Objectives:CD4+ T cells play key roles in regulating immune responses during pregnancy; therefore, we aimed to understand the CD4+ T-cell surface proteome and transcriptome during pregnancy. Methods:CD4+ T cells were analysed in blood and decidua from term pregnancies (> 37 weeks) and non-pregnant blood. > 350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with > 130 CITE-seq barcoded antibodies. Results:Surface protein screening identified changes to ILT4/CD85d, CD9, IFN-γ receptor β-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T-cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. Conclusions:Our findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications, including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

Timely and accurate nucleic acid extraction is a critical bottleneck in point-of-care (POC) molecular diagnostics, particularly for low-abundance cell-free nucleic acids (DNA, RNA, miRNA) in plasma. We present a fully finger-actuated microfluidic device for power-free, pipette-free, nucleic acid extraction. The prototype device integrates finger-actuated valves, pump and blister reagent storage, enabling a complete, intuitive, "sample-to-eluate" workflow, including lysis, binding, washing and elution via manual actuation in approximately 35 minutes. The device operation was benchmarked against a commercial magnetic bead-based manual benchtop and an automated robotic workflow. In DNA-spiked aqueous samples (5-20 ng mL-1), the device demonstrated recoveries of 84.6%, against 90.7% on the bench. Optimisation of magnetic bead volume identified 50 μL as the threshold for maximal recovery while minimising reagent consumption. Recovery from human pooled plasma samples were found to be on average 18, 62, and 552% higher than that obtained with the control bench extraction on initial sample volumes of 100, 75 and 50 μL, respectively. Coefficient of variation (CV%) for all techniques remained within reported laboratory ranges (20-80%). The device's clinical utility was piloted using patient samples positive for cytomegalovirus (CMV), monkeypox virus (MPXV) or chikungunya (CHIKV). The device achieved successful qPCR amplification for all targets, with viral DNA Cq values within as little as 0.3 cycles of the standard protocol. This instrument-free, manual circulating nucleic acid extraction tool could help bridge the gap between lateral flow based nucleic acid amplification technologies and real-world decentralised diagnostics, offering a scalable solution for infectious disease management in resource-limited settings.

BACKGROUND:Culture remains essential for antimicrobial resistance profiling for gonorrhoea. However, the sensitivity of culture for gonorrhoea is low. This study aimed to compare positivity rates of culture for Neisseria gonorrhoeae using a gonococcus (GC) bi-plate containing selective and non-selective agar versus a single GC plate containing selective agar only, and assess the impact of clinician inoculation training and timing between a nucleic acid amplification test (NAAT) and culture on culture positivity. METHODS:A cross-sectional study was conducted at Melbourne Sexual Health Centre between April and June 2021. Clients undergoing gonorrhoea testing provided samples for both NAAT and culture, with alternating use of GC bi-plates and single GC plates over 2-week intervals. Clinicians received training on inoculation techniques before the study. A retrospective analysis of culture positivity before training was also performed. Culture positivity was compared between plate types, pre- and post-training, and across different time gaps between NAAT and culture. RESULTS:Among 276 clients included, there was no difference in culture positivity between GC bi-plate and single plate across all anatomical sites (P = 0.439). Oropharyngeal culture had the lowest positivity rates (44.3% bi-plate vs 36.2% single plate, P = 0.382). There was no significant difference in culture positivity before and after training staff on inoculation technique (P = 0.782), nor if the cultures were performed 1-5 days between positive NAAT and culture compared with 6-14 days. CONCLUSION:This study found no significant difference between GC bi-plate and GC single plate in detecting N. gonorrhoeae. Inoculation training and delayed culture post-NAAT did not significantly impact culture positivity. Improving culture methods remains critical for antimicrobial resistance surveillance.

