To Örebro University

Sexually transmitted Chlamydia trachomatis infections remain common globally and most frequently are asymptomatic. The 2025 European C. trachomatis guideline provides up-to-date guidance regarding indications for testing and treatment of C. trachomatis infections. It includes advice on urogenital and extragenital C. trachomatis testing including the use of self-collected specimens; recommendation to use only validated NAATs for diagnosis; and recommendation to treat all C. trachomatis infections with doxycycline as first line in preference to single-dose azithromycin regimens. The absence of evidence and limited value of broad screening in asymptomatic populations for C. trachomatis infections is also discussed.

OBJECTIVES: The decennial National Surveys of Sexual Attitudes and Lifestyles (Natsal) provide general population prevalence estimates in Britain for key sexually transmitted infections (STIs) through biosampling. Since methodological choices can impact acceptability and response rates, we evaluated processes for Natsal-4, including face-to-face and remote interview arrangements, non-return of test results and vaginal swab collection in two pilot studies.

METHODS: The pilots were conducted during June to August 2021 and February to March 2022. Participants aged 16-59 years were invited to provide urine samples (cisgender men and trans/gender diverse) or three vaginal swabs (cisgender women; urine was requested if vaginal swabs were declined) following interview. Samples were self-collected at home and posted to the laboratory by the interviewer if the interview was face to face, or by the participant if they preferred to collect the sample later or the interview was remote. Process feedback was collected after the first pilot via qualitative interviews with participants and after both pilots through informal interviewer debriefing.

RESULTS: Of 261 participants interviewed (pilot 1=130; pilot 2=131), 161 (62%) consented to biosampling, of which 129 (49%) provided samples. A sample was received from 78/153 (51%) of women, of whom 60 (77%) provided vaginal swabs and 18 (23%) provided a urine sample. A urine sample was received from 51/108 (47%) cisgender men or trans/gender diverse participants. All samples collected immediately after face-to-face interviews were received (n=77), while 64% of samples from participants consenting to post samples after face-to-face interviews and 60% after remote interviews were received. Process feedback confirmed our methods were broadly acceptable.

CONCLUSIONS: We demonstrated that our approach to biosampling and STI testing for a national sexual health survey was reasonably acceptable and feasible in the period coming out the COVID-19 pandemic. Self-collection of vaginal swabs for research, which provide higher testing sensitivity than urine, was feasible and acceptable in a home setting.

The continued emergence of Neisseria gonorrhoeae strains that express resistance to multiple antibiotics, including the last drug for empiric monotherapy (ceftriaxone), necessitates the development of new treatment options to cure gonorrheal infections. Toward this goal, we recently reported that corallopyronin A (CorA), which targets the switch region of the β' subunit (RpoC) of bacterial DNA-dependent RNA polymerase (RNAP), has potent anti-gonococcal activity against a panel of multidrug-resistant clinical strains. Moreover, in that study, CorA could eliminate gonococcal infection of primary human epithelial cells and gonococci in a biofilm state. To determine if N. gonorrhoeae could develop high-level resistance to CorA in a single step, we sought to isolate spontaneous mutants expressing any CorA resistance phenotypes. However, no single-step mutants with high-level CorA resistance were isolated. High-level CorA resistance could only be achieved in this study through a multi-step pathway involving over-expression of the MtrCDE drug efflux pump and single amino acid changes in the β and β' subunits (RpoB and RpoC, respectively) of RNAP. Molecular modeling of RpoB and RpoC interacting with CorA was used to deduce how the amino acid changes in RpoB and RpoC could influence gonococcal resistance to CorA. Bioinformatic analyses of whole genome sequences of clinical gonococcal isolates indicated that the CorA resistance determining mutations in RpoB/C, identified herein, are very rare (≤ 0.0029%), suggesting that the proposed pathway for resistance is predictive of how this phenotype could potentially evolve if CorA is used therapeutically to treat gonorrhea in the future. IMPORTANCE: The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

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.

INTRODUCTION: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global public health concern and enhanced global gonococcal AMR surveillance is imperative. As in many African countries, regular, representative and quality-assured gonococcal AMR is lacking in Ethiopia. We describe the AMR in gonococcal isolates from five cities across Ethiopia, 2021-22, and patient epidemiological data.

METHODS: Urethral discharge from males and cervical discharge from females were collected from October 2021 to September 2022. Epidemiological data were collected using a questionnaire. MIC determination (ETEST; eight antimicrobials) was performed on gonococcal isolates and EUCAST breakpoints (v13.1) were used.

