Sequencing directly from antigen-detection rapid diagnostic tests in Belgium, 2022: a gamechanger in genomic surveillance?

BackgroundLateral flow antigen-detection rapid diagnostic tests (Ag-RDTs) for viral infections constitute a fast, cheap and reliable alternative to nucleic acid amplification tests (NAATs). Whereas leftover material from NAATs can be employed for genomic analysis of positive samples, there is a paucity of information on whether viral genetic characterisation can be achieved from archived Ag-RDTs.AimTo evaluate the possibility of retrieving leftover material of several viruses from a range of Ag-RDTs, for molecular genetic analysis.MethodsArchived Ag-RDTs which had been stored for up to 3 months at room temperature were used to extract viral nucleic acids for subsequent RT-qPCR, Sanger sequencing and Nanopore whole genome sequencing. The effects of brands of Ag-RDT and of various ways to prepare Ag-RDT material were evaluated.ResultsSARS-CoV-2 nucleic acids were successfully extracted and sequenced from nine different brands of Ag-RDTs for SARS-CoV-2, and for five of these, after storage for 3 months at room temperature. The approach also worked for Ag-RDTs for influenza virus (n = 3 brands), as well as for rotavirus and adenovirus 40/41 (n = 1 brand). The buffer of the Ag-RDT had an important influence on viral RNA yield from the test strip and the efficiency of subsequent sequencing.ConclusionOur finding that the test strip in Ag-RDTs is suited to preserve viral genomic material, even for several months at room temperature, and therefore can serve as source material for genetic characterisation could help improve global coverage of genomic surveillance for SARS-CoV-2 as well as for other viruses.

Multicenter Diagnostic Evaluation of a Novel Coronavirus Antigen Lateral Flow Test among Symptomatic Individuals in Brazil and the United Kingdom.

The COVID-19 pandemic has led to the commercialization of many antigen-based rapid diagnostic tests (Ag-RDTs), requiring independent evaluations. This report describes the clinical evaluation of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom. The collected samples (446 nasal swabs from Brazil and 246 nasopharyngeal samples from the UK) were analyzed by the Ag-RDT and compared to reverse transcription-quantitative PCR (RT-qPCR). Analytical evaluation of the Ag-RDT was performed using direct culture supernatants of SARS-CoV-2 strains from the wild-type (B.1), Alpha (B.1.1.7), Delta (B.1.617.2), Gamma (P.1), and Omicron (B.1.1.529) lineages. An overall sensitivity and specificity of 88.2% (95% confidence interval [CI], 81.3 to 93.3) and 100.0% (95% CI, 99.1 to 100.0), respectively, were obtained for the Brazilian and UK cohorts. The analytical limit of detection was determined as 1.0 × 103 PFU/mL (Alpha), 2.5 × 102 PFU/mL (Delta), 2.5 × 103 PFU/mL (Gamma), and 1.0 × 103 PFU/mL (Omicron), giving a viral copy equivalent of approximately 2.1 × 104 copies/mL, 9.0 × 105 copies/mL, 1.7 × 106 copies/mL, and 1.8 × 105 copies/mL for the Ag-RDT, respectively. Overall, while a higher sensitivity was claimed by the manufacturers than that found in this study, this evaluation finds that the Ag-RDT meets the WHO minimum performance requirements for sensitivity and specificity of COVID-19 Ag-RDTs. This study illustrates the comparative performance of the Hotgen Ag-RDT across two global settings and considers the different approaches in evaluation methods. IMPORTANCE Since the beginning of the SARS-CoV-2 pandemic, we have witnessed growing numbers of antigen rapid diagnostic tests (Ag-RDTs) being brought to market. In the United Kingdom, this was somewhat controlled indirectly as the government offered free tests from a small number of companies. However, as this has now ceased, individuals are responsible for their own acquisition of test kits. Similarly in Brazil, as of January 2022, pharmacies and other health care retailers are permitted to sell Ag-RDTs directly to the community. Many of these Ag-RDTs have not been externally evaluated, and results are not readily available to the public. Thus, there is now a need for a transparent evaluation of Ag-RDTs with both analytical and clinical evaluation. We present an independent review of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom.

