RESUMEN
BackgroundReliable point-of-care (POC) diagnostics not requiring laboratory infrastructure could be a game changer in the COVID-19 pandemic, particularly in the Global South. We assessed performance, limit of detection and ease-of-use of three antigen-detecting, rapid POC tests (Ag-RDT) for SARS-CoV-2. MethodsThis prospective, multi-centre diagnostic accuracy study recruited participants suspected to have SARS-CoV-2 in Germany and the UK. Paired nasopharyngeal swabs (NP) or NP and/or oropharyngeal swabs (OP) were collected from participants (one for clinical RT-PCR and one for Ag-RDT). Performance of each of three Ag-RDTs was compared to RT-PCR overall, and according to predefined subcategories e.g. cycle threshold (CT)-value, days from symptoms onset, etc. In addition, limited verification of the analytical limit-of-detection (LOD) was determined. To understand the usability a System Usability Scale (SUS) questionnaire and ease-of-use (EoU) assessment were performed. ResultsBetween April 17th and August 25th, 2020, 2417 participants were enrolled, with 70 (3.0%) testing positive by RT-PCR. The best-performing test (SD Biosensor, Inc. STANDARD Q) was 76x6% (95% Confidence Interval (CI) 62x8-86x4) sensitive and 99x3% (CI 98x6-99x6) specific. A sub-analysis showed all samples with RT-PCR CT-values <25 were detectable by STANDARD Q. The test was considered easy-to-use (SUS 86/100) and suitable for POC. Bioeasy and Coris showed specificity of 93x1% (CI 91x0%-94x8%) and 95x8% (CI 93x4%-97x4%), respectively, not meeting the predefined target of [≥]98%. ConclusionThere is large variability in performance of Ag-RDT with SD Biosensor showing promise. Given the usability at POC, this test is likely to have impact despite imperfect sensitivity; however further research and modelling are needed.
RESUMEN
Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and alternative, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.
RESUMEN
The COVID-19 pandemic caused by the novel SARS-CoV-2 virus poses a significant public-health problem. In order to control the pandemic, rapid tests for detecting existing infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) hold outstanding promise towards greatly simplified and broadly applicable testing methods. RT-LAMP assays appear more robust than qPCR-based methods and only require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP protocol using clinical SARS-CoV-2 samples and also established a protocol that does not require prior RNA isolation ("swab-to-RT-LAMP"). Our study is based on several hundred clinical patient samples with a wide range of viral loads, thus allowing, for the first time, to accurately determine the sensitivity and specificity of the RT-LAMP assay for the detection of SARS-CoV-2 in patients. We found that RT-LAMP can reliably detect SARS-CoV-2 samples with a qPCR threshold cycle number (CT value) of up to 30 in the standard RT-qPCR assay. We used both, either purified RNA or direct pharyngeal swab specimens and showed that RT-LAMP assays have, despite a decreased sensitivity compared to RT-qPCR, excellent specificity. We also developed a multiplexed LAMP-sequencing protocol as a diagnostic and validation procedure to detect and record the outcome of RT-LAMP assays. LAMP-sequencing is fully scalable and can assess the results of thousands of LAMP reactions in parallel. Finally, we propose applications of RT-LAMP based assays for SARS-CoV-2 detection.