Objectives The study aimed to explore the acceptability of reducing the frequency of asymptomatic Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) screening among gay, bisexual, and other men who have sex with men (GBMSM)(Although the term GBMSM is used for convenience, the study also includes nonbinary people who were assigned male at birth who have sex with men.). Additionally, it sought to identify barriers and facilitators to implementing such changes and to develop potential interventions that could support a shift in current screening guidelines. Methods This qualitative study explored stakeholder perspectives on reducing screening frequency and identified potential interventions that could support future guideline changes of this kind. Semistructured interviews were conducted with 22 GBMSM and 8 professional stakeholders. Data were thematically analysed using the Capabilty, Opportunity, Motivation - Behaviour (COM-B) and Theoretical Domains Framework (TDF). TDF domains were mapped to behaviour change techniques to inform intervention development. Candidate interventions were refined based on acceptability, practicability, effectiveness, affordability, side effects, equity. Results Overall, GBMSM stakeholder responses to discontinuing asymptomatic Ng and Ct screening tended to be negative, while professional stakeholder opinions were mixed. Reducing the recommended screening frequency to 6 monthly was generally more acceptable to both groups. Barriers and facilitators to guideline changes included issues of knowledge and trust, social influence and identity, context and resources, concerns about consequences and emotional responses and habit. Ten candidate interventions were suggested. These involve providing information, social support, behavioural substitutions and feedback as well as facilitating discussions to resolve concerns. Conclusion Any reduction in the recommended frequency of asymptomatic screening will encounter a range of interrelated barriers, including knowledge gaps, social influences and emotional factors. We identified evidence-based interventions that could improve acceptance and minimise unintended consequences. Future research should incorporate stakeholder workshops to refine these strategies.

Globally, the utility of pathogen genomics for public health was highlighted by the COVID-19 pandemic. Approaches to enhance coordination and improve implementation of pathogen genomics for public health are needed. The Communicable Diseases Genomics Network (CDGN) was established in 2015 in Australia. The network, embedded at the public health laboratory interface and supported by the Australian Government, has facilitated a coordinated model in Australia for public health pathogen genomics. CDGN activities have facilitated pilot projects to demonstrate use cases, harmonisation of data sharing and governance arrangements, outbreak and pandemic response, translational research, policy development and workforce capacity building. The impact of CDGN has been enabling the significant progress towards public health genomics implementation in Australia, and providing a model that could be applied in other federated settings, aligned with international best practice.

Understanding host factors driving asymptomatic versus severe disease outcomes is of key importance if we are to control emerging and re-emerging viral infections. HLA-B*15:01 has been associated with asymptomatic SARS-CoV-2 infection in nonhospitalized individuals of European ancestry, with protective immunity attributed to preexisting cross-reactive CD8+ T-cells directed against HLA-B*15:01-restricted Spike-derived S919-927 peptide (B15/S919+CD8+ T-cells). However, fundamental questions remained on the abundance and clonotypic nature of CD8+ T-cell responses in HLA-B*15:01-positive patients who succumbed to life-threatening COVID-19. Here, we analyzed B15/S919+CD8+ T-cell responses in COVID-19 patients from independent HLA-typed COVID-19 patient cohorts across three continents, Australia, Asia and Europe. We assessed B15/S919+CD8+ T-cells in COVID-19 patients across disease outcomes ranging from asymptomatic to hospitalized critical illness. We found that severe/critical COVID-19 patients mounted B15/S919+CD8+ T-cell responses lacking a highly expanded key public B15/S919+CD8+ T-cell receptor (TCR; TRAV9-2/TRBV7-2) which recurred across multiple individuals in COVID-19 patients with a mild disease. Instead, B15/S919+CD8+ T-cell responses in life-threatening disease had a prevalence of an alternate TCR clonotypic motif (TRAV38-2/DV8/TRBV20-1), potentially contributing, at least in part, to why B15/S919+CD8+ T-cells in severe COVID-19 patients were less protective. Interestingly, the frequency, memory phenotype, and activation profiles of circulating B15/S919+CD8+ T-cells did not differ across disease severity. Moreover, B15/S919+CD8+ T-cells were better maintained into convalescence compared to other SARS-CoV-2-specificities. Our study thus provides evidence on the differential nature of the TCR clonal repertoire in 22.37% of HLA-B*15:01-positive COVID-19 patients who developed severe or critical disease in our cohorts, comparing to HLA-B*15:01-expressing individuals with mild COVID-19.

