key: cord-0689535-y93bnynm
authors: Korenkov, M.; Poopalasingam, N.; Madler, M.; Vanshylla, K.; Eggeling, R.; Wirtz, M.; Fish, I.; Dewald, F.; Gieselmann, L.; Lehmann, C.; Faetkenheuer, G.; Gruell, H.; Pfeifer, N.; Heger, E.; Klein, F.
title: Reliable assessment of in vitro SARS-CoV-2 infectivity by a Rapid Antigen Detection Test
date: 2021-04-06
journal: nan
DOI: 10.1101/2021.03.30.21254624
sha: 6405752ee81eaa20a1cb5015aef7420e64dd4ccc
doc_id: 689535
cord_uid: y93bnynm

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising candidates for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is yet to be fully determined. Two combined oro- and nasopharyngeal swabs were collected from individuals at a routine SARS-CoV-2 diagnostic center. Side-by-side evaluations of RT-qPCR and RADT as well as live virus cultures of positive samples were performed to determine the sensitivity of the Standard Q COVID-19 Ag Test (SD Biosensor/Roche) in detecting SARS-CoV-2-infected individuals with cultivable virus. A total of 2,028 samples were tested and 118 virus cultures inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag Test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86% and 99.89%. For adjusted Ct values <20, <25, and <30 the RADT reached sensitivities of 100%, 98.15%, and 88.64%, respectively. All 29 culture positive samples were detected by RADT. While overall sensitivity was low, Standard Q COVID-19 RADT reliably detected patients with high RNA loads. Additionally, negative RADT results fully corresponded with the lack of viral cultivability in Vero E6 cells. These results indicate that RADT can be a valuable tool for the detection of individuals that are likely to transmit SARS-CoV-2. RADT testing could therefore guide public health testing strategies to combat the COVID-19 pandemic.

Timely diagnosis of SARS-CoV-2 infection with subsequent contact tracing and rapid isolation is a critical public health strategy to contain the current COVID-19 pandemic (1) (2) (3) . The current gold standard of SARS-CoV-2 testing is based on real-time reverse-transcription-PCR (RT-qPCR) (4) .

However, despite high sensitivity and specificity, RT-qPCR is less suited for rapid point-of-care identification of infectious individuals, as RT-qPCR is also able to detect non-replicating virus RNA (5) (6) (7) . Therefore, there is a need for an inexpensive alternative testing method to directly detect infectious individuals which can be deployed widely without the use of specialized equipment (8, 9) .

One promising approach is the use of lateral flow immunochromatographic assays commonly referred to as rapid antigen detection tests (RADT) designed to detect viral antigens. RADT are of particular use for community-based screenings due to low turnaround times and feasibility for onsite testing (10, 11) . Different tests have already been approved for clinical use, however, performance studies under real-life conditions evaluating the quality of different RADT are limited. In these studies, reported test characteristics, such as sensitivity, varied greatly depending on cohort composition (24.3-89%) . While RADT showed better performance for high RNA load samples, specificity in general, however, remained high (92-100%) (12) (13) (14) (15) .

As high RNA loads are typically associated with a higher probability of infectiousness, the diagnostic performance of RADT in the context of infectivity models is yet to be determined (16) (17) (18) . Therefore, there is a need for large-scale field studies with a focus on virus cultivability to be able to appropriately interpret RADT results. Here, we combine side-by-side evaluation of RADT under real-life conditions in a routine diagnostic center with the cultivability of live virus from RT-qPCR positive individuals to determine the sensitivity of the Standard Q COVID-19 Ag Test (SD Biosensor/Roche) for detecting SARS-CoV-2 samples with cultivable virus in Vero E6 cells.

To validate RADT performance, paired oro-and nasopharyngeal swabs were collected and tested using both RT-qPCR and RADT. RT-qPCR positive samples were additionally cultivated in Vero E6 cells to determine the ability of RADT to detect infectious samples (Fig. 1A) . A total of 2,032 samples was tested with both RT-qPCR and RADT out of which 4 were excluded due to 3 missing results and 1 incorrect appliance of the RADT leaving 2,028 (99.80%) samples from 1,849 individuals eligible for analysis (Fig. 1B) . SARS-CoV-2 was detected in 210 samples by RT-qPCR representing a study prevalence of 10.36%. At the time of sampling 866 (42.70%) swabs were taken from symptomatic individuals, while 810 (39.94%) specimens were collected from asymptomatic individuals. For 352 (17.35%) samples symptom status was unknown at the time of analysis. 599 (69.17%) symptomatic individuals reported up to 3 symptoms, 247 (28.52%) more than 3 and for 20 samples (2.31%) the number of symptoms was not reported. In our cohort 141 (7.62%) samples were obtained from participants who were tested twice or more (Fig. 1C ). 1,239 (61.09%) samples were taken from female and 789 (38.91%) from male individuals. Participants had a median age of 32.25 years (IQR: 26.14 -43.12) (Fig. 1D) . The median Ct value determined by RT-qPCR was 31.49 (IQR: 24.19 -34.16) (Fig. 1E) . 126 (60%) of 210 RT-qPCR positive and 2 RT-qPCR inconclusive samples were cultivated in Vero E6 cells on the same day of sample collection. Of those 8 (4.80%) were excluded due to culture contaminations (6) or negative RT-qPCR results upon retesting (2) (Fig. 1B) . A detailed cohort description can be found in the table S1.

