Comparative Longitudinal Serological Study of Anti-SARS-CoV-2 Antibody Profiles in People with COVID-19.

Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.

Surveillance testing using salivary RT-PCR for SARS-CoV-2 in managed quarantine facilities in Australia: A laboratory validation and implementation study.

Background: Regular repeat surveillance testing is a strategy to identify asymptomatic individuals with SARS-CoV-2 infections in high-risk work settings to prevent onward community transmission. Saliva sampling is less invasive compared to nasal/oropharyngeal sampling, thus making it suitable for regular testing. In this multi-centre evaluation, we aimed to validate RT-PCR using salivary swab testing of SARS-CoV-2 for large-scale surveillance testing and assess implementation amongst staff working in the hotel quarantine system in Victoria, Australia. Methods: A multi-centre laboratory evaluation study was conducted to systematically validate the in vitro and clinical performance of salivary swab RT-PCR for implementation of SARS-CoV-2 surveillance testing. Analytical sensitivity for multiple RT-PCR platforms was assessed using a dilution series of known SARS-CoV-2 viral loads, and assay specificity was examined using a panel of viral pathogens other than SARS-CoV-2. In addition, we tested capacity for large-scale saliva testing using a four-sample pooling approach, where positive pools were subsequently decoupled and retested. Regular, frequent self-collected saliva swab RT-PCR testing was implemented for staff across fourteen quarantine hotels. Samples were tested at three diagnostic laboratories validated in this study, and results were provided back to staff in real-time. Findings: The agreement of self-collected saliva swabs for RT-PCR was 84.5% (95% CI 68.6 to 93.8) compared to RT-PCR using nasal/oropharyngeal swab samples collected by a healthcare practitioner, when saliva samples were collected within seven days of symptom onset. Between 7th December 2020 and 17th December 2021, almost 500,000 RT-PCR tests were performed on saliva swabs self-collected by 102 staff working in quarantine hotels in Melbourne. Of these, 20 positive saliva swabs were produced by 13 staff (0.004%). The majority of staff that tested positive occurred during periods of community transmission of the SARS-CoV-2 Delta variant. Interpretation: Salivary RT-PCR had an acceptable level of agreement compared to standard nasal/oropharyngeal swab RT-PCR within early symptom onset. The scalability, tolerability and ease of self-collection highlights utility for frequent or repeated testing in high-risk settings, such as quarantine or healthcare environments where regular monitoring of staff is critical for public health, and protection of vulnerable populations. Funding: This work was funded by the Victorian Department of Health.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

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.

Use of emerging testing technologies and approaches for SARS-CoV-2: review of literature and global experience in an Australian context.

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.

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

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

Evaluation of 6 Commercial SARS-CoV-2 Serology Assays Detecting Different Antibodies for Clinical Testing and Serosurveillance.

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.

Infrared Based Saliva Screening Test for COVID-19.

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.

Robust correlations across six SARS-CoV-2 serology assays detecting distinct antibody features.

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.

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses.

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3+cTFH1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.

Detection of SARS-CoV-2 in saliva: implications for specimen transport and storage.

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.

Sample pooling on the Cepheid Xpert® Xpress SARS-CoV-2 assay.

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 4 and 6. 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 values ranging between 20 and 28 and all were detected qualitatively when contained in pools of 4 or 6 samples. For these samples, pooling had a median change in cycle threshold value of 2.0 in pools of 4, and of 2.9 in pools of 6. With the use of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay, pooling of 4 or 6 samples may be an effective strategy to increase testing capacity.

COVID-19 in Australia: our national response to the first cases of SARS-CoV-2 infection during the early biocontainment phase.

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 the patient cohort was 42 years (interquartile range (IQR), 24-53 years) with six men and five women. Eight (72.7%) patients 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 over time as the outbreak evolved. Future research should extend this Australian case series to examine global epidemiological variation of this novel infection.

Sample pooling is a viable strategy for SARS-CoV-2 detection in low-prevalence settings.

The 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 containment measures. The aims of this study were to investigate the viability of sample pooling as a strategy for increasing test throughput and conserving PCR reagents; and to report our early experience with pooling of clinical samples. A 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. Sensitivity 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. Pooling is a viable strategy for high-throughput testing of SARS-CoV-2 in low-prevalence settings.

Tracking the COVID-19 pandemic in Australia using genomics.

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

Implementation and evaluation of a novel real-time multiplex assay for SARS-CoV-2: in-field learnings from a clinical microbiology laboratory.

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.

Evaluation of Serological Tests for SARS-CoV-2: Implications for Serology Testing in a Low-Prevalence Setting.

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.

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.