDA/CE approved Nucleic Acid Amplification Tests (NAATs) at the laboratory. Clinical sites and reference laboratories participated in an internal and external quality control programme. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values for each anatomical site were estimated using a meta-analytic approach. RESULTS: One thousand seven hundred two MSM were recruited across all clinical sites for a total of 5049 biological specimens. NG and CT were respectively detected in 274 and 287 of samples. Overall, the NG POCT sensitivity and specificity was 91.43% and 99.75% in urine (LR + 372.80, LR- 0.09), 89.68% and 99.55% in rectal specimens (LR + 197.30, LR- 0.10) and 75.87% and 98.77% at the pharynx respectively (LR + 61.94, LR- 0.24). The CT component of the POCT sensitivity was 84.82% and specificity 99.63% in urine (LR + 228.68, LR- 0.15), 78.07% and 99.19% respectively on rectal site (LR + 96.23, LR-0.22), 67.79% and 99.88% respectively at pharyngeal site (LR + 554.89, LR- 0.32). 95.95% of MSM reported to be willing to wait for POCT results and no provider reported difficulties in terms of performance or interpretation of the results of the Xpert CT/NG. CONCLUSION: Rapid turnaround time, ease of use and high acceptability make the Xpert CT/NG testing system a strategic tool for increasing testing frequency, reaching those not yet tested and offering the possibility of immediate treatment if needed. The assay showed good negative likelihood ratios and confirms its use to rule out CT/NG infections. Sensitivity varied across sites and pathogens. Periodic staff training at the testing sites should be mandatory.

A multi-country comparative study of two treponemal tests for the serodiagnosis of syphilis amongst men who have sex with men (MSM): Chemo-luminescent assay vs Treponema pallidum particle agglutination assay.

INTRODUCTION: International guidelines recommend routine screening for syphilis (aetiological agent: Treponema pallidum subspecies pallidum) amongst key populations and vulnerable populations using tests detecting treponemal and non-treponemal antibodies. Whilst treponemal tests have high sensitivities and specificities, they differ regarding subjective or objective interpretation, throughput and workload. Chemiluminescence immunoassays (CLIAs) are cost- and time-effective automated methods for detecting treponemal antibodies. The Treponema pallidum particle agglutination assay (TPPA) has been considered the "gold standard" treponemal assay, however, this includes a highly manual procedure, low throughput and subjective interpretation. The present multi-country study evaluated the ADVIA Centaur® Syphilis CLIA (Siemens Healthcare) assay compared to the reference SERODIA-TP·PA® (Fujirebio Diagnostics) for the serodiagnosis of syphilis amongst men who have sex with men (MSM). METHOD: 1,485 MSM were enrolled in Brighton (UK), Malta, and Verona (Italy) as part of a larger WHO multi-country and multi-site ProSPeRo study. Ethical approval was obtained. Serum was tested with the ADVIA Centaur® Syphilis CLIA assay and SERODIA-TP·PA®, in accordance with the manufacturers' instructions, for a first round of validation. A second round of validation was carried out for discrepant results that were additionally tested with both Western Blot (Westernblot EUROIMMUN®) and an Immunoblot (INNO-LIA, Fujirebio Diagnostics). Sensitivity, specificity, positive and negative predictive value (PPV and NPV), likelihood ratios (positive/negative), and the Diagnostic Odds Ratio (DOR)/pre-post-test probability (Fagan's nomogram) were calculated. RESULTS: Out of 1,485 eligible samples analysed in the first phase, the SERODIA-TP·PA® identified 360 positive and 1,125 negative cases. The ADVIA Centaur® Syphilis CLIA assay (Siemens) identified 366 positives, missclassifying one TPPA-positive sample. In the second phase, the ADVIA Centaur® Syphilis CLIA resulted in 1 false negative and 4 false positive results. Considering the syphilis study prevalence of 24% (95% CI: 22-26.7), The sensitivity of the ADVIA Centaur® Syphilis CLIA assay was 99.7% (95% CI: 98.5-100), and the specificity was 99.4% (95% CI: 98.7-99.7). The ROC area values were 0.996 (95% CI: 0.992-0.999), and both the PPV and NPV values were above 98% (PPV 98.1%, 95% CI: 96.1-99.2; NPV 99.9%, 95% CI: 99.5-100). CONCLUSIONS: The ADVIA Centaur® Syphilis CLIA assay showed similar performance compared to the SERODIA-TP·PA®. Considering the study is based on QUADAS principles and with a homogeneous population, results are also likely to be generalisable to MSM population but potentially not applicable to lower prevalence populations routinely screened for syphilis. The automated CLIA treponemal assay confirmed to be accurate and appropriate for routine initial syphilis screening, i.e. when the reverse testing algorithm is applied.

Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

Independent clinic-based evaluation of point-of-care testing for the screening of Chlamydia trachomatis, Neisseria gonorrhoea and Trichomonas vaginalis in women-at-risk in Australia, Guatemala, Morocco, and South Africa.

BACKGROUND: In 2018, the World Health Organization commenced a multi-country validation study of the Cepheid GeneXpert for a range of molecular-based point-of-care (POC) tests in primary care settings. One study arm focused on the evaluation of POC tests for screening 'women at risk' for chlamydia (CT), gonorrhoea (NG) and trichomonas (TV) in four countries - Australia, Guatemala, Morocco and South Africa. METHODS: Study participants completed a pre-test questionnaire which included demographics, clinical information and general questions on POC testing (POCT). Two vaginal swab samples (either self-collected or clinician collected) from each patient were tested on the GeneXpert at the POC and at a reference laboratory using quality-assured nucleic acid amplification tests (NAATs). RESULTS: One thousand three hundred and eighty-three women were enrolled: 58.6% from South Africa, 29.2% from Morocco, 6.2% from Guatemala, and 6.0% from Australia. 1296 samples for CT/NG and 1380 samples for TV were tested by the GeneXpert and the reference NAAT. The rate of unsuccessful tests on the GeneXpert was 1.9% for CT, 1.5% for NG and 0.96% for TV. The prevalence of CT, NG and TV was 31%, 13% and 23%, respectively. 1.5% of samples were positive for all three infections; 7.8% were positive for CT and NG; 2.4% were positive for NG and TV; and 7.3% were positive for CT and TV. Compared to reference NAATs, pooled estimates of sensitivity for the GeneXpert tests were 83.7% (95% confidence intervals 69.2-92.1) for CT, 90.5% (85.1-94.1) for NG and 64.7% (58.1-70.7) for TV (although estimates varied considerably between countries). Estimates for specificity were ≥96% for all three tests both within- and between-countries. Pooled positive and negative likelihood ratios were: 32.7 ([CI] 21.2-50.5) and 0.17 (0.08-0.33) for CT; 95.3 (36.9-245.7) and 0.10 (0.06-0.15) for NG; and 56.5 (31.6-101.1) and 0.35 (0.27-0.47) for TV. CONCLUSION: This multi-country evaluation is the first of its kind world-wide. Positive likelihood ratios, as well as specificity estimates, indicate the GeneXpert POC test results for CT, NG and TV were clinically acceptable for ruling in the presence of disease. However, negative likelihood ratios and variable sensitivity estimates from this study were poorer than expected for ruling out these infections, particularly for TV. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee, as well as local ethics committees from all participating countries.

Important considerations regarding the widespread use of doxycycline chemoprophylaxis against sexually transmitted infections.

Rates of sexually transmitted infections (STIs) continue to rise across the world and interventions are essential to reduce their incidence. Past and recent studies have indicated this may be achieved using doxycycline post-exposure prophylaxis (PEP) and this has sparked considerable interest in its use. However, many unanswered questions remain as to its long-term effects and particularly potentially negative impact on human microbiomes and antimicrobial resistance among STIs, other pathogens, and commensals. In this review, we discuss seven areas of concern pertaining to the widespread use of doxycycline PEP.

High-level in vitro resistance to gentamicin acquired in a stepwise manner in Neisseria gonorrhoeae.

OBJECTIVES: Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. METHODS: Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MIC = 4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. RESULTS: WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520I + R635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. CONCLUSIONS: We describe the first high-level gentamicin-resistant gonococcal isolate (MIC = 128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.

Genomic surveillance and antimicrobial resistance determinants in Neisseria gonorrhoeae isolates from Uganda, Malawi and South Africa, 2015-20.

