Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance (preprint)

The capacity to undertake whole genome sequencing (WGS) in public health laboratories (PHLs) has grown rapidly in response to COVID-19, and SARS-CoV-2 genomic data has been invaluable for managing the pandemic. The public health response has been further supported by the rapid upgrade and implementation of laboratory and bioinformatic resources. However, there remains a high degree of variability in methods and capabilities between laboratories. In addition to evolving methodology and improved understanding of SARS-CoV-2, public health laboratories have become strained during surges in case numbers, adding to the difficulty of ensuring the highest data accuracy. Here, we formed a national working group comprised of laboratory scientists and bioinformaticians from Australia and New Zealand to improve data concordance across PHLs. Through investigating discordant sequence data from Australia's first external SARS-CoV-2 WGS proficiency testing program (PTP), we show that most discrepancies in genome assessment arose from intrahost variation. While others could be remedied using reasonable, parsimonious bioinformatic quality control. Furthermore, we demonstrate how multidisciplinary national working groups can inform guidelines in real time for bioinformatic quality acceptance criteria. Provision of technical feedback allows laboratory improvement during a pandemic in real time, enhancing public health responses.

Genomics-Informed Responses in the Elimination of COVID-19 in Australia (preprint)

BACKGROUND: A cornerstone of Australia’s ability to control COVID-19 has been effective border control, using an extensive supervised quarantine program. However, a rapid recrudescence in COVID-19 cases was observed in the state of Victoria in June 2020. Here, we describe the genomic findings that located the source of this second wave as a breach in supervised hotel quarantine and demonstrate the successful elimination of COVID-19 for a second time in Australia.METHODS: Genome sequencing was performed on all available SARS-CoV-2-positive samples in Victoria and integrated genomic and epidemiological investigation undertaken.RESULTS: At 31st January 2021, 20,451 COVID-19 cases were reported in Victoria; samples were sequenced from 75% of cases (15,431/20,451). Genomics revealed 98% (10,426/10,646) of locally-acquired cases during the second wave were derived from a single incursion from hotel quarantine, with the outbreak strain rapidly detected in other Australian states and territories. Phylodynamic analyses indicated an epidemic growth rate comparable to emerging variants, such as B.1.1.7 in the United Kingdom. Strict public health interventions resulted in the elimination of the outbreak strain by 29th October 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread.CONCLUSIONS: Rapid escalation of clonal outbreaks can occur from even a single breach of control practices, as revealed through our genomic ‘enhanced outbreak-detection' system. The subsequent elimination and rapid control of new SARS-CoV-2 incursions reinforce that decisive public health responses to emergent cases are effective even with high epidemic growth rates, and “elimination” should be favored in settings where this is achievable.FUNDING STATEMENT: The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and the Victorian Infectious Diseases Reference Laboratory (VIDRL) at The Doherty Institute are funded by the Victorian Government. This work was supported by the National Health and Medical Research Council, Australia (NHMRC); Partnership Grant (APP1149991), Investigator Grant to BPH (APP1196103), Investigator Grant to DAW (APP1174555), Research Fellowship to TPS (APP1105525), MRFF COVID-19 Genomics Grant (MRF9200006).DECLARATION OF INTERESTS: None to declare. ETHICS APPROVAL STATEMENT: Data were collected in accordance with the Victorian Public Health and Wellbeing Act 2008. Ethical approval was received from the University of Melbourne Human Research Ethics Committee (study number 1954615.3).

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 (preprint)

Background: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, no 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 78.9% (duration 1 to 33 days) to 100% in those within 7 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.Declaration of Interests: All authors: no conflicts.Ethics Approval Statement: Ethics review and study approval was provided by Monash Health Human Research and Ethics Committee (RES-20-0000-678A) and local Governance approval was provided by Melbourne Health and Austin Health Offices for Research.

The Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework: An Implementation Science Approach to Evaluating Pathogen Whole Genome Sequencing in Public Health (preprint)

Background Pathogen whole genome sequencing (WGS) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding: (i) the optimal models for integration of genomic data into epidemiological investigations, and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. Methods We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis and SARS-CoV-2. Results The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. Conclusions Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts. 

Evaluation of serological tests for SARS-CoV-2: Implications for serology testing in a low-prevalence setting (preprint)

Background: Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little pre-market validation. Methods: Performance characteristics for five PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralisation test (sVNT). Results: Sensitivities for PoCT ranged from 51.8% (95% CI 43.1 to 60.4%) to 67.9% (95% CI 59.4-75.6%), and specificities from 95.6% (95% CI 89.2-98.8%) to 100.0% (95% CI 96.1-100.0%). Overall ELISA sensitivity for either IgA or IgG detection was 67.9% (95% CI 59.4-75.6), increasing to 93.8% (95% CI 85.0-98.3%) for samples >14 days post symptom onset. Overall, sVNT sensitivity was 60.9% (95% CI 53.2-68.4%), rising to 91.2%% (95% CI 81.8-96.7%) for samples collected >14 days post-symptom onset, with a specificity 94.4% (95% CI 89.2-97.5%), Conclusion: Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in the reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.

Tracking the COVID-19 pandemic in Australia using genomics (preprint)

BACKGROUND: Whole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODS: Cases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTS: Between 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 samples were included in genomic analyses. Sequenced samples from Australia were representative of the global diversity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified; these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential samples from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing. CONCLUSIONS: Our data provide a comprehensive framework for the use of SARS-CoV-2 genomics in public health responses. The application of genomics to rapidly identify SARS-CoV-2 transmission chains will become critically important as social restrictions ease globally. Public health responses to emergent cases must be swift, highly focused and effective.

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA (preprint)

2.IntroductionThe 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. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd 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 as little as 1 L 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{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay 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 minutes (SD {+/-}7 minutes). 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. ConclusionWith a simplified workflow, 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. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.