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However, the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore, non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers, including agalactosylated IgG, interleukin (IL)-6, IL-18, and an expanded population of CD11c−CD21− double negative 3 (DN3) B cells were observed in STM patients and were associated with impaired neutralization. In contrast, mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses, suggesting potential avenues for immunomodulation via rational vaccine design.

Background Quality assurance programs (QAPs) are used to evaluate the analytical quality of a diagnostic test and provide feedback to improve quality processes in testing. Rapid diagnostic tests were used in both laboratory and non-laboratory settings to diagnose COVID-19, although varied in reported performance. We aimed to design and implement a QAP for antigen rapid diagnostic tests (Ag-RDTs) for COVID-19 in Cambodia, Lao PDR, and Papua New Guinea. Methods Inactivated SARS-CoV-2 material derived from cell culture of Wildtype, Delta, and Omicron isolates were used to manufacture 435 quality control (QC) panels (consisting of a single positive and negative sample) and 36 external quality assessment (EQA) panels for use across 15 sites in accordance with provided training and protocols. Results In total, 369 QC results and 112 EQA results were submitted by electronic reporting or paper forms by two countries. 19·3% (34/176) positive and 99·5% (192/193) negative samples were correctly reported in the QC, and 37·5% (30/80) positive and 97·5% (39/40) negative samples were correctly reported in the EQA. Conclusion This QAP demonstrates the importance of operator training and the design of QAP materials, which may have contributed to the high rate of false-negative interpretations in known COVID-19 positive samples. The implementation of this project, to our knowledge the first of its kind in the region and one of only a few globally, emphasised the importance of quality assurance principles, including non-laboratory community testing sites, to promote diagnostic quality for COVID-19 testing.

Abstract Background Point-of-care testing (PoCT) is an increasingly important diagnostic tool in the healthcare system for accessible pathology testing in hospital, primary care, and community care settings. Clear regulation and governance models are important to ensure quality of PoCT results for patient care. Methods This review aimed to identify existing regulation and guidelines for management of PoCT and how this has been implemented within Australian healthcare services. We conducted a search of academic publications in PubMed and grey literature (national, state, and independent organisations) and other publicly available information from internet searches for governance of PoCT in Australia. Relevant data from these sources were extracted and narratively synthesised. Results Forty-seven sources (17 studies from PubMed, 30 grey literature) were included in the final review. Of the grey literature sources, fifteen current PoCT governance documents comprising of six standards, five guidelines and four frameworks at the international, national and jurisdictional level were included with an increasing number of grey literature sources since the onset of the COVID-19 pandemic in 2020. The seventeen included research articles were categorised according to implementation barriers and facilitators with the themes of workforce, clinical governance, PoCT workflow, and cost. An understanding of the clinical and cultural context for PoCT was the most frequently reported facilitator of PoCT, while the most frequently reported barrier was related to inadequate data management. Conclusion This review demonstrated limited and inconsistent sources on regulatory and governance models for implementing and managing PoCT in Australia. Identified PoCT programs showcased diverse implementation and governance models to support quality PoCT, with few reporting formal accreditation. Streamlined, practical regulation and governance for PoCT may increase adoption across healthcare settings while ensuring quality results and meeting the needs of patients and healthcare practitioners.

Serovars of Salmonella are significant bacterial pathogens and are leading contributors to the global burden of diarrhoeal disease. Salmonella pathogenicity islands (SPIs) are essential for the survival and success of this genus, enabling colonisation, invasion, and survival in hostile environments. While genomics has transformed efforts to understand the evolution, dissemination, and antimicrobial resistance of members, its use to explore virulence determinants that contribute to the pathogenicity of specific organisms and severity of infection remains varied. Here, we discuss the importance of SPIs to the evolution of Salmonella, the implications in the shift of identification of SPIs from molecular microbiology to genomic-based approaches, and examine current efforts to explore the distribution and prevalence of SPIs in large-scale datasets of Salmonella genomes.