OBJECTIVES: Global antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae is essential. In 2017-18, only five (10.6%) countries in the WHO African Region reported to the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP). Genomics enhances our understanding of gonococcal populations nationally and internationally, including AMR strain transmission; however, genomic studies from Africa are extremely scarce. We describe the gonococcal genomic lineages/sublineages, including AMR determinants, and baseline genomic diversity among strains in Uganda, Malawi and South Africa, 2015-20, and compare with sequences from Kenya and Burkina Faso. METHODS: Gonococcal isolates cultured in Uganda (n = 433), Malawi (n = 154) and South Africa (n = 99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (n = 159), Burkina Faso (n = 52) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: Resistance to ciprofloxacin was high in all countries (57.1%-100%). All isolates were susceptible to ceftriaxone, cefixime and spectinomycin, and 99.9% were susceptible to azithromycin. AMR determinants for ciprofloxacin, benzylpenicillin and tetracycline were common, but rare for cephalosporins and azithromycin. Most isolates belonged to the more antimicrobial-susceptible lineage B (n = 780) compared with the AMR lineage A (n = 141), and limited geographical phylogenomic signal was observed. CONCLUSIONS: We report the first multi-country gonococcal genomic comparison from Africa, which will support the WHO GASP and WHO enhanced GASP (EGASP). The high prevalence of resistance to ciprofloxacin (and empirical use continues), tetracycline and benzylpenicillin, and the emerging resistance determinants for azithromycin show it is imperative to strengthen the gonococcal AMR surveillance, ideally including genomics, in African countries.

Performance of the first commercial dual resistance assay, AmpliSens Mycoplasma genitalium-ML/FQ-Resist-FL, for detection of potential macrolide and quinolone resistance-associated mutations and prevalence of M. genitalium resistance mutations in St. Petersburg, Russia.

OBJECTIVES: Antimicrobial resistance in Mycoplasma genitalium (MG) is a poorly surveyed and controlled global health concern. We evaluated the first commercial dual resistance assay, AmpliSens M. genitalium-ML/FQ-Resist-FL assay, for detection of potential macrolide and quinolone resistance-associated mutations (MRAMs and QRAMs, respectively) and estimated the prevalence of these mutations in MG in St. Petersburg, Russia. METHODS: Urogenital samples positive (n=145 from 2007 to 2020) and negative (n=56 from 2021) for MG in routine diagnostics were retrospectively analysed using the AmpliSens M. genitalium-ML/FQ-Resist-FL assay (Central Research Institute of Epidemiology, Moscow, Russia) and Sanger sequencing for validation. RESULTS: The AmpliSens M. genitalium-ML/FQ-Resist-FL assay detected potential MRAMs and QRAMs with sensitivities of 100% (CI95% 83.9 to 100) and 92.3% (CI95% 66.7 to 99.6) and specificities of 99.2% (CI95% 95.6 to 100) and 100% (CI95% 97.2 to 100), respectively, in clinical specimens with ≥1000 MG geq/mL. In total, MRAMs were detected in 13.8% (CI95% 9.1 to 20.3) of samples, with 23S rRNA A2058G being the most prevalent mutation (45.0% (CI95% 25.8 to 65.8)). QRAMs were found in 9.0% (CI95% 5.3 to 14.7) of samples, with S83I the most frequent mutation (53.8% (CI95% 29.1 to 76.8)). Dual resistance was observed in 5.5% (CI95% 2.8 to 10.5) of samples. Potential MRAM and dual resistance rates significantly increased over time: from 0% in 2007-2008 to 25% (p trend =0.0009) and 10% (p trend =0.0447), respectively, in 2018-2020. QRAM rate appeared to increase (from 0% to 13%), but significance was not reached (p trend =0.0605). CONCLUSIONS: The rapid increase in MG antimicrobial resistance in St. Petersburg, especially prominent for MRAMs, necessitates implementation of macrolide resistance-guided therapy in Russia. The first commercial dual resistance assay, AmpliSens M. genitalium-ML/FQ-Resist-FL assay, was sensitive and specific for detection of potential MRAMs and QRAMs and could be valuable in macrolide resistance-guided therapies and possibly for surveillance of QRAMs. International surveillance of antimicrobial resistance-associated mutations in MG, further research into clinical relevance of several parC mutations and novel treatments are essential.

Cost-effectiveness of testing for Mycoplasma genitalium among men who have sex with men in Australia.

