key: cord-0927951-jfgmboxo
authors: Kobayashi, Ryo; Murai, Ryosei; Moriai, Mikako; Nirasawa, Shinya; Yonezawa, Hitoshi; Kondoh, Takashi; Saeki, Masachika; Yakuwa, Yuki; Sato, Yuki; Katayama, Yuki; Nakafuri, Hirotaka; Kitayama, Ikumi; Asanuma, Koichi; Fujiya, Yoshihiro; Takahashi, Satoshi
title: Evaluation of false positives in the SARS-CoV-2 quantitative antigen test
date: 2021-06-25
journal: J Infect Chemother
DOI: 10.1016/j.jiac.2021.06.019
sha: 27c79a87ee60741fb722795679a29cd068a76101
doc_id: 927951
cord_uid: jfgmboxo

Introduction Highly sensitive reagents for detecting SARS-CoV-2 antigens have been developed for accurate and rapid diagnosis till date. In this study, we aim to clarify the frequency of false-positive reactions and reveal their details in SARS-CoV-2 quantitative antigen test using an automated laboratory device. Methods Nasopharyngeal swab samples (n=4,992) and saliva samples (n=5,430) were collected. We measured their SARS-CoV-2 antigen using Lumipulse® Presto SARS-CoV-2 Ag and performed a nucleic acid amplification test (NAAT) using the AmpdirectTM 2019 Novel Coronavirus Detection Kit as needed. The results obtained from each detection test were compared accordingly. Results There were 304 nasopharyngeal samples and 114 saliva samples were positive in the Lumipulse® Presto SARS-CoV-2 Ag test. All positive nasopharyngeal samples in the antigen test were also positive for NAAT. In contrast, only three (2.6%) of all the positive saliva samples in the antigen test were negative for NAAT. One showed no linearity with a dilute solution in the dilution test. Additionally, the quantitative antigen levels of all the three samples did not decrease after reaction with the anti-SARS-CoV-2 antibody. Conclusions The judgment difference between the quantitative antigen test and NAAT seemed to be caused by non-specific reactions in the antigen test. Although the high positive and negative predictive value of this quantitative antigen test could be confirmed, we should consider the possibility of false-positives caused by non-specific reactions and understand the characteristics of antigen testing. We recommend that repeating centrifugation before measurement, especially in saliva samples, should be performed appropriately.

Highly sensitive reagents for detecting SARS-CoV-2 antigens have been developed for accurate and rapid diagnosis till date. In this study, we aim to clarify the frequency of false-positive reactions and reveal their details in SARS-CoV-2 quantitative antigen test using an automated laboratory device.

Nasopharyngeal swab samples (n=4,992) and saliva samples (n=5,430) were collected.

We measured their SARS-CoV-2 antigen using Lumipulse ® Presto SARS-CoV-2 Ag and performed a nucleic acid amplification test (NAAT) using the Ampdirect TM 2019 Novel Coronavirus Detection Kit as needed. The results obtained from each detection test were compared accordingly.

There were 304 nasopharyngeal samples and 114 saliva samples were positive in the Lumipulse ® Presto SARS-CoV-2 Ag test. All positive nasopharyngeal samples in the antigen test were also positive for NAAT. In contrast, only three (2.6%) of all the positive saliva samples in the antigen test were negative for NAAT. One showed no linearity with a dilute solution in the dilution test. Additionally, the quantitative antigen levels of all the three samples did not decrease after reaction with the anti-SARS-CoV-2 antibody.

The judgment difference between the quantitative antigen test and NAAT seemed to be caused by non-specific reactions in the antigen test. Although the high positive and negative predictive value of this quantitative antigen test could be confirmed, we should consider the possibility of false-positives caused by non-specific reactions and J o u r n a l P r e -p r o o f INTRODUCTION Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes the atypical pneumonia known as coronavirus disease (COVID-19), emerged in late 2019 in Wuhan, China [1] . The World Health Organization has declared COVID-19 a public health emergency of international concern and a very high-risk assessment at the global level [2, 3] .

