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.

BACKGROUND: Infection with Trichomonas vaginalis (TV) is the most prevalent curable sexually transmitted infection (STI) globally and is associated with prelabour rupture of membranes, preterm delivery, and low birthweight. Point-of-care (POC) testing for TV during pregnancy may facilitate rapid antenatal case detection and treatment. This study, part of the World Health Organization's global ProSPeRo study, aimed to evaluate the performance of OSOM® Trichomonas Rapid Test, an antigen-based POC test, against a reference nucleic acid amplification test (NAAT) among pregnant women in Zambia. We also assessed the operational characteristics and patient acceptability of the POC test, within the context of WHO's target product profiles for STI POC tests.

METHODS: We enrolled pregnant women attending four health centres in Nchelenge, Zambia, for antenatal care between 15 February and 26 May 2023. Vaginal swabs for the TV POC test and a reference NAAT (Aptima® Trichomonas vaginalis assay) were obtained. POC test results were read independently by two study staff members. Study staff filled a questionnaire on the operational characteristics of the POC test, and participants were asked about their willingness to wait for results.

RESULTS: Paired POC and reference test samples were collected from 1,015 participants. Overall, 23.0% (233/1015) tested positive for TV by NAAT, and 15.3% (155/1015) tested positive by the POC test, with three inconclusive results. The overall sensitivity and specificity of the POC test were 66.4% (95% confidence intervals [CI] 57.7-74.1%) and 99.6% (95% CI: 98.8-99.9%), respectively. Sensitivity was higher among those with TV-associated symptoms compared to those without (83.6% versus 60.4%, relative ratio 1.39, 95% CI 1.14-1.68). Inter-rater agreement was 99.7% (Cohen's Kappa 0.989). The study staff (n = 14) found the test easy to use and interpret, with most staff (12/14) reporting results were available within 25 min.

CONCLUSION: Overall, the TV POC test showed lower sensitivity than WHO's 85% target, but exceeded the 99% specificity target. Among symptomatic pregnant women, sensitivity nearly reached the WHO target. The assay was user-friendly, required minimal training, and delivered results quickly. Further studies are needed to determine the optimal antenatal settings for this technology.

TRIAL REGISTRATION: PACTR202302766902029.

BACKGROUND: Over the past 5 years, since publication of the initial review, studies have provided additional data on macrolide and fluoroquinolone resistance in Mycoplasma genitalium, including data from regions previously lacking this information. We aimed to provide contemporary estimates of macrolide and fluoroquinolone resistance in M genitalium to inform national, regional, and global treatment guidelines.

METHODS: This is an update of a previous systematic review and meta-analysis, which was performed up to Jan 7, 2019. In this update, we searched PubMed, Embase, and MEDLINE from Jan 1, 2018, to April 18, 2023, for published studies reporting macrolide, fluoroquinolone, or dual-class (macrolide and fluoroquinolone) resistance in M genitalium. Data were combined with the previous meta-analysis to examine resistance prevalence in M genitalium samples collected up to and including 2021. Random-effects meta-analyses were used to calculate summary estimates of prevalence. Subgroup analyses by WHO region and four time periods (before 2012 to 2018-21) were performed. This study was registered with PROSPERO, number CRD42021273340.

FINDINGS: 166 studies (59 from the previous search period reporting data from M genitalium samples collected between 2003 and 2017, and 107 from the updated search period reporting data from M genitalium samples collected between 2005 and 2021) were included: 157 reporting macrolide resistance (41 countries; 22 974 samples), 89 reporting fluoroquinolone resistance (35 countries; 14 165 samples), and 74 reporting dual-class resistance (34 countries; 11 070 samples). In 2018-21, the overall prevalence of macrolide, fluoroquinolone, and dual-class resistance were 33·3% (95% CI 27·2-39·7), 13·3% (10·0-17·0), and 6·5% (4·0-9·4), respectively. Over time, there was a slight, although not statistically significant, decline in macrolide resistance in the Western Pacific and the Americas, but there was an increase in macrolide resistance in the European region. Fluoroquinolone resistance was highest in the Western Pacific and increased in the European non-Nordic region. ParC S83I was the most common variant associated with fluoroquinolone resistance, increasing from 0% (95% CI <0·0001-0·30) before 2012 to 7·3% (4·7-10·3) in 2018-21; ptrend=0·055.

INTERPRETATION: Macrolide and fluoroquinolone resistance in M genitalium requires ongoing international surveillance, use of resistance assays for optimal antibiotic stewardship, and novel treatment options. FUNDING: Australian Research Council.

OBJECTIVES: Doxycycline post-exposure prophylaxis (doxycycline-PEP) can reduce incident cases of syphilis, chlamydia and possibly gonorrhoea especially among men who have sex with men with recent bacterial sexually transmitted infections (STIs). Owing to potential implementation of doxycycline-PEP internationally, global tetracycline/doxycycline resistance data for contemporary Neisseria gonorrhoeae isolates has become imperative. We report tetracycline resistance data for gonococcal isolates (n = 2993) from eight WHO Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) countries in three WHO regions in 2021-2024, i.e. to estimate potential impact of doxycycline-PEP on the incident gonorrhoea cases in these WHO EGASP countries.