The Standard Q COVID-19 Ag Test yielded a positive result in 92 (4.54%) and a negative result in 1,936 (95.46%) of all samples ( Fig. 2A) 29 ,17% and 9.68% for 6 log10, 5 log10 and 4 log10 copies/ml, respectively (Fig. 2D ). We conclude that observed RADT sensitivity declines at adjusted Ct values above 27 or below 6 log10 copies/ml but reliably detects samples with higher RNA loads.

Data on symptoms was obtained and analyzed for 1,676 (82.64%) out of 2,028 samples. 130 high RNA loads, respectively. The virus culture assay shows 90% and 50% PPR for an adjusted Ct value around 21.45 or 7.31 log10 copies/ml and 23 or 6.8 log10 copies/ml, respectively. RADT show a PPR of 90% and 50% at an adjusted Ct value of 24.7 or 6.24 log10 copies/ml and 29.0 or 4.78 log10 copies/ml, respectively (Fig. 4h) . In summary these data show that a negative RADT result can reliably predict non-infectiousness in our culture Vero E6 assay.

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To evaluate whether repeated testing of several individuals affect our conclusions, additional statistical analyses were carried out (see Material and Methods). The results shown in fig. S2 confirm conclusions from Fig. 2B , 2D and 4H, respectively. P-values for Table 2 and Table S1 and S2 were calculated with Fisher's exact test. Correction for repeated RADT measurements was 

RADT are cheap and fast diagnostic tools that can be immediately performed at the point of care.

Here, we present comprehensive data on the use of Standard Q COVID-19 RADT for high throughput testing of a large cohort tested under real-life conditions in a SARS-CoV-2 outpatient diagnostic center.

Implementation of the RADT on top of routine diagnostic was completed without much difficulty as the rapid feasibility made it easy to run multiple tests in parallel for a single operator. Upon following the manufacturer's instructions there were only two cases in which the result could not be read out clearly. As a consequence, we were able to conduct 2,028 paired RT-qPCR and RADT tests directly on site and cultivate virus on the same day without prior sample freezing. While sensitivities of other RADT vary between 24% and 93% in different studies (12, (19) (20) (21) , reported Standard Q RADT sensitivities are mostly in the range from 68% to 90% (13, 22, 23) . The overall diagnostic sensitivity observed in our study was 42.86%. However, the investigated cohort of nonhospitalized patients was to a large extend comprised of individuals with adjusted Ct values over All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.03.30.21254624 doi: medRxiv preprint 30 (122/210) who were still detected in RT-qPCR due to persisting viral RNA. Stratification by RNA load revealed that the Standard Q RADT performed reliably for patients with viral loads over 5.4 log10 or below Ct 27, which is in accordance to recent studies (12, 14, 24) . Since a virus concentration of 6 log10 copies/ml is commonly suspected to be the threshold for contagiousness of the patient, we aimed to investigate the correlation between RADT result and SARS-CoV-2 in vitro infectivity (17, 18) .

In contrast to highly sensitive nucleic acid-based detection methods that do not specifically test for intact viral particles required for transmission, viral culture is a frequently used, albeit laborious, method to determine the presence of infectious virus in clinical samples (25) (26) (27) (28) . Although the detection of viable virus in cell culture models is strong evidence of infectiousness, a negative result does not eliminate the possibility of human transmission (11, 29) . Moreover, the validity of viral culture as a surrogate for infectivity may depend on the susceptibility of the cell line used (26) (27) (28) . However, loss of infectious titer in classical Vero E6 cells has been associated with a lack of transmission despite detection of viral RNA in preclinical models (30) .

To investigate whether the Standard Q RADT might be able to reliably detect culture-positive samples in Vero E6 cells, we attempted virus isolation from samples positive for SARS-CoV-2 RNA. All samples from which virus could be recovered had previously tested positive in RADT.

Moreover, none of the samples tested negative by RADT contained infectious virus determined by cell culture. Furthermore, when taking symptom duration into account, we detected no positive culture after 6 days of symptom onset indicating a decreased probability of recovering viable virus as symptom duration increases (18, 25, (31) (32) (33) ; at the same time the RADT identified positive All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.03.30.21254624 doi: medRxiv preprint samples for up to 9 days. While some groups have described virus isolation from samples above Ct 30 (27, 34) , here, positive cultures were only observed from samples with higher RNA loads which was in accordance with previous observations (10, 24, 32, 35) . Taking the time of suspected exposure and duration of symptoms into account (27, 29) , our results suggest that RADT testing is of potential use for determining infectivity at the time of sampling.

