Comprehensive observational study evaluating the enduring effectiveness of 4CMenB, the meningococcal B vaccine against gonococcal infections in the Northern Territory and South Australia, Australia: study protocol.

INTRODUCTION: The effectiveness of antibiotics for treating gonococcal infections is compromised due to escalating antibiotic resistance; and the development of an effective gonococcal vaccine has been challenging. Emerging evidence suggests that the licensed meningococcal B (MenB) vaccine, 4CMenB is effective against gonococcal infections due to cross-reacting antibodies and 95% genetic homology between the two bacteria, Neisseria meningitidis and Neisseria gonorrhoeae, that cause the diseases. This project aims to undertake epidemiological and genomic surveillance to evaluate the long-term protection of the 4CMenB vaccine against gonococcal infections in the Northern Territory (NT) and South Australia (SA), and to determine the potential benefit of a booster vaccine doses to provide longer-term protection against gonococcal infections. METHODS AND ANALYSES: This observational study will provide long-term evaluation results of the effectiveness of the 4CMenB vaccine against gonococcal infections at 4-7 years post 4CMenB programme implementation. Routine notifiable disease notifications will be the basis for assessing the impact of the vaccine on gonococcal infections. Pathology laboratories will provide data on the number and percentage of N. gonorrhoeae positive tests relative to all tests administered and will coordinate molecular sequencing for isolates. Genome sequencing results will be provided by SA Pathology and Territory Pathology/New South Wales Health Pathology, and linked with notification data by SA Health and NT Health. There are limitations in observational studies including the potential for confounding. Confounders will be analysed separately for each outcome/comparison. ETHICS AND DISSEMINATION: The protocol and all study documents have been reviewed and approved by the SA Department for Health and Well-being Human Research Ethics Committee (HREC/2022/HRE00308), and the evaluation will commence in the NT on receipt of approval from the NT Health and Menzies School of Health Research Human Research Ethics Committee. Results will be published in peer-reviewed journals and presented at scientific meetings and public forums.

Multicentre double-blind randomised placebo-controlled trial evaluating the efficacy of the meningococcal B vaccine, 4CMenB (Bexsero), against Neisseria gonorrhoeae infection in men who have sex with men: the GoGoVax study protocol.

INTRODUCTION: Gonorrhoea, the sexually transmissible infection caused by Neisseria gonorrhoeae, has a substantial impact on sexual and reproductive health globally with an estimated 82 million new infections each year worldwide. N. gonorrhoeae antimicrobial resistance continues to escalate, and disease control is largely reliant on effective therapy as there is no proven effective gonococcal vaccine available. However, there is increasing evidence from observational cohort studies that the serogroup B meningococcal vaccine four-component meningitis B vaccine (4CMenB) (Bexsero), licensed to prevent invasive disease caused by Neisseria meningitidis, may provide cross-protection against the closely related bacterium N. gonorrhoeae. This study will evaluate the efficacy of 4CMenB against N. gonorrhoeae infection in men (cis and trans), transwomen and non-binary people who have sex with men (hereafter referred to as GBM+). METHODS AND ANALYSIS: This is a double-blind, randomised placebo-controlled trial in GBM+, either HIV-negative on pre-exposure prophylaxis against HIV or living with HIV (CD4 count >350 cells/mm3), who have had a diagnosis of gonorrhoea or infectious syphilis in the last 18 months (a key characteristic associated with a high risk of N. gonorrhoeae infection). Participants are randomised 1:1 to receive two doses of 4CMenB or placebo 3 months apart. Participants have 3-monthly visits over 24 months, which include testing for N. gonorrhoeae and other sexually transmissible infections, collection of demographics, sexual behaviour risks and antibiotic use, and collection of research samples for analysis of N. gonorrhoeae-specific systemic and mucosal immune responses. The primary outcome is the incidence of the first episode of N. gonorrhoeae infection, as determined by nucleic acid amplification tests, post month 4. Additional outcomes consider the incidence of symptomatic or asymptomatic N. gonorrhoeae infection at different anatomical sites (ie, urogenital, anorectum or oropharynx), incidence by N. gonorrhoeae genotype and antimicrobial resistance phenotype, and level and functional activity of N. gonorrhoeae-specific antibodies. ETHICS AND DISSEMINATION: Ethical approval was obtained from the St Vincent's Hospital Human Research Ethics Committee, St Vincent's Hospital Sydney, NSW, Australia (ref: 2020/ETH01084). Results will be disseminated in peer-reviewed journals and via presentation at national and international conferences. TRIAL REGISTRATION NUMBER: NCT04415424.