METHODS: WHO EGASP isolates cultured from men with urethral discharge in Cambodia (n = 482), Indonesia (n = 101), Malawi (n = 121), The Philippines (n = 843), South Africa (n = 597), Thailand (n = 250), Uganda (n = 350) and Vietnam (n = 249) in 2021-2024 were examined. MICs (mg/L) of tetracycline were determined using Etest.

RESULTS: The tetracycline resistance (range) using the current EUCAST (MIC > 0.5 mg/L) and CLSI (MIC > 1 mg/L) clinical resistance breakpoints in the eight WHO EGASP countries was 92.2% (83.5%-99.6%) and 80.6% (66.3%-98.6%), respectively. Using a previous minocycline-PEP resistance breakpoint (MIC > 2 mg/L) and breakpoint for high-level plasmid (tetM)-mediated tetracycline resistance (MIC > 8 mg/L), the tetracycline resistance (range) was 77.3% (47.4%-98.6%) and 74.3% (31.3%-98.6%), respectively.

CONCLUSIONS: The exceedingly high levels of gonococcal tetracycline resistance (independent of resistance breakpoint used) in the eight WHO EGASP countries elucidate that doxycycline-PEP will unlikely significantly reduce the gonorrhoea cases in these countries. Furthermore, doxycycline-PEP might rapidly select for additional gonococcal strains with tetracycline resistance (low- and high-level) and MDR/XDR strains, i.e. because these strains are mostly resistant to tetracycline.

BACKGROUND: As syphilis rates have increased globally, chancroid has dramatically declined as a cause of genital ulcer disease (GUD).

METHODS: We recruited patients ≥18 years presenting to an STI clinic with GUD from Lilongwe, Malawi from November 2019 - April 2022. Lesion exudates were tested by darkfield microscopy (DFM) and polymerase chain reaction (PCR) for Treponema pallidum (TP), and by PCR for Haemophilus ducreyi (HD), herpes simplex virus (HSV) and Chlamydia trachomatis (CT). We evaluated the sensitivity and specificity of DFM relative to TP PCR, the distribution of GUD etiologies by PCR, and the performance of our HD PCR relative to Allplex Genital Ulcer assay (Seegene Inc) using the Cohen's kappa statistic.

RESULTS: We enrolled 568 participants; the median age was 27 years (interquartile range: 23, 34), 61% (345/564) were men, and 13% (60/464) were living with HIV or newly diagnosed with HIV. DFM identified TP in 55 (10%) of participants, with a sensitivity and specificity of 12% and 94%, respectively. PCR identified TP in 367 (65%), HD in 128 (23%), HSV in 98 (17%), and CT in 36 (6%) of participants with only 1/36 (2.8%) with serovar L1, L2 or L3 consistent with lymphogranuloma venereum; no etiology was identified in 48 (8%). External validation confirmed the high HD prevalence (Cohen's kappa 0.78, 89% agreement).

CONCLUSION: Syphilis and chancroid are common etiologies of GUD in Malawi. Our findings underscore the value of highly sensitive molecular diagnostic methods to periodically assess GUD causes among STI patients in countries using syndromic management.

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.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is compromising gonorrhoea treatment, and enhanced N. gonorrhoeae AMR and genome-based epidemiological surveillance is imperative. Molecular tests are replacing N. gonorrhoeae culture internationally, excluding possibilities to perform WGS. We describe and evaluate a novel approach using a custom SureSelectXTHS Target-Enrichment probe panel automated on the Magnis NGS Prep System and Illumina sequencing to generate accurate N. gonorrhoeae genomes directly from clinical urogenital and extragenital specimens.

METHODS: One hundred thirteen clinical N. gonorrhoeae-positive APTIMA Combo 2 (AC2) specimens (with 89 linked N. gonorrhoeae isolates) were included. DNA was extracted using QIAsymphony DSP Virus/Pathogen kit. Amplisens multiplex RT-PCR assay (AM-PCR) identified 105 (92.9%) of the AC2 specimens as N. gonorrhoeae positive, which were further examined. Sequence libraries for AC2 specimens were prepared on the Magnis NGS Prep System using the Magnis SureSelectXTHS Reagent kit for Illumina paired-end platforms. Paired-end sequencing was performed on Illumina platforms. RESULTS: Seventy-four of the 105 (70.5%) AC2 samples remained N. gonorrhoeae positive with a cycle threshold <20 in the AM-PCR and subjected to SureSelectXTHS target enrichment and subsequently Illumina WGS. Seventy-two (97.3%) of all target-enriched specimens were successfully genome-sequenced. All linked AC2 specimens and N. gonorrhoeae isolates from the same anatomical site had identical AMR determinants and molecular epidemiological sequence types.

CONCLUSIONS: We show that custom SureSelectXTHS target enrichment automated on the Magnis NGS Prep System, followed by Illumina sequencing, enables culture-independent genome-based surveillance of N. gonorrhoeae AMR and molecular epidemiology. This novel methodological advancement provides an efficient and accurate WGS of N. gonorrhoeae directly from clinical urogenital and extragenital NAAT specimens.

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.