This study, however, is subject to some limitations. Although the examined single-center study population was large, our cohort might not be considered representative of the general population due to young age and disproportionate gender distribution. The data on symptoms and their duration are only reliable to a limited extent, since they were retrospectively analyzed from mostly self-reported symptoms entered into a web tool. Furthermore, instead of a nasal swab, we used an oro-and nasopharyngeal swab to investigate RADT performance, which impedes the feasibility for the general public.

In combating overdispersed SARS-CoV-2 transmission, rapid detection and isolation of highly infectious individuals is a primary goal (8, 9, (36) (37) (38) (39) (40) . In our investigation the Standard Q RADT was able to reliably detect high viral load as well as all culture positive samples. Therefore, this test could be used as a fast surrogate marker for viral cultivation in order to identify and prevent SARS-CoV-2 transmissions by highly infectious individuals. Although less sensitive than RT-qPCR, RADT could compensate for this disadvantage through easy and feasible mass screenings (8, 41) . Furthermore, one might suspect that RT-qPCR positive, but RADT negative individuals do not pose a high risk of transmissions, since all samples remained culture negative in our experimental setup. However, individual results must be interpreted with caution as SARS-CoV-All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.03.30.21254624 doi: medRxiv preprint 2 infection could remain undetected in early stages. Simple to perform and applicable anywhere, RADT enable mass testing as a complementary method to RT-qPCR to more effectively combat the SARS-CoV-2 pandemic.

Between 26 th October 2020 and 08 th January 2021 all enrolled participants were tested for SARS- were retrospectively analyzed including clinical data retrieved from a symptoms diary webtool that all individuals registering for a SARS-CoV-2 test are asked to complete. Due to a number of implausible self-reported entries, only webtool entries not older than a week from the time of testing were included into symptom analysis. Patients were categorized as symptomatic, if reported symptom duration at the time of testing was ≤ 14 days and one of the following symptoms was found: fever, cough, rhinorrhea, nausea, diarrhea, shortness of breath, and/or a new olfactory or taste disorder. All rights reserved. No reuse allowed without permission.

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The Standard Q COVID-19 Ag Test (SD Biosensor Inc., Suwon-si, Republic of Korea/Hoffmann La Roche AG, Basel, Switzerland) was performed according to the manufacturer's instructions using the enclosed dry swab with one modification (Noble Biosciences, Inc., Hwaseong-si, Republic of Korea). Instead of a nasopharyngeal swab, a combined oro-and nasopharyngeal swab was performed to ensure comparability with RT-qPCR. Operating instructions in brief: The collected swab was mixed in the provided tube of collection medium and 3 drops were applied through a nozzle cap onto the test strip. Results were read out visually after 15-20 minutes by medically trained and instructed personnel. In accordance with the manufacturer's reference guide, faint lines were considered positive if the control line was also present.

RT-qPCR was performed using different SARS-CoV-2 RNA detection protocols that were normalized according to the same standard. The following SARS-CoV-2 detection protocols were utilized: (1) Nucleic acid extraction was done for 935 (46.10%) samples using the MagNA Pure (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Sensitivity and Specificity with 95% confidence intervals (CI) as well as positive and negative prediction values were calculated using the RT-qPCR as a reference. Culture and RADT results were evaluated by a contingency table and a p-value was calculated with Fisher's exact test. Mann-Whitney U-test (MWU) was used to compare differences between medians. P values <0.05 were considered significant. Probit regression was carried out using a generalized linear model (Rfunction glm) with probit link function. To correct for repeated measurements from the same All rights reserved. No reuse allowed without permission.

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The Institutional Review Board of the University of Cologne acknowledged and approved the study under application number 21-1039. Table S1 . Additional Cohort Description Table S2 . RADT results by symptoms and follow-up testing All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.03.30.21254624 doi: medRxiv preprint

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Acknowledgments: We thank all study participants who devoted time to our research; members of the Klein Laboratory and the University Hospital Cologne's Institute of Virology and SARS-CoV-2 diagnostic center for continuous support and helpful discussions

Nadine Henn for lab management and assistance; and the Becker Laboratory, Marburg, for sharing Vero E6 cells. Funding: Bundesministerium für Bildung und Forschung grant 01KX2021

MK, NP Funding acquisition: NPfeifer, FK Project administration: MK, NP Supervision: EH, Npfeifer, FK Writing -original draft: MK, NP Writing -review & editing

The Rapid Antigen Detection Tests investigated in this study were kindly provided by Hoffmann-La Roche AG (Basel, Switzerland).Data and materials availability: All data, code and materials used in the analysis will be made available upon publication. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.