Five Antigen Tests for SARS-CoV-2: Virus Viability Matters

Abstract

Antigen testing for SARS-CoV-2 (AGT) is generally considered inferior to RT-PCR testing in terms of sensitivity. However, little is known about the infectiousness of RT-PCR positive patients who pass undetected by AGT. In a screening setting for mildly symptomatic or asymptomatic patients with high COVID-19 prevalence (30–40%), 1141 patients were tested using one of five AGTs and RT-PCR. Where the results differed, virus viability in the samples was tested on cell culture (CV-1 cells). The test battery included AGTs by JOYSBIO, Assure Tech, SD Biosensor, VivaChek Biotech and NDFOS. Sensitivities of the ATGs compared to RT-PCR ranged from 42% to 76%. The best test yielded a 76% sensitivity, 97% specificity, 92% positive, and 89% negative predictive values, respectively. However, in the best performing ATG tests, almost 90% of samples with “false negative” AGT results contained no viable virus. Corrected on the virus viability, sensitivities grew to 81%–97% and, with one exception, the tests yielded high specificities >96%. Performance characteristics of the best test after adjustment were 96% sensitivity, 97% specificity, 92% positive, and 99% negative predictive values (high prevalence population). We, therefore, believe that virus viability should be considered when assessing the AGT performance. Also, our results indicate that a well-performing antigen test could in a high-prevalence setting serve as an excellent tool for identifying patients shedding viable virus. We also propose that the high proportion of RT-PCR-positive samples containing no viable virus in the group of “false negatives” of the antigen test should be further investigated with the aim of possibly preventing needless isolation of such patients.

Performance of Seven SARS-CoV-2 Self-Tests Based on Saliva, Anterior Nasal and Nasopharyngeal Swabs Corrected for Infectiousness in Real-Life Conditions: A Cross-Sectional Test Accuracy Study

“…Here, we have to point out that we have detected viable virus in 20% of samples with C t between 30 and 35, and surprisingly, even in 8% of tested samples with C t between 35 and 40 (please note that only 488 samples with discrepant results between RAT and qPCR were analyzed using cell culture). This supports our opinion voiced in our previous papers [ 7 , 8 ] that simply reducing the C t threshold for classifying patients as positive (i.e., the method obviously often employed by the manufacturers when performing their validation studies) is not the way to go for validation. This is also supported by the study on the relationship between virus viability and C t threshold/number of RNA copies in the sample by La Scola [ 11 ] who found viable virus up to C t threshold 33 (interestingly, as much as 50% of samples at C t 32 contained viable virus).…”

“…Tests using other sampling methods than NPS were highly inferior to the NPS-based test, the performance of which meets the WHO/ECDC criteria and is in line with the better tests evaluated in our previous study [ 7 ]. This does not necessarily mean that all self-tests using saliva or nasal swabs are so vastly inferior, but the fact that all these tests failed definitely makes one doubt the effectiveness of these tests in the high-capacity setting in general.…”

“…In this study, we have evaluated the real-world performances of seven RATs in a setting of a high-capacity testing center using our previously proposed method [7,8]. Briefly, the reasoning is that the principal aim of RATs is to identify infectious patients, while PCR can detect dead viral particles that may have been excreted from the organism during recovery, killed by good mucosal immunity or even got to the nasal mucosa already dead (e.g., on dust particles).…”

“…We aimed to compare the diagnostic performance of RATs utilizing three sampling methods (NPS-1 test, ANS-2 tests and saliva-4 tests). Moreover, in line with our previously described method [7,8] (see also a brief summary of the reasoning in Discussion), virus culture was performed in all samples where qPCR and RAT results differed to correct the RATs performances on infectiousness.…”

“…Low C t values below 20 or even 25 will be captured by a majority of tests approved in the EU; however, higher C t values, which (i) are in many countries still considered proof of positivity (for example, up to 40 in the Czech Republic) and (ii) which can still identify infectious patients, are detected much less reliably even with NPS-based RATs. This fact is likely often used by manufacturers who claim extremely high sensitivities based on pre-selection of patients with low C t values while real-world performances are usually much worse (e.g., [ 2 , 7 ]).…”