BACKGROUND:Neisseria gonorrhoeae is a human pathogen of major public health importance due to its increasing global prevalence and antimicrobial resistance (AMR). Evidence suggests that oropharyngeal infection plays a key role in N gonorrhoeae transmission and AMR; however, our understanding of oropharyngeal gonorrhoea pathogenesis is poor. A controlled human infection model (CHIM) for oropharyngeal gonorrhoea will improve understanding of infection and accelerate urgently needed novel gonorrhoea prevention and therapeutic strategies. As the first step in the development of this CHIM, we describe a systematic approach to CHIM strain selection that leverages genomics and clinical data. METHODS:In this genomics-based analysis, we applied a systematic N gonorrhoeae challenge strain selection strategy incorporating genomic and clinical data to a primary dataset of clinical isolates of N gonorrhoeae collected from adult patients in Victoria, Australia, between Jan 1 and Dec 31, 2017, and July 1, 2019, and June 30, 2021. This selection strategy used clinical, phenotypic, and genomic characteristics to define a set of eight criteria that aimed to ensure the contemporary global clinical relevance of the candidate strains; select strains that would be applicable for the assessment of current and future gonorrhoea vaccines; and maximise participant safety by reducing the risk of disseminated gonococcal infection and clinically significant AMR. We applied these criteria to our primary dataset to generate a panel of potential challenge strains. From this final dataset of potential challenge strains, we predetermined that we would select up to ten isolates to proceed to the next stage of detailed phenotypic characterisation for final N gonorrhoeae CHIM strain selection. FINDINGS:5881 isolates comprised the primary dataset. After application of the selection criteria, most of the isolates (5795 [98·6%] of 5881) were excluded, mostly due to having clinically significant AMR and poor contemporary global clinical relevance. The remaining 86 N gonorrhoeae challenge strain candidates comprised five multilocus sequence types and six N gonorrhoeae multiantigen sequence types, many of which were represented by a single isolate. Of these 86 strains, five isolates were selected to maximise coverage of the phylogenetically distinct groups within the 86 candidate challenge strains and ensure representation of strains collected from various anatomical sites. INTERPRETATION:We transparently describe a novel, systematic, and rational genomics-based strategy for oropharyngeal gonorrhoea CHIM strain selection that improves the efficiency and transparency of CHIM strain selection and enables identification of contemporary and clinically relevant potential challenge strains. A final N gonorrhoeae challenge strain will be selected from the subset of five shortlisted candidates after detailed phenotypic assessment. FUNDING:Medical Research Future Fund, Australian National Health and Medical Research Council and Australian Government Research Training Program.

Introduction Substantial population-level variation in vaccine-specific antibody responses has been observed following global coronavirus disease 2019 (COVID-19) vaccination efforts. Beyond the influence of clinical and demographic features, immunogenetic variation is suggested to underlie divergent serological responses following COVID-19 vaccination of distinct populations. Methods Immunoglobulin G1 (IgG1) allotypic markers (G1m) for 121 COVID-19 vaccinated healthy adults were genotyped via Sanger sequencing. Vaccine-specific IgG and Fc gamma receptor (FcγR) engagement were characterised via bead-based multiplex array. Results Following two COVID-19 vaccine doses, G1m1,17+/+ compared to G1m-1,3+/+ vaccinees had increased IgG and FcγR engagement specific for the antigenically conserved SARS-CoV-2 Spike 2 (S2) domain. IgG targeting antigenically novel SARS-CoV-2 receptor binding domain (RBD) trended higher in G1m1,17+/+ vaccinees, facilitating increased RBD-specific FcγR2a-R131 and FcγR2b binding. Conclusion Primary COVID-19 vaccination induced increased S2-specific IgG in G1m1,17+/+ vaccinees, facilitating enhanced anti-viral FcγR engagement and suggesting immunogenetics may be a valuble consideration for next-generation vaccine design.