Zimbabwe's emergency response to COVID-19: Enhancing access and accelerating COVID-19 testing as the first line of defense against the COVID-19 pandemic.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spreads rapidly, causing outbreaks that grow exponentially within a short period before interventions are sought and effectively implemented. Testing is part of the first line of defense against Corona Virus Disease of 2019 (COVID-19), playing a critical role in the early identification and isolation of cases to slow transmission, provision of targeted care to those affected, and protection of health system operations. Laboratory tests for COVID-19 based on nucleic acid amplification techniques were rapidly developed in the early days of the pandemic, but such tests typically require sophisticated laboratory infrastructure and skilled staff. In March 2020, Zimbabwe confirmed its first case of COVID-19; this was followed by an increase in infection rates as the pandemic spread across the country, thus increasing the demand for testing. One national laboratory was set to test all the country's COVID-19 suspect cases, building pressure on human and financial resources. Staff burnout and longer turnaround times of more than 48 h were experienced, and results were released late for clinical relevance. Leveraging on existing PCR testing platforms, including GeneXpert machines, eased the pressure for a short period before facing the stockout of SARs-CoV-2 cartridges for a long time, leading to work overload at a few testing sites contributing to long turnaround times. On September 11, WHO released the interim guidance to use antigen rapid diagnostic test as a diagnostic tool. The Zimbabwe laboratory pillar quickly adopted it and made plans for its implementation. The National Microbiology Reference Laboratory verified the two emergency-listed kits, the Panbio Abbott and the Standard Q, Biosensor, and they met the WHO minimum performance of ≥97% specificity and ≥80% sensitivity. Decentralizing diagnostic testing leveraging existing human resources became a game-changer in improving COVID-19 containment measures. Task shifting through training on Antigen rapid diagnostic tests (Ag-RDT) commenced, and testing was decentralized to all the ten provinces, from 1 central testing laboratory to more than 1,000 testing centers. WhatsApp platforms made it easier for data to be reported from remote areas. Result turnaround times were improved to the same day, and accessibility to testing was enhanced.

Performance evaluation of antigen detection rapid diagnostic test (Ag-RDT) for COVID-19 diagnosis in a primary healthcare center during the Shanghai COVID-19 quarantine period.

BACKGROUND: Rapid and accurate detection of SARS-CoV-2 infection is the cornerstone of prompt patient care. However, the reliability of the antigen rapid diagnostic test (Ag-RDT) in the diagnosis of SARS-CoV-2 infection remains inconclusive. METHODS: We conducted a field evaluation of Ag-RDT performance during the Shanghai Coronavirus disease 2019 (COVID-19) quarantine and screened 7225 individuals visiting our Emergency Department. 83 asymptomatic SARS-CoV-2 (+) individuals were enrolled in the current study. Simultaneously, Ag-RDT was performed to evaluate its testing performance. RESULTS: For the Ag-RDT(-) cases, the average cycle threshold (Ct) values of the N gene were 27.26 ± 4.59, which were significantly higher than the Ct value (21.9 ± 4.73) of the Ag-RDT(+) individuals (p < 0.0001). The overall sensitivity of Ag-RDT versus that of RT-PCR was 43.37%. The Ag-RDT(+) individuals regarding the N gene's Ct value were 16 cases in the < 20 range, 12 in 20-25, 5 in 25-30, and 3 in 30-35. The corresponding sensitivity was 84.21%, 52.17%, 21.74% and 16.67%, respectively. Meanwhile, sampling had a straight specificity of 100% regardless of the Ct value. CONCLUSIONS: The Ag-RDT were extremely sensitive in asymptomatic COVID-19 individuals with a Ct value < 20.

