To Örebro University

We report the complete reference genome sequences of three Haemophilus ducreyi isolates (ITG3105, CIP76118, and TMA3542), generated using Oxford Nanopore and Illumina sequencing, and hybrid assemblies. Each genome has a closed circular chromosome; ITG3105 and TMA3542 also have plasmids. These reference genomes support genomic, epidemiological, and antimicrobial resistance surveillance of H. ducreyi.

Modern medicine relies heavily on effective antimicrobial therapies. However, rapid emergence and global spread of antimicrobial resistance (AMR) threaten the treatment and control of bacterial infections. Neisseria gonorrhoeae, the causative agent of gonorrhoea, has an extraordinary capacity to acquire and develop resistance to all antimicrobials introduced for treatment. The aim of this study was to gain insight into the evolution and global emergence of AMR over the past century. This thesis further investigates whether the observed genomic data patterns are sufficiently informative for accurate prediction of antimicrobial susceptibility that can directly inform treatment.

By applying whole-genome sequencing (WGS) to global, historical, and contemporary gonococcal collections spanning all continents and isolated from the pre-antibiotic era to the present day, several questions can be addressed. The data revealed that the global gonococcal population is divided into two distinct lineages with different evolutionary strategies. Furthermore, temporal analysis demonstrates that the modern N. gonorrhoeae is younger than previously presumed and that antimicrobial exposure has been a major driver of the evolution of this species.

The accumulated knowledge base of phenotypic and especially genomic AMR generated in this work is compiled and integrated within a dedicated analytical framework, SensiTyper, demonstrating that WGS-based approaches can infer antimicrobial susceptibility and recommend susceptibility-guided individualised treatment strategies.

OBJECTIVES: The spread of ceftriaxone-resistant Neisseria gonorrhoeae is threatening the last option for gonorrhoea treatment, ceftriaxone. Gepotidacin, the first-in-class triazaacenaphthylene bacterial topoisomerase type IIA inhibitor, recently showed non-inferiority compared to ceftriaxone-azithromycin for treatment of uncomplicated urogenital gonorrhoea in a Phase 3 randomized controlled trial. We evaluated the in vitro susceptibility to gepotidacin in clinical gonococcal isolates (n = 2912), including 125 (4.3%) ceftriaxone-resistant isolates, collected 2021-24 in eight WHO Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) countries in three WHO regions.

METHODS: Isolates from Cambodia (n = 474), Indonesia (n = 107), Malawi (n = 111), the Philippines (n = 817), South Africa (n = 578), Thailand (n = 249), Uganda (n = 342) and Vietnam (n = 234) were examined. MICs of gepotidacin were determined using agar dilution. Gepotidacin target genes (gyrA and parC) were examined with Illumina sequencing.

RESULTS: Gepotidacin showed high in vitro activity, with MICs ranging from <0.016 to 4 mg/L. The modal MIC was 0.5 mg/L, MIC₅₀ 0.5 mg/L and MIC₉₀ 1 mg/L. Minor variations in the MIC distributions across countries were observed. ParC D86N, which in suboptimal gepotidacin concentrations predisposes for resistance development, was found in 35.5% of isolates.

CONCLUSIONS: We show that the in vitro susceptibility to gepotidacin in N. gonorrhoeae isolates, including 4.3% ceftriaxone-resistant isolates, collected 2021-24 in eight WHO EGASP countries, including five Asian countries, is high. Our findings support gepotidacin's continued clinical development, registration and introduction as a novel oral treatment for gonorrhoea. However, as with all new novel antimicrobials, cautious and optimal introduction, and surveillance of phenotypic and genomic susceptibility to gepotidacin internationally, pre- and post-licencing, should accompany any clinical implementation.

BACKGROUND: Zoliflodacin, a first-in-class oral bacterial, DNA gyrase (GyrB) inhibitor, showed non-inferiority to ceftriaxone combined with azithromycin in a recent large international, phase 3, randomised controlled trial for treatment of uncomplicated urogenital gonorrhoea. The aim of this study was to describe the microbiological and whole-genome sequencing (WGS) analyses of paired baseline (pre-treatment) and test-of-cure (TOC) gonococcal isolates from the zoliflodacin phase 3, randomised controlled trial to further characterise and evaluate the protocol-specified microbiological failures with zoliflodacin (n=22) or ceftriaxone and azithromycin (n=1).

