Search

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 1 March and 25 March 2020. All positive samples and a random subset of negative samples were sent to a reference laboratory for confirmation. In total, 2673 samples were analysed using the Coronavirus Typing assay. The predominant sample type was a combined nasopharyngeal/throat swab (2640/2673; 98.8%). Fifty-four patients were positive for SARS-CoV-2 (2.0%) 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 laboratory gold 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 In many countries, standard regulatory requirements for the introduction of new assays have been replaced by emergency authorisations and it is critical that laboratories share their post-market validation experiences, as the consequences of widespread introduction of a suboptimal assay for SARS-CoV-2 are profound. Here, we share our in-field experience, and encourage other laboratories to follow suit.

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., Genome sequencing can be used to infer pathogen transmission dynamics and inform public health responses. Here, the authors sequence >1,200 SARS-CoV-2 samples from Victoria, Australia and find genomic support for the effectiveness of social restrictions in reducing transmission.

Objectives To design and evaluate 3D‐printed nasal swabs for collection of samples for SARS‐CoV‐2 testing. Design An iterative design process was employed. Laboratory evaluation included in vitro assessment of mock nasopharyngeal samples spiked with two different concentrations of gamma‐irradiated SARS‐CoV‐2. A prospective clinical study compared SARS‐CoV‐2 and human cellular material recovery by 3D‐printed swabs and standard nasopharyngeal swabs. Setting, participants Royal Melbourne Hospital, May 2020. Participants in the clinical evaluation were 50 hospital staff members attending a COVID‐19 screening clinic and two inpatients with laboratory‐confirmed COVID‐19. Intervention In the clinical evaluation, a flocked nasopharyngeal swab sample was collected with the Copan ESwab and a mid‐nasal sample from the other nostril was collected with the 3D‐printed swab. Results In the laboratory evaluation, qualitative agreement with regard to SARS‐CoV‐2 detection in mock samples collected with 3D‐printed swabs and two standard swabs was complete. In the clinical evaluation, qualitative agreement with regard to RNase P detection (a surrogate measure of adequate collection of human cellular material) in samples collected from 50 hospital staff members with standard and 3D‐printed swabs was complete. Qualitative agreement with regard to SARS‐CoV‐2 detection in three pairs of 3D‐printed mid‐nasal and standard swab samples from two inpatients with laboratory‐confirmed SARS‐CoV‐2 was also complete. Conclusions Using 3D‐printed swabs to collect nasal samples for SARS‐CoV‐2 testing is feasible, acceptable to patients and health carers, and convenient.

Objectives To describe the first isolation and sequencing of SARS‐CoV‐2 in Australia and rapid sharing of the isolate. Setting SARS‐CoV‐2 was isolated from a 58‐year‐old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. Major outcomes Clinical course and laboratory features of the first reported case of COVID‐19 (the illness caused by SARS‐CoV‐2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. Results A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS‐CoV‐2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS‐CoV‐2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS‐CoV‐2 genomes. Within 24 hours of isolation, the first Australian SARS‐CoV‐2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. Conclusions The ability to rapidly identify, propagate, and internationally share our SARS‐CoV‐2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

BackgroundRapid 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 against the currently circulating delta variant, including an assessment of culture infectivity.MethodsAnalytical sensitivity was evaluated against SARS-CoV-2 B.1.617.2 (Delta), reported as TCID50/mL, cycle threshold (Ct) 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.ResultsNineteen kits consistently detected SARS-CoV-2 antigen equivalent to 1.3 × 106 copies/mL (5.8 × 103 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%) concordant, 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 concordance was 97.6% (95% CI, 86.3% to 100.0%) compared to cell culture positivity.ConclusionsAntigen test positivity correlated with positive viral culture, suggesting antigen test results may determine SARS-CoV-2 transmission risk. Analytical sensitivity varied considerably between kits highlighting the need for ongoing systematic post-market evaluation to inform test selection and deployment.

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.

