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There have been significant advances in the prevention and management of Ebola virus disease (EVD) caused by Zaire Ebola virus (ZEBOV), including the development of two effective vaccines, rVSV-ZEBOV and Ad26.ZEBOV/MVA-BN-Filo. In addition, ZEBOV monoclonal antibodies have become first-line therapy for EVD. However, the 2022-23 outbreak of Sudan Ebola virus (SUDV) in Uganda has highlighted the gap in current therapies and vaccines, whose efficacy is uncertain against non-ZEBOV species. Health-care and laboratory staff working in EVD treatment centres or Ebola virus diagnostic and research laboratories face unique risks relating to potential occupational exposure to Ebola viruses. Given the substantial morbidity and mortality associated with EVD, facilities should have strategies in place to manage occupational exposures, including consideration of post-exposure therapies. In this Review, we discuss currently available evidence for prevention and post-exposure prophylaxis of EVD, including therapies currently under evaluation for SUDV.

OBJECTIVES: To ensure accurate diagnosis of infectious diseases, preanalytical factors should be considered when assessing specimen quality and subsequent test result. Accordingly, we aimed to systematically assess the effect of storage time, temperature and buffer on the analytical sensitivity of detecting the sexually transmitted pathogen, Neisseria gonorrhoeae across multiple molecular diagnostic platforms. METHODS: Cultured N. gonorrhoeae was spiked into generic and commercial storage buffers and stored at four temperatures and five time points, ranging from -20°C to 37°C, over 30 days. Samples were processed using the Alinity m STI, Xpert CT/NG and Aptima Combo 2 nucleic acid amplification assays and an in-house quantitative PCR assay. A reduction in analytical sensitivity was defined as a significant (p<0.05) increase in cycle threshold (Ct) value relative to control samples. RESULTS: In total, 2756 samples were processed, with N. gonorrhoeae detected in 99.2% of samples. With respect to time, analytical sensitivity was maintained from day 2 (113/120; 94.2%) up to day 30 (110/120; 91.7%) relative to baseline samples. With respect to temperature, analytical sensitivity was maintained from -20°C (147/150; 98.0%) up to 37°C (136/150; 90.7%) relative to baseline samples. Generic buffers, Viral Transport Medium and Amies Liquid Media showed a reduction in analytical sensitivity compared with their commercial counterparts, Aptima Multitest Swab Transport Media and Abbott Alinity transport buffer using select diagnostic assays; this reduction appeared temperature dependent, with the largest differences in median Ct values observed at 37°C (p<0.05). CONCLUSIONS: Increased prevalence of sample self-collection for sexually transmitted infections (STIs) warrants an evaluation of preanalytical sample storage variables on diagnostic testing performance. Here, across a range of time points, temperatures and storage buffers, N. gonorrhoeae was successfully detected, supporti

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Neisseria gonorrhoeae infection is an important public health issue, with an annual global incidence of 87 million. N. gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health and may rarely cause life-threatening disease. Global rates of N. gonorrhoeae infection have increased over the past 20 years. Importantly, rates of antimicrobial resistance to key antimicrobials also continue to increase, with the United States Centers for Disease Control and Prevention identifying drug-resistant N. gonorrhoeae as an urgent threat to public health. This review summarizes the current evidence for N. gonorrhoeae vaccines, including historical clinical trials, key N. gonorrhoeae vaccine preclinical studies, and studies of the impact of Neisseria meningitidis vaccines on N. gonorrhoeae infection. A comprehensive survey of potential vaccine antigens, including those identified through traditional vaccine immunogenicity approaches, as well as those identified using more contemporary reverse vaccinology approaches, are also described. Finally, the potential epidemiological impacts of a N. gonorrhoeae vaccine and research priorities for further vaccine development are described.

BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche device

BACKGROUND: Current clinical care for common bacterial STIs (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG)) involves empiric antimicrobial therapy when clients are symptomatic, or if asymptomatic, waiting for laboratory testing and recall if indicated. Near-to-patient testing (NPT) can improve pathogen-specific prescribing and reduce unnecessary or inappropriate antibiotic use in treating sexually transmitted infections (STI) by providing same-day delivery of results and treatment. METHODS: We compared the economic cost of NPT to current clinic practice for managing clients with suspected proctitis, non-gonococcal urethritis (NGU), or as an STI contact, from a health provider's perspective. With a microsimulation of 1000 clients, we calculated the cost per client tested and per STI- and pathogen- detected for each testing strategy. Sensitivity analyses were conducted to assess the robustness of the main outcomes. Costs are reported as Australian dollars (2023). RESULTS: In the standard care arm, cost per client tested for proctitis, NGU in men who have sex with men (MSM) and heterosexual men were the highest at $247.96 (95% Prediction Interval (PI): 246.77-249.15), $204.23 (95% PI: 202.70-205.75) and $195.01 (95% PI: 193.81-196.21) respectively. Comparatively, in the NPT arm, it costs $162.36 (95% PI: 161.43-163.28), $158.39 (95% PI: 157.62-159.15) and $149.17 (95% PI: 148.62-149.73), respectively. Using NPT resulted in cost savings of 34.52%, 22.45% and 23.51%, respectively. Among all the testing strategies, substantial difference in cost per client tested between the standard care arm and the NPT arm was observed for contacts of CT or NG, varying from 27.37% to 35.28%. CONCLUSION: We found that NPT is cost-saving compared with standard clinical care for individuals with STI symptoms and sexual contacts of CT, NG, and MG.