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.

Comparing Polymerase Chain Reaction Testing of Nasopharyngeal Swab and Lower Respiratory Tract Specimens for the Diagnosis of Pneumocystis jirovecii Pneumonia.

Using nasopharyngeal (NP) swab samples instead of lower respiratory tract specimens for polymerase chain reaction (PCR) to diagnose Pneumocystis jirovecii pneumonia (PJP) may be better tolerated and improve diagnostic accessibility. In this 2-year Australian retrospective cohort study of patients with clinically suspected PJP, P jirovecii PCR on NP swab samples had perfect specificity but low sensitivity (0.66).

Achievement of therapeutic antibiotic exposures using Bayesian dosing software in critically unwell children and adults with sepsis.

PURPOSE: Early recognition and effective treatment of sepsis improves outcomes in critically ill patients. However, antibiotic exposures are frequently suboptimal in the intensive care unit (ICU) setting. We describe the feasibility of the Bayesian dosing software Individually Designed Optimum Dosing Strategies (ID-ODS™), to reduce time to effective antibiotic exposure in children and adults with sepsis in ICU. METHODS: A multi-centre prospective, non-randomised interventional trial in three adult ICUs and one paediatric ICU. In a pre-intervention Phase 1, we measured the time to target antibiotic exposure in participants. In Phase 2, antibiotic dosing recommendations were made using ID-ODS™, and time to target antibiotic concentrations were compared to patients in Phase 1 (a pre-post-design). RESULTS: 175 antibiotic courses (Phase 1 = 123, Phase 2 = 52) were analysed from 156 participants. Across all patients, there was no difference in the time to achieve target exposures (8.7 h vs 14.3 h in Phase 1 and Phase 2, respectively, p = 0.45). Sixty-one courses in 54 participants failed to achieve target exposures within 24 h of antibiotic commencement (n = 36 in Phase 1, n = 18 in Phase 2). In these participants, ID-ODS™ was associated with a reduction in time to target antibiotic exposure (96 vs 36.4 h in Phase 1 and Phase 2, respectively, p < 0.01). These patients were less likely to exhibit subtherapeutic antibiotic exposures at 96 h (hazard ratio (HR) 0.02, 95% confidence interval (CI) 0.01-0.05, p < 0.01). There was no difference observed in in-hospital mortality. CONCLUSIONS: Dosing software may reduce the time to achieve target antibiotic exposures. It should be evaluated further in trials to establish its impact on clinical outcomes.

Determining Pneumocystis jirovecii Colonisation from Infection Using PCR-Based Diagnostics in HIV-Negative Individuals.

