A generic, scalable, and rapid TR-FRET -based assay for SARS-CoV-2 antigen detection

The ongoing COVID-19 pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple method for the detection of SARS-CoV-2 in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Forster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2 the limits of detection were established as 25 pg of NP or 20 infectious viral units (i.u.), and 875 pg of SP or 625 i.u. of SARS-CoV-2. Testing RT-PCR positive (n=48, with cycle threshold [Ct] values from 11 to 30) or negative (n=96) nasopharyngeal swabs, we showed that the assay yields positive results for all samples with Ct values of <25 and a single RT-PCR negative sample. We determined the presence of infectious virus in the RT-PCR-positive nasopharyngeal swabs by virus isolation, and observed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation, and 77.1% (37/48) and 99.0% (95/96) in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity and is relatively simple to set up as an "in-house" test. The assay principle as such is applicable to other viral infections, and could also be readily adapted to a massively high throughput testing format.

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients

ImportanceUnderstanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has practical implications for patient management in healthcare facilities. ObjectiveTo determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR testing. DesignA retrospective study on case series from 4 March - 15 April 2020. SettingA population-based study conducted in primary and tertiary care in the Helsinki Capital Region, Finland. ParticipantsAdults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, and who had sufficient data for grading of clinical suspicion of COVID-19 in their medical records were eligible. All 1,194 inpatients admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals was sampled from epidemiological line lists by systematic quasi-random sampling. Altogether 83 eligible outpatients (4.6%) and 3 inpatients (0.3%) were excluded due to insufficient data for grading of clinical suspicion. ExposuresHigh clinical suspicion for COVID-19 was used as the reference standard for the RT-PCR test. Patients were considered to have high clinical suspicion of COVID-19 if the physician in charge recorded the suspicion on clinical grounds, or the patient fulfilled specifically defined clinical and exposure criteria. Main measuresSensitivity of SARS-CoV-2 RT-PCR by using manually curated clinical characteristics as the gold standard. ResultsThe study population included 1,814 outpatients (mean [SD] age, 45.4 [17.2] years; 69.1% women) and 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women). The sensitivity (95% CI) for laboratory confirmed cases, i.e. repeatedly tested patients were as follows: 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the following sensitivity values (95% CI) were observed: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. Conclusions and relevanceThe clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the clinical sensitivity of SARS-CoV-2 RT-PCR test? FindingsIn this population-based retrospective study on medical records of 1,814 outpatients and 1,194 inpatients, the clinical sensitivity of SARS-CoV-2 RT-PCR was 47.3-89.9%. MeaningThe false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests.

SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility

Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert(R) Xpress SARS-CoV-2 and Mobidiag Novodiag(R) Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag(R) test in tertiary care emergency departments was assessed. In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert(R), whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag(R). Rapid SARS-CoV-2 testing with Novodiag(R) was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag(R) and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag(R) and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag(R), but positive with the reference method with late Ct values. On average, a test result using Novodiag(R) was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.