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Objectives: This study aimed to validate and implement a rapid screening assay for molecular detection of the penA -60 allele that is associated with ceftriaxone resistance in Neisseria gonorrhoeae for use on both isolate lysates and clinical specimen DNA extracts., Methods: A N. gonorrhoeae penA real-time (RT)-PCR was adapted to include a species-specific pap confirmation target and a commercially available internal control to monitor for PCR inhibition.The modified assay was validated using N. gonorrhoeae -positive (n=24) and N. gonorrhoeae -negative (n=42) clinical specimens and isolate lysates. The panel included seven samples with resistance conferred by penA alleles targeted by the assay and four samples with different penA alleles. The feasibility of using the penA RT-PCR for molecular surveillance was assessed using clinical specimens from 54 individuals attending a London sexual health clinic who also had a N. gonorrhoeae isolate included in the 2020 Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP)., Results: The assay correctly identified N. gonorrhoeae specimens (n=7) with penA -60/64 alleles targeted by the assay. No penA false negatives/positives were detected, giving the penA target of the assay a sensitivity, specificity, positive and negative predicted values (PPV, NPV) of 100% (95% CIs; sensitivity; 56.1-100%, specificity; 93.6-100%, PPV; 56.1-100%, NPV; 93.6-100%).No cross-reactivity with other Neisseria species or other urogenital pathogens was detected. The N. gonorrhoeae target ( pap ) was detected in 73 out of 78 of the N. gonorrhoeae -positive specimens, resulting in 92.6% sensitivity (95% CI 83.0% to 97.3%), 100% specificity (95% CI 75.9% to 100%) and PPV, and a NPV of 89.4% (95% CI 52.5% to 90.9%). No penA -59/60/64 alleles were detected within the clinical specimens from the GRASP 2020 feasibility molecular surveillance study (n=54 individuals)., Conclusion: The implementation of this PCR assay for patient management, public health and surveillance purposes enables the rapid detection of gonococcal ceftriaxone resistance conferred by the most widely circulating penA alleles., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Quarterly STI screening is recommended for high-risk gay, bisexual and other men who have sex with men (MSM) in the UK, but frequent antibiotic exposure could potentially increase the risk of antimicrobial resistance (AMR) developing in Neisseria gonorrhoeae . We investigated whether repeat diagnosis of gonorrhoea in those attending sexual health services (SHS) was associated with reduced antimicrobial susceptibility., Methods: Antimicrobial susceptibility data relating to the most recent gonorrhoea diagnosis for each individual included in the Gonococcal Resistance to Antimicrobials Surveillance Programme (2015-2019) were matched to their historical records in the national GUMCAD STI surveillance data set (2012-2019). The number of gonorrhoea diagnoses in the previous 3 years was calculated for each SHS attendee. Logistic regression was used to examine the associations between the number of diagnoses and reduced susceptibility to ceftriaxone (minimum inhibitory concentration (MIC) >0.03 mg/L), cefixime (MIC >0.06 mg/L) and azithromycin (MIC >0.25 mg/L) at the time of the latest diagnosis., Results: Of 6161 individuals included in the analysis, 3913 (63.5%) were MSM, 1220 (19.8%) were heterosexual men and 814 (13.2%) were women. Among MSM, 2476 (63.3%) had 1 past gonorrhoea diagnosis, 1295 (33.1%) had 2-4, 140 (3.6%) 5-9, and 2 (0.1%) ≥10. Most women and heterosexual men (91.7%) had one past gonorrhoea diagnosis; none had more than four. Reduced ceftriaxone and cefixime susceptibility was more common among MSM with two to four gonorrhoea diagnoses (3.8% and 5.8%, respectively) compared with those with one (2.2% and 3.9%, respectively). After adjusting for potential confounding, this association remained (adjusted OR: 1.59, 95% CI 1.07 to 2.37, p=0.02; adjusted OR: 1.54, 95% CI 1.11 to 2.14, p=0.01). No evidence was found for any other associations., Conclusions: Among MSM, repeat diagnosis of gonorrhoea may be associated with reduced ceftriaxone and cefixime susceptibility. As these are last-line therapies for gonorrhoea, further research is needed to assess the impact of intensive STI screening on AMR., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: This study sought to provide data on the prevalence of macrolide (23S rRNA) and fluoroquinolone ( parC ) resistance-associated mutations seen in Mycoplasma genitalium -positive specimens received in the UK national reference laboratory., Methods: In total, 2580 clinical specimens from patients with suspected or confirmed M. genitalium infection were received at the national reference laboratory between September 2017 and