Assessment of twenty-two SARS-CoV-2 rapid antigen tests against SARS-CoV-2: A laboratory evaluation study (preprint)

Background Rapid antigen testing is widely used as a way of scaling up population-level testing. To better inform antigen test deployment in Australia, we evaluated 22 commercially available antigen tests, including an assessment of culture infectivity. Methods Analytical sensitivity was evaluated against SARS-CoV-2 B.1.617.2 (Delta), reported as TCID50/mL, cycle threshold (Ct) value and viral load (RNA copies/mL). Specificity was assessed against non-SARS-CoV-2 viruses. Clinical sensitivity and correlation with cell culture infectivity was assessed using the Abbott PanBio COVID-19 Ag test. Results Nineteen kits consistently detected SARS-CoV-2 antigen equivalent to 1.3x10^6 copies/mL (5.8x10^3 TCID50/mL). Specificity for all kits was 100%. Compared to RT-PCR the Abbott PanBio COVID-19 Ag test was 52.6% (95% CI, 41.6% to 63.3%) sensitive, with a 50% detection probability for infectious cell culture at 5.9 log10 RNA copies/mL (95% CI, 5.3 to 6.5 log10 copies/mL). Antigen test sensitivity was 97.6% (95% CI, 86.3% to 100.0%) compared to positive infectious in cell culture. Conclusions Antigen test positivity correlated with positive viral culture, suggesting antigen test results may determine SARS-CoV-2 transmission risk. Sensitivity varied considerably between test kits and highlights the need for ongoing systematic post-market evaluation, providing valuable information to help guide antigen test selection and deployment.

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).

Evaluation of six commercial SARS-CoV-2 Enzyme-Linked Immunosorbent assays for clinical testing and serosurveillance. (preprint)

Background: Serological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. As we move into the era of vaccines, the detection of neutralising antibody will become increasingly important. Many serological tests have been developed under emergency use authorization, but their reliability remains unclear. Methods: We evaluated the performance of six commercially-available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test, for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or neutralising antibodies and a subset of results were compared to microneutralisation. Results: For sera collected > 14 days post-symptom onset the Wantai total Ab performed best with highest sensitivity 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript Surrogate Virus Neutralization Test, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA and 83.3% for Wantai IgM. Specificity for the best performing assay was 99.5% and for the lowest 97.1%. Conclusion: Wantai ELISA, detecting total immunoglobulins against SARS-CoV-2 receptor binding domain, had the best performance. Antibody target, timing and longevity of the immune response, and the objectives of testing should be considered in test choice. ELISAs should be used within a confirmatory testing algorithm to ensure reliable results. ELISAs provide high quality results, with flexibility for test numbers without the need for manufacturer specific analyzers. Key words: SARS-CoV-2, COVID-19, serology, ELISA, antibody, neutralising.

Sample Pooling is a Viable Strategy for SARS-CoV-2 Detection in Low-Prevalence Settings (preprint)

BACKGROUNDThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly increased demand on laboratory throughput and reagents for nucleic acid extraction and polymerase chain reaction (PCR). Reagent shortages may limit the expansion of testing required to scale back isolation measures. AIMTo investigate the viability of sample pooling as a strategy for increasing test throughput and conserving PCR reagents; to report our early experience with pooling of clinical samples. METHODSA pre-implementation study was performed to assess the sensitivity and theoretical efficiency of two, four, and eight-sample pools in a real-time reverse transcription PCR-based workflow. A standard operating procedure was developed and implemented in two laboratories during periods of peak demand, inclusive of over 29,000 clinical samples processed in our laboratory. RESULTSSensitivity decreased (mean absolute increase in cycle threshold value of 0.6, 2.3, and 3.0 for pools of two, four, and eight samples respectively) and efficiency increased as pool size increased. Gains from pooling diminished at high disease prevalence. Our standard operating procedure was successfully implemented across two laboratories. Increased workflow complexity imparts a higher risk of errors, and requires risk mitigation strategies. Turnaround time for individual samples increased, hence urgent samples should not be pooled. CONCLUSIONSPooling is a viable strategy for high-throughput testing of SARS-CoV-2 in low-prevalence settings.