OBJECTIVES: Amino acid variations across more than 30 immunoglobulin (Ig) allotypes may introduce structural changes that influence recognition by anti-Ig detection reagents, consequently confounding interpretation of antibody responses, particularly in genetically diverse cohorts. Here, we assessed a panel of commercial monoclonal anti-IgG1 clones for capacity to universally recognise two dominant IgG1 haplotypes (G1m-1,3 and G1m1,17). METHODS: Four commercial monoclonal anti-human IgG1 clones were assessed via ELISAs and multiplex bead-based assays for their ability to bind G1m-1,3 and G1m1,17 IgG1 variants. Detection antibodies were validated against monoclonal IgG1 allotype standards and tested for capacity to recognise antigen-specific plasma IgG1 from G1m-1,3 and G1m1,17 homozygous and heterozygous SARS-CoV-2 BNT162b2 vaccinated (n = 28) and COVID-19 convalescent (n = 44) individuals. An Fc-specific pan-IgG detection antibody corroborated differences between hinge- and Fc-specific anti-IgG1 responses. RESULTS: Hinge-specific anti-IgG1 clone 4E3 preferentially bound G1m1,17 compared to G1m-1,3 IgG1. Consequently, SARS-CoV-2 Spike-specific IgG1 levels detected in G1m1,17/G1m1,17 BNT162b2 vaccinees appeared 9- to 17-fold higher than in G1m-1,3/G1m-1,3 vaccinees. Fc-specific IgG1 and pan-IgG detection antibodies equivalently bound G1m-1,3 and G1m1,17 IgG1 variants, and detected comparable Spike-specific IgG1 levels between haplotypes. IgG1 responses against other human coronaviruses and influenza were similarly poorly detected by 4E3 anti-IgG1 in G1m-1,3/G1m-1,3 subjects. CONCLUSION: Anti-IgG1 clone 4E3 confounds assessment of antibody responses in clinical cohorts owing to bias towards detection of G1m1,17 IgG1 variants. Validation of anti-Ig clones should include evaluation of binding to relevant antibody variants, particularly as the role of immunogenetics upon humoral immunity is increasingly explored in diverse populations.

BACKGROUND: Empiric treatment of sexually transmitted infections can cause unnecessary antibiotic use. We determined if near-to-patient-testing (NPT) for Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium (MG) improved antibiotic-use for a range of clinical presentations. METHODS: Clients attending with non-gonococcal urethritis (NGU), proctitis, as STI-contacts, or for an MG-test-of-cure (MG-TOC) between March and December 2021 were recruited. Participants received near-to-patient-testing (NPT-group) for the three STIs using the GeneXpert® System (Cepheid), and concurrent routine-testing by transcription-mediated-amplification (TMA; Aptima, Hologic). Antibiotic-use among NGU or proctitis cases in the NPT-group was compared to clinic-controls undergoing routine-testing only. The proportion in the NPT-group who notified partners <24 hrs of their STI-specific result was calculated. FINDINGS: Among 904 consults by 808 NPT-participants, ≥1 STI was detected in 63/252 (25.0%) with NGU, 22/51 (43.1%) with proctitis, and 167/527 (31.7%) STI-contacts. MG was detected among 35/157 (22.3%) MG-TOC consults. Among NGU and proctitis cases, fewer in the NPT-group received empiric treatment compared to clinic-controls (29.4% [95% CI: 24.3-34.9%] vs 83.8% [95% CI: 79.2-87.8%], p < 0.001), resulting in more NPT-group cases appropriately treated (STI-specific drug/no drug appropriately; 80.9% [95% CI: 76.0-85.1%] vs 33.0% [95% CI: 27.7-38.6%], p < 0.001) and fewer mistreated (incorrect drug/treated but pathogen-negative; 17.8% [13.7-22.6%] vs 61.4% [55.6-66.9%], p < 0.001). Of 167/264 in the NPT-group with an STI who responded regarding partner-notification, 95.2% notified all/some partners; 85.9% notified them <24 hrs of the STI-specific result. INTERPRETATION: Near-to-patient-testing significantly improved antibiotic use and a high proportion of individuals rapidly notified partners of STI-specific results, highlighting the broad benefits of timely diagnostic str

There is increasing debate regarding the harms and benefits of frequent asymptomatic screening for Chlamydia trachomatis and Neisseria gonorrhoeae in men who have sex with men (MSM). One concern is that frequent asymptomatic screening could result in increased antimicrobial resistance in an array of sexually acquired infections and other pathogens, due to selection pressure exerted by frequent broad-spectrum antimicrobial usage within some sexual networks. Here, we outline the harms and benefits of frequent C trachomatis and N gonorrhoeae screening in MSM in high-income settings and propose that screening frequency be reduced. We describe the evidence gaps that should be further explored to better understand the implications of reducing the frequency of asymptomatic C trachomatis and N gonorrhoeae screening in MSM and the surveillance systems that should be in place to prepare for such changes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following Omicron BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months after infection. Both BA.1 and BA.2 infections robustly boosted neutralization activity against the infecting strain while expanding breadth against BA.4, although neutralization activity was substantially reduced for the more recent XBB and BQ.1.1 strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modeling of neutralization titers predicts that protection from symptomatic reinfection against antigenically similar strains will be durable but is undermined by new emerging strains with further neutralization escape.

Early detection and treatment of syphilis will reduce the infectious period and transmission. We aimed to determine whether screening men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP) for syphilis using Treponema pallidum polymerase chain reaction (PCR) could detect syphilis before the appearance of syphilis antibodies in serology. MSM attending 3-monthly PrEP clinic visits in Melbourne, Australia, were screened with a PCR assay targeting the polA gene of T. pallidum from an anal swab and an oral rinse between November 2019 and March 2020. Participants were serologically screened for syphilis using chemiluminescence immunoassay. A total of 309 asymptomatic participants provided an anal swab and oral rinse sample for T. pallidum PCR screening. Two syphilis cases (0.6%) were detected: one man had a positive serology only; another man had T. pallidum detected by PCR from an anal swab and a positive serology. PCR positivity was 0.3% (n = 1) for anal swabs and 0% (n = 0) for oral rinse. In this study, T. pallidum PCR screening at routine PrEP clinic visits did not identify additional cases of early syphilis over serological screening performed at these visits. IMPORTANCE With the ongoing syphilis epidemic in men who have sex with men (MSM), we investigated the role of using Treponema pallidum polymerase chain reaction (PCR) testing at the oral cavity and anus in MSM taking pre-exposure prophylaxis for the early detection of syphilis. We evaluated whether the PCR tests from these mucosal sites can detect syphilis infection early, before the development of syphilis antibodies in serology. Our study found two syphilis cases among 309 MSM, and only one syphilis case had a positive anal PCR swab, although serology was positive. We conclude that additional PCR testing is likely to be expensive and would not be cost effective for individuals who regularly screen for syphilis. However, future studies with a larger sample size are required.

