Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents.

This study presents the synthesis of four series of novel hybrid chalcones (20,21)a-g and (23,24)a-g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28-33)a-g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d-g, 24a-g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 µM and LC50 values in the range of 4.09 µM to >100 µM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25-62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 µg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.

Evaluation of a new CT/NG/TV/MG Real-Time PCR Kit (Vircell) versus the Allplex STI Essential Assay (Seegene) for the diagnosis of sexually transmitted infections.

Sexually transmitted infections (STI) are a public health problem. Real-time PCR assays are the most sensitive test for screening and diagnosis of these infections. The aim of this study was to evaluate a new CT/NG/TV/MG Real-Time PCR (RT-PCR) kit (Vircell) for the detection of Chamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis for the diagnosis of sexual transmitted infections using the Allplex STI Essential Assay (Seegene) as the reference's method. A total of 497 samples from different anatomical sites (endocervical, urethral, rectal, pharyngeal and urine) were analysed from October 2022 to February 2023. A total of 108 (21.73 %) and 106 (21.33 %) positive samples were found for any of the assays used. The most commonly detected pathogen was N. gonorrhoeae (52 samples; 10.46 %), and the least commonly detected was T. vaginalis (three samples; 0.60 %). The anatomical site with the highest prevalence of micro-organisms was a non-urogenital site, the pharynx (26 positive samples; 5.23 %). Using the Allplex STI Essential Assay (Seegene) as the reference method, the diagnosis performance showed that the average specificity of CT/NG/TV/MG RT-PCR Kit (Vircell) was 99.84 % and the sensitivity was 99.53 %. The overall concordance was k=0.98 (CI95 %; 0.96-1). In conclusion, the CT/NG/TV/MG RT-PCR Kit (Vircell) assay shows a good sensitivity and specificity and constitutes a promising and additional alternative to routine procedures for distinct types of clinical specimen in diagnosis STI.

Australian Gonococcal Surveillance Programme, 1 January to 31 March 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 January to 31 March 2023.

Australian Gonococcal Surveillance Programme, 1 April to 30 June 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 April to 30 June 2023.

Australian Gonococcal Surveillance Programme, 1 July to 30 September 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 July to 30 September 2023.

Target-Mediated Fluoroquinolone Resistance in Neisseria gonorrhoeae: Actions of Ciprofloxacin against Gyrase and Topoisomerase IV.

Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wild-type gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water-metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wild-type and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.

Screening for Chlamydia and Gonorrhea in Youth Correctional Facilities, Utah, USA.

We reviewed data obtained in October 2021-May 2023 from youth who reported a history of sexual activity upon admission to 1 of 12 juvenile justice facilities in Utah, USA, that offered screening for Chlamydia trachomatis and Neisseria gonorrhoeae. Urinalysis revealed C. trachomatis positivity of 10.77%, N. gonorrhoeae positivity of 1.08%, and coinfection C. trachomatis N. gonorrhoeae) of 0.90%. Prevalence of infection was similar for youths in rural and urban facilities. A total of 12.01% of those identifying as male and 14.01% of those identifying as female tested positive for C. trachomatis, N. gonorrhoeae, or coinfection. Of young adults who tested positive, 74.65% received their results while incarcerated, all of whom accepted treatment. Our research underscores the feasibility of providing prompt C. trachomatis/N. gonorrhoeae screening and treatment in juvenile correctional facilities. The pervasiveness of infection emphasizes the urgent need for early identification and treatment for C. trachomatis and N. gonorrhoeae in incarcerated youth nationwide.

45 years of tetracycline post exposure prophylaxis for STIs and the risk of tetracycline resistance: a systematic review and meta-analysis.

There is considerable interest in the use of doxycycline post exposure prophylaxis (PEP) to reduce the incidence of bacterial sexually transmitted infections (STIs). An important concern is that this could select for tetracycline resistance in these STIs and other species. We searched PubMed and Google Scholar, (1948-2023) for randomized controlled trials comparing tetracycline PEP with non-tetracycline controls. The primary outcome was antimicrobial resistance (AMR) to tetracyclines in all bacterial species with available data. Our search yielded 140 studies, of which three met the inclusion criteria. Tetracycline PEP was associated with an increasedprevalence of tetracycline resistance in Neisseria gonorrhoeae, but this effect was not statistically significant (Pooled OR 2.3, 95% CI 0.9-3.4). PEP had a marked effect on the N. gonorrhoeae tetracycline MIC distribution in the one study where this was assessed. Prophylactic efficacy was 100% at low MICs and 0% at high MICs. In the one study where this was assessed, PEP resulted in a significant increase in tetracycline resistance in commensal Neisseria species compared to the control group (OR 2.9, 95% CI 1.5-5.5) but no significant effect on the prevalence of tetracycline resistance in Staphylococcus aureus. The available evidence suggests that PEP with tetracyclines could be associated with selecting tetracycline resistance in N. gonorrhoeae and commensal Neisseria species.