Emerging testing technologies for detection of SARS-CoV-2 include those that are rapid and can be used at point-of-care (POC), and those facilitating high throughput laboratory-based testing. Tests designed to be performed at POC (such as antigen tests and molecular assays) have the potential to expedite isolation of infectious patients and their contacts, but most are less sensitive than standard-of-care reverse transcription polymerase chain reaction (RT-PCR). Data on clinical performance of the majority of emerging assays are limited with most evaluations performed on contrived or stored laboratory samples. Further evaluations of these assays are required, particularly when performed at POC on symptomatic and asymptomatic patients and at various time-points after symptom onset. A few studies have so far shown several of these assays have high specificity. However, large prospective evaluations are needed to confirm specificity, particularly before the assays are implemented in low prevalence settings or asymptomatic populations. High throughput laboratory-based testing includes the use of new sample types (e.g., saliva to increase acceptability) or innovative uses of existing technology (e.g., sample pooling). Information detailing population-wide testing strategies for SARS-COV-2 is largely missing from peer-reviewed literature. Logistics and supply chains are key considerations in any plan to 'scale up' testing in the Australian context. The strategic use of novel assays will help strike the balance between achieving adequate test numbers without overwhelming laboratory capacity. To protect testing of high-risk populations, the aims of testing with respect to the phase of the pandemic must be considered.

Summary: 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

We report the kinetics of immune responses 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

We present a case of late symptom onset of COVID-19 infection 72 days after initial diagnosis in an immunocompromised 53-year-old man. SARS-CoV-2 was cultured from his sputum sample at this time, and genomic sequencing suggested reinfection was unlikely. After receipt of convalescent plasma, SARS-CoV-2 became undetectable by PCR 111 days after diagnosis, although SARS-CoV-2 antibodies remained not detectable. This case posed difficult public health management issues in a low prevalence COVID-19 setting as the person required extended home isolation given his prolonged SARS-CoV-2 PCR detection.

Background On 31 December 2019, the World Health Organization recognised clusters of pneumonia‐like cases due to a novel coronavirus disease (COVID‐19). COVID‐19 became a pandemic 71 days later. Aim To report the clinical and epidemiological features, laboratory data and outcomes of the first group of 11 returned travellers with COVID‐19 in Australia. Methods This is a retrospective, multi‐centre case series. All patients with confirmed COVID‐19 infection were admitted to tertiary referral hospitals in New South Wales, Queensland, Victoria and South Australia. Results The median age of our patient cohort was 42 years (IQR, 24–53 years) with six men and five women. Eight patients (72.7%) had returned from Wuhan, one from Shenzhen, one from Japan, and one from Europe. Possible human‐to‐human transmission from close family contacts in gatherings overseas occurred in two cases. Symptoms on admission were fever, cough and sore throat (n = 9, 81.8%). Co‐morbidities included hypertension (n = 3, 27.3%) and hypercholesterolaemia (n = 2, 18.2%). No patients developed severe acute respiratory distress nor required intensive care unit admission or mechanical ventilation. After a median hospital stay of 14.5 days (IQR, 6.75–21), all patients were discharged. Conclusions This is a historical record of the first COVID‐19 cases in Australia during the early biocontainment phase of the national response. These findings were invaluable for establishing early inpatient and outpatient COVID‐19 models of care and informing the management of COVID‐19 overtime as the outbreak evolved. Future research should extend this Australian case series to examine global epidemiological variation of this novel infection. This article is protected by copyright. All rights reserved.

Saliva has recently been proposed as a suitable specimen for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Use of saliva as a diagnostic specimen may present opportunities for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing in remote and low-resource settings. Determining the stability of SARS-CoV-2 RNA in saliva over time is an important step in determining optimal storage and transport times. We undertook an in vitro study to assess whether SARS-CoV-2 could be detected in contrived saliva samples. The contrived saliva samples comprised 10 ml pooled saliva spiked with gamma-irradiated SARS-CoV-2 to achieve a concentration of 2.58×104 copies ml SARS-CoV-2, which was subsequently divided into 2 ml aliquots comprising: (i) neat saliva; and a 1 : 1 dilution with (ii) normal saline; (iii) viral transport media, and (iv) liquid Amies medium. Contrived samples were made in quadruplicate, with two samples of each stored at either: (i) room temperature or (ii) 4 °C. SARS-CoV-2 was detected in all SARS-CoV-2 spiked samples at time point 0, day 1, 3 and 7 at both storage temperatures using the N gene RT-PCR assay and time point 0, day 1 and day 7 using the Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) RT-PCR assay. The ability to detect SARS-CoV-2 in saliva over a 1 week period is an important finding that presents further opportunities for saliva testing as a diagnostic specimen for the diagnosis of SARS-CoV-2.

