State-wide genomic epidemiology investigations of COVID-19 in healthcare workers in 2020 Victoria, Australia: Qualitative thematic analysis to provide insights for future pandemic preparedness.

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.

Genomic characterisation reveals a dominant lineage of SARS-CoV-2 in Papua New Guinea.

The coronavirus disease pandemic has highlighted the utility of pathogen genomics as a key part of comprehensive public health response to emerging infectious diseases threats, however, the ability to generate, analyse, and respond to pathogen genomic data varies around the world. Papua New Guinea (PNG), which has limited in-country capacity for genomics, has experienced significant outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with initial genomics data indicating a large proportion of cases were from lineages that are not well defined within the current nomenclature. Through a partnership between in-country public health agencies and academic organisations, industry, and a public health genomics reference laboratory in Australia a system for routine SARS-CoV-2 genomics from PNG was established. Here we aim to characterise and describe the genomics of PNG's second wave and examine the sudden expansion of a lineage that is not well defined but very prevalent in the Western Pacific region. We generated 1797 sequences from cases in PNG and performed phylogenetic and phylodynamic analyses to examine the outbreak and characterise the circulating lineages and clusters present. Our results reveal the rapid expansion of the B.1.466.2 and related lineages within PNG, from multiple introductions into the country. We also highlight the difficulties that unstable lineage assignment causes when using genomics to assist with rapid cluster definitions.

Lab-in-a-van: Rapid SARS-CoV-2 testing response with a mobile laboratory.

BACKGROUND: High testing rates and rapid contact tracing have been key interventions to control COVID-19 in Victoria, Australia. A mobile laboratory (LabVan), for rapid SARS-CoV-2 diagnostics, was deployed at sites deemed critical by the Victorian State Department of Health as part of the response. We describe the process of design, implementation, and performance benchmarked against a central reference laboratory. METHODS: A BSL2 compliant laboratory, complete with a class II biological safety cabinet, was built within a Mercedes-Benz Sprinter Panel Van. Swabs were collected by on-site collection teams, registered using mobile internet-enabled tablets and tested using the Xpert® Xpress SARS-CoV-2 assay. Results were reported remotely via HL7 messaging to Public Health Units. Patients with negative results were automatically notified by mobile telephone text messaging (SMS). FINDINGS: A pilot trial of the LabVan identified a median turnaround time (TAT) from collection to reporting of 1:19 h:mm (IQR 0:18, Range 1:03-18:32) compared to 9:40 h:mm (IQR 8:46, Range 6:51-19:30) for standard processing within the central laboratory. During deployment in nine rural and urban COVID-19 outbreaks the median TAT was 2:18 h:mm (IQR 1:18, Range 0:50-16:52) compared to 19:08 h:mm (IQR 5:49, Range 1:36-58:52) for samples submitted to the central laboratory. No quality control issues were identified in the LabVan. INTERPRETATION: The LabVan is an ISO15189 compliant testing facility fully operationalized for mobile point-of-care testing that significantly reduces TAT for result reporting, facilitating rapid public health actions. FUNDING: This work was supported by the Department of Health, Victoria State Government, Australia.

COVID-19: Integrating genomic and epidemiological data to inform public health interventions and policy in Tasmania, Australia.

OBJECTIVE: We undertook an integrated analysis of genomic and epidemiological data to investigate a large health-care-associated outbreak of coronavirus disease 2019 (COVID-19) and to better understand the epidemiology of COVID-19 cases in Tasmania, Australia. METHODS: Epidemiological data collected on COVID-19 cases notified in Tasmania between 2 March and 15 May 2020, and positive samples of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or RNA extracted from the samples were included. Sequencing was conducted by tiled amplicon polymerase chain reaction with ARTIC v1 or v3 primers and Illumina sequencing. Consensus sequences were generated, sequences were aligned to a reference sequence and phylogenetic analysis was performed. Genomic clusters were determined and integrated with epidemiological data to provide additional information. RESULTS: All 231 COVID-19 cases notified in Tasmania during the study period and 266 SARS-CoV-2-positive samples, representing 217/231 (94%) notified cases, were included; 184/217 (84%) were clustered, 21/217 (10%) were unique and 12/217 (6%) could not be sequenced. Genomics confirmed the presence of seven clusters already identified through epidemiological links, clarified transmission networks in which the epidemiology had been unclear and identified one cluster that had not previously been recognized. DISCUSSION: Genomic analysis provided useful additional information on COVID-19 in Tasmania, including evidence of a large health-care-associated outbreak linked to an overseas cruise, the probable source of infection in cases with no previously identified epidemiological link and confirmation that there was no identified community transmission from other imported cases. Genomic insights are an important component of the response to COVID-19, and continuing genomic surveillance is warranted.