RESULTS: From 1142 urogenital swab samples, 299 species-identified gonococcal isolates were identified; 78.3% were from males and 21.7% from females. The median age for males and females was 25 and 23 years, respectively. Most isolates (61.2%) were identified in Addis Ababa, followed by Gondar (11.4%), Adama (10.4%), Bahir Dar (10.0%) and Jimma (7.0%). The resistance level to ciprofloxacin, tetracycline and benzylpenicillin was 97.0%, 97.0% and 87.6%, respectively, and 87.6% of isolates were producing β-lactamase. All isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. Recommended therapy [ceftriaxone (250 mg) plus azithromycin (1 g)] was used for 84.2% of patients.

CONCLUSIONS: We present the first national quality-assured gonococcal AMR data from Ethiopia. Resistance levels to ciprofloxacin, tetracycline and benzylpenicillin were exceedingly high. However, all isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. In Ethiopia, it is essential to strengthen the gonococcal AMR surveillance by including further epidemiological data, more isolates from different cities, and WGS.

Sexually transmitted infections (STIs) have been part of human life since ancient times, and their symptoms affect quality of life, and sequelae are common. Socioeconomic and behavioural trends affect the prevalence of STIs, but the discovery of antimicrobials gave hope for treatment, control of the spread of infection and lower rates of sequelae. This has to some extent been achieved, but increasing antimicrobial resistance and increasing transmission in high-risk sexual networks threaten this progress. For Neisseria gonorrhoeae, the only remaining first-line treatment (with ceftriaxone) is at risk of becoming ineffective, and for Mycoplasma genitalium, for which fewer alternative antimicrobial classes are available, incurable infections have already been reported. For Chlamydia trachomatis, in vitro resistance to first-line tetracyclines and macrolides has never been confirmed despite decades of treatment of this highly prevalent STI. Similarly, Treponema pallidum, the cause of syphilis, has remained susceptible to first-line penicillin.

BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology.

METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined.

FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found.

INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe.

Background: Bacterial vaginosis (BV) is a common vaginal disorder among women of reproductive age, and BV can be associated with adverse pregnancy outcomes and enhanced acquisition and transmission of STIs/HIV. The present study aimed to determine the prevalence of BV using the Nugent score and sociodemographic factors associated with BV among women in Maputo, Mozambique, and to evaluate the performance of the AmpliSens® Florocenosis/Bacterial vaginosis-FRT PCR kit versus Nugent score for diagnosis of BV.

Material and Methods: Vaginal swabs were collected from 886 non-pregnant symptomatic women during their visit to the Mavalane Health area in Maputo, Mozambique from February 2018 to January 2019. BV was diagnosed by Nugent score. The AmpliSens®Florocenosis/Bacterial vaginosis-FRT PCR kit (InterLabService, Moscow, Russia) was evaluated for BV diagnosis. HIV was detected using Determine HIV1/2 (Alere Medical Co. Ltd, Chiba, Japan) plus Uni-Gold HIV1/2 (Trinity Biotech, Ireland). The chisquare test was used to estimate associations between categorical variables.

Results: The prevalence of BV by PCR, Nugent score, and HIV was 47.2%, 39.1%, and 22.5%, respectively. Of those with BV, 52% were HIV-positive and 48% HIV-negative (p < 0.001). The highest proportion of women was under 24 years old (38.1%), single (49.5%), with secondary education (53.5%), and living in rural areas (55.4%). BV was associated with young age at first sexual intercourse (44.5%) (χ2 = 17.47, p=< 0.001), condom use (43.3%) (χ2 =3.7, p= 0.05), and no use of contraceptives (49%) (χ2= 13.6, p=0.02). In real-time PCR, a higher proportion of BV cases (47.2%) were detected. However, 12.5% of women had an unknown vaginal dysbiosis. The sensitivity and specificity of the PCR were 99.6% and 82.2%, respectively. Using the PCR as a reference test, the sensitivity and specificity of the Nugent score were 86.2% and 99.4%, respectively. The concordance of both tests was κ=0.825 (95% CI, 0.78 - 0.86), p<0.001.

Conclusions: A high prevalence of BV was associated with young age at first sexual intercourse, condom and contraceptive use among women in Maputo, Mozambique. The AmpliSens® Florocenosis/Bacterial vaginosis-FRT PCR assay detected more BV-positive cases than the Nugent score and needs further evaluation in other settings.

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.

Ceftriaxone-resistant Neisseria gonorrhoeae strains, mostly associated with Asia, threaten gonorrhea treatment. We report the reference genomes of two ceftriaxone-resistant isolates found in routine surveillance in Bangkok, Thailand. The genomes belonged to the more antimicrobial-susceptible genomic lineage B, illustrating that both ceftriaxone-resistant strains and the mosaic penA-60.001 ceftriaxone-resistance determinant are spreading.