November 2018. M. genitalium -positive clinical specimens were identified using a reverse transcription-PCR targeting two M. genitalium genes: MgPa and gap . Resistance-associated single nucleotide poylmorphisms were sought in all positive specimens by sequence analysis of the 23S rRNA and parC genes., Results: Eighteen per cent (458 of 2580) of clinical specimens were positive for M. genitalium and 389 had sequence data for both macrolide and fluoroquinolone resistance markers. Of these, 71% (275 of 389) had macrolide resistance-associated mutations, 8% (31 of 389) had fluoroquinolone resistance-associated mutations (S83I/R and D87Y/N) and 7% (26 of 389) had mutations associated with resistance to both antimicrobials. Only 28% (108 of 389) had no mutations associated with resistance to either class of antibiotic. Five specimens had mutations of unknown clinical significance in the parC gene (eg, G81C and S83N)., Conclusions: Mutations associated with resistance to macrolides were very frequent. By contrast, susceptibility to the second-line treatment, moxifloxacin (a fluoroquinolone), was estimated at 92% based on the absence of resistance-associated mutations. The few specimens with mutations of unknown clinical significance in the parC gene were excluded from the analysis and so the actual level of fluoroquinolone susceptibility may be slightly lower than that reported here. Surveillance of antimicrobial resistance in M. genitalium is imperative for this to remain a treatable infection., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: A multicentre, randomised non-inferiority trial compared the efficacy and safety of 14 days of ofloxacin and metronidazole (standard-of-care (SoC)) versus a single dose of intramuscular ceftriaxone followed by 5 days of azithromycin and metronidazole (intervention arm (IA)) in women with mild-to-moderate pelvic inflammatory disease (PID)., Methods: Women with a clinical diagnosis of PID presenting at sexual health services were randomised to the SoC or IA arms. Treating clinicians and participants were not blinded to treatment allocation but the clinician performing the assessment of primary outcome was blinded. The primary outcome was clinical cure defined as ≥70% reduction in the modified McCormack pain score at day 14-21 after starting treatment. Secondary outcomes included adherence, tolerability and microbiological cure., Results: Of the randomised population 72/153 (47.1%) reached the primary end point in the SoC arm, compared with 68/160 (42.5%) in the IA (difference in cure 4.6% (95% CI -15.6% to 6.5%). Following exclusion of 86 women who were lost to follow-up, attended outside the day 14-21 follow-up period, or withdrew consent, 72/107 (67.3%) had clinical cure in the SoC arm compared with 68/120 (56.7%) in the IA, giving a difference in cure rate of 10.6% (95% CI -23.2% to 1.9%). We were unable to demonstrate non-inferiority of the IA compared with SoC arm. Women in the IA took more treatment doses compared with the SoC group (113/124 (91%) vs 75/117 (64%), p=0.0001), but were more likely to experience diarrhoea (61% vs 24%, p<0.0001). Of 288 samples available for analysis, Mycoplasma genitalium was identified in 10% (28/288), 58% (11/19) of which had baseline antimicrobial resistance-associated mutations., Conclusion: A short-course azithromycin-based regimen is likely to be less effective than the standard treatment with ofloxacin plus metronidazole. The high rate of baseline antimicrobial resistance supports resistance testing in those with M. genitalium infection to guide appropriate therapy., Trial Registration Number: 2010-023254-36., Competing Interests: Competing interests: JR reports personal fees from GSK Pharma, Hologic Diagnostics, Mycovia and Janssen Pharma as well as ownership of shares in GSK Pharma and AstraZeneca Pharma; and is author of the UK and European Guidelines on Pelvic Inflammatory Disease; is a Member of the European Sexually Transmitted Infections Guidelines Editorial Board; is a Member of the National Institute for Health Research Funding Committee (Health Technology Assessment programme); was previously a Member of the National Institute for Health Research HTA Primary Care, Community and Preventative Interventions Panel (2013-2016). He is an NIHR Journals Editor and associate editor of Sexually Transmitted Infections journal. He is an officer of the British Association for Sexual Health and HIV (vice-president), and the International Union against Sexually Transmitted Infections (treasurer), and a charity trustee of the Sexually Transmitted Infections Research Foundation., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Mycoplasma genitalium is a common sexually transmitted infection. Treatment guidelines focus on those with symptoms and sexual contacts, generally with regimens including doxycycline