Chlamydia trachomatis and Neisseria gonorrhoeae rectal infections: Interplay between rectal microbiome, HPV infection and Torquetenovirus.

Men having sex with men (MSM) represent a key population, in which sexually transmitted rectal infections (STIs) caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and high-risk HPV (HR-HPV) are very common and linked to significant morbidity. Investigating the anorectal microbiome associated with rectal STIs holds potential for deeper insights into the pathogenesis of these infections and the development of innovative control strategies. In this study, we explored the interplay at the rectal site between C. trachomatis, N. gonorrhoeae, HR-HPV infection, and the anorectal microbiome in a cohort of 92 MSM (47 infected by CT and/or NG vs 45 controls). Moreover, we assessed the presence of Torquetenovirus (TTV), a non-pathogenic endogenous virus, considered as a possible predictor of immune system activation. We found a high prevalence of HR-HPV rectal infections (61%), especially in subjects with a concurrent CT/NG rectal infection (70.2%) and in people living with HIV (84%). In addition, we observed that TTV was more prevalent in subjects with CT/NG rectal infections than in non-infected ones (70.2% vs 46.7%, respectively). The anorectal microbiome of patients infected by CT and/or NG exhibited a reduction in Escherichia, while the presence of TTV was significantly associated with higher levels of Bacteroides. We observed a positive correlation of HR-HPV types with Escherichia and Corynebacterium, and a negative correlation with the Firmicutes phylum, and with Prevotella, Oscillospira, Sutterella. Our findings shed light on some of the dynamics occurring within the rectal environment involving chlamydial/gonococcal infections, HPV, TTV, and the anorectal microbiome. These data could open new perspectives for the control and prevention of STIs in MSM.

Acceptability and Usability of Self-Sampling for the Detection of Sexually Transmitted Infections Among Transgender Women: The TransOdara Multicentric Study in Brazil.

BACKGROUND: The effective testing of sexually transmitted infections (STIs) requires sampling from potential infection sites. This study aimed to assess the choice, satisfaction, and performance of self-collected samples (SCS) from potential infection sites for STI testing among transgender women in Brazil. METHODS: TransOdara was a multicentric, cross-sectional STI prevalence study conducted in 5 Brazilian cities. Using respondent-driven sampling, 1317 transgender women 18 years or older were recruited. Participants completed interviewer-led questionnaires and provided swab samples from multiple sites (anorectal, oropharyngeal, genital) for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and human papillomavirus (HPV) testing. Participants were given a choice of SCS or provider-collected samples (PCS) at each site. RESULTS: Most participants selected SCS for anorectal (74.9%; 95% confidence interval [CI], 72.4-77.3) and genital (72.7%; 95% CI, 70.2-75.1) sites, whereas fewer chose for oropharyngeal samples (49.8%; 95% CI, 47.0-52.6). For future testing, most participants expressed a preference for SCS for genital (72.2%; 95% CI, 69.5-74.7) and anorectal (70.2%; 95% CI, 67.6-72.7) sites. There was no significant difference in the positive test results for CT and NG between SCS and PCS at anorectal and oropharyngeal sites, or for HPV at anorectal and genital (penile or neovaginal) sites. CONCLUSIONS: This study demonstrated a high level of acceptability and usability of self-sampling for STI testing among transgender women. A preference for SCS was evident at the anorectal and genital sites, and the results of SCS were comparable to those of PCS. The findings suggest that multisite STI testing utilizing self-collection methods as a provided option can be effectively integrated into sexual health services for transgender women.

Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C.

Introduction: Outer membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined. Methods: Ten paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays. Results and discussion: Post-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, ß-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design.

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.

Epidemiology of gonorrhoea: systematic review, meta-analyses, and meta-regressions, World Health Organization European Region, 1949 to 2021.