Implementation and evaluation of a novel real-time multiplex assay for SARS-CoV-2: In-field learnings from a clinical microbiology laboratory (preprint)

The unprecedented scale of testing required to effectively control the coronavirus disease (COVID-19) pandemic has necessitated urgent implementation of rapid testing in clinical microbiology laboratories. To date, there are limited data available on the analytical performance of emerging commercially available assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and integration of these assays into laboratory workflows. Here, we performed a prospective validation study of a commercially available assay, the AusDiagnostics Coronavirus Typing (8-well) assay. Respiratory tract samples for SARS-CoV-2 testing were collected between 1st March and 25th March 2020. All positive samples and a random subset of negative samples were sent to a reference laboratory for confirmation. In total, 2,673 samples were analyzed using the Coronavirus Typing assay. The predominant sample type was a combined nasopharyngeal/throat swab (2,640/2,673; 98.8%). Fifty-four patients were positive for SARS-CoV-2 (0.02%) using the Coronavirus Typing assay; 53/54 (98.1%) positive results and 621/621 (100%) negative results were concordant with the reference laboratory. Compared to the reference standard, sensitivity of the Coronavirus Typing assay for SARS-CoV-2 was 100% [95% CI 93.2%-100%], specificity 99.8% [95% CI 99.1%-100%], positive predictive value 98.1% (95% CI 90.2%-99.7%] and negative predictive value 100% [95% CI 99.4%-100%]. In many countries, standard regulatory requirements for the introduction of new assays have been replaced by emergency authorizations and it is critical that laboratories share their post-market validation experiences, as the consequences of widespread introduction of a sub-optimal assay for SARS-CoV-2 are profound. Here, we share our in-field experience, and encourage other laboratories to follow suit.

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.

Direct RNA sequencing and early evolution of SARS-CoV-2 (preprint)

Fundamental aspects of SARS-CoV-2 biology remain to be described, having the potential to provide insight to the response effort for this high-priority pathogen. Here we describe the first native RNA sequence of SARS-CoV-2, detailing the coronaviral transcriptome and epitranscriptome, and share these data publicly. A data-driven inference of viral genetic features and evolutionary rate is also made. The rapid sharing of sequence information throughout the SARS-CoV-2 pandemic represents an inflection point for public health and genomic epidemiology, providing early insights into the biology and evolution of this emerging pathogen.

Breadth of concomitant immune responses underpinning viral clearance and patient recovery in a non-severe case of COVID-19 (preprint)

We report the kinetics of the immune response in relation to clinical and virological features of a patient with mild-to-moderate coronavirus disease-19 (COVID-19) requiring hospitalisation. Increased antibody-secreting cells, follicular T-helper cells, activated CD4+ and CD8+ T-cells and IgM/IgG SARS-CoV-2-binding antibodies were detected in blood, prior to symptomatic recovery. These immunological changes persisted for at least 7 days following full resolution of symptoms, indicating substantial anti-viral immunity in this non-severe COVID-19.

Breadth of concomitant immune responses underpinning viral clearance and patient recovery in a non-severe case of COVID-19 (preprint)

We report the kinetics of the immune response in relation to clinical and virological features of a patient with mild-to-moderate coronavirus disease-19 (COVID-19) requiring hospitalisation. Increased antibody-secreting cells, follicular T-helper cells, activated CD4+ and CD8+ T-cells and IgM/IgG SARS-CoV-2-binding antibodies were detected in blood, prior to symptomatic recovery. These immunological changes persisted for at least 7 days following full resolution of symptoms, indicating substantial anti-viral immunity in this non-severe COVID-19.Authors Irani Thevarajan and Thi HO Nguyen contributed equally to this work.