Molecular detection of ceftriaxone resistance in Neisseria gonorrhoeae clinical specimens: a tool for public health control.

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.

Is there an association between previous infection with Neisseria gonorrhoeae and gonococcal AMR? A cross-sectional analysis of national and sentinel surveillance data in England, 2015-2019.

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.

No widespread dissemination of Chlamydia trachomatis diagnostic-escape variants and the impact of Neisseria gonorrhoeae positivity on the Aptima Combo 2 assay.

OBJECTIVES: A Finnish Chlamydia trachomatis (CT) new variant was detected in 2019 that escaped detection in the Hologic Aptima Combo 2 (AC2) assay due to a C1515T mutation in the CT 23S rRNA target region. Reflex testing of CT-negative/CT-equivocal specimens as well as those positive for Neisseria gonorrhoeae (NG) with the Hologic Aptima CT (ACT) assay was recommended to identify any CT variants. METHODS: From June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed. RESULTS: A total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens. CONCLUSIONS: No widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

Detection of markers predictive of macrolide and fluoroquinolone resistance in Mycoplasma genitalium from patients attending sexual health services.

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.

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.

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).

Antimicrobial resistance in Neisseria gonorrhoeae isolates from foreign-born population in the European Gonococcal Antimicrobial Surveillance Programme.

OBJECTIVES: International spread has contributed substantially to the high prevalence of antimicrobial resistant (AMR) Neisseria gonorrhoeae infections worldwide. We compared the prevalence of AMR gonococcal isolates among native persons to foreign-born (reporting country different from country of birth) persons, and describe the epidemiological and clinical characteristics of foreign-born patients and their associations to AMR. METHODS: We analysed isolates and patient data reported to the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) 2010-2014 (n=9529). RESULTS: Forty-three per cent of isolates had known country of birth and 17.2% of these were from persons born abroad. Almost 50% of foreign-born were from the WHO European Region (13.1% from non-European Union [EU] and the European Economic Area [EEA] countries). Compared with isolates from natives, isolates from foreign-born had a similar level (p>0.05) of azithromycin resistance (7.5% vs 7.2%), ciprofloxacin resistance (50.0% vs 46.3%) and of decreased susceptibility to ceftriaxone (1.9% vs 2.8%); a lower rate of cefixime resistance (5.7% vs 3.6%, p=0.02), and a higher proportion of isolates producing penicillinase (8.4% vs 11.7%, p=0.02). Among isolates from persons born outside EU/EEA, the level of decreased susceptibility to ceftriaxone was higher (1.8% vs 3.5%, p=0.02), particularly in those from the WHO Eastern Mediterranean Region and non-EU/EEA WHO European countries (1.9% vs 9.6% and 8.7%, respectively, p<0.01). In multivariable analysis, foreign-born patients with AMR isolates were more likely to be from non-EU/EEA WHO European countries (adjusted OR [aOR]: 3.2, 95% CI 1.8 to 5.8), WHO Eastern Mediterranean countries (aOR: 1.8, 95% CI 1.1 to 3.3) and heterosexual males (aOR: 1.8, 95% CI 1.2 to 2.7). CONCLUSIONS: Importation of AMR strains remains an important threat in the EU/EEA. Research to improve understanding of sexual networks within foreign born and sexual tourism populations could help to inform effective tailor-made interventions. The Euro-GASP demonstrates the public health value of quality-assured surveillance of gonococcal AMR and the need for strengthened AMR surveillance, particularly in the non-EU/EEA WHO European Region.

Antimicrobial resistance in Mycoplasma genitalium sampled from the British general population.

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.

Substantial underdiagnosis of lymphogranuloma venereum in men who have sex with men in Europe: preliminary findings from a multicentre surveillance pilot.

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.

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance.

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.