Evaluation of the Panbio COVID-19 Antigen Rapid Diagnostic Test in Subjects Infected with Omicron Using Different Specimens.

Community testing is a crucial tool for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.529) raised concerns about its primary site of replication, impacting sample collection and its detectability by rapid antigen tests. We tested the performance of the Panbio antigen rapid diagnostic test (Ag-RDT) using nasal and oral specimens for COVID-19 diagnosis in 192 symptomatic individuals, with quantitative reverse transcription-PCR (RT-qPCR) of nasopharyngeal samples as a control. Variant of concern (VOC) investigation was performed with the 4Plex SARS-CoV-2 screening kit. The SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) Ag-RDT. Our data showed good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that the Panbio COVID-19 antigen rapid diagnostic test does not provide good sensitivity with oral swabs for Omicron Ag-RDT detection. IMPORTANCE This study showed that the antigen rapid test for COVID19 worked fine using nasal swabs when it was utilized in patients infected with the Omicron variant, showing a concordance with PCR in 93% of patients tested. The nasal swab yielded more reliable results than the oral swab when an antigen rapid diagnosis test (the Panbio COVID-19 antigen rapid diagnostic test) was used in patients infected with the Omicron variant.

Evaluation of FAST COVID-19 SARS-CoV-2 Antigen Rapid Test Kit for Detection of SARS-CoV-2 in Respiratory Samples from Mildly Symptomatic or Asymptomatic Patients.

Molecular tests are the gold standard to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but are associated with a diagnostic delay, while antigen detection tests can generate results within 20 min even outside a laboratory. In order to evaluate the accuracy and reliability of the FAST COVID-19 SARS-CoV-2 Antigen Rapid Test Kit (Ag-RDT), two respiratory swabs were collected simultaneously from 501 patients, with mild or no coronavirus disease 2019 (COVID-19)-related symptoms, and analyzed with both the Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) and the FAST COVID-19 SARS-CoV-2 Antigen Rapid Test. Results were then compared to determine clinical performance in a screening setting. We measured a precision of 97.41% (95% CI 92.42-99.15%) and a recall of 98.26% (95% CI 93.88-99.25%), with a specificity of 99.22% (95% CI 97.74-99.74%), a negative predictive value of 99.48% (95% CI 97.98-99.87%), and an overall accuracy of 99.00% (95% CI 97.69-99.68%). Concordance was described by a Kappa coefficient of 0.971 (95% CI 0.947-0.996). Considering short lead times, low cost, and opportunities for decentralized testing, the Ag-RDT test can enhance the efforts to control SARS-CoV-2 spread in several settings.

Development of a risk assessment profile tool to determine appropriate use of SARS-CoV-2 rapid antigen detection tests for different activities and events in Ireland, since October 2021.

We describe the development of a risk assessment profile tool that incorporates data from multiple domains to help determine activities and events where rapid antigen detection tests (Ag-RDT) could be used to screen asymptomatic individuals to identify infectious cases as an additional mitigation measure to reduce transmission of SARS-CoV-2. The tool aims to stratify, in real time, the overall risk of SARS-CoV-2 transmission associated with common activities and events, and this can be matched to an appropriate Ag-RDT testing protocol.

Viable Severe Acute Respiratory Syndrome Coronavirus 2 Isolates Exhibit Higher Correlation With Rapid Antigen Assays Than Subgenomic RNA or Genomic RNA.