Viral Genomics to Inform Infection-control Response in Occupational Coronavirus Disease 2019 (COVID-19) Transmission.

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.

Genomics-informed responses in the elimination of COVID-19 in Victoria, Australia: an observational, genomic epidemiological study.

BACKGROUND: A cornerstone of Australia's ability to control COVID-19 has been effective border control with an extensive supervised quarantine programme. However, a rapid recrudescence of COVID-19 was observed in the state of Victoria in June, 2020. We aim to describe the genomic findings that located the source of this second wave and show the role of genomic epidemiology in the successful elimination of COVID-19 for a second time in Australia. METHODS: In this observational, genomic epidemiological study, we did genomic sequencing of all laboratory-confirmed cases of COVID-19 diagnosed in Victoria, Australia between Jan 25, 2020, and Jan 31, 2021. We did phylogenetic analyses, genomic cluster discovery, and integrated results with epidemiological data (detailed information on demographics, risk factors, and exposure) collected via interview by the Victorian Government Department of Health. Genomic transmission networks were used to group multiple genomic clusters when epidemiological and genomic data suggested they arose from a single importation event and diversified within Victoria. To identify transmission of emergent lineages between Victoria and other states or territories in Australia, all publicly available SARS-CoV-2 sequences uploaded before Feb 11, 2021, were obtained from the national sequence sharing programme AusTrakka, and epidemiological data were obtained from the submitting laboratories. We did phylodynamic analyses to estimate the growth rate, doubling time, and number of days from the first local infection to the collection of the first sequenced genome for the dominant local cluster, and compared our growth estimates to previously published estimates from a similar growth phase of lineage B.1.1.7 (also known as the Alpha variant) in the UK. FINDINGS: Between Jan 25, 2020, and Jan 31, 2021, there were 20 451 laboratory-confirmed cases of COVID-19 in Victoria, Australia, of which 15 431 were submitted for sequencing, and 11 711 met all quality control metrics and were included in our analysis. We identified 595 genomic clusters, with a median of five cases per cluster (IQR 2-11). Overall, samples from 11 503 (98·2%) of 11 711 cases clustered with another sample in Victoria, either within a genomic cluster or transmission network. Genomic analysis revealed that 10 426 cases, including 10 416 (98·4%) of 10 584 locally acquired cases, diagnosed during the second wave (between June and October, 2020) were derived from a single incursion from hotel quarantine, with the outbreak lineage (transmission network G, lineage D.2) rapidly detected in other Australian states and territories. Phylodynamic analyses indicated that the epidemic growth rate of the outbreak lineage in Victoria during the initial growth phase (samples collected between June 4 and July 9, 2020; 47·4 putative transmission events, per branch, per year [1/years; 95% credible interval 26·0-85·0]), was similar to that of other reported variants, such as B.1.1.7 in the UK (mean approximately 71·5 1/years). Strict interventions were implemented, and the outbreak lineage has not been detected in Australia since Oct 29, 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. INTERPRETATION: Our study highlights how rapid escalation of clonal outbreaks can occur from a single incursion. However, strict quarantine measures and decisive public health responses to emergent cases are effective, even with high epidemic growth rates. Real-time genomic surveillance can alter the way in which public health agencies view and respond to COVID-19 outbreaks. FUNDING: The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.

Defective Severe Acute Respiratory Syndrome Coronavirus 2 Immune Responses in an Immunocompromised Individual With Prolonged Viral Replication.

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.

Adding saliva testing to oropharyngeal and deep nasal swab testing increases PCR detection of SARS-CoV-2 in primary care and children.

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.

Optimal preparation of SARS-CoV-2 viral transport medium for culture.

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.

Multi-site assessment of rapid, point-of-care antigen testing for the diagnosis of SARS-CoV-2 infection in a low-prevalence setting: A validation and implementation study.

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.

Tracking the COVID-19 pandemic in Australia using genomics.

Genomic sequencing has significant potential to inform public health management for SARS-CoV-2. Here we report high-throughput genomics for SARS-CoV-2, sequencing 80% of cases in Victoria, Australia (population 6.24 million) between 6 January and 14 April 2020 (total 1,333 COVID-19 cases). We integrate epidemiological, genomic and phylodynamic data to identify clusters and impact of interventions. The global diversity of SARS-CoV-2 is represented, consistent with multiple importations. Seventy-six distinct genomic clusters were identified, including large clusters associated with social venues, healthcare and cruise ships. Sequencing sequential samples from 98 patients reveals minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicates a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after implementing travel restrictions and physical distancing. Our data provide a concrete framework for the use of SARS-CoV-2 genomics in public health responses, including its use to rapidly identify SARS-CoV-2 transmission chains, increasingly important as social restrictions ease globally.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.