Highlights • Clinical samples with ‘neat’ Ct values between 20 and 28 were all detected in pools of six. • Testing in a pool of six led to a mean drop of cycle threshold value of 2.9 • Samples with viral load around the assay's limit of detection may be missed by pooling. • All confirmed negative samples were also negative when tested in pools of six., The COVID-19 pandemic has placed unprecedented global demand on laboratory supplies required for testing. Sample pooling has been investigated by laboratories as a strategy to preserve testing capacity. We evaluate the performance of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay for testing samples in pools of four and six. Clinical samples containing SARS-CoV-2, and confirmed negative clinical samples were used to create sample pools. Clinical samples had ‘neat’ Xpert® E gene cycle threshold (Ct) values ranging between 20 and 28 and all were detected qualitatively when contained in pools of four or six samples. For these samples, pooling had a median change in Ct value of 2.0 in pools of four, and of 2.9 in pools of six. With the use of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay, pooling of four or six samples may be an effective strategy to increase testing capacity.

BackgroundSerological 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.MethodsWe 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.ResultsFor 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%.ConclusionWantai 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.

Background Serological testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the coronavirus disease 2019 (COVID-19) pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. Methods We evaluated the performance of 6 commercially available enzyme-linked immunosorbent assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared with a cell culture–based microneutralization (MN) assay. We tested sera from patients with prior reverse transcription polymerase chain reaction–confirmed SARS-CoV-2 infection, prepandemic sera, and potential cross-reactive sera from patients with other non-COVID-19 acute infections. Results For sera collected >14 days post–symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% CI, 94.6%–100%), followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 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% for the Wantai total Ab, and for the lowest-performing assay it was 97.1% for sVNT (as per the Instructions for Use [IFU]). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG, and sVNT (as per IFU) with 97%, 97% and 95%, respectively; Wantai IgM had the poorest agreement at 93%. Conclusions Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture–based neutralization assay showed good result correlation between all assays. However, correlation between the cell-based neutralization test and some assays detecting Ab’s not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimize serological test algorithms for assessing antibody responses post–SARS-CoV-2 infection or vaccination.

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).

Objectives As the world transitions into a new era of the COVID‐19 pandemic in which vaccines become available, there is an increasing demand for rapid reliable serological testing to identify individuals with levels of immunity considered protective by infection or vaccination. Methods We used 34 SARS‐CoV‐2 samples to perform a rapid surrogate virus neutralisation test (sVNT), applicable to many laboratories as it circumvents the need for biosafety level‐3 containment. We correlated results from the sVNT with five additional commonly used SARS‐CoV‐2 serology techniques: the microneutralisation test (MNT), in‐house ELISAs, commercial Euroimmun‐ and Wantai‐based ELISAs (RBD, spike and nucleoprotein; IgG, IgA and IgM), antigen‐binding avidity, and high‐throughput multiplex analyses to profile isotype, subclass and Fc effector binding potential. We correlated antibody levels with antibody‐secreting cell (ASC) and circulatory T follicular helper (cTfh) cell numbers. Results Antibody data obtained with commercial ELISAs closely reflected results using in‐house ELISAs against RBD and spike. A correlation matrix across ten measured ELISA parameters revealed positive correlations for all factors. The frequency of inhibition by rapid sVNT strongly correlated with spike‐specific IgG and IgA titres detected by both commercial and in‐house ELISAs, and MNT titres. Multiplex analyses revealed strongest correlations between IgG, IgG1, FcR and C1q specific to spike and RBD. Acute cTfh‐type 1 cell numbers correlated with spike and RBD‐specific IgG antibodies measured by ELISAs and sVNT. Conclusion Our comprehensive analyses provide important insights into SARS‐CoV‐2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS‐CoV‐2‐specific humoral responses., We describe robust correlations across six SARS‐CoV‐2 serology assays detecting distinct antibody features. Our comprehensive analyses provide important insights into SARS‐CoV‐2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS‐CoV‐2‐specific humoral responses.