BackgroundEpidemiology of Neisseria gonorrhoeae (NG) infection remains inadequately understood.AimWe aimed to characterise NG epidemiology in Europe.MethodsWe used Cochrane and PRISMA guidelines to systematically review, report, synthesise and analyse NG prevalence data from 1949 to 30 September 2021. Random-effects meta-analyses estimated pooled prevalence. Meta-regression analyses investigated associations and sources of heterogeneity.ResultsThe 844 included publications yielded 1,573 prevalence measures. Pooled prevalence of current urogenital infection was 1.0% (95% CI: 0.7-1.2%) among general populations, 3.2% (95% CI: 1.8-4.8%) among female sex workers, 4.9% (95% CI: 4.2-5.6%) among sexually transmitted infection clinic attendees and 12.1% (95% CI: 8.8-15.8%) among symptomatic men. Among men who have sex with men, pooled prevalence was 0.9% (95% CI: 0.5-1.4%), 5.6% (95% CI: 3.6-8.1%), and 3.8% (95% CI: 2.5-5.4%), respectively, for current urogenital, anorectal or oropharyngeal infection. Current urogenital, anorectal or oropharyngeal infection was 1.45-fold (95% CI: 1.19-1.77%), 2.75-fold (95% CI: 1.89-4.02%) and 2.64-fold (95% CI: 1.77-3.93%) higher among men than women. Current urogenital infection declined 0.97-fold (95% CI: 0.96-0.98%) yearly, but anorectal and oropharyngeal infection increased (1.02-fold; 95% CI: 1.01-1.04% and 1.02-fold; 95% CI: 1.00-1.04%), respectively.ConclusionsNeisseria gonorrhoeae epidemiology in Europe has distinct and contrasting epidemiologies for vaginal sex transmission in heterosexual sex networks vs anal and oral sex transmission in MSM sexual networks. Increased transmission may facilitate drug-resistant strain emergence. Europe is far from achieving the World Health Organization target of 90% incidence reduction by 2030.

Antimicrobial resistance and heterogeneity of Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in Lusaka, Zambia.

BACKGROUND: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae is a threat to public health as strains have developed resistance to antimicrobials available for the treatment of gonorrhea. Whole genome sequencing (WGS) can detect and predict antimicrobial resistance to enhance the control and prevention of gonorrhea. Data on the molecular epidemiology of N. gonorrhoeae is sparse in Zambia. This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending sexually transmitted infection (STI) clinics in Lusaka, Zambia. METHODS: A cross-sectional study that sequenced 38 N. gonorrhoeae isolated from 122 patients with gonorrhea from 2019 to 2020 was conducted. The AMR profiles were determined by the E-test, and the DNA was extracted using the NucliSens easyMaG magnetic device. Whole genome sequencing was performed on the Illumina NextSeq550 platform. The Bacterial analysis pipeline (BAP) that is readily available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 was used for the identification of the species, assembling the genome, multi-locus sequence typing (MLST), detection of plasmids and AMR genes. Phylogeny by single nucleotide polymorphisms (SNPs) was determined with the CCphylo dataset. RESULTS: The most frequent STs with 18.4% of isolates each were ST7363, ST1921 and ST1582, followed by ST1583 (13%), novel ST17026 (7.9%), ST1588 (7.9%), ST1596 (5.3%), ST11181 (5.3%), ST11750 (2.6/%) and ST11241 (2.6%) among the 38 genotyped isolates. The blaTeM-1B and tetM (55%) was the most prevalent combination of AMR genes, followed by blaTeM-1B (18.4%), tetM (15.8%), and the combination of blaTeM-1B, ermT, and tetL was 2.6% of the isolates. The AMR phenotypes were predicted in ciprofloxacin, penicillin, tetracycline, azithromycin, and cefixime. The combination of mutations 23.7% was gryA (S91F), parC (E91G), ponA (L421) and rpsJ (V57M), followed by 18.4% in gyrA (S91F), ponA (L421P), rpsJ (V57M), and 18.4% in gyrA (D95G, S91F), ponA (L421P), and rpsJ (V57M). The combinations in gyrA (D95G, S91F) and rpsJ (V57M), and gyrA (D95G, S91F), parC (E91F), ponA (L421P) and rpsJ (V57M) were 13.2% each of the isolates. Plasmid TEM-1 (84.2%), tetM (15.8%), and gonococcal genetic island (GGI) was detected in all isolates. CONCLUSION: This study revealed remarkable heterogeneity of N. gonorrhoeae with blaTEM-1, tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline, and ciprofloxacin demanding revision of the standard treatment guidelines and improved antimicrobial stewardship in Zambia.