METHODS: In this retrospective, genomic, observational study, results from antimicrobial susceptibility testing (agar dilution method) of isolates (n=960; 936 baseline isolates from 763 participants and 24 TOC isolates [23 with a paired baseline isolate in the same anatomical site] from 20 participants) collected during the zoliflodacin phase 3, randomised controlled trial done in 16 outpatient clinics in Belgium, the Netherlands, South Africa, Thailand, and the USA (Nov 6, 2019-March 16, 2023) are described. WGS analysis was performed on paired baseline and TOC isolates from participants with microbiological failures (zoliflodacin 44 isolates [19 participants]; ceftriaxone and azithromycin two isolates [one participant]), and the three baseline isolates with highest zoliflodacin minimum inhibitory concentration (MIC 0·5 mg/L).

FINDINGS: All isolates were inhibited by the same zoliflodacin concentrations (MICs ≤0·008 to 0·5 mg/L) as wild-type strains cultured internationally in 2013-23. In participants with a microbiological failure after zoliflodacin treatment (n=22, 19 participants), zoliflodacin MIC values for baseline and TOC isolates were similar, and resistance selection was lacking. WGS showed that five (23%) of 22 infections (95% CI 10-43 [in four participants]) of zoliflodacin microbiological failures had different strains at TOC versus baseline. In 17 zoliflodacin microbiological failures (15 participants), isolates at baseline and TOC were indistinguishable. 13 of these 17 microbiological failures, corresponding to 59% (95% CI 39-77; 13 of 22) of all zoliflodacin microbiological failures, were in urogenital or rectal sites in 11 participants and the isolates had zoliflodacin MICs less than or equal to 0·008 to 0·25 mg/L. The single microbiological failure after ceftriaxone and azithromycin treatment had different strains at TOC versus at baseline. No sequenced isolates had mutations associated with elevated zoliflodacin MICs.

INTERPRETATION: In the zoliflodacin phase 3, randomised controlled trial, 23% of the zoliflodacin microbiological failures and the single ceftriaxone and azithromycin microbiological failure had different gonococcal strains at TOC versus baseline, which suggests reinfections and not treatment failures. In addition, 59% of the zoliflodacin microbiological failures, all in anogenital sites, had no obvious microbiological explanation based on the low zoliflodacin MICs, previous pharmacodynamic studies, and no evidence of resistance selection after zoliflodacin therapy. A reinfection as the cause for these microbiological failures could not be excluded. We recommend that WGS is implemented in future randomised controlled trials for gonorrhoea treatment to further evaluate possible microbiological failures, exclude reinfections (to avoid underestimating the cure rates), and characterise antimicrobial resistance determinants. 

Introduction: Syphilis is a leading cause of adverse pregnancy outcomes, excessively affecting sub-Saharan Africa. Early diagnosis and treatment are crucial to prevent mother-to-child transmission. The aims of the present study were to assess prevalence and epidemiological and clinical correlates of active syphilis diagnosed using serological [treponemal and non-treponemal (lipoidal)] assays and the research-use-only (RUO) Aptima Treponema pallidum assay [Hologic; transcription-mediated amplification (TMA) assay] among pregnant women attending four antenatal care facilities in Nchelenge, Zambia in 2023.

Methods: Syphilis serology was performed using rapid diagnostic test [RDT; Core tests (R) ONE STEP Syphilis Test Kit (Core Technology, Atlanta, USA)]; positive samples were further evaluated by rapid plasma reagin [RPR; RPR Carbon Antigen Reagent (Eurocarb Products Ltd., Bristol, United Kingdom)] test. Active syphilis was defined as a positive RDT plus a positive RPR test, or a positive RUO Aptima T. pallidum assay. T. pallidum and other non-viral STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis) were detected in clinician-collected vaginal swab samples using Aptima assays on the Panther system (Hologic). HIV testing was performed with the Determine HIV Test Kit, and bacterial vaginosis (BV) was diagnosed using Nugent's score. Active syphilis correlates were identified using univariable and multivariable logistic regression.