BACKGROUND: Pneumocystis jirovecii pneumonia is increasingly diagnosed with highly sensitive PCR diagnostics in immunocompromised, HIV-negative individuals. We assessed the performance of our in-house quantitative PCR with the aim to optimise interpretation. METHODS: Retrospective audit of all positive P. jirovecii qPCRs on induced sputum or BAL fluid at a single centre from 2012 to 2023. Medical and laboratory records were analysed and people with HIV were excluded. Cases were categorised as colonisation, high-probability PCP or uncertain PCP infection against a clinical gold standard incorporating clinico-radiological data. Quantitative PCR assay targeting the 5s gene was utilised throughout the time period. RESULTS: Of the 82 positive qPCRs, 28 were categorised as high-probability PCP infection, 30 as uncertain PCP and 24 as colonisation. There was a significant difference in qPCR values stratified by clinical category but not respiratory sample type. Current assay performance with a cutoff of 2.5 × 105 copies/mL had a sensitivity of 50% (95% CI, 30.65-69.35%) and specificity of 83.33% (95% CI, 62.62-95.26%). Youden Index calculated at 6.5 × 104 copies/mL had a sensitivity of 75% (56.64-87.32%, 95% CI) and specificity of 66.67% (46.71-82.03%, 95% CI). High and low cutoffs were explored. Significant variables associated with infection were age > 70 years old, the presence of fever, hypoxia or ground glass changes. CONCLUSIONS: A single qPCR cutoff cannot reliably determine P. jirovecii infection from colonisation. Low and high cutoffs are useful, however, a large "possible infection" cohort will remain where interpretation of clinic-radiological factors remains essential. Standardisation of assays with prospective validation in specific immunocompromised groups will allow greater generalisability and allow large-scale prospective assay validation to be performed.

Investigating oral human papillomavirus co-infection with Neisseria gonorrhoeae and Chlamydia trachomatis.

Compared to cervical cancer, little is known about human papillomavirus (HPV)-driven oropharyngeal cancer and their cofactors. Here, we investigated potential associations between Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) with oral HPV and HPV persistence, which are known cofactors in cervical carcinogenesis, and also play a role in HPV-driven oropharyngeal cancer. Saliva samples (n = 547) from 312 people were tested for CT and NG and whom had previously been tested for oral HPV infection in a longitudinal study. Eight participants were positive for CT (2.6%) and one for NG (0.3%). Six of these nine participants were also positive for oral HPV in at least one of their samples. We found no significant associations between HPV, CT, or NG infection in the saliva samples analyzed. These preliminary data suggest CT and NG have little influence on oral HPV-positivity and persistence in a general population. However, larger studies focusing on 'at risk' population cohorts are necessary to assess potential associations between oral sexually transmissible infections and oral HPV infections, and their outcomes.

Maximizing the Neisseria gonorrhoeae confirmatory rate and the genotypic detection of ciprofloxacin resistance for samples screened with cobas CT/NG.

Supplementary nucleic acid amplification testing for Neisseria gonorrhoeae (NG) is widely used to circumvent specificity problems associated with extragenital sites. Here, we compared different supplementary approaches for confirming NG-positive samples from the cobas 4800 CT/NG (c4800) and cobas 6800 CT/NG (c6800) assays using the ResistancePlusGC (RP-GC) assay, which in addition to detecting NG, also predicts ciprofloxacin susceptibility via NG gyrA characterization. Two different nucleic acid extraction techniques were investigated for RP-GC detection; extracts from c4800 (c4800-RP-GC) and MagNA Pure 96 (MP96-RP-GC). NG-positive (n = 300) and -negative (n = 150) samples in cobas PCR media from routine c4800 testing were retrospectively retested with c4800, c6800, c4800-RP-GC, and MP96-RP-GC. Selected samples were also tested with Xpert CT/NG (Xpert) for discrepant analysis. The gyrA status was compared to ETEST ciprofloxacin susceptibility or non-susceptibility for recovered isolates (n = 63). Extragenital confirmatory rates were higher for MP96-RP-GC (131/140; 93.6%) compared to c4800-RP-GC (126/146; 86.3%), albeit not significantly (P = 0.6677). Of 9 samples testing positive by c6800 and negative by MP96-RP-GC, 7/9 (77.8%) were also negative by Xpert. By contrast, the number of samples returning a valid gyrA status was significantly (P = 0.0003) higher for MP96-RP-GC (270/293; 92.2%) compared to c4800-RP-GC (245/298; 82.2%). The overall MP96-RP-GC gyrA status correlated 98.4% (61/62) with the reported ciprofloxacin sensitive (35/36; 97.2%) or non-susceptible (26/26; 100%) phenotype. Improved RP-GC confirmatory rates and reported gyrA status were observed using MP96 nucleic acids compared to c4800 extracts. The data further highlight the ongoing need for NG supplemental testing for oropharyngeal samples.