OBJECTIVES: Mycoplasma genitalium (MG) disproportionately affects men who have sex with men (MSM). We determined the cost-effectiveness of different testing strategies for MG in MSM, taking a healthcare provider perspective. METHODS: We used inputs from a dynamic transmission model of MG among MSM living in Australia in a decision tree model to evaluate the impact of four testing scenarios on MG incidence: (1) no one tested; (2) symptomatic MSM; (3) symptomatic and high-risk asymptomatic MSM; (4) all MSM. We calculated the incremental cost-effectiveness ratios (ICERs) using a willingness-to-pay threshold of $A30 000 per quality-adjusted life year (QALY) gained. We explored the impact of adding an antimicrobial resistance (AMR) tax (ie, additional cost per antibiotic consumed) to identify the threshold, whereby any testing for MG is no longer cost-effective. RESULTS: Testing only symptomatic MSM is the most cost-effective (ICER $3677 per QALY gained) approach. Offering testing to all MSM is dominated (ie, higher costs and lower QALYs gained compared with other strategies). When the AMR tax per antibiotic given was above $150, any testing for MG was no longer cost-effective. CONCLUSION: Testing only symptomatic MSM is the most cost-effective option, even when the potential costs associated with AMR are accounted for (up to $150 additional cost per antibiotic given). For pathogens like MG, where there are anticipated future costs related to AMR, we recommend models that test the impact of incorporating an AMR tax as they can change the results and conclusions of cost-effectiveness studies.

Sexual and reproductive health in Britain during the first year of the COVID-19 pandemic: cross-sectional population survey (Natsal-COVID-Wave 2) and national surveillance data.

OBJECTIVES: To assess sexual behaviour, and sexual and reproductive health (SRH) outcomes, after 1 year of the COVID-19 pandemic in Britain. METHODS: 6658 participants aged 18-59 and resident in Britain completed a cross-sectional web-panel survey (Natsal-COVID-Wave 2, March-April 2021), 1 year after the first lockdown. Natsal-COVID-2 follows the Natsal-COVID-Wave 1 survey (July-August 2020) which captured impacts in the initial months. Quota-based sampling and weighting resulted in a quasi-representative population sample. Data were contextualised with reference to the most recent probability sample population data (Natsal-3; collected 2010-12; 15 162 participants aged 16-74) and national surveillance data on recorded sexually transmitted infection (STI) testing, conceptions, and abortions in England/Wales (2010-2020). The main outcomes were: sexual behaviour; SRH service use; pregnancy, abortion and fertility management; sexual dissatisfaction, distress and difficulties. RESULTS: In the year from the first lockdown, over two-thirds of participants reported one or more sexual partners (women 71.8%; men 69.9%), while fewer than 20.0% reported a new partner (women 10.4%; men 16.8%). Median occasions of sex per month was two. Compared with 2010-12 (Natsal-3), we found less sexual risk behaviour (lower reporting of multiple partners, new partners, and new condomless partners), including among younger participants and those reporting same-sex behaviour. One in 10 women reported a pregnancy; pregnancies were fewer than in 2010-12 and less likely to be scored as unplanned. 19.3% of women and 22.8% of men were distressed or worried about their sex life, significantly more than in 2010-12. Compared with surveillance trends from 2010 to 2019, we found lower than expected use of STI-related services and HIV testing, lower levels of chlamydia testing, and fewer conceptions and abortions. CONCLUSIONS: Our findings are consistent with significant changes in sexual behaviour, SRH, and service uptake in the year following the first lockdown in Britain. These data are foundational to SRH recovery and policy planning.

How did the COVID-19 pandemic affect access to condoms, chlamydia and HIV testing, and cervical cancer screening at a population level in Britain? (Natsal-COVID).