Background: Rapid identification and effective isolation are crucial for curbing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To meet this requirement, antigen-detection rapid diagnostic tests (Ag-RDTs) are essential. Methods: Between February 2020 and August 2020 we performed a cohort study of patients with confirmed COVID-19. The clinical performance of Ag rapid fluorescence immunoassay (FIA) and Ag Gold was evaluated and compared in parallel with genomic and subgenomic real-time reverse transcription-polymerase chain reaction (rRT-PCR) and cell culture-based assays. Results: In total, 150 samples were tested. Of these, 63 serial samples were obtained from 11 patients with SARS-CoV-2 and 87 from negative controls. Serial respiratory samples were obtained 2 days prior to symptom onset (-2) up to 25 days post-symptom onset. Overall, for rRT-PCR-positive samples (n = 51), the detection sensitivity of Ag rapid FIA and Ag Gold was 74.5% and 53.49%, respectively, with a specificity of 100%; however, for samples with low cycle threshold (Ct) values, Ag rapid FIA and Ag Gold exhibited a sensitivity of 82.61% (Ct ≤ 30, 5.6 log10RNA copies/mL) and 80% (Ct ≤ 25, 6.9 log10RNA copies/mL), respectively. Despite low analytical sensitivity, both Ag-RDTs detected 100% infection in cell culture-positive samples (n = 15) and were highly effective in distinguishing viable samples from those with subgenomic RNA (66.66%). For both Ag-RDTs, all samples that yielded discordant results (rRT-PCR + ve/Ag-RDT -ve) were also negative by culture. Conclusion: The data suggest that Ag-RDTs reliably detect viable SARS-CoV-2; thus, they may serve as an important tool for rapid detection of potentially infectious individuals.

Diagnostic Performance of Automated SARS-CoV-2 Antigen Assay in Nasal Swab during COVID-19 Vaccination Campaign.

BACKGROUND: To control the spread of the pandemic brought about by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is necessary to have an automated reliable diagnostic assay. To date, the RT-PCR (RT-qPCR) has been the recommended laboratory method to diagnose SARS-CoV-2 infection, but there is a need for more automated and reliable tests. The aim of this real-life study was to assess the diagnostic performance of DiaSorin's LIAISON SARS-CoV-2 antigen (Ag) chemiluminescence immunoassay in detecting SARS-CoV-2 in vaccinated and unvaccinated individuals. METHODS: A prospective study was performed on 300 nasopharyngeal swabs randomly collected from 31 May to 6 July 2021. Nasopharyngeal samples were assayed with DiaSorin's LIAISON SARS-CoV-2 Ag and TaqPath™ COVID-19 multiplex RT-qPCR. RESULTS: Of 300 participants, 150 had a RT-qPCR confirmed SARS-CoV-2 infection of whom 113 (75.33%) were also detected by the DiaSorin LIAISON SARS-CoV-2 Ag. Taking RT-qPCR as a reference, the sensitivity and specificity of the DiaSorin LIAISON SARS-CoV-2 Ag assay were evaluated as 75.33% (95% CI = 67.64-82) and 100% (95% CI = 97.57-100), respectively. When a viral load cut-off was applied for high viral load (median cycle threshold (Ct) < 18.57), the overall sensitivity was increased to 96.55% (95% CI = 88.09-99.58). Interestingly, median RT-qPCR Ct and SARS-CoV-2 Ag values were similar between fully vaccinated and unvaccinated subjects. CONCLUSIONS: Automated, quantitative LIAISON SARS-CoV-2 Ag assay shows good performance to identify SARS-CoV-2-infected individuals with moderate to high viral loads. LIAISON SARS-CoV-2 Ag testing could be used as frontline testing for COVID-19 diagnosis and be more suitable for large utilization.

Postmortem Antigen-Detecting Rapid Diagnostic Tests to Predict Infectivity of SARS-CoV-2-Associated Deaths.

We investigated the infectivity of 128 severe acute respiratory disease coronavirus 2-associated deaths and evaluated predictive values of standard diagnostic procedures. Maintained infectivity (20%) did not correlate with viral RNA loads but correlated well with anti-S antibody levels. Sensitivity >90% for antigen-detecting rapid diagnostic tests supports their usefulness for assessment.

The Comparative Clinical Performance of Four SARS-CoV-2 Rapid Antigen Tests and Their Correlation to Infectivity In Vitro.

Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8-82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.