Evaluation of Illumina® COVIDSeq™ as a tool for Omicron SARS-CoV-2 characterisation.

The continued emergence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires ongoing genetic surveillance to support public health responses. The expansion of reliable next generation sequence (NGS) platforms has enabled the rapid characterisation of the constant emergence of new SARS-CoV-2 variants using nasopharyngeal swab specimens. Several studies have assessed the ability of COVIDSeq to type earlier SARS-CoV-2 strains (pre-Delta) rapidly and successfully, however, there is limited data showing suitability against Omicron variants. In the present study, we evaluated the performance of the Illumina COVIDSeq Assay as a streamlined amplicon-based NGS platform for detection and typing of Omicron variants. Our results demonstrate the high performance of SARS-CoV-2 sequencing using the COVIDSeq approach, with good repeatability, reproducibility and sensitivity for samples approaching CT 31. The COVIDSeq approach was 100% concordant with samples previously characterized by sequencing methods. The quick library preparation process and high throughput kit made it ideal for reflex testing, with a total time required for sequencing and analysis of approximately two days. This study demonstrates the effectiveness and versatility of the amplicon-based NGS characterisation method for SARS-CoV-2, providing a foundation for further research and development of custom-designed amplicon panels targeting different microorganisms.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

Application of the ViroKey® SQ FLEX assay for detection of cytomegalovirus antiviral resistance.

BACKGROUND: Cytomegalovirus (CMV) is a viral infection which establishes lifelong latency, often reactivating and causing disease in immunosuppressed individuals, including haematopoietic stem cell transplant (HSCT) recipients. Treatment can be problematic due to antiviral resistance which substantially increases the risk of patient mortality. Diagnostic testing capabilities for CMV antiviral resistance in Australia and elsewhere have traditionally relied on gene-specific Sanger sequencing approaches, however, are now being superseded by next generation sequencing protocols. OBJECTIVE: Provide a snapshot of local mutations and explore the feasibility of the ViroKeyࣨ® SQ FLEX Genotyping Assay (Vela Diagnostics Pty Ltd) by examining sequencing success. METHOD: Performed sequencing on adult (n = 38) and paediatric (n = 81) plasma samples, over a large range of viral loads (above and below the assay recommended threshold of ≥1,000 International Units (IU)/mL; noting most of our paediatric samples have loads <1,000 IU/mL). RESULTS: Eleven test runs (including three repeat runs; 14 to 15 samples per run) were conducted, and four runs were deemed valid. The overall individual sample success rate for the four evaluable test runs was 71.2% (42/59 samples); 80.4% (37/46) samples ≥1,000 IU/mL were valid. Ten clinically important antiviral resistance mutations were detected, the most common being A594V in the UL97 gene, found in 6 (5%) samples. CONCLUSIONS: A range of technical issues were experienced, however with improvement this platform could be a useful addition to routine pathology workflows, providing timely antiviral resistance results for patients undergoing HSCT.

A universal reagent for detection of emerging diseases using bioengineered multifunctional yeast nanofragments.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

Antimicrobial susceptibility assays for Neisseria gonorrhoeae: a proof-of-principle population-based retrospective analysis.