The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.

BACKGROUND: The first mpox case was reported in May 2022 in Australia. Most cases have been diagnosed in men who have sex with men (MSM). This study aimed to examine community understanding of mpox, attitudes towards vaccination, and potential changes in sexual practices surrounding the mpox outbreak among MSM and transgender people in Victoria, Australia. METHODS: Participants were recruited from sexual health clinics and communities in Victoria, Australia, in August-October 2022. Participants were asked about their understanding and knowledge of mpox, vaccination uptake and intentions to change sexual practices. Univariable and multivariable logistic regression was performed to examine the factors associated with mpox vaccine uptake. RESULTS: Most participants (97.8%, 525/537) had heard about mpox and 10.5% (55/525) knew someone who had had mpox. Of the 12 mpox knowledge questions, the median score of correct answers was 10 (IQR=8-11) out of a maximum of 12. More than a third (36.6%, 191/522) had been vaccinated against mpox. MSM who had a good knowledge of mpox had the highest odds of receiving mpox vaccine compared with those who had poor knowledge (aOR=4.05; 95% CI: 1.54-10.61). To prevent mpox, half reported they would reduce having sex with casual partners, stop having chemsex (used drugs for the purpose of sex), stop attending sex-on-premises-venues, and stop having group sex. A quarter reported they would increase condom use for anal sex. CONCLUSIONS: One-third of high-risk participants and a substantial proportion of participants intended to reduce or stop certain practices, which may explain the large reduction in mpox cases.

BACKGROUND: In-depth immunogenicity studies of tixagevimab-cilgavimab (T-C) are lacking, including following breakthrough coronavirus disease 2019 (COVID-19) in vaccinated patients with hematologic malignancy (HM) receiving T-C as pre-exposure prophylaxis. METHODS: We performed a prospective, observational cohort study and detailed immunological analyses of 93 patients with HM who received T-C from May 2022, with and without breakthrough infection, during a follow-up period of 6 months and dominant Omicron BA.5 variant. RESULTS: In 93 patients who received T-C, there was an increase in Omicron BA.4/5 receptor-binding domain (RBD) immunoglobulin G (IgG) antibody titers that persisted for 6 months and was equivalent to 3-dose-vaccinated uninfected healthy controls at 1 month postinjection. Omicron BA.4/5 neutralizing antibody was lower in patients receiving B-cell-depleting therapy within 12 months despite receipt of T-C. COVID-19 vaccination during T-C treatment did not incrementally improve RBD or neutralizing antibody levels. In 16 patients with predominantly mild breakthrough infection, no change in serum neutralization of Omicron BA.4/5 postinfection was detected. Activation-induced marker assay revealed an increase in CD4+ (but not CD8+) T cells post infection, comparable to previously infected healthy controls. CONCLUSIONS: Our study provides proof-of-principle for a pre-exposure prophylaxis strategy and highlights the importance of humoral and cellular immunity post-breakthrough COVID-19 in vaccinated patients with HM.

BACKGROUND: In the 2022 mpox outbreak, several studies have explored longitudinal DNA shedding of mpox virus (MPXV) using PCR. However, there are fewer studies assessing infectivity in cell culture, and, by inference, MPXV transmissibility. Such information could help inform infection control and public health guidelines. AIMS AND METHODS: The aim of this study was to correlate cell culture infectivity of clinical samples with viral loads in clinical samples. Between May to October 2022, clinical samples from different body sites sent to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for MPXV PCR detection were cultured in Vero cells as a surrogate for infectivity. RESULTS: In the study period, 144 samples from 70 patients were tested by MPXV PCR. Viral loads in skin lesions were significantly higher than those in throat or nasopharyngeal samples (median Ct 22.0 vs 29.0, p = 0.0013 and median Ct 22.0 vs 36.5, p = 0.0001, respectively). Similarly, viral loads were significantly higher in anal samples compared to throat or nasopharyngeal samples (median Ct 20.0 vs. 29.0, p=<0.0001 and median Ct 20.0 vs. 36.5, p=<0.0001, respectively). Viral culture was successfully performed in 80/94 samples. Using logistic regression analysis, 50% of the samples were positive in viral culture at Ct 34.1 (95% confidence intervals 32.1-37.4). CONCLUSIONS: Our data further validate recent findings showing that samples with a higher MPXV viral load are more likely to demonstrate infectivity in cell culture. Although the presence of infectious virus in cell culture may not directly translate with clinical transmission risk, our data may be used as an adjunct help inform guidelines on testing and isolation policies in individuals with mpox.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Zanubrutinib-treated and treatment-naïve patients with chronic lymphocytic leukaemia (CLL) or Waldenstrom's macroglobulinaemia were recruited in this prospective study to comprehensively profile humoral and cellular immune responses to COVID-19 vaccination. Overall, 45 patients (median 72 years old) were recruited; the majority were male (71%), had CLL (76%) and were on zanubrutinib (78%). Seroconversion rates were 65% and 77% following two and three doses, respectively. CD4+ and CD8+ T-cell response rates increased with third dose. In zanubrutinib-treated patients, 86% developed either a humoral or cellular response. Patients on zanubrutinib developed substantial immune responses following two COVID-19 vaccine doses, which further improved following a third dose.

Quantitative polymerase chain reaction (qPCR) is a gold standard method for the detection and quantification of pathogenic organisms. Standard qPCR is inexpensive, sensitive and highly specific to the pathogen of interest. While qPCR assays can be multiplexed to allow the detection of multiple organisms in one reaction, it is prohibitively labour intensive to screen large numbers of samples for several pathogens at the same time. The TaqMan Array Card (TAC) is a cost-effective and accurate technique that expands the number of assays that can be simultaneously performed on a sample, with no increase in set-up time and only small reductions in sensitivity. This approach is highly beneficial in settings where there is a need to monitor a large panel of pathogens. We illustrate the application of TAC to the monitoring of gastrointestinal pathogens, which span viral, bacterial, protist and helminth taxa. This protocol outlines the laboratory set-up of a TaqMan Array Card, and some recommended data processing steps to aid in accurate interpretation of the results. A video protocol is additionally provided to assist in the use of the technique.•The TAC is designed primarily for gene expression assays, but has recently been utilised in several studies for pathogen detection in human clinical samples.•We expand the use of TAC for pathogen detection across human, animal and environmental sample types, and have developed a protocol and guidelines for the processing and interpretation of results that circumvents issues with the automated outputs.•This technique is applicable to pathogen or organism detection in any context, if quality nucleic acid extracts can be obtained from the sample type of interest.

