Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control (preprint)

Throughout the current SARS-CoV-2 pandemic, limited diagnostic testing capacity prevented sentinel testing of the population, demonstrating the need for novel testing strategies and infrastructures. Here, we describe the set-up of an alternative testing platform, which allows scalable surveillance testing as an acute pandemic response tool and for pandemic preparedness purposes, exemplified by SARS-CoV-2 diagnostics in an academic environment. The testing strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated 96-well plate-based RNA extraction, and viral RNA detection using a semi-quantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable to RT-quantitative polymerase chain reaction (RT-qPCR). We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, LAMP assay analysis by colorimetry or by sequencing (LAMP-seq), and communication of results to participants and the health authorities. Using large sample sets including longitudinal sample series we evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. We performed >35,000 tests during the pandemic, with an average turnover time of fewer than 6 hours from sample arrival at the test station to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics. As RT-LAMP-based testing requires advanced, but non-specialized laboratory equipment, it is independent of potentially limiting clinical diagnostics supply chains.

Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test (preprint)

BackgroundIn 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies demonstrated for SARS-CoV-2 Ag-RDTs nasal sampling to be a comparable and reliable alternative for nasopharyngeal (NP) sampling. MethodsWe conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio Ag-RDT (the second WHO-listed SARS-CoV-2 Ag-RDT, distributed by Abbott). We calculated positive and negative percent agreement and, compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR), sensitivity and specificity for both sampling techniques. ResultsA SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0% - 94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5% - 99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2% - 92.3%) and 88.9% (40/45; CI 76.5% - 95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1% - 99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7% - 99.8%) for both, NMT and NP sampling. ConclusionFor the Panbio Ag-RDT supervised NMT self-sampling yields to results comparable to NP sampling. This suggests that nasal self-sampling could be used for scale-up population testing.

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2 (preprint)

BackgroundRapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity ([≤]85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and MethodsThis multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. ResultsStudy conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load [≥] 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. ConclusionThe LumiraDx assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

Evaluation of the accuracy and ease-of-use of Abbott PanBio - A WHO emergency use listed, rapid, antigen-detecting point-of-care diagnostic test for SARS-CoV-2 (preprint)

BackgroundDiagnostics are essential for controlling the pandemic. Identifying a reliable and fast diagnostic is needed to support testing. We assessed performance and ease-of-use of the Abbott PanBio antigen-detecting rapid diagnostic test (Ag-RDT). MethodsThis prospective, multi-centre diagnostic accuracy study enrolled at two sites in Germany. Following routine testing with RT-PCR, a second study-exclusive swab was performed for Ag-RDT testing. Routine swabs were nasopharyngeal (NP) or combined NP/oropharyngeal (OP) whereas the study-exclusive swabs were NP. To evaluate performance, sensitivity and specificity were assessed overall and in predefined sub analyses accordingly to cycle-threshold values, days of symptoms, disease severity and study site. Additionally, an ease-of-use assessment and System Usability Scale (SUS) were performed. Findings1108 participants were enrolled between Sept 28 and Oct 30, 2020. Of these, 106 (9{middle dot}6%) were PCR-positive. The Abbott PanBio detected 92/106 PCR-positive participants with a sensitivity of 86{middle dot}8% (95% CI: 79{middle dot}0% - 92{middle dot}0%) and a specificity of 99{middle dot}9% (95% CI: 99{middle dot}4%-100%). The sub analyses indicated that sensitivity was 95{middle dot}8% in CT-values <25 and within the first seven days from symptom onset. The test was characterized as easy to use (SUS: 86/100) and considered suitable for point-of- care settings. InterpretationThe Abbott PanBio Ag-RDT performs well for SARS-CoV-2 testing in this large manufacturer independent study, confirming its WHO recommendation for Emergency Use in settings with limited resources. FundingThe Foundation of Innovative New Diagnostics supplied the test kits for the study. The internal funds from the Heidelberg University as well as the Charite Berlin supported this study.

Evaluation of the accuracy, ease of use and limit of detection of novel, rapid, antigen-detecting point-of-care diagnostics for SARS-CoV-2 (preprint)

Abstract Background: Reliable 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 (LOD) and ease-of-use of three antigen-detecting, rapid POC diagnostics (Ag-RDT) for SARS-CoV-2. Methods: This prospective, multi-centre diagnostic accuracy study, recruited participants suspected to have SARS-CoV2 in Germany and UK. Paired nasopharyngeal swabs (NP) or NP and/or oropharyngeal swabs (OP) were collected from participants (one for clinical real-time reverse transcription polymerase chain reaction (RT-PCR) and one for Ag-RDT testing). 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 symptom onset, etc. In addition, limited verification of analytical limit-of-detection (LOD) was determined. To understand the usability of each Ag-RDT a System Usability Scale (SUS) questionnaire and ease-of-use assessment were performed. Results: Between 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 76.6% [95% Confidence Interval (CI) 62.8-86.4] sensitive and 99.3% [CI 98.6-99.6] 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 93.1% [CI 91.0%-94.8%] and 95.8% [CI 93.4%-97.4%], respectively, not meeting the predefined target of [≥]98%. Conclusion: There is large variability in performance of Ag-RDT tests with one test showing promise. Given the usability at POC, these tests are likely to have impact despite imperfect sensitivity; however further research and modelling are needed.