BACKGROUND: Neisseria gonorrhoeae treatment guided by molecular antimicrobial susceptibility assays could improve treatment options and antimicrobial stewardship; however, few commercial assays are available. We aimed to investigate antimicrobial susceptibility of N gonorrhoeae isolates in New South Wales, Australia, and estimate the potential usefulness of hypothetical combinations of rapid molecular antimicrobial susceptibility assays. METHODS: In this proof-of-principle, population-based, retrospective analysis, we assessed N gonorrhoeae susceptibility data for ceftriaxone, azithromycin, ciprofloxacin, and penicillin. Isolates were previously collected as part of the Australian Gonococcal Surveillance Programme between Jan 1, 2008, and Dec 31, 2019. All cultured N gonorrhoeae isolates with susceptibility data to all four antimicrobials were included. However, only one isolate was included if several isolates originated from the same individual within 13 days of the previous isolate originating from that individual, and there were less than two standard double-dilution minimum inhibitory concentrations between the isolates. We assessed the use of different combinations of hypothetical antimicrobial susceptibility assays and treatment combinations in terms of their ability to minimise overall ceftriaxone use, and use specifically in isolates with decreased susceptibility to ceftriaxone, compared with standard non-assay-guided empirical ceftriaxone treatment. FINDINGS: We included 23 089 N gonorrhoeae isolates. The prevalence of antimicrobial sensitivity fluctuated significantly during the study. Isolates with decreased susceptibility to ceftriaxone were more likely to be resistant to one or more antimicrobials than isolates without decreased susceptibility (782 [98·6%] of 793 vs 10 661 [47·8%] of 22 296), particularly ciprofloxacin (p<0·0001) and penicillin (p<0·0001). Compared with empirical ceftriaxone treatment, we estimated that strategies based on the use of hypothetical antimicrobial susceptibility would reduce ceftriaxone use (p<0·0001). However, because of co-resistance, most assay-directed treatment strategies, including those involving use of assays for two antibiotics, would result in only moderate reductions in ceftriaxone use among isolates with decreased susceptibility to ceftriaxone. INTERPRETATION: Individualised treatment guided by molecular antimicrobial susceptibility diagnostics could help to reduce overall ceftriaxone use in gonorrhoea. However, the use of these assays needs to be informed by the non-random nature of co-resistance among circulating N gonorrhoeae strains. FUNDING: Australian Government and Queensland Government.

Cytomegalovirus in children undergoing haematopoietic stem cell transplantation: a diagnostic and therapeutic approach to antiviral resistance.

Cytomegalovirus (CMV) is a ubiquitous virus which causes a mild illness in healthy individuals. In immunocompromised individuals, such as children receiving haematopoietic stem cell transplantation, CMV can reactivate, causing serious disease and increasing the risk of death. CMV can be effectively treated with antiviral drugs, but antiviral resistance is an increasingly common complication. Available therapies are associated with adverse effects such as bone marrow suppression and renal impairment, making the choice of appropriate treatment challenging. New agents are emerging and require evaluation in children to establish their role. This review will discuss established and emerging diagnostic tools and treatment options for CMV, including antiviral resistant CMV, in children undergoing haematopoietic stem cell transplant.

Decreased Neisseria gonorrhoeae genotypic diversity following COVID-19 restrictions in Queensland, Australia 2020.

We investigated the potential effects of COVID-19 public health restrictions on the prevalence and distribution of Neisseria gonorrhoeae (NG) genotypes in our Queensland isolate population in the first half of the year 2020. A total of 763 NG isolates were genotyped to examine gonococcal strain distribution and prevalence for the first 6 months of 2020, with 1 January 2020 to 31 March 2020 classified as 'pre' COVID-19 restrictions (n = 463) and 1 April 2020 to 30 June 2020 classified as 'post' COVID-19 restrictions (n = 300). Genotypes most prevalent 'pre' restrictions remained proportionally high 'post' restrictions, with some significantly increasing 'post' restrictions. However, genotype diversity was significantly reduced 'post' restrictions. Overall, it seems public health restrictions (9-10 weeks) were not sufficient to affect rates of infection or reduce the prevalence of well-established genotypes in our population, potentially due to reduced access to services or health-seeking behaviours.