BACKGROUND: Typhoid fever, a systemic infection caused by Salmonella enterica serovar Typhi, remains a considerable public health threat in impoverished regions within many low- and middle-income settings. However, we still lack a detailed understanding of the emergence, population structure, molecular mechanisms of antimicrobial resistance (AMR), and transmission dynamics of S. Typhi across many settings, particularly throughout the Asia-Pacific islands. Here we present a comprehensive whole genome sequence (WGS) based overview of S. Typhi populations circulating in Papua New Guinea (PNG) over 30 years. PRINCIPLE FINDINGS: Bioinformatic analysis of 86 S. Typhi isolates collected between 1980-2010 demonstrated that the population structure of PNG is dominated by a single genotype (2.1.7) that appears to have emerged in the Indonesian archipelago in the mid-twentieth century with minimal evidence of inter-country transmission. Genotypic and phenotypic data demonstrated that the PNG S. Typhi population appears to be susceptible to former first line drugs for treating typhoid fever (chloramphenicol, ampicillin and co-trimoxazole), as well as fluoroquinolones, third generation cephalosporins, and macrolides. PNG genotype 2.1.7 was genetically conserved, with very few deletions, and no evidence of plasmid or prophage acquisition. Genetic variation among this population was attributed to either single point mutations, or homologous recombination adjacent to repetitive ribosomal RNA operons. SIGNIFICANCE: Antimicrobials remain an effective option for the treatment of typhoid fever in PNG, along with other intervention strategies including improvements to water, sanitation and hygiene (WaSH) related infrastructure and potentially the introduction of Vi-conjugate vaccines. However, continued genomic surveillance is warranted to monitor for the emergence of AMR within local populations, or the introduction of AMR associated genotypes of S. Typhi in this setting.

BACKGROUND: The incidence of syphilis has increased markedly in the past decade in high-income countries, including Australia. To date, however, genomic studies of Treponema pallidum have focused mainly on the northern hemisphere. Here, we aimed to characterise the lineages of T pallidum driving the current syphilis epidemic in Australia. METHODS: In this genomic epidemiological analysis, using phylogenomic and phylodynamic analyses, we analysed 456 high-quality T pallidum genomes collected from clinical samples in Australia between Oct 19, 2005, and Dec 31, 2020, and contextualised this information with publicly available sequence data. We also performed detailed genomic characterisation of putative antimicrobial resistance determinants, in addition to correlating single-locus typing of the TP0548 allele with the T pallidum phylogeny. FINDINGS: Phylogenomic analyses identified four major sublineages circulating in Australia and globally, two belonging to the SS14 lineage, and two belonging to the Nichols lineage. Australian sublineages were further delineated into twelve subgroups, with five of the six largest subgroups associated with men who have sex with men, and the sixth lineage was predominantly associated with heterosexual people. Most Australian T pallidum genomes (398 [87%] of 456) were genotypically macrolide resistant, and TP0548 typing correlated significantly with T pallidum genomic subgroups. INTERPRETATION: These findings show that the current syphilis epidemic in Australia is driven by multiple lineages of T pallidum, rather than one distinct outbreak. Major subgroups of T pallidum in Australia have emerged within the past 30 years, are closely related to global lineages, and circulate across different sexual networks. In conjunction with improved testing and treatment, these data could better inform the control of syphilis in Australia. FUNDING: National Health and Medical Research Council, Australian Research Council.

BACKGROUND: Australia's response to the coronavirus disease 2019 (COVID-19) pandemic relies on widespread availability of rapid, accurate testing and reporting of results to facilitate contact tracing. The extensive geographical area of Australia presents a logistical challenge, with many of the population located distant from a laboratory capable of robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. A strategy to address this is the deployment of a mobile facility utilizing novel diagnostic platforms. This study aimed to evaluate the feasibility of a fully contained transportable SARS-CoV-2 testing laboratory using a range of rapid point-of-care tests. METHOD: A 20 ft (6.1 m) shipping container was refurbished (GeneWorks, Adelaide, South Australia) with climate controls, laboratory benches, hand-wash station and a class II biosafety cabinet. Portable marquees situated adjacent to the container served as stations for registration, sample acquisition and personal protective equipment for staff. Specimens were collected and tested on-site utilizing either the Abbott ID NOW or Abbott Panbio rapid tests. SARS-CoV-2 positive results from the rapid platforms or any participants reporting symptoms consistent with COVID-19 were tested on-site by GeneXpert Xpress RT-PCR. All samples were tested in parallel with a standard-of-care RT-PCR test (Panther Fusion SARS-CoV-2 assay) performed at the public health reference laboratory. In-laboratory environmental conditions and data management-related factors were also recorded. RESULTS: Over a 3 week period, 415 participants were recruited for point-of-care SARS-CoV-2 testing. From time of enrolment, the median result turnaround time was 26 min for the Abbott ID NOW, 32 min for the Abbott Panbio and 75 min for the Xpert Xpress. The environmental conditions of the refurbished shipping container were found to be suitable for all platforms tested, although humidity may have produced condensation within the c

The COVID-19 pandemic has driven demand for integrated genomics, resulting in fast-tracked development of AusTrakka, Australia’s pathogen genomics platform. This facilitated rapid data sharing, democratised access to computational and bioinformatic resources and expertise, and achieved national real-time genomic surveillance.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.