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.

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 premarket validation. Methods Performance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT). Results Sensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%–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%). ELISA sensitivity for 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. sVNT sensitivity was 60.9% (95% CI, 53.2%–68.4%), rising to 91.2% (95% CI, 81.8%–96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%–97.5%). Conclusions 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 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., Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings where positive predictive values are low.

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.

BackgroundRobust 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.MethodsPerformance 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).ResultsSensitivities 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%),ConclusionPerformance 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.

2.AbstractIntroductionThe 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 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 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.

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.

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.

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.

SARS-CoV-2 causes a spectrum of illness, ranging from asymptomatic to severe COVID-19. As the immunological basis for severity remains ill-defined, we analysed 78 SARS-CoV-2-infected individuals at acute and/or convalescent timepoints, up to 102 days post-symptom onset, quantifying 184 innate and adaptive immunological parameters. Acute COVID-19 in hospitalised patients was associated with high levels of IL-6, IL-18 and IL-10, elevated neutrophil-to-lymphocyte and neutrophil-to-T cell ratios, and high proportions of activated CD38+ neutrophils, CD38+ eosinophils, CD38+ HLA-DRlo monocytes, CD38+CD56dim NK cells, CD38+ γδ T cells, antibody-secreting cells, CD38+ICOS+ circulating T follicular helper (cTFH) cells, CD38+HLA-DR+CD4+ T cells, effector CD27-CD45RA+ and CD38+ CD8+ T cells. During convalescence, elevated seroconversion and neutralising antibodies were prominent and correlated with acute CXCR3+ cTFH cell activation. Strikingly, intensive care unit (ICU) patients with severe COVID-19 displayed higher levels of soluble IL-6R, IL-18, and hyperactivation of innate, adaptive and myeloid compartments than ward patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients during acute and convalescent phases of SARS-CoV-2 infection, and integrate key cellular pathways of complex perturbed immune networks that underpin severe COVID-19, providing important insights into potential biomarkers and immunotherapies. Funding: This work was supported by the Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grant to KK (#1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of the Hong Kong Special Administrative Region, China (#T11-712/19-N) to KK, the Jack Ma Foundation to KK, KS, DIG and AWC, the a2 milk foundation to KS, the Victorian Government MRFF award (#2002073) to SJK, DIG and AKW, MRFF Award (#1202445) to KK, NHMRC program grant 1149990 (SJK), NHMRC project grant 1162760 (AKW), NHMRC Program Grant (#1071916) to KK and DCJ, NHMRC Program Grant (1113293) to DIG, the Merridew Foundation to NJCK. KK is supported by NHMRC Senior Research Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship (#1137285), KS by an NHMRC Investigator grant (#1177174), SYCT by a NHMRC Career Development Fellowship (#1145033), DIG by an NHMRC Senior Principal Research Fellowship (#1117766) and SJK by NHMRC Senior Principal Research Fellowship (#1136322). SRL is supported by an NHMRC program grant (#1149990) and practitioner fellowship (#1135851). LH and WZ are supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne. XJ is supported by China Scholarship Council-University of Melbourne joint Scholarship. CES has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (#792532). JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. TMA and FM-W are International Society for Advancement of Cytometry Marylou Ingram Scholars. GHP is supported by the Australian Government Research Training Program (RTP) Scholarship. JAJ is supported by an NHMRC Early Career Fellowship (ECF) (#1123673). EBC. is supported by a NHMRC Peter Doherty Fellowship (#1091516). KK and AC were supported by the University of Melbourne Dame Kate Campbell Fellowship. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: The study was approved by the Alfred Hospital (#280/14), Melbourne Health (HREC/17/MH/53), Monash Health (HREC/15/MonH/64/2016.196), The University of Melbourne (#2056689, #2056761, #1442952, #1955465 and #1443389) and James Cook University (H7886) Human Research Ethics Committees.