SUMMARYLaboratory-acquired infections (LAIs), particularly those from high-risk viruses, pose significant threats to exposed individuals and to the general public. In this review, we evaluate the existing evidence for viral LAI prevention, including available vaccinations, post-exposure prophylaxis (PEP), and follow-up procedures following occupational exposure to Risk Group 3 and 4 viral infectious agents within clinical testing laboratories. This review provides guidance on the therapeutic options and follow-up, all essential for preparedness planning and timely management in the event of exposure.

Infection with Neisseria gonorrhoeae, the causative agent of gonorrhea, causes significant morbidity worldwide and can have long-term impacts on reproductive health. The greatest global burden of gonorrhea occurs in low- and middle-income settings. Global public health significance is increasing due to rising antimicrobial resistance, which threatens future gonorrhea management. The oropharynx is an important asymptomatic reservoir for gonorrhea transmission and a high-risk site for development of antimicrobial resistance and treatment failure. Controlled human infection model (CHIM) studies using N gonorrhoeae may provide a means to accelerate the development of urgently needed therapeutics, vaccines, and other biomedical prevention strategies. A gonorrhea urethritis CHIM has been used since the 1980s with no reported serious adverse events. Here, we describe the rationale for an oropharyngeal gonorrhea CHIM, including analysis of potential ethical issues that should inform the development of this novel study design.

Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .

Background:Earlier syphilis detection is needed to reduce infectiousness and transmission and to improve control. Polymerase chain reaction (PCR) for Treponema pallidum is highly sensitive for detecting primary syphilis but is not often widely available or used. The aim of this systematic review and meta-analysis was to investigate the proportion of PCR-positive, seronegative primary syphilis infections when serology was performed at clinical presentation. Methods:We undertook a systematic review and meta-analysis to identify studies of patients presenting with primary syphilis where T pallidum PCR was performed on the primary syphilis lesion and serology for syphilis was performed on the same occasion. The review was conducted according to the Cochrane protocol. Medline, Embase, and PubMed were searched from 1 January 2000 to 27 November 2022 (date of search). Only studies published in English were included. A pooled estimate of the proportion of PCR-positive, seronegative primary infections was calculated via a random effects model. Results:Of 2571 studies identified, 8 met inclusion criteria and were included. This contributed to 758 individuals with T pallidum PCR-positive primary lesions who had serology performed at the same initial visit. Among these, a pooled estimate of 10% (95% CI, 6%-13%; 73/758; I 2 = 65%, P < .01) was negative on all serologic markers, ranging between 4% (95% CI, 0%-7%; 4/108) and 20% (95% CI, 10%-29%; 14/71). Conclusions:T pallidum was detected by PCR in 10% cases, which would have been missed if serology alone was used. T pallidum PCR is important for optimizing early detection of primary syphilis.

Background:Self-testing for SARS-CoV-2 infection using lateral flow devices (LFDs) was a key component of the COVID-19 pandemic response; however, LFD performance has shown a high degree of variability. Between August 2020 and July 2023, the UK Health Security Agency (UKHSA), including predecessor organisations, undertook a three-phase SARS-CoV-2 test development and evaluation programme to independently evaluate commercially available SARS-CoV-2 LFDs, incorporating standardised laboratory assessment of test sensitivity. Here we describe results from a comparison of UKHSA laboratory assessment findings with manufacturer-reported LFD sensitivity data. Methods:The UKHSA assessed the sensitivity of LFDs, by laboratory testing of surplus clinical samples from a secondary healthcare setting. These data were compared with manufacturer-reported clinical sensitivity data and analytical sensitivity (limit of detection [LOD; 50% tissue culture infectious dose [TCID50]/mL]) from LFD instructions for use (IFU). Findings:UKHSA-determined LFD sensitivity ranged from 32 to 83%. Of 86 LFDs assessed, 73 included device sensitivity data in the manufacturers' IFU that claimed clinical sensitivity ≥85%, and 49 claimed clinical sensitivity ≥95%. No evidence of correlation was observed between manufacturer-reported test sensitivity and UKHSA determined test sensitivity, and no evidence of correlation was observed between manufacturer-reported test LOD and UKHSA-determined test sensitivity. Interpretation:Laboratory evaluation found no evidence of correlation between manufacturer-reported SARS-CoV-2 LFD sensitivity data and UKHSA laboratory-determined sensitivity, supporting previous reports of discrepancies. Our findings suggest that manufacturer-reported performance data and claims for SARS-CoV-2 LFDs should be interpreted with caution and support the need for independent monitoring and testing, and standardisation of analysis methodologies. Funding:This study was funded by UK Department of Health and Social Care; UK Health Security Agency (formerly Public Health England and the National Health Service Test and Trace); and the University of Oxford NIHR Biomedical Research Centre.