Results: In total, 996 pregnant women were included in the analysis, of whom 136 women (13.7%) had an active syphilis infection. Of these women with active syphilis, 117 (86.0%) were positive in serology only, 15 (11.0%) in serology plus RUO Aptima T. pallidum assay, and four (2.9%) in RUO Aptima T. pallidum assay only. Rates of other current STIs and reproductive tract infections included: BV (23.3%), T. vaginalis (22.7%), M. genitalium (12.7%), HIV (8.7%), N. gonorrhoeae (8.4%), and C. trachomatis (7.4%). Secundigravidity, history of stillbirth, current concomitant M. genitalium, N. gonorrhoeae, T. vaginalis, and HIV infections were significant risk factors of active syphilis. Advanced gestational age (from 28 to 39 weeks) at enrolment was associated with significantly lower risk of syphilis positivity.

Discussion: A remarkably high burden of syphilis and high co-infection rates with HIV and other non-viral STIs in pregnant women in Zambia underscore the urgent need for integrating syphilis and HIV universal screening programs, as well as to consider concurrent testing for selected other non-viral STIs. Where feasible due to the increased cost, supplementing serological screening with a highly sensitive and specific molecular test such as the RUO Aptima T. pallidum assay for suspected early syphilis can ensure timely detection of both seroconverted and seronegative early syphilis cases, early treatment and effective prevention of mother-to-child transmission.

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.

Resistance to the last treatment option for gonorrhoea, ceftriaxone is a major public health concern globally, which poses a serious threat to the currently recommended ceftriaxone monotherapy. We elucidate the emergence, evolution and temporal spread of ceftriaxone-resistant gonococcal strains in China, using whole-genome sequencing of 357 ceftriaxone-resistant isolates from eight provinces during 2002-2022. We describe distinct differences between ceftriaxone-resistant gonococcal strains in China and global isolates. The resistance levels for the 357 isolates for cefixime, azithromycin, ciprofloxacin, benzylpenicillin, tetracycline, and spectinomycin were 92.4%, 17.0%, 100%, 84.9%, 92.7%, and 0.8%, respectively. Before 2019, ceftriaxone-resistant isolates in China mainly harboured non-mosaic penA alleles like penA-13, penA-12, penA-18 or mosaic penA-10. From 2019, ceftriaxone-resistant isolates with mosaic penA-60.001 increased rapidly, becoming the dominant penA allele. Meanwhile, penA-60.001 was horizontally transferred from MLST ST1903 strains to strains of other MLST STs that were prevalent in China. Notably, the MLST ST7365 isolates with penA-60.001 allele had higher ceftriaxone MICs than isolates of the typical ST1903 FC428 clone. This may be attributed to a main clade of MLST ST7365 strains in China exhibiting elevated ceftriaxone MICs, and the temporal phylogeny showed this clade emerged around 1994. In summary, we reveal the unique genomic and evolutionary patterns of ceftriaxone-resistant gonococcal strains in China, and the 357 not previously reported isolates substantially increase the number of global ceftriaxone-resistant isolates. Our results suggest potential evolutionary pathways and directions of ceftriaxone-resistant strains, and provide valuable data for examining genomic epidemiology, antimicrobial resistance determinants and formulating strategies for surveillance and interventions.

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.

Screening of 54,837 gonococcal genomes identified 12 new variants lacking one (n = 9) or both (n = 3) of the Xpert CT/NG assay's gonococcal targets. In total, 17 diagnostic-escape variants occurred across five countries and multiple genomic lineages; phylogenomic analysis revealed both ancestral and strain-specific recombination events. Xpert CT/NG diagnostic escape remains rare (0.026%) but illustrates recurrent recombination in gonococci. This emphasises the necessity of continuous external quality assessments, supplementary testing of gonococcal-positive molecular screening samples, and appropriate genomic and epidemiologic surveillance.