and/or azithromycin as first-line and moxifloxacin as second-line treatment. We investigated the prevalence of antimicrobial resistance (AMR)-conferring mutations in M. genitalium among the sexually-active British general population., Methods: The third national survey of sexual attitudes and lifestyles (Natsal-3) is a probability sample survey of 15 162 men and women aged 16-74 years in Britain conducted during 2010-12. Urine test results for M. genitalium were available for 4507 participants aged 16-44 years reporting > 1 lifetime sexual partner. In this study, we sequenced regions of the 23S rRNA and parC genes to detect known genotypic determinants for resistance to macrolides and fluoroquinolones respectively., Results: 94% (66/70) of specimens were re-confirmed as M. genitalium positive, with successful sequencing in 85% (56/66) for 23S rRNA and 92% (61/66) for parC genes. Mutations in 23S rRNA gene (position A2058/A2059) were detected in 16.1% (95%CI: 8.6% to 27.8%) and in parC (encoding ParC D87N/D87Y) in 3.3% (0.9%-11.2%). Macrolide resistance was more likely in participants reporting STI diagnoses (past 5 years) (44.4% (18.9%-73.3%) vs 10.6% (4.6%-22.6%); p=0.029) or sexual health clinic attendance (past year) (43.8% (23.1%-66.8%) vs 5.0% (1.4%-16.5%); p=0.001). All 11 participants with AMR-conferring mutations had attended sexual health clinics (past 5 years), but none reported recent symptoms., Conclusions: This study highlights challenges in M. genitalium management and control. Macrolide resistance was present in one in six specimens from the general population in 2010-2012, but no participants with AMR M. genitalium reported symptoms. Given anticipated increases in diagnostic testing, new strategies including novel antimicrobials, AMR-guided therapy, and surveillance of AMR and treatment failure are recommended., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Objectives: Understanding the public health impact of lymphogranuloma venereum (LGV) in Europe is hampered by inadequate diagnostics and surveillance systems in many European countries. We developed and piloted LGV surveillance in three European countries without existing systems and performed a preliminary investigation of LGV epidemiology, where little evidence currently exists., Methods: We recruited STI or dermatovenereology clinics and associated laboratories serving men who have sex with men (MSM) in Austria, Croatia and Slovenia, using the UK for comparison. We undertook centralised LGV testing of Chlamydia trachomatis (CT)-positive rectal swabs collected between October 2016 and May 2017 from MSM attending these clinics. Stored specimens from Austria (2015-2016) and Croatia (2014) were also tested. Clinical and sociodemographic data were collected using a standardised proforma. The ompA gene of LGV-positive specimens was sequenced., Results: In total, 500 specimens from CT-positive MSM were tested, and LGV positivity was 25.6% (128/500; 95% CI 22.0% to 29.6%) overall, and 47.6% (79/166; 40.1% to 55.2%) in Austria, 20.0% (3/15; 7.1% to 45.2%) in Croatia, 16.7% (1/6; 3.0% to 56.4%) in Slovenia and 14.4% (45/313; 10.9% to 18.7 %) in the UK. Proformas were completed for cases in Croatia, Slovenia and in the UK; proformas could not be completed for Austrian cases, but limited data were available from line listings. Where recorded, 83.9% (78/93) of LGV-CT cases were HIV-positive compared with 65.4% (149/228) of non-LGV-CT cases; MSM with LGV-CT were more likely to have proctitis (Austria, 91.8% vs 40.5%, p<0.001; Croatia, 100% vs 25%, p=0.04; UK, 52.4% vs 11.7%, p<0.001) than those with non-LGV-CT. Six different ompA sequences were identified, including three new variants; the L2 ompA sequence predominated (58.6%, 51/87)., Conclusions: LGV is substantially underdiagnosed in MSM across Europe. Unified efforts are needed to overcome barriers to testing, establish effective surveillance, and optimise diagnosis, treatment and prevention., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To compare performance of the ResistancePlus kit (SpeeDx, Australia) with in-house methods for the detection of Mycoplasma genitalium- specific DNA and mutations associated with resistance to macrolide antimicrobials, directly from clinical specimens., Methods: Assay specificity and sensitivity was analysed using DNA from 46 non- M. genitalium organisms and standard curve analysis, respectively. A panel of archived DNA extracted from 97 M. genitalium -positive clinical specimens, for which the macrolide susceptibility genotype had been previously determined, were tested on the assay and results compared., Results: Final analytical specificity was 100%. Sensitivity was detected to at least 140 genome copies/µL. The assay detected M. genitalium in 92/97 (94.9%, 95% CI 88.4% to 98.3%) previously positive specimens. The genetic