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 40 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. The 2017 meeting was held from September 25–27 in Melbourne, Australia, and was hosted by the Peter Doherty Institute for Infection and Immunity and the Institut Pasteur. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including the recent Zika epidemic, infections by human papillomavirus, influenza, HIV, hepatitis C, HTLV-1, and chikungunya viruses, and new and emerging viruses in the Australasia region. Plans for the 2018 meeting also are noted., Highlights • The GVN is an international research network comprised of 40 Centers of Excellence and 6 Affiliates in 24 countries. • The 2017 Global Virus Network (GVN) Meeting was held in Melbourne, Australia from September 25–27. • New data were presented on various aspects of medical virology, therapies, and emerging viruses in the Australasia region. • International collaboration is critical to developing new and effective viral vaccines and therapeutics. • The 2018 international GVN meeting will be held on November 28–30 in Annecy, France.

Investigating fever in the returned traveller can be difficult and costly. Dengue is one of the most frequently reported aetiologies. NS1 is a non-structural dengue virus protein detectable during acute infection. The aim of this report is to describe the utility of the Platelia Dengue NS1 antigen enzyme immunoassay (EIA) for detection of dengue in a non-endemic region compared to a composite gold standard of contemporaneous molecular testing and seroconversion. We performed a retrospective analysis of all dengue serology tests from 6 February 2012 to 5 December 2018. Dengue serology and in-house flavivirus molecular results were identified using the laboratory information management system. Dengue serology was performed using the Bio-Rad Platelia Dengue NS1 antigen EIA, and Abbott Panbio Dengue IgG and IgM EIA. True positive NS1 result was defined as positive molecular test within one week of the positive NS1 result or seroconversion within 120 days. NS1 negative samples that remained negative to all dengue markers on repeat more than 10 and up to 120 days after were labelled as true negatives. More than 75% of cases had a serology pattern consistent with primary dengue. Sensitivity and specificity of NS1 Ag EIA was 96.4% (95% CI 92.3-98.7%) and 98.4% (95% CI 94.5-99.8%), respectively. Performance was poorer in serotype 4 infections (sensitivity 50%). Platelia Dengue NS1 antigen EIA test performance in the returned traveller cohort fulfils the remit as a single diagnostic test for acute dengue infection.

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans.

We describe a fatal case of Japanese encephalitis virus infection following short-term travel to Thailand. Viral RNA was detected in urine and whole blood out to 26 and 28 days, respectively, after the onset of symptoms. Live virus was isolated from a urine specimen from day 14.

The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.

Background. During 2001–2008, the Victorian Infectious Diseases Reference Laboratory (VIDRL) prepared and provided a measles and rubella proficiency test panel for distribution to the World Health Organization (WHO) measles and rubella network laboratories as part of their annual laboratory accreditation assessment.Methods. Panel test results were forwarded to VIDRL, and results from 8 consecutive years were analyzed. We assessed the type of assays used and results achieved on the basis of the positive and negative interpretation of submitted results, by year and WHO region, for measles and rubella.Results. Over time, there has been a noticeable increase in laboratory and WHO regional participation. For all panels, the proportion of laboratories in all WHO regions using the WHO-validated Dade Behring assay for measles and rubella-specific IgM antibodies ranged from 35% to 100% and 59% to 100%, respectively. For all regions and years, the proportion of laboratories obtaining a pass score ranged from 87% to 100% for measles and 93% to 100% for rubella.Conclusions. During 2001–2008, a large proportion of laboratories worldwide achieved and maintained a pass score for both measles and rubella. Measles and rubella proficiency testing is regarded as a major achievement for the WHO measles and rubella laboratory program.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

Virus can replicate in several common cell lines, sometimes without cytopathic effect., Given the potential for laboratory-associated severe acute respiratory syndrome–associated coronavirus (SARS-CoV) infections, we must know which cell lines are susceptible to the virus. We investigated 21 cell lines routinely used for virus isolation or research. After infection with SARS-CoV, cells were observed for cytopathic effects, and quantitative real-time polymerase chain reaction was used to measure ongoing viral replication. An indirect immunofluorescence assay was also used as a confirmatory test. The study identified 10 new cell lines capable of supporting the replication of SARS-CoV and confirmed the susceptibility of 4 cell lines previously reported. This study shows that SARS-CoV can be isolated in several cell lines commonly used for diagnostic or research purposes. It also shows that SARS-CoV can achieve high titers in several cell lines, sometimes in the absence of specific cytopathic effects.