BACKGROUND: Group A Streptococcus (GAS) causes superficial pharyngitis and skin infections as well as serious autoimmune sequelae such as acute rheumatic fever (ARF) and subsequent rheumatic heart disease. ARF pathogenesis remains poorly understood. Immune priming by repeated GAS infections is thought to trigger ARF, and there is growing evidence for the role of skin infections in this process. METHODS: We utilized our recently developed 8-plex immunoassay, comprising antigens used in clinical serology for diagnosis of ARF (SLO, DNase B, SpnA), and 5 conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD, Group A carbohydrate), to characterize antibody responses in sera from New Zealand children with a range of clinically diagnosed GAS disease: ARF (n = 79), GAS-positive pharyngitis (n = 94), GAS-positive skin infection (n = 51), and matched healthy controls (n = 90). RESULTS: The magnitude and breadth of antibodies in ARF was very high, giving rise to a distinct serological profile. An average of 6.5 antigen-specific reactivities per individual was observed in ARF, compared to 4.2 in skin infections and 3.3 in pharyngitis. CONCLUSIONS: ARF patients have a unique serological profile, which may be the result of repeated precursor pharyngitis and skin infections that progressively boost antibody breadth and magnitude.

Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.

Rapid diagnosis and whole genome sequencing confirmed a case of monkeypox in an HIV-positive individual receiving antiretroviral therapy. The patient had a normal CD4+ T-cell count and suppressed HIV viral load and presented with a genital rash in Melbourne, Australia after return from Europe in May 2022. He subsequently developed systemic illness and disseminated rash and 11 days after symptom onset, he was hospitalised to manage painful bacterial cellulitis of the genital area.

Vaccination against SARS-CoV-2 protects from infection and improves clinical outcomes in breakthrough infections, likely reflecting residual vaccine-elicited immunity and recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and cellular immunity after vaccination of seropositive individuals and after Delta or Omicron breakthrough infection in vaccinated individuals. Early longitudinal sampling revealed the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titers. While vaccination of seropositive individuals resulted in robust recall of humoral and T cell immunity, recall of vaccine-elicited responses was delayed and variable in magnitude during breakthrough infections and depended on the infecting variant of concern. While the delayed kinetics of immune recall provides a potential mechanism for the lack of early control of viral replication, the recall of antibodies coincided with viral clearance and likely underpins the protective effects of vaccination against severe COVID-19.

BACKGROUND: Whole genome sequencing (WGS) is increasingly used by tuberculosis (TB) programs to monitor Mycobacterium tuberculosis (Mtb) transmission. We aimed to characterise the molecular epidemiology of TB and Mtb transmission in the low-incidence setting of Victoria, Australia, and assess the utility of WGS. METHODS: WGS was performed on all first Mtb isolates from TB cases from 2017 to 2020. Potential clusters (≤12 single nucleotide polymorphisms [SNPs]) were investigated for epidemiological links. Transmission events in highly-related (≤5 SNPs) clusters were classified as likely or possible, based on the presence or absence of an epidemiological link, respectively. Case characteristics and transmission settings (as defined by case relationship) were summarised. Poisson regression was used to examine associations with secondary case number. FINDINGS: Of 1844 TB cases, 1276 (69.2%) had sequenced isolates, with 182 (14.2%) in 54 highly-related clusters, 2-40 cases in size. Following investigation, 140 cases (11.0% of sequenced) were classified as resulting from likely/possible local-transmission, including 82 (6.4%) for which transmission was likely. Common identified transmission settings were social/religious (26.4%), household (22.9%) and family living in different households (7.1%), but many were uncertain (41.4%). While household transmission featured in many clusters (n = 24), clusters were generally smaller (median = 3 cases) than the fewer that included transmission in social/religious settings (n = 12, median = 7.5 cases). Sputum-smear-positivity was associated with higher secondary case numbers. INTERPRETATION: WGS results suggest Mtb transmission commonly occurs outside the household in our low-incidence setting. Further work is required to optimise the use of WGS in public health management of TB. FUNDING: The Victorian Tuberculosis Program receives block funding for activities including case management and contact tracing from the Victorian Departmen

BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) remain an inequitable cause of avoidable suffering and early death in many countries, including among Indigenous Māori and Pacific populations in New Zealand. There is a lack of robust evidence on interventions to prevent ARF. This study aimed to identify modifiable risk factors, with the goal of producing evidence to support policies and programs to decrease rates of ARF. METHODS: A case-control study was undertaken in New Zealand using hospitalised, first episode ARF cases meeting a standard case-definition. Population controls (ratio of 3:1) were matched by age, ethnicity, socioeconomic deprivation, location, sex, and recruitment month. A comprehensive, pre-tested questionnaire was administered face-to-face by trained interviewers. FINDINGS: The study included 124 cases and 372 controls. Multivariable analysis identified strong associations between ARF and household crowding (OR 3·88; 95%CI 1·68-8·98) and barriers to accessing primary health care (OR 2·07; 95% CI 1·08-4·00), as well as a high intake of sugar-sweetened beverages (OR 2·00; 1·13-3·54). There was a marked five-fold higher ARF risk for those with a family history of ARF (OR 4·97; 95% CI 2·53-9·77). ARF risk was elevated following self-reported skin infection (aOR 2·53; 1·44-4·42) and sore throat (aOR 2·33; 1·49-3·62). INTERPRETATION: These globally relevant findings direct attention to the critical importance of household crowding and access to primary health care as strong modifiable causal factors in the development of ARF. They also support a greater focus on the role of managing skin infections in ARF prevention. FUNDING: This research was funded by the Health Research Council of New Zealand (HRC) Rheumatic Fever Research Partnership (supported by the New Zealand Ministry of Health, Te Puni Kōkiri, Cure Kids, Heart Foundation, and HRC) award number 13/959.