BACKGROUND:Non-typhoidal Salmonella is a globally important bacterial pathogen, typically associated with foodborne gastrointestinal infection. Some non-typhoidal Salmonella serovars can also colonise typically sterile sites in people to cause invasive non-typhoidal Salmonella disease. Salmonella enterica serovar Panama is responsible for a substantial number of cases of human bloodstream infection, but despite its global dissemination, numerous outbreaks, and a reported association with invasive non-typhoidal Salmonella disease, S enterica serovar Panama (S Panama) is understudied. We aimed to describe the genomic epidemiology and evolutionary history of S Panama to provide a vital baseline of understanding for this globally important serovar. METHODS:In this genomic epidemiology study, we analysed S Panama genomes derived from historical collections, national surveillance datasets, and publicly available epidemiological and whole-genome sequencing data which span the years 1931-2019. Maximum likelihood and Bayesian phylodynamic approaches were used to investigate population structure and evolutionary history and to infer geotemporal dissemination. A combination of different bioinformatic approaches with short-read and long-read data were used to characterise geographical and clade-specific trends in antimicrobial resistance (AMR) and genetic markers for invasiveness. FINDINGS:We analysed 836 S Panama genomes, of which 559 (67%) were sequenced as part of this study. The collection represents all inhabited continents and includes isolates collected between 1931 and 2019. We identified the presence of four geographically linked S Panama clades (C1 [ie, the Latin America and the Caribbean clade; n=338], C2 [ie, the European clade; n=124], C3 [ie, the Martinique clade; n=131], and C4 [ie, the Asia and Oceania clade; n=104]) and regional trends in AMR profiles. Most isolates (715 [86%] of 836) were pan-susceptible to antibiotics and belonged to clades circulating in Latin America and the Caribbean (64%, n=458). Most antibiotic-resistant isolates in our collection (113 [93%] of 121) fell within clades C4 (ie, the Asia and Oceania clade) and C2 (ie, the European clade), the latter of which had the highest invasiveness index values based on the conservation of 196 extraintestinal predictor genes. INTERPRETATION:This first large-scale phylogenetic analysis of S Panama has revealed important information about the population structure, AMR, global ecology, and genetic markers of invasiveness of the identified genomic subtypes. Our findings provide an important baseline for understanding S Panama infection. The presence of multidrug-resistant clades with elevated invasiveness index values should be monitored through ongoing surveillance, as such clades could pose an increased public health risk. FUNDING:UK Research and Innovation Global Challenges Research Fund and Biotechnology and Biological Sciences Research Council, UK Medical Research Council, Wellcome Trust, John Lennon Memorial Scholarship, Institut Pasteur, Santé publique France, Fondation Le Roch-Les Mousquetaires, Investissement d'Avenir Programme, and Australian National Health and Medical Research Council.