The nucleic acid amplification test (NAAT) is the gold standard and is mostly used to diagnose COVID-19. However, this method requires several hours and a special technique to detect the nucleic acids. Moreover, specialized instruments and expertise are required to perform these tests [4, 5] . Other approaches have been developed to diagnose COVID-19 by targeting SARS-CoV-2 antigen. Recently, a new quantifying reagent that can detect SARS-CoV-2 antigens has been developed in order to perform a rapid and accurate detection. The performance of a quantitative antigen test was excellent enough for real-life clinical practice in terms of sensitivity and specificity [6] . In addition, recent studies have revealed the utility of the SARS-CoV-2 quantitative antigen test [7, 8] .

However, the method for detection was based on chemiluminescent enzyme immunoassay (CLEIA), which may potentially give rise to the rare phenomenon of false-positives [9] . In this study, we aimed to clarify the frequency of false reactions under our unique premeasured management for detecting SARS-CoV-2 antigen. Nasopharyngeal swabs from patients at the public health center of Sapporo City were collected using UTM or sodium chloride solution. All saliva samples were collected in sterile tubes. All samples were tested for SARS-CoV-2 antigen as soon as they arrived at our laboratory and were preserved at −80 °C after testing.

We measured the SARS-CoV-2 quantitative antigen using Lumipulse ® Presto SARS-CoV-2 Ag (Fujirebio Inc., Tokyo, Japan). The Lumipulse ® Presto SARS-CoV-2

Ag was analyzed using a fully automated Lumipulse ® L2400 (Fujirebio Inc., Tokyo, Japan). In nasopharyngeal samples, preserved solutions were tested after centrifugation at 2,000 × g for 5 min. On the other hand, saliva samples were diluted 2-fold with phosphate buffered saline (PBS) and centrifuged at 20,000 × g for 10 min to remove cells and debris.

Immediately after this procedure, the supernatant was separated into other tubes and centrifuged at 2,000 × g for 5 min and tested accordingly. All nasopharyngeal swabs were judged as positive or judgment pending or negative by the antigen level (negative: less J o u r n a l P r e -p r o o f than 1.34 pg/mL, judgment pending: from 1.34 to less than 10.00 pg/mL, and positive: over 10.0 pg/mL) according to the manufacturer's protocol. Similarly, because saliva samples were diluted 2-fold with PBS, all saliva samples were judged by the antigen level in consideration of dilution factor (negative: less than 0.67 pg/mL, judgment pending: from 0.67 to less than 4.00 pg/mL, and positive: over 4.00 pg/mL). When the result of first test was within the judgment pending level, the sample was tested after re-centrifuged at 2,000 x g for 5 minutes regardless of sample species. We judged the sample by the result of retest. On the other hand, we evaluated our unique judgment pending level by lowering the lower limit of judgment pending level to 1.00 pg/mL in nasopharyngeal swab samples and 0.50 pg/mL in saliva samples. In the results, samples with a positive SARS-CoV-2 antigen test and negative NAAT, were defined as false-positives accordingly.

The SARS-CoV-2 nucleic acid test was performed on a LightCycler480 System (Roche, Basel, Switzerland) using the Ampdirect TM 2019 Novel Coronavirus Detection Kit (Shimadzu Corporation, Kyoto, Japan). This test could be performed without extraction of ribonucleic acid. Samples were mixed with an equal amount of the sample treatment reagent and heated at 90 ° C for 5 minutes, and then, 10 µL of them were used.

All assays were performed according to the manufacturer's protocol, and the samples were judged as positive or negative based on the amplification curve; when the amplification curve was recognized to rise during the assay, it was judged as positive. (Table 1a) and 114 saliva samples (Table 1b) (Table 3 ). This nasopharyngeal sample was collected 14 days after symptom onset, and the details of the saliva samples were unclear.

