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.

SARS-CoV-2 infection results in immune responses in the respiratory tract and peripheral blood that suggest mechanisms of disease severity.

Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples correlates with RBD-specific IgM and IgG levels. Cytokines/chemokines vary between respiratory samples and plasma, indicating that inflammation should be assessed in respiratory specimens to understand immunopathology. IFN-α2 and IL-12p70 in endotracheal aspirate and neutralization in sputum negatively correlate with duration of hospital stay. Diverse immune subsets are detected in respiratory samples, dominated by neutrophils. Importantly, dexamethasone treatment does not affect humoral responses in blood of COVID-19 patients. Our study unveils differential immune responses between respiratory samples and blood, and shows how drug therapy affects immune responses during COVID-19.

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.

Staff to staff transmission as a driver of healthcare worker infections with COVID-19.

BACKGROUND: High rates of healthcare worker (HCW) infections due to COVID-19 have been attributed to several factors, including inadequate personal protective equipment (PPE), exposure to a high density of patients with COVID-19, and poor building ventilation. We investigated an increase in the number of staff COVID-19 infections at our hospital to determine the factors contributing to infection and to implement the interventions required to prevent subsequent infections. METHODS: We conducted a single-centre retrospective cohort study of staff working at a tertiary referral hospital who tested positive for SARS-CoV-2 between 25 January 2020 and 25 November 2020. The primary outcome was the source of COVID-19 infection. RESULTS: Of 45 staff who returned a positive test result for SARS-CoV-2, 19 were determined to be acquired at our hospital. Fifteen (15/19; 79% [95% CI: 54-94%]) of these were identified through contact tracing and testing following exposures to other infected staff and were presumed to be staff-to-staff transmission, including an outbreak in 10 healthcare workers (HCWs) linked to a single ward that cared for COVID-19 patients. The staff tearoom was identified as the likely location for transmission, with subsequent reduction in HCW infections and resolution of the outbreak following implementation of enhanced control measures in tearoom facilities. No HCW contacts (0/204; 0% [95% CI: 0-2%]) developed COVID-19 infection following exposure to unrecognised patients with COVID-19. CONCLUSION: Unrecognised infections among staff may be a significant driver of HCW infections in healthcare settings. Control measures should be implemented to prevent acquisition from other staff as well as patient-staff transmission.

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.

COVID-MATCH65-A prospectively derived clinical decision rule for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: We report on the key clinical predictors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and present a clinical decision rule that can risk stratify patients for COVID-19. DESIGN, PARTICIPANTS AND SETTING: A prospective cohort of patients assessed for COVID-19 at a screening clinic in Melbourne, Australia. The primary outcome was a positive COVID-19 test from nasopharyngeal swab. A backwards stepwise logistic regression was used to derive a model of clinical variables predictive of a positive COVID-19 test. Internal validation of the final model was performed using bootstrapped samples and the model scoring derived from the coefficients, with modelling performed for increasing prevalence. RESULTS: Of 4226 patients with suspected COVID-19 who were assessed, 2976 patients underwent SARS-CoV-2 testing (n = 108 SARS-CoV-2 positive) and were used to determine factors associated with a positive COVID-19 test. The 7 features associated with a positive COVID-19 test on multivariable analysis were: COVID-19 patient exposure or international travel, Myalgia/malaise, Anosmia or ageusia, Temperature, Coryza/sore throat, Hypoxia-oxygen saturation < 97%, 65 years or older-summarized in the mnemonic COVID-MATCH65. Internal validation showed an AUC of 0.836. A cut-off of ≥ 1.5 points was associated with a 92.6% sensitivity and 99.5% negative predictive value (NPV) for COVID-19. CONCLUSIONS: From the largest prospective outpatient cohort of suspected COVID-19 we define the clinical factors predictive of a positive SARS-CoV-2 test. The subsequent clinical decision rule, COVID-MATCH65, has a high sensitivity and NPV for SARS-CoV-2 and can be employed in the pandemic, adjusted for disease prevalence, to aid COVID-19 risk-assessment and vital testing resource allocation.

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.

Duration of Respiratory and Gastrointestinal Viral Shedding in Children With SARS-CoV-2: A Systematic Review and Synthesis of Data.

BACKGROUND: Children with coronavirus disease 2019 (COVID-19) are more likely to have mild or no symptoms compared with adults and may represent important vectors for transmitting the virus. Little is known about the duration of respiratory and gastrointestinal viral shedding in children with COVID-19. OBJECTIVE: To determine the average shedding times of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the respiratory and gastrointestinal tracts in children. METHODS: We performed a systematic search of Ovid MEDLINE, Embase and Cochrane CENTRAL databases for studies reporting real-time reverse transcriptase polymerase chain reaction (rt-PCR) results in children with COVID-19, then extracted and synthesized data on duration of viral shedding from symptom onset in respiratory and gastrointestinal samples. RESULTS: Based on data compiled from 69 pediatric cases, the duration of viral shedding through the respiratory tract is up to 24 days from symptom onset with a mean of 11.1 ± 5.8 days. Of the children who underwent testing with stool PCR, rectal swab or anal swab, 86% returned a positive result. The mean duration of viral shedding via the gastrointestinal tract was 23.6 ± 8.8 days from symptom onset. In 89% of cases, viral shedding via the gastrointestinal tract persisted after nasopharyngeal or throat swabs became negative, for as long as 4 weeks. CONCLUSIONS: To our knowledge, this is the first attempt to systematically review the duration of respiratory and gastrointestinal viral shedding of SARS-CoV-2 in pediatric patients. These findings may have important implications for infection control strategies during the COVID-19 pandemic.