key: cord-0994929-n7xu9840
authors: Yokota, I.; Sakurazawa, T.; Sugita, J.; Iwasaki, S.; Yasuda, K.; Yamashita, N.; Fujisawa, S.; Nishida, M.; Konno, S.; Teshima, T.
title: Performance of qualitative and quantitative antigen tests for SARS-CoV-2 in early symptomatic patients using saliva
date: 2020-11-10
journal: nan
DOI: 10.1101/2020.11.06.20227363
sha: 71f9c59f55ee2fa1d89bb70ff539082e4e653deb
doc_id: 994929
cord_uid: n7xu9840

Background: The rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent need for the prevention and containment of disease outbreaks in communities. Although the gold standard is polymerase chain reaction (PCR), antigen tests such as immunochromatographic assay (ICA) and chemiluminescent enzyme immunoassay (CLEIA) that can yield results within 30 minutes. Methods: We evaluated performance of ICA and CLEIA using 34 frozen PCR-positive specimens (17 saliva and 17 nasopharyngeal swab) and 307 PCR-negative samples. Results: ICA detected SARS-CoV-2 in only 14 (41%) samples, with positivity of 24% in saliva and 59% in NPS. Notably, ICA detected SARS-CoV-2 in 5 (83%) of 6 samples collected within 4 days after symptom onset. CLEIA detected SARS-CoV-2 in 31 (91%) samples, with positivity of 82% in saliva and 100% in NPS. CLEIA was negative in 3 samples with low viral load by PCR. Conclusions: These results suggest that use of ICA should be limited to earlier time after symptom onset and CLEIA is more sensitive and can be used in situations where quick results are required.

Rapid detection of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the prevention and containment of coronavirus disease-19 outbreaks in communities and hospitals. Currently, the "gold standard" of viral detection is quantitative reverse transcriptase polymerase chain reaction (PCR) using nasopharyngeal swab samples (NPS) [1, 2] . NPS sampling requires specialized medical personnel with protective equipment, posing risks of viral transmission to healthcare workers, and false-negative results may occur due to deficiency in sampling technique [2, 3] .

Self-collected saliva can be as effective as traditional nasal swabbing, making a major step for a type of screening, which is much faster, and less instructive and expensive [4] [5] [6] .

Although PCR is highly accurate and reliable, it is time-consuming as a screening test.

Viral antigen detection tests such as a rapid antigen test (immunochromatographic assay, ICA) and chemiluminescent enzyme immunoassay (CLEIA) are much more easy and yield results quickly than PCR [7] [8] [9] [10] [11] . In this study, we evaluated the utility of ICA and CLEIA in comparison with PCR using self-collected saliva and NPS.

We screened 34 SARS-CoV-2 positive samples (17 NPS and 17 saliva samples) as established by PCR and 307 negative saliva samples. All positive samples were obtained from symptomatic patients with COVID-19 and all the negative samples were obtained from asymptomatic persons at screening. All the samples had been frozen and thawed before analysis. This study was approved by the Institutional Ethics Board (Hokkaido University Hospital Division of Clinical Research Administration Number: 020-0116), and informed consent was obtained from all patients.

Saliva and NPS samples were collected as previously described [12] . Saliva samples were diluted 4-fold with phosphate buffered saline and centrifuged at 20,000 × g for 5 min at All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint 4 4 o C to remove cells and debris [12] . Following thawing, samples were centrifuged at 2,000 × g for 5 min at 4 o C to remove cells and debris. Each sample was divided for application to four testing.

RNA was extracted using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany).

PCR tests were performed as described [12] , according to the manual by National Institute of 

ICA was performed using Espline SARS-CoV-2 (Fujirebio, Tokyo, Japan) according to the manufacturer's instructions. In brief, samples were mixed with the sample preparation mixture. The mixture (200μl) and 2 drops of buffer was added to the ample port and the results were interpreted after incubation for 30 minutes.