Rapid antigen diagnostic tests (Ag-RDTs) that quickly and accurately identify SARS-CoV-2 are an essential part of the COVID-19 response, but multiple factors can affect the validity of Ag-RDTs results. In Cambodia, several commercial Ag-RDTs have become available since the COVID-19 outbreak, but quality control (QC) and external quality assurance (EQA) of these rapid tests have yet to be fully and systematically implemented. We collaborated with laboratory experts in Australia and piloted an EQA programme of the commonly used COVID-19 Ag-RDTs at the University of Health Sciences’ MERIEUX Laboratory (Tier 1 site—responsible for the in-country receipt and distribution of QA material) and four other participating laboratories (Tier 2—healthcare facility based) between November 2021 and November 2022. The preimplementation training including the Khmer-translated documentation was conducted virtually for Tier 1 laboratories and in-person for Tier 2 laboratories. All QC (n=290) and EQA (n=60) specimens were distributed to the laboratories and testing was performed according to the frequency of Ag-RDTs use in each laboratory. All National Reference Laboratory-provided EQA and QC specimens were tested and results were submitted via the EDCNet portal using QR code scanning. The Tier 1 laboratory reported 100% concordance with the EQA reference result, while some of Tier 2 laboratories’ results were discordant. While continued capacity building and support with troubleshooting have been key to the successful EQA piloting at the UHS laboratory, the programme experienced delays in the shipping/delivery of EQA and QC panels due to customs and border requirements, which could have hindered implementation and potentially impacted the quality of the QA materials. The pilot EQA programme demonstrated potential scalability and provided data on the reliability of test results at the site. However, to ensure sustainability and practicability of this activity, in-country EQA panel preparation may need to be considered.

ABSTRACT The Treponema pallidum particle agglutination (TPPA) assay is no longer available in some settings. Here, we report the results of a clinical laboratory evaluation of alternative treponemal assays, including three T. pallidum hemagglutination assays (TPHAs), a T. pallidum IgG enzyme-linked immunosorbent assay (ELISA), and a detailed laboratory evaluation of the only TPHA with Australian regulatory approval. The clinical laboratory evaluation comprised 300 sera (120 TPPA reactive and 180 TPPA non-reactive) collected from individuals ≥18 years between 1 June 2021 and 1 June 2023. Median age was 33, 92% were from a sexually transmitted infection clinic, 71% were male, 15.3% were living with HIV and 5% were pregnant. Twenty-four percent had active untreated syphilis, 25% had prior treated syphilis, and 50.7% had no syphilis. Compared to TPPA, positive percent agreement (PPA) was 99.2% (95% confidence interval [CI], 95.4–99.9%), 100% (95% CI, 96.9–100%), 99.2% (95% CI, 95.4–99.9%), and 93.3% (95% CI, 87.4–96.6%), respectively, for the Arlington, Fortress, and Randox TPHAs, and Euroimmun IgG ELISA. Negative percent agreement (NPA) was 97.8% (95% CI, 94.4–99.1%) for each TPHA and 98.9% (95% CI, 96.0–99.8%) for the Euroimmun IgG ELISA. Clinical sensitivity of the Arlington and Fortress TPHAs for active untreated syphilis infection was equivalent to TPPA at 97.2% (95% CI, 90.4–99.5%) and clinical specificity of these assays was 99.3% (95% CI, 96.3–100%). In the evaluation of the NewBio TPHA, comprising 322 clinical serum and plasma samples and 22 quality assurance panel samples; overall PPA compared to TPPA was 100% (95% CI, 97.5–100%) and NPA 98.9% (95% CI, 94.1–98.9%). TPHAs are an acceptable alternative for confirmatory treponemal serology. IMPORTANCE Rates of syphilis, including congenital syphilis, are increasing globally, resulting in substantial morbidity and neonatal mortality. A key pillar of syphilis control is timely and accurate diagnosis. Serology is the primary diagnostic test for syphilis. Serological testing for syphilis has been impacted by a withdrawal of the Treponema pallidum particle agglutination (TPPA) assay from several geographical regions, including Australia and Europe. Here, we describe the clinical and laboratory performance of alternative treponemal serological assays, with a focus on alternative agglutination assays ( Treponema pallidum hemagglutination assays, TPHAs) as these assays comprise alternative antigens to commercial treponemal immunoassays, require a small volume of sample input and do not require specific instrumentation. This study demonstrates that TPHAs have excellent clinical and analytical performance characteristics and provides confidence that these assays are an acceptable alternative in settings that no longer have access to the TPPA.