Vaginal dysbiosis seems associated with hrHPV infection in women attending the Dutch Cervical Cancer Screening Program.

Human papillomavirus (HPV) is a sexually transmitted virus, which infects approximately 80% of all men and women at some time in their lives. Usually, the infection is resolved successfully by the body's immune system. Persistent infection with high-risk HPV (hrHPV) is necessary but not sufficient for cervical cancer development, and additional factors, such as the vaginal microbiome (vaginome), are thought to be involved. The aim of this study is to investigate whether either vaginal dysbiosis (imbalance in vaginal bacterial composition) or sexually transmitted pathogens, e.g., Chlamydia trachomatis (CT), are possible cofactors for hrHPV infection and HPV-induced cervical dysplasia in asymptomatic women attending the Dutch Cervical Cancer Screening Program. In this study, 492 hrHPV-positive and 500 hrHPV-negative cervical smears from women attending the Screening Program were included. Age and cytology were known for the hrHPV-positive samples. All cervical smears were diluted in Aptima® specimen transfer medium and tested with Aptima® transcription-mediated amplification assays targeting CT, Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Candida spp. (CS), C. glabrata (CG), Trichomonas vaginalis (TV), and bacterial vaginosis (BV). The prevalences of CT, NG, MG, CS, CG, TV, and BV in this cohort were found to be 1.9%, 0.0%, 1.7%, 5.4%, 1.4%, 0.1%, and 27.2%, respectively. When comparing HPV groups, it was found that CT, MG, and BV had a significantly higher prevalence in hrHPV-positive smears as compared with hrHPV-negative samples (for all p < 0.001). No significant differences were found when comparing different age groups and cytology outcomes. In conclusion, vaginal dysbiosis seems associated with hrHPV infection in women attending the Dutch Cervical Cancer Screening Program.

Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing.

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Extragenital Sexually Transmitted Infections Among High-Risk Men Who Have Sex With Men in Johannesburg, South Africa.

BACKGROUND: In South Africa, extragenital etiological sexually transmitted infection (STI) screening among men who have sex with men (MSM) is not routinely available. We aimed to determine the prevalence of STI pathogens at rectal and pharyngeal sites, syphilis seroprevalence, and associated risk factors among a selection of high-risk MSM without symptomatic urethritis attending a men's health clinic in Johannesburg, South Africa. METHODS: A cross-sectional study was conducted in 2022. Enrolled clients self-reported demographic, sexual behavioral risks, and clinical information. Client or clinician-collected rectal and pharyngeal swabs were tested for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis. C. trachomatis-positive rectal samples were reflex tested for lymphogranuloma venereum. Blood specimens were screened for syphilis. Univariate and multivariate regression models were used to determine factors independently associated with the presence of an extragenital STI or syphilis. RESULTS: Among the 97 participants (median age, 29 years), 24.7% had an extragenital STI and 9.4% had high nontreponemal antibody titers (rapid plasma reagin ≥1:16). Rectal STIs were detected in 26.4% participants: N. gonorrhoeae (14.3%), C. trachomatis (9.9%), and M. genitalium (5.5%). Pharyngeal STIs were less prevalent (4.1%). Overall, the prevalence of any STI was 41%. Sex under the influence of drugs (adjusted odds ratio, 4.94; 95% confidence interval, 1.56-15.69) and engaging in condomless receptive anal intercourse with a casual partner (adjusted odds ratio, 8.36; 95% confidence interval, 1.73-40.28) were independent risk factors for having an extragenital STI. CONCLUSIONS: The high burden of extragenital STIs and active syphilis in asymptomatic MSM underscores the importance of routine etiological screening in this key population, as the syndromic approach would not enable detection or treatment of these infections.

Point-of-care testing and treatment of sexually transmitted and genital infections to improve birth outcomes in high-burden, low-resource settings (WANTAIM): a pragmatic cluster randomised crossover trial in Papua New Guinea.