BACKGROUND: Regular repeat surveillance testing is a strategy to identify asymptomatic individuals with SARS-CoV-2 infections in high-risk work settings to prevent onward community transmission. Saliva sampling is less invasive compared to nasal/oropharyngeal sampling, thus making it suitable for regular testing. In this multi-centre evaluation, we aimed to validate RT-PCR using salivary swab testing of SARS-CoV-2 for large-scale surveillance testing and assess implementation amongst staff working in the hotel quarantine system in Victoria, Australia. METHODS: A multi-centre laboratory evaluation study was conducted to systematically validate the in vitro and clinical performance of salivary swab RT-PCR for implementation of SARS-CoV-2 surveillance testing. Analytical sensitivity for multiple RT-PCR platforms was assessed using a dilution series of known SARS-CoV-2 viral loads, and assay specificity was examined using a panel of viral pathogens other than SARS-CoV-2. In addition, we tested capacity for large-scale saliva testing using a four-sample pooling approach, where positive pools were subsequently decoupled and retested. Regular, frequent self-collected saliva swab RT-PCR testing was implemented for staff across fourteen quarantine hotels. Samples were tested at three diagnostic laboratories validated in this study, and results were provided back to staff in real-time. FINDINGS: The agreement of self-collected saliva swabs for RT-PCR was 84.5% (95% CI 68.6 to 93.8) compared to RT-PCR using nasal/oropharyngeal swab samples collected by a healthcare practitioner, when saliva samples were collected within seven days of symptom onset. Between 7th December 2020 and 17th December 2021, almost 500,000 RT-PCR tests were performed on saliva swabs self-collected by 102 staff working in quarantine hotels in Melbourne. Of these, 20 positive saliva swabs were produced by 13 staff (0.004%). The majority of staff that tested positive occurred during periods of community tra

BACKGROUND: Group A streptococcal (GAS) infections can trigger an immune-mediated response resulting in acute rheumatic fever (ARF). The role of social and environmental risk factors for GAS pharyngitis and skin infections are not well understood. This study aimed to identify factors associated with GAS pharyngitis and skin infections, and to determine if these are the same as those for ARF. METHODS: A case-control study, including 733 children aged 5-14 years, was undertaken between March 2018 and October 2019 in Auckland, New Zealand. Healthy controls (n = 190) and symptomatic cases including GAS pharyngitis (n = 210), GAS seronegative carriers (n = 182), and GAS skin infections (n = 151) were recruited. Trained interviewers administered a comprehensive, pre-tested, face-to-face questionnaire. FINDINGS: Multivariable analysis identified strong associations between barriers to accessing primary healthcare and having GAS pharyngitis (adjusted OR 3·3; 95% CI 1·8-6·0), GAS carriage (aOR 2·9; 95% CI 1·5-6·0) or a GAS skin infection (aOR 3·5; 95% CI 1·6-7·6). Children who had GAS skin infections were more likely than all other groups to report living in a crowded home (aOR 1·9; 95% CI 1·0-3·4), have Māori or Pacific grandparents (aOR 3·0; 95% CI 1·2-7·6), a family history of ARF (aOR 2·2; 95% CI 1·1-4·3), or having a previous diagnosis of eczema (aOR 3·9; 95% CI 2·2-6·9). INTERPRETATION: Reducing barriers to accessing primary healthcare (including financial restrictions, the inability to book an appointment, lack of transport, and lack of childcare for other children) to treat GAS pharyngitis and skin infections could potentially reduce these infections and lead to a reduction in their sequelae, including ARF. These strategies should be co-designed and culturally appropriate for the communities being served and carefully evaluated. FUNDING: This work was supported by the Health Research Council of New Zealand (HRC), award number 16/005.

BACKGROUND: COVID-19 has affected many healthcare workers (HCWs) globally. We performed state-wide SARS-CoV-2 genomic epidemiological investigations to identify HCW transmission dynamics and provide recommendations to optimise healthcare system preparedness for future outbreaks. METHODS: Genome sequencing was attempted on all COVID-19 cases in Victoria, Australia. We combined genomic and epidemiologic data to investigate the source of HCW infections across multiple healthcare facilities (HCFs) in the state. Phylogenetic analysis and fine-scale hierarchical clustering were performed for the entire dataset including community and healthcare cases. Facilities provided standardised epidemiological data and putative transmission links. FINDINGS: Between March-October 2020, approximately 1,240 HCW COVID-19 infection cases were identified; 765 are included here, requested for hospital investigations. Genomic sequencing was successful for 612 (80%) cases. Thirty-six investigations were undertaken across 12 HCFs. Genomic analysis revealed that multiple introductions of COVID-19 into facilities (31/36) were more common than single introductions (5/36). Major contributors to HCW acquisitions included mobility of staff and patients between wards and facilities, and characteristics and behaviours of patients that generated numerous secondary infections. Key limitations at the HCF level were identified. INTERPRETATION: Genomic epidemiological analyses enhanced understanding of HCW infections, revealing unsuspected clusters and transmission networks. Combined analysis of all HCWs and patients in a HCF should be conducted, supported by high rates of sequencing coverage for all cases in the population. Established systems for integrated genomic epidemiological investigations in healthcare settings will improve HCW safety in future pandemics. FUNDING: The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.

BACKGROUND: Early, rapid detection of SARS-CoV-2 is essential in healthcare settings in order to implement appropriate infection control precautions and rapidly assign patients to care pathways. Rapid testing methods, such as SARS-CoV-2 rapid antigen testing (RAT) may improve patient care, despite a lower sensitivity than real-time PCR (RT-PCR) testing. METHODS: Patients presenting to an Emergency Department (ED) in Melbourne, Australia, were risk-stratified for their likelihood of active COVID-19 infection, and a non-randomised cohort of patients were tested by both Abbott Panbio™ COVID-19 Ag test (RAT) and SARS-CoV-2 RT-PCR. Patients with a positive RAT in the 'At or High Risk' COVID-19 group were moved immediately to a COVID-19 ward rather than waiting for a RT-PCR result. Clinical and laboratory data were assessed to determine test performance characteristics; and length of stay in the ED was compared for the different patient cohorts. FINDINGS: Analysis of 1762 paired RAT/RT-PCR samples demonstrated an overall sensitivity of 75.5% (206/273; 95% CI: 69·9-80·4) for the Abbott Panbio™ COVID-12 Ag test, with specificity of 100% (1489/1489; 95% CI: 99·8-100). Sensitivity improved with increasing risk for COVID-19 infection, from 72·4% (95% CI: 52·8-87·3) in the 'No Risk' cohort to 100% (95% CI: 29·2-100) in the 'High Risk' group. Time in the ED for the 'At/High Risk' group decreased from 421 minutes (IQR: 281, 525) for those with a positive RAT result to 274 minutes (IQR:140, 425) for those with a negative RAT result, p = 0.02. INTERPRETATION: The positive predictive value of a positive RAT in this setting was high, allowing more rapid instigation of COVID-19 care pathways and an improvement in patient flow within the ED. FUNDING: Royal Melbourne Hospital, Melbourne, Australia.