Lumipulse SARS-CoV-2 Ag kit® (Fujirebio, Tokyo, Japan) is a sandwich CLEIA using monoclonal antibodies that recognize SARS-CoV-2 N-Ag on LUMIPULSE G1200 automated machine (Fujirebio). 100 µL of saliva diluted with PBS was analyzed to measure N-Ag levels according to the manufacturer's instructions. In brief, the treatment solution and the specimen were consecutively aspirated using a single-use tip and was dispensed into suspension of magnetic beads coated with the monoclonal antibody to SARS-CoV-2 N-Ag. After 10-minute incubation followed by a wash-step, alkaline phosphatase-conjugated anti-SARS-CoV-2 monoclonal antibody were added and incubated for another 10 minutes. After a second All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint 5 wash-step, AMPPD substrate solution was added and developed for 5 minutes. The amounts of SARS-CoV-2 N-Ag were calibrated with the developed chemiluminescence signals. The calibrator of this assay contains the recombinant SARS-CoV-2 N antigen standardized with the recombinant SARS-CoV-2 antigen. Antigen levels of 0.67 pg/mL or greater were defined as positive. (Table 1) . Particularly, the virus was positive in only 24% (95%CI: 7-50%) in saliva in contrast to 59% (95%CI: 33-82%) positivity in NPS. It should be noted that ICA was positive in 5 (83%, 95%CI: 36-100%) out of 6 samples collected within 4 days after symptom onset and in 9 (32%, 95%CI: 16-52%) of 28 samples collected thereafter. In NPS, it was positive in 9 (82%, 95%CI: 48-98%) out of 11 samples collected within 10 days after symptom onset, but in 1 (17%, 95%CI: 0-64%) of 6 samples collected thereafter. In saliva, all 3 samples collected at 2-4 days after symptom onset were positive, but only 1 (7%, 95%CI: 0-34%) of 14 samples collected All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. 

Our results suggest that ICA could be used only within 4 days after symptom onset using both NPS and self-collected saliva. However, ICA is not reliable in samples collected thereafter with significant concerns of high false-negative rates. Sensitivity of ICA markedly decreased over time after symptom onset, particularly in saliva. It is well documented that SARS-CoV-2 tends to persist longer in NPS than in saliva [12, 13] . It is thus reasonable to speculate that lower sensitivity of saliva ICA is due to late sampling rather than the difference in All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint antigen load between saliva and NPS. Overall, sensitivity of ICA using NPS was 59%, which was similar to 51-67.5% sensitivity of rapid antigen test using NPS for influenza [14] [15] [16] [17] . It should be noted that antigen testing of influenza using NPS is also recommended to perform within 3 days after symptom onset [17] . ICA is suitable for point-of-care testing and therefore these results should be confirmed in larger cohort.

In contrast, CLEIA is much more reliable and accurate in both NPS and saliva than ICA, with high correlation observed between antigen concentrations and RNA load by PCR.

However, there were three PCR-positive but CLEIA-negative samples. These samples were all saliva collected at 7, 12, and 14 days after symptom onset. We therefore recommend using saliva for CLEIA only in patients who developed symptoms within a week. Moreover, in these samples, Ct values were 32.4-33.8 by PCR. A "positive" PCR result does not necessarily indicate presence of live virus [18] , and recent studies showing patients with Ct values above 33-34 by PCR not to be contagious [19, 20] . Vice versa, there were 2 (0.65%) of 307 samples were PCR-negative but CLEIA-positive with high antigen concentrations of 8.45 and 24.23 pg/mL. Reexamination of these specimens confirmed CLEIA-positivity. This could reflect a false positive CLEIA, but the possibility of false negative PCR result cannot be completely ruled out and the clinical implication of this discrepancy remains to be elucidated.

Measuring antigen concentrations by CLEIA is a novel approach to viral detection with equivalent utility compared to PCR. To improve diagnostic accuracy, two-step testing with initial CLEIA and secondary nucleic acid amplification tests (NAAT) is reasonable. Thus, the great advantage of having a rapid and accurate CLEIA is the ability for primary screening to establish a "gray zone" reserving definitive diagnosis by confirmatory NAAT testing. Reverse transcriptase loop-mediated isothermal amplification (LAMP) [7] has become the second most common NAAT after PCR with several advantages over PCR: rapid turn-around time within 30 minutes, ease of implementation, and potential utility at point of care using a simple device [6, 9, [21] [22] [23] [24] [25] . We recently reported that LAMP had equivalent efficacy to PCR using saliva in both All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint asymptomatic persons and symptomatic patients [6] . Thus, LAMP is currently being used at the international airport quarantine as the confirmatory NAAT testing of CLEIA in Japan.

Antigen detection of SARS-CoV-2 yield results quickly. However, use of a rapid antigen test should be limited to within a few days after symptom onset. A quantitative antigen test using both nasopharyngeal and saliva specimens shows high concordance with PCR and can be used in situations where quick results are required. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint 

Upper and lower panels show frequency of PCR-positivity and PCR-negativity, respectively, according to CLEIA antigen concentrations. Frequencies in each range were shown above/below histogram. All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint 

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All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint 15 Figure 3 .All rights reserved. No reuse allowed without permission. preprint (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 this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227363 doi: medRxiv preprint