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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.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.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.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.The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.

Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. Methods VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. Results The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. Discussion The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.

Current microbiological methods lack the resolution to accurately identify multidrug-resistant organism (MDRO) transmission, however, whole genome sequencing can identify highly-related patient isolates providing opportunities for precision infection control interventions. We investigated the feasibility and potential impact of a prospective multi-centre genomics workflow for hospital infection control.We conducted a prospective genomics implementation study across eight Australian hospitals over 15 months (2017,2018), collecting all clinical and screening isolates from inpatients withIn total, 2275 isolates were included from 1970 patients, predominantly ESBL-Ec (40·8%) followed by MRSA (35·6%),Implementation of a multi-centre genomics-informed infection control workflow is feasible and identifies many unrecognised MDRO transmissions. This provides critical opportunities for interventions to improve patient safety in hospitals.Melbourne Genomics Health Alliance (supported by State Government of Victoria, Australia), and National Health and Medical Research Council (Australia).

Healthcare workers are at increased risk of occupational transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report 2 instances of healthcare workers contracting SARS-CoV-2 despite no known breach of personal protective equipment. Additional specific equipment cleaning was initiated. Viral genomic sequencing supported this transmission hypothesis and our subsequent response.

Background The World Health Organization (WHO) coordinates the Global Invasive Bacterial Vaccine-Preventable Diseases (IB-VPD) Surveillance Network to support vaccine introduction decisions and use. The network was established to strengthen surveillance and laboratory confirmation of meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Methods Sentinel hospitals report cases of children Results From 2014 through 2019, >137 000 suspected meningitis cases were reported by 58 participating countries, with 44.6% (n = 61 386) reported from countries in the WHO African Region. More than half (56.6%, n = 77 873) were among children Conclusions This multitier, global surveillance network has supported countries in detecting and serotyping the 3 principal invasive bacterial pathogens that cause pediatric meningitis. Streptococcus pneumoniae was the most common bacterial pathogen detected globally despite the growing number of countries that have nationally introduced PCV. The large proportions of deaths due to S. pneumoniae reflect the high proportion of meningitis cases caused by this pathogen. This global network demonstrated a strong correlation between PCV introduction status and reduction in the proportion of pneumococcal meningitis infections caused by vaccine serotypes. Maintaining case-based, active surveillance with laboratory confirmation for prioritized vaccine-preventable diseases remains a critical component of the global agenda in public health. The World Health Organization (WHO)-coordinated Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network reported data from 2014 to 2019, contributing to the estimates of the disease burden and serotypes of pediatric meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2–specific antibody responses were absent and SARS-CoV-2–specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses., We describe defects in severe acute respiratory syndrome coronavirus 2–specific immune responses and persistent viral replication in a patient with lymphoma. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

Objective To compare the concordance and acceptability of saliva testing with standard‐of‐care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) in children and in general practice. Design Prospective multicentre diagnostic validation study. Setting Royal Children’s Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July–October 2020. Participants 1050 people who provided paired saliva and oropharyngeal‐nasal swabs for SARS‐CoV‐2 testing. Main outcome measures Numbers of cases in which SARS‐CoV‐2 was detected in either specimen type by real‐time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. Results SARS‐CoV‐2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58–84%) for saliva and 63% (95% CI, 49–76%) for oropharyngeal‐nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70–95%) for saliva and 63% (95% CI, 45–79%) for oropharyngeal‐nasal swabs. Adding saliva testing to standard‐of‐care oropharyngeal‐nasal swab testing increased overall case detection by 59% (95% CI, 29–95%). Providing saliva was preferred to an oropharyngeal‐nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). Conclusion In children over 10 years of age and adults, saliva testing alone may be suitable for SARS‐CoV‐2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal‐nasal swab testing for increasing case detection.

Background: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, few studies have assessed the performance and implementation of rapid antigen-based SARS-CoV-2 testing in a setting with a low prevalence of COVID-19 infections, such as Australia. Methods: This study recruited participants presenting for COVID-19 testing at three Melbourne metropolitan hospitals during a period of low COVID-19 prevalence. The Abbott PanBioTM COVID-19 Ag point-of-care test was performed alongside RT-PCR. In addition, participants with COVID-19 notified to the Victorian Government were invited to provide additional swabs to aid validation. Implementation challenges were also documented. Findings: The specificity of the Abbott PanBioTM COVID-19 Ag test was 99.96% (95% CI 99.73 - 100%). Sensitivity amongst participants with RT-PCR-confirmed infection was dependent upon the duration of symptoms reported, ranging from 77.3% (duration 1 to 33 days) to 100% in those within seven days of symptom onset. A range of implementation challenges were identified which may inform future COVID-19 testing strategies in a low prevalence setting. Interpretation: Given the high specificity, antigen-based tests may be most useful in rapidly triaging public health and hospital resources while expediting confirmatory RT-PCR testing. Considering the limitations in test sensitivity and the potential for rapid transmission in susceptible populations, particularly in hospital settings, careful consideration is required for implementation of antigen testing in a low prevalence setting. Funding: This work was funded by the Victorian Department of Health and Human Services. The funder was not involved in data analysis or manuscript preparation.

Background Multiresistant organisms (MROs) pose a critical threat to public health. Population-based programs for control of MROs such as carbapenemase-producing Enterobacterales (CPE) have emerged and evaluation is needed. We assessed the feasibility and impact of a statewide CPE surveillance and response program deployed across Victoria, Australia (population 6.5 million). Methods A prospective multimodal intervention including active screening, carrier isolation, centralized case investigation, and comparative pathogen genomics was implemented. We analyzed trends in CPE incidence and clinical presentation, risk factors, and local transmission over the program’s first 3 years (2016–2018). Results CPE case ascertainment increased over the study period to 1.42 cases/100 000 population, linked to increased screening without a concomitant rise in active clinical infections (0.45–0.60 infections/100 000 population, P = .640). KPC-2 infection decreased from 0.29 infections/100 000 population prior to intervention to 0.03 infections/100 000 population in 2018 (P = .003). Comprehensive case investigation identified instances of overseas community acquisition. Median time between isolate referral and genomic and epidemiological assessment for local transmission was 11 days (IQR, 9–14). Prospective surveillance identified numerous small transmission networks (median, 2; range, 1–19 cases), predominantly IMP and KPC, with median pairwise distance of 8 (IQR, 4–13) single nucleotide polymorphisms; low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted response. Conclusions We demonstrate the value of centralized CPE control programs to increase case ascertainment, resolve risk factors, and identify local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globally., A statewide multimodal intervention for the control of carbapenemase-producing Enterobacterales was associated with a significant increase in case ascertainment, with no rise in clinical infections. Timely prospective epidemiological and genomic surveillance identified numerous small local transmission networks permitting rapid response.

Improved knowledge of factors that promote outbreaks of enteric pathogens among men who have sex with men (MSM) could enable targeted public health interventions. We detected enteric pathogens in 57 of 519 (11%) asymptomatic MSM, and we found that enteric pathogen detection was associated with both oroanal sex (rimming) and group sex.

Background The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis. Methods Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains). Results We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains. Conclusions The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated “reverted mutant” strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1–3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease.

BackgroundUntil recently,Klebsiella pneumoniaecarbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.MethodsAll KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.ResultsInitial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.ConclusionA real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50–60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Background:\ud \ud Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D).\ud \ud Methodology/Principal Findings:\ud \ud Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.\ud \ud Conclusions/Significance:\ud \ud This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.