BACKGROUND: Mycoplasma genitalium (MG) infection is challenging to cure because of rising antimicrobial resistance and limited treatment options. METHODS: This was a prospective evaluation of the efficacy and tolerability of resistance-guided combination antimicrobial therapy for MG treatment at Melbourne Sexual Health Centre (August 2019-December 2020). All patients received 7 days of doxycycline before combination therapy based on the macrolide-resistant profile. Macrolide-susceptible infections received combination doxycycline + azithromycin (1 g, day 1; 500 mg, days 2-4) and macrolide-resistant infections combination doxycycline + moxifloxacin (400 mg daily for 7 days). Adherence and adverse effects were recorded at test-of-cure, recommended 14-28 days after antimicrobial completion. Sequencing was performed to determine the prevalence of single nucleotide polymorphisms (SNPs) in the parC gene and their association with moxifloxacin treatment outcomes in macrolide-resistant infections. RESULTS: Of 100 patients with macrolide-susceptible MG treated with doxycycline + azithromycin, 93 were cured (93.0%; 95% confidence interval [CI], 86.1-97.1). Of 247 patients with macrolide-resistant MG receiving doxycycline + moxifloxacin, 210 were cured (85.0%; 95% CI, 80.0-89.2). parC sequencing was available for 164 (66%) macrolide-resistant infections; 29% had SNPs at parC S83 or D87 (23% S83I). The absence of SNPs at parC S83/D87 was associated with 98.3% cure (95% CI, 93.9-99.8) following doxycycline + moxifloxacin. The presence of the parC S83I-SNP was associated with failure in 62.5% (95% CI, 45.8-77.3). Side effects were common (40%-46%) and predominantly mild and gastrointestinal. CONCLUSIONS: Combination doxycycline + azithromycin achieved high cure for macrolide-susceptible infections. However, in the context of a high prevalence of the parC S83I mutation (23%) in macrolide-resistant infections, doxycycline + moxifloxacin cured only 85%. Infections that were wild-typ

INTRODUCTION: Neisseria gonorrhoeae infections are common and incidence increasing. Oropharyngeal infections are associated with greater treatment failure compared with other sites and drive transmission to anogenital sites through saliva. Gonococcal resistance is increasing and new treatments are scarce, therefore, clinicians must optimise currently available and emerging treatments in order to have efficacious therapeutic options. This requires pharmacokinetic data from the oral cavity/oropharynx, however, availability of such information is currently limited. METHODS AND ANALYSIS: Healthy male volunteers (participants) recruited into the study will receive single doses of either ceftriaxone 1 g, cefixime 400 mg or ceftriaxone 500 mg plus 2 g azithromycin. Participants will provide samples at 6-8 time points (treatment regimen dependent) from four oral sites, two oral fluids, one anorectal swab and blood. Participants will complete online questionnaires about their medical history, sexual practices and any side effects experienced up to days 5-7. Saliva/oral mucosal pH and oral microbiome analysis will be undertaken. Bioanalysis will be conducted by liquid chromatography-mass spectrometry. Drug concentrations over time will be used to develop mathematical models for optimisation of drug dosing regimens and to estimate pharmacodynamic targets of efficacy. ETHICS AND DISSEMINATION: This study was approved by Royal Melbourne Hospital Human Research Ethics Committee (60370/MH-2021). The study results will be submitted for publication in peer-reviewed journals and reported at conferences. Summary results will be sent to participants requesting them. All data relevant to the study will be included in the article or uploaded as supplementary information. TRIAL REGISTRATION NUMBER: ACTRN12621000339853.

Mouthwash is a commonly used product and has been proposed as an alternative intervention to prevent gonorrhea transmission. However, the long-term effects of mouthwash on the oral microbiota are largely unknown. We investigated the impact of 12 weeks of daily mouthwash use on the oropharyngeal microbiota in a subset of men who have sex with men who participated in a randomized trial comparing the efficacy of two alcohol-free mouthwashes for the prevention of gonorrhea. We characterized the oropharyngeal microbiota using 16S rRNA gene sequencing of tonsillar fossae samples collected before and after 12 weeks of daily use of Listerine mouthwash or Biotène dry mouth oral rinse. Permutational multivariate analysis of variance (PERMANOVA) was used to assess differences in oropharyngeal microbiota composition following mouthwash use. Differential abundance testing was performed using ALDEx2, with false-discovery rate correction. A total of 306 samples from 153 men were analyzed (Listerine, n = 78 and Biotène, n = 75). There was no difference in the overall structure of the oropharyngeal microbiota following Listerine or Biotène use (PERMANOVA P = 0.413 and P = 0.331, respectively). Although no bacterial taxa were significantly differentially abundant following Listerine use, we observed a small but significant decrease in the abundance of both Streptococcus and Leptotrichia following Biotène use. Overall, our findings suggest that daily use of antiseptic mouthwash has minimal long-term effects on the composition of the oropharyngeal microbiota. IMPORTANCE Given the role of the oral microbiota in human health, it is important to understand if and how external factors influence its composition. Mouthwash use is common in some populations, and the use of antiseptic mouthwash has been proposed as an alternative intervention to prevent gonorrhea transmission. However, the long-term effect of mouthwash use on the oral microbiota composition is largely unknown. We found that da