macrolide susceptibility assigned was concordant with previous results in 85/92 (92.4%, 95% CI 85.0% to 96.9%) specimens or 85/97 (87.6%, 95% CI: 79.4% to 93.4%) when the false-negative specimens were included. On seven (7/92, 7.6%) occasions, resistant specimens were called susceptible. Further testing resolved discrepancies for all but five (5.2%) specimens., Conclusions: The ResistancePlus assay generally performed well in comparison to methods currently employed at the reference laboratory. It detected a range of different mutations; however, a small number of specimens that were genotyped as macrolide resistant by Sanger sequencing were either not detected by the assay or were genotyped as susceptible. This could impact on treatment outcomes if assay results were used for patient management., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objectives: Resistance to both macrolides and fluoroquinolones has been reported in Mycoplasma genitalium; however, due to limited diagnostics, studies are often small and confined to specific geographical areas. This study sought to determine the rate of predicted resistance in M. genitalium -positive specimens referred for diagnostic testing., Methods: Seventy-four M. genitalium -positive specimens, referred to the national reference laboratory (2010-2013) from 19 centres across England, were blinded and anonymised. Specimens were examined for markers predictive of resistance to macrolides and fluoroquinolones using PCR followed by sequence analysis of 23 S rRNA gene, or gyrA and parC , respectively., Results: 23 S rRNA gene PCR sequencing revealed that 82.4% (61/74) of specimens harboured a single nucleotide polymorphism (SNP) associated with macrolide resistance. Differences were observed between the rates of predicted macrolide resistance in male (95.1% (58/61)) and female (23.1% (3/13)) patients (P = <0.001). By contrast, all specimens for which sequencing data were available (73/74) yielded wild-type gyrA sequences; and 58/61 (95.1%) had wild-type parC genes. Three specimens (3/61 4.9%) had SNPs in the parC gene associated with fluoroquinolone treatment failure, and all three also had predicted resistance to macrolides., Conclusions: Eighty-two per cent and 4.9% of M. genitalium specimens had SNPs associated with macrolide and fluoroquinolone resistance, respectively. Due to lack of widespread availability of testing for M. genitalium in the UK, this study sample was likely to be sourced from patients who may have already failed first-line macrolide therapy. Nevertheless, this study highlights the need for both greater access to M. genitalium diagnostics and genetic antimicrobial resistance testing., Competing Interests: Competing interests: Since the laboratory work described in this report was completed, AMRHAI has received funding from SpeeDx for a kit evaluation. HF has participated in the Consensus Steering Group for Mycoplasma genitalium diagnosis in the UK, arranged and funded by Hologic., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Introduction: Nucleic acid amplification tests, with their ability to detect very small amounts of nucleic acid, have become the principle diagnostic tests for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in many sexual health clinics. The aim of this study was to investigate the extent of surface contamination with CT and GC within a city centre sexual health clinic and to evaluate the potential for contamination of containers used for the collection of self-taken swabs., Method: Surface contamination with CT and GC was assessed by systematically sampling 154 different sites within one clinic using transcription-mediated amplification (TMA), quantitative PCR and culture. The caps of containers used by patients to collect self-taken samples were also tested for CT and GC using TMA., Results: Of the 154 sites sampled, 20 (13.0%) tested positive on TMA. Of these, five (3.2%) were positive for CT alone, 11 (7.1%) for GC alone and four (2.6%) for both CT and GC. The proportion of GC TMA-positive test results differed by gender, with 11 (18.3%) positive results from the male patient clinic area compared with one (1.6%) from the female area (p=0.002). Positive samples were obtained from a variety of locations in the clinic, but the patient toilets were more likely to be contaminated than examination rooms (p=0.015). Quantitative PCR and culture assays were negative for all samples. 46 caps of the containers used for self-taken swabs were negative for both CT and GC on TMA testing., Conclusions: Surface contamination with chlamydial and gonococcal rRNA can occur within sexual health clinics, but the quantity of nucleic acid detected is low and infection risk to patients and staff is small. There remains a potential risk of contamination of patient samples leading to false-positive results.