We evaluated the accuracy of the automated quantitative antigen test, Lumipulse ® Presto SARS-CoV-2 Ag. While this reagent is useful for clinical laboratory tests because of its high positive and negative predictive value, false-positive results were potentially caused by non-specific reactions because this reagent is based on the CLEIA method that detects the reactivity of the antigen-antibody. Nevertheless, it was found that the false-positive rate of this test was much lower than that of the qualitative rapid antigen test that had been used worldwide. [10] . We observed three false-positive samples due to non-specific reactions among the 114 positive saliva samples (2.6%). In the cases of false-positive samples, dilution linearity and absorption tests are generally useful for confirmation of accurate judgment. It has been reported that non-specific reactions tend to be diminished by dilution [11] . In this study, one of the three samples did not show dilution linearity by dedicating the dilute solution, and all the three samples were judged as negative by the absorption test. Quantitative SARS-CoV-2 antigen measurement is an excellent clinical test because the frequency of false-positives is not high compared with other tests based on the CLEIA method [12] . Because 67 of 183 samples within judgment pending level were judged as negative after re-centrifugation, repeating centrifugation would be possible to make the reduction of the false-positive reaction caused by impurities of samples and lead to decrease in performing of unnecessary NAAT [13] [14] .

In real-life clinical practice, it would be a potentially useful test in a facility that cannot perform NAAT. However, all false-positive reactions were observed in the saliva samples.

It was considered that saliva would be more likely to cause a false-positive reaction because saliva contained more impurities than the nasopharyngeal swabs. Therefore, care should be taken when measuring the saliva samples.

Lumipulse ® Presto SARS-CoV-2 Ag reagent has the judgment pending level in the range of 1.34 to 10.00 pg/mL for nasopharyngeal swabs and 0.67 to 4.00 pg/mL for saliva samples. If the measured level of the sample is within these ranges, re-measurement after centrifugation is recommended. If the measured level after repeat centrifugation is not nearly changed, a confirmation test by NAAT is recommended by the manufacturer. There was a significant difference in antigen levels between the NAAT-positive and NAAT-negative groups in the judgment pending level according to the manufacturer's protocol; however, it seemed to be difficult to judge positive or negative based only on the antigen level. Therefore, we also evaluated the set value of this judgment pending level. The positive rate by NAAT in the judgment pending group was 37.5% (27/72) in nasopharyngeal swabs and 75.0% (30/44) in saliva samples respectively.

We further investigated by lowering the lower limit of judgment pending level to 1.00 pg/mL in nasopharyngeal swab samples and 0.50 pg/mL in saliva samples. Fifty nasopharyngeal swab samples and 20 saliva samples were newly classified into our unique judgment pending level, and one nasopharyngeal sample and three saliva samples were positive by NAAT. One nasopharyngeal swab sample was collected 14 days after symptom onset. We reported that the results of NAAT did not match with the results of the antigen test 13 days after symptom onset [6] . We considered that the antigen level would decrease earlier than the RNA load in the nasopharynx. On the other hand, the Ct values by NAAT of the three saliva samples were more than 33.35, in N2, and they might not possibly maintain infectivity [15] . If we obtained positive results in the NAAT and negative results in the antigen test, it was considered that most samples obtained from the patients have passed approximately 2 weeks after onset or have a low viral load [6] . On the other hand, from the viewpoint of diagnostic tests for infectious diseases, it is J o u r n a l P r e -p r o o f important to detect positives at an early stage of infection [16] . For the purpose of confirming the spread of infectious disease including the early stage of infection and asymptomatic patients, which would have a low viral load, it might be better to consider the lower limit of judgment pending level in nasopharyngeal swab samples and saliva samples.

This study had some limitations. All samples judged as negative with the lower limit of our unique definition were not tested by NAAT. In saliva samples, we could not obtain the details of suspected patient information because the samples were gathered out of the hospital. Unfortunately, we could not determine the cause of the non-specific reactions. In general, various substances in the body may potentially cause such non-specific reactions.

In conclusion, the quantitative SARS-CoV-2 antigen test reagent, Lumipulse ® Presto SARS-CoV-2 Ag reagent, has a highly positive and negative predictive value. We suggest the lower limit of judgment pending level may be reconsidered to detect positives at the early stage of infection. However, we should note that false-positive reactions rarely occur, particularly in saliva samples. Repeating centrifugation is the useful method for reduction of false-positive reactions. J o u r n a l P r e -p r o o f 

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