Non-typhoidal Salmonella (NTS) is the second most common cause of foodborne bacterial gastroenteritis in Australia with antimicrobial resistance (AMR) increasing in recent years. Whole-genome sequencing (WGS) provides opportunities for in silico detection of AMR determinants. The objectives of this study were two-fold: (1) establish the utility of WGS analyses for inferring phenotypic resistance in NTS, and (2) explore clinically relevant genotypic AMR profiles to third generation cephalosporins (3GC) in NTS lineages. The concordance of 2490 NTS isolates with matched WGS and phenotypic susceptibility data against 13 clinically relevant antimicrobials was explored. In silico serovar prediction and typing was performed on assembled reads and interrogated for known AMR determinants. The surrounding genomic context, plasmid determinants and co-occurring AMR patterns were further investigated for multidrug resistant serovars harbouring bla CMY-2, bla CTX-M-55 or bla CTX-M-65. Our data demonstrated a high correlation between WGS and phenotypic susceptibility testing. Phenotypic-genotypic concordance was observed between 2440/2490 (98.0 %) isolates, with overall sensitivity and specificity rates >98 % and positive and negative predictive values >97 %. The most common AMR determinants were bla TEM-1, sul2, tet(A), strA-strB and floR. Phenotypic resistance to cefotaxime and azithromycin was low and observed in 6.2 % (151/2486) and 0.9 % (16/1834) of the isolates, respectively. Several multi-drug resistant NTS lineages were resistant to 3GC due to different genetic mechanisms including bla CMY-2, bla CTX-M-55 or bla CTX-M-65. This study shows WGS can enhance existing AMR surveillance in NTS datasets routinely produced in public health laboratories to identify emerging AMR in NTS. These approaches will be critical for developing capacity to detect emerging public health threats such as resistance to 3GC.

INTRODUCTION: Acute rheumatic fever (ARF) is usually considered a consequence of group A streptococcus (GAS) pharyngitis, with GAS skin infections not considered a major trigger. The aim was to quantify the risk of ARF following a GAS-positive skin or throat swab. METHODS: This retrospective analysis used pre-existing administrative data. Throat and skin swab data (1 866 981 swabs) from the Auckland region, New Zealand and antibiotic dispensing data were used (2010-2017). Incident ARF cases were identified using hospitalisation data (2010-2018). The risk ratio (RR) of ARF following swab collection was estimated across selected features and timeframes. Antibiotic dispensing data were linked to investigate whether this altered ARF risk following GAS detection. RESULTS: ARF risk increased following GAS detection in a throat or skin swab. Māori and Pacific Peoples had the highest ARF risk 8-90 days following a GAS-positive throat or skin swab, compared with a GAS-negative swab. During this period, the RR for Māori and Pacific Peoples following a GAS-positive throat swab was 4.8 (95% CI 3.6 to 6.4) and following a GAS-positive skin swab, the RR was 5.1 (95% CI 1.8 to 15.0). Antibiotic dispensing was not associated with a reduction in ARF risk following GAS detection in a throat swab (antibiotics not dispensed (RR: 4.1, 95% CI 2.7 to 6.2), antibiotics dispensed (RR: 4.3, 95% CI 2.5 to 7.4) or in a skin swab (antibiotics not dispensed (RR: 3.5, 95% CI 0.9 to 13.9), antibiotics dispensed (RR: 2.0, 95% CI 0.3 to 12.1). CONCLUSIONS: A GAS-positive throat or skin swab is strongly associated with subsequent ARF, particularly for Māori and Pacific Peoples. This study provides the first population-level evidence that GAS skin infection can trigger ARF.

Understanding the relative contribution of different between-farm transmission pathways is essential in guiding recommendations for mitigating disease spread. This study investigated the association between contact pathways linking poultry farms in New Zealand and the genetic relatedness of antimicrobial resistant Campylobacter jejuni Sequence Type 6964 (ST-6964), with the aim of identifying the most likely contact pathways that contributed to its rapid spread across the industry. Whole-genome sequencing was performed on 167C. jejuni ST-6964 isolates sampled from across 30 New Zealand commercial poultry enterprises. The genetic relatedness between isolates was determined using whole genome multilocus sequence typing (wgMLST). Permutational multivariate analysis of variance and distance-based linear models were used to explore the strength of the relationship between pairwise genetic associations among the C. jejuni isolates and each of several pairwise distance matrices, indicating either the geographical distance between farms or the network distance of transportation vehicles. Overall, a significant association was found between the pairwise genetic relatedness of the C. jejuni isolates and the parent company, the road distance and the network distance of transporting feed vehicles. This result suggests that the transportation of feed within the commercial poultry industry as well as other local contacts between flocks, such as the movements of personnel, may have played a significant role in the spread of C. jejuni. However, further information on the historical contact patterns between farms is needed to fully characterise the risk of these pathways and to understand how they could be targeted to reduce the spread of C. jejuni.

Introduction. Mycoplasma genitalium is a sexually transmitted pathogen with increasing resistance to first- and second-line antimicrobials. The 'near-patient test' ResistancePlus MG FleXible (SpeeDx) detects M. genitalium plus four macrolide resistance mutations (MRMs), facilitating same-day patient follow up.Hypothesis/Gap Statement. This assay has not been assessed on freshly collected samples.Aim. Our goal was to evaluate the performance of the ResistancePlus MG FleXible test against the standard of care open platform test.Methods. ResistancePlus MG FleXible (analysed on the Cepheid GeneXpert platform) was evaluated on freshly collected samples and compared to the standard of care open platform test ResistancePlus MG (SpeeDx) analysed on the LightCycler 480 II (Roche).Results. For 270 valid tests, ResistancePlus MG FleXible yielded a high positive per cent agreement (PPA) of 94.1% [96/102; 95 % confidence interval (CI): 87.6-97.8 %] and negative per cent agreement (NPA) of 95.2% (160/168; 95 % CI: 90.8-97.9%) for M. genitalium detection compared to the reference assay (kappa for test concordance of 0.89; 95 % CI: 0.83-0.95). Performance was similar across different sample types. For the detection of MRMs, ResistancePlus MG FleXible had a PPA of 97.1% (66/68; 95% CI: 89.8-99.6) and NPA of 78.6% (22/28; 95 % CI: 59.0-91.7), with test comparison kappa of 0.79 (95 % CI: 0.65-0.93). Notably, of six discordant results (i.e. determined to be wild type by the reference assay), five were positive for MRMs by Sanger sequencing, indicating that the ResistancePlus MG FleXible assay has an improved performance for mutation detection.Conclusion. ResistancePlus MG FleXible had comparable test performance for M. genitalium detection as the open platform assay, with improved detection of MRMs. The ResistancePlus MG FleXible 'near-patient' assay can deliver a rapid result to expedite appropriate treatment.