OBJECTIVES: To investigate how differential access to key interventions to reduce STIs, HIV and their sequelae changed during the COVID-19 pandemic. METHODS: British participants (18-59 years) completed a cross-sectional web survey 1 year (March-April 2021) after the initial lockdown in Britain. Quota-based sampling and weighting resulted in a quasi-representative population sample. We compared Natsal-COVID data with Natsal-3, a household-based probability sample cross-sectional survey (16-74 years) conducted in 2010-2012. Reported unmet need for condoms because of the pandemic and uptake of chlamydia testing/HIV testing/cervical cancer screening were analysed among sexually experienced participants (18-44 years) (n=3869, Natsal-COVID; n=8551, Natsal-3). ORs adjusted for age and other potential confounders describe associations with demographic and behavioural factors. RESULTS: In 2021, 6.9% of women and 16.2% of men reported unmet need for condoms because of the pandemic. This was more likely among participants: aged 18-24 years, of black or black British ethnicity, and reporting same-sex sex (past 5 years) or one or more new relationships (past year). Chlamydia and HIV testing were more commonly reported by younger participants, those reporting condomless sex with new sexual partners and men reporting same-sex partners; a very similar distribution to 10 years previously (Natsal-3). However, there were differences during the pandemic, including stronger associations with chlamydia testing for men reporting same-sex partners; with HIV testing for women reporting new sexual partners and with cervical screening among smokers. CONCLUSIONS: Our study suggests differential access to key primary and secondary STI/HIV prevention interventions continued during the first year of the COVID-19 pandemic. However, there was not strong evidence that differential access has changed during the pandemic when compared with 2010-2012. While the pandemic might not have exacerbated inequalities in access to primary and secondary prevention, it is clear that large inequalities persisted, typically among those at greatest STI/HIV risk.

Projecting the development of antimicrobial resistance in Neisseria gonorrhoeae from antimicrobial surveillance data: a mathematical modelling study.

BACKGROUND: The World Health Organization recommends changing the first-line antimicrobial treatment for gonorrhoea when ≥ 5% of Neisseria gonorrhoeae cases fail treatment or are resistant. Susceptibility to ceftriaxone, the last remaining treatment option has been decreasing in many countries. We used antimicrobial resistance surveillance data and developed mathematical models to project the time to reach the 5% threshold for resistance to first-line antimicrobials used for N. gonorrhoeae. METHODS: We used data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales from 2000-2018 about minimum inhibitory concentrations (MIC) for ciprofloxacin, azithromycin, cefixime and ceftriaxone and antimicrobial treatment in two groups, heterosexual men and women (HMW) and men who have sex with men (MSM). We developed two susceptible-infected-susceptible models to fit these data and produce projections of the proportion of resistance until 2030. The single-step model represents the situation in which a single mutation results in antimicrobial resistance. In the multi-step model, the sequential accumulation of resistance mutations is reflected by changes in the MIC distribution. RESULTS: The single-step model described resistance to ciprofloxacin well. Both single-step and multi-step models could describe azithromycin and cefixime resistance, with projected resistance levels higher with the multi-step than the single step model. For ceftriaxone, with very few observed cases of full resistance, the multi-step model was needed to describe long-term dynamics of resistance. Extrapolating from the observed upward drift in MIC values, the multi-step model projected ≥ 5% resistance to ceftriaxone could be reached by 2030, based on treatment pressure alone. Ceftriaxone resistance was projected to rise to 13.2% (95% credible interval [CrI]: 0.7-44.8%) among HMW and 19.6% (95%CrI: 2.6-54.4%) among MSM by 2030. CONCLUSIONS: New first-line antimicrobials for gonorrhoea treatment are needed. In the meantime, public health authorities should strengthen surveillance for AMR in N. gonorrhoeae and implement strategies for continued antimicrobial stewardship. Our models show the utility of long-term representative surveillance of gonococcal antimicrobial susceptibility data and can be adapted for use in, and for comparison with, other countries.

Multidrug-resistant Neisseria gonorrhoeae isolate SE690: mosaic penA-60.001 gene causing ceftriaxone resistance internationally has spread to the more antimicrobial-susceptible genomic lineage, Sweden, September 2022.

We report a ceftriaxone-resistant, multidrug-resistant urogenital Neisseria gonorrhoeae in a female sex worker in Sweden, September 2022, who was treated with ceftriaxone 1 g, but did not return for test-of-cure. Whole genome sequencing of isolate SE690 identified MLST ST8130, NG-STAR CC1885 (new NG-STAR ST4859) and mosaic penA-60.001. The latter, causing ceftriaxone resistance in the internationally spreading FC428 clone, has now also spread to the more antimicrobial-susceptible genomic lineage B, showing that strains across the gonococcal phylogeny can develop ceftriaxone resistance.