BACKGROUND: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and bacterial vaginosis have been associated with adverse maternal and perinatal outcomes, but there is conflicting evidence on the benefits of antenatal screening and treatment for these conditions. We aimed to determine the effect of antenatal point-of-care testing and immediate treatment of C trachomatis, N gonorrhoeae, T vaginalis, and bacterial vaginosis on preterm birth, low birthweight, and other adverse maternal and perinatal outcomes compared with current standard of care, which included symptom-based treatment without laboratory confirmation. METHODS: In this pragmatic cluster randomised crossover trial, we enrolled women (aged ≥16 years) attending an antenatal clinic at 26 weeks' gestation or earlier (confirmed by obstetric ultrasound), living within approximately 1 h drive of a study clinic, and able to provide reliable contact details at ten primary health facilities and their catchment communities (clusters) in Papua New Guinea. Clusters were randomly allocated 1:1 to receive either the intervention or control (standard care) in the first phase of the trial. Following an interval (washout period) of 2-3 months at the end of the first phase, each cluster crossed over to the other group. Randomisation was stratified by province. Individual participants were informed about trial group allocation only after completing informed consent procedures. The primary outcome was a composite of preterm birth (livebirth before 37 weeks' gestation), low birthweight (<2500 g), or both, analysed according to the intention-to-treat population. This study is registered with ISRCTN Registry, ISRCTN37134032, and is completed. FINDINGS: Between July 26, 2017, and Aug 30, 2021, 4526 women were enrolled (2210 [63·3%] of 3492 women in the intervention group and 2316 [62·8%] of 3687 in the control group). Primary outcome data were available for 4297 (94·9%) newborn babies of 4526 women. The proportion of preterm birth, low birthweight, or both, in the intervention group, expressed as the mean of crude proportions across clusters, was 18·8% (SD 4·7%) compared with 17·8% in the control group (risk ratio [RR] 1·06, 95% CI 0·78-1·42; p=0·67). There were 1052 serious adverse events reported (566 in the intervention group and 486 in the control group) among 929 trial participants, and no differences by trial group. INTERPRETATION: Point-of-care testing and treatment of C trachomatis, N gonorrhoeae, T vaginalis, and bacterial vaginosis did not reduce preterm birth or low birthweight compared with standard care. Within the subgroup of women with N gonorrhoeae, there was a substantial reduction in the primary outcome. FUNDING: UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; UK Medical Research Council; the Wellcome Trust; the Australian National Health and Medical Research Council; and Swiss National Science Foundation.

Characteristics of reproductive tract infections caused by common pathogens among the outpatients of reproductive medicine center in Putian: retrospective study.

BACKGROUND: This study aims to explore the infection and age distribution of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Herpes simplex virus type II (HSV II) among the outpatients of Reproductive Medicine Center in Putian, Fujian Province to provide a clinical basis for the early diagnosis and treatment of various reproductive tract diseases and infertility in this region. METHODS: A total of 1736 samples of secretions and exfoliated cervical cells were collected from the outpatients of the Reproductive Medicine Center of the Affiliated Hospital of Putian University from December 2021 to April 2023. The infections of UU, CT, NG and HSVII were detected by real-time fluorescence polymerase chain reaction (PCR), and the infection statuses of the patients with different genders, ages and diagnoses were analysed. RESULTS: Among the 1736 patients, 611 were male and 1125 were female. The male patients had higher UU infection rate but lower HSV II infection rate than the female patients. No significant difference in CT and NG infection rates was observed between the genders. The CT infection rate gradually decreased with the increase in the age. The difference in UU, NG and HSV II infection rates among the different age groups was not statistically significant. For UU infection, the male infertile patients had the highest rate of 37.72% (172/456). Meanwhile, the differences in CT, NG and HSV II infection rates among the different diagnosis groups were not statistically significant. Among the male and female infertile patients, the CT infection rate was the highest in the 21-25 years of age group at 11.11% (2/18) and 9.47% (9/95), respectively. No statistically significant difference in UU, CT, NG and HSV II infection rates was observed among the different age groups of patients diagnosed in relation to the family planning guidance and between the male and female patients with other diagnoses results. CONCLUSIONS: This study showed that UU was the most frequently identified pathogen in infertile men in Putian, Fujian Province. The CT infection rate was the highest in people under 20 years old, and the infection showed a tendency toward young individuals. Therefore, the publicity of sexual health knowledge must be strengthened, and the prevention and treatment of venereal diseases among young and middle-aged people must be improved. Moreover, the pathogen infection is related to infertility to a certain extent, which is conducive to clinical diagnosis and treatment.