key: cord-258250-zueo1xfa
authors: Hirotsu, Yosuke; Maejima, Makoto; Shibusawa, Masahiro; Nagakubo, Yuki; Hosaka, Kazuhiro; Amemiya, Kenji; Sueki, Hitomi; Hayakawa, Miyoko; Mochizuki, Hitoshi; Tsutsui, Toshiharu; Kakizaki, Yumiko; Miyashita, Yoshihiro; Yagi, Shintaro; Kojima, Satoshi; Omata, Masao
title: Comparison of Automated SARS-CoV-2 Antigen Test for COVID-19 Infection with Quantitative RT-PCR using 313 Nasopharyngeal Swabs Including from 7 Serially Followed Patients
date: 2020-08-12
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.08.029
sha: 
doc_id: 258250
cord_uid: zueo1xfa

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR) in routine clinical practice. In the current pandemic situation, a more rapid and high-throughput method is in growing demand. Here, we validated the performance of a new antigen test (LUMIPULSE) based on the chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients, 231 individual samples from 4 infected patients and 215 non-infected individuals) were analyzed for SARS-CoV-2 by quantitative RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. We determined the cutoff value for antigen detection using receiver operating characteristic curve analysis and compared the antigen test performance with that of RT-qPCR. Further, we compared the viral loads and antigen levels in serial samples from seven infected patients. When using RT-qPCR as the reference, the antigen test exhibited 55.2% sensitivity and 99.6% specificity with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, respectively. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline along with viral clearance. This gradual decline was in contrast with the abrupt “positive-to-negative” and “negative-to-positive” status changes observed with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients.

In the current pandemic situation, a more rapid and high-throughput method is in growing demand. Here, we validated the performance of a new antigen test were analyzed for SARS-CoV-2 by quantitative RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. We determined the cutoff value for antigen detection using receiver operating characteristic curve analysis and compared the antigen test performance with that of RT-qPCR. Further, we compared the viral loads and antigen levels in serial samples from seven infected patients. When using RT-qPCR as the reference, the antigen test exhibited 55.2% sensitivity and 99.6% specificity with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, respectively. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline along with viral clearance. This gradual decline was in contrast with the abrupt "positive-to-negative" and "negative-to-positive" status changes observed with RT-qPCR, particularly in the late phase of infection. In summary, Within a few months, SARS-CoV-2 had spread around the world, threatening human life [2] . To date, 11 million individuals have been infected with SARS-CoV-2 and 0.52 million patients have died from coronavirus disease 2019 (COVID-19) [2] . As Japan continues to battle the COVID-19 epidemic with a second wave, more than one thousand newly infected patients have been confirmed daily. In Japan, 38,687 individuals were infected with SARS-CoV-2 and 1,012 patients passed away by 3th, August, 2020.

J o u r n a l P r e -p r o o f 5 The World Health Organization (WHO) has raised a global warning and announced the need for a test system for COVID-19-suspected patients [3] .

SARS-CoV-2 is now known to also be spread by infected people who experience only mild symptoms or are asymptomatic carriers [4] [5] [6] . Therefore, there is a need to expand testing to asymptomatic individuals depending on the regional situation. Furthermore, there are concerns that environmental contamination is resulting in further spread of the virus, particularly in hospitals [7, 8] .

In routine clinical practice, SARS-CoV-2 infection is determined by reverse-transcription PCR (RT-PCR) analysis [9] . The RT-PCR test is conducted using different types of specimens including sputum, nasopharyngeal swabs, pharyngeal swabs, saliva, stool, bronchoalveolar lavage fluid, and endotracheal aspirate fluid [10] [11] [12] . As we and other group previously reported, using a pooling strategy with RT-PCR is one of the most effective methods for screening individuals, realizing savings in terms of time, reagents, and cost [13, 14] .

However, the RT-PCR test is not rapid (it typically takes 3-4 h), and it requires specialized laboratory equipment and skilled technicians, while antigen J o u r n a l P r e -p r o o f tests are a simple method that can be performed routinely in clinical laboratories [15, 16] . Antigen tests have been widely applied to detect infection with viruses other than SARS-CoV-2 [16] . Therefore, the development of a more costeffective and high-throughput test system is important for preventing viral spread and monitoring the level of infection in COVID-19 patients.

Here, we present a newly developed SARS-CoV-2 antigen test system based on the chemiluminescence enzyme immunoassay (CLEIA). We compared the quantitative RT-PCR (RT-qPCR) results for viral load with the CLEIA results for antigen level following testing of 313 nasopharyngeal swabs.

Moreover, we examined the antigen levels in a series of samples collected from hospitalized patients with COVID-19 infection.

We collected 313 nasopharyngeal swabs from individuals at Yamanashi Central Hospital. All samples were obtained using cotton swabs and viral transport media in UTM® (Copan Diagnostics, Murrieta, CA, USA). The viral J o u r n a l P r e -p r o o f transport media were stored at 4°C until nucleic acids extraction. Total nucleic acids were extracted within 2 hours after swab collecting. (Thermo Fisher Scientific) as previously described [14, 17] . Briefly, we added 200 µL of viral transport media, 5 µL of proteinase K, 265 μL binding solution, 10 μL total nucleic acid-binding beads, 0.5 mL wash buffer, and 0.5-1 mL of 80% ethanol to each well of a deep-well 96-well plate. Nucleic acids were eluted with 70 μL elution solution. Total nucleic acids were immediately subjected to the following RT-qPCR test and residual samples were stored at −80°C.

According to the protocol developed by the National Institute of Infectious Diseases (NIID) in Japan [18] , we performed one-step RT-qPCR to detect SARS-CoV-2 [19] . A threshold cycle (Ct) value was assigned to each PCR reaction and the amplification curve was visually assessed. According to the national protocol (version 2.9.1), we deemed a sample to be positive when a visible amplification plot was observed, whereas a sample was deemed negative when no amplification was observed. The absolute copy number of the viral load was determined using the Ct value of the AccuPlex SARS-CoV-2 reference (SeraCare, Milford, MA, USA).

The remaining viral transport media from each nasopharyngeal swab was frozen after RT-qPCR. These samples were sent to an outside laboratory (Fujirebio, Inc., Tokyo, Japan). Once thawed, the viral transport medium was viscous; hence, samples were centrifuged at 1,300 ×g for 10 min and the supernatants were used for subsequent analysis.

We used 100 µL of the supernatant per sample of thawed viral transport media from each nasopharyngeal swab to measure the antigen level with the LUMIPULSE SARS-CoV-2 Ag kit (Fujirebio) on the LUMIPULSE G600II automated immunoassay analyzer (Fujirebio) based on the CLEIA method.

In this assay, the treatment solution and the sample were consecutively aspirated using a single tip. The mixture was dispensed into the anti-SARS-CoV-2 Ag monoclonal antibody-coated magnetic particle solution and then incubated for 10 minutes at 37°C. After the first wash step, alkaline phosphatase-conjugated anti-SARS-CoV-2 Ag monoclonal antibody was then added and incubated for 10 minutes at 37°C. After another wash step, the substrate solution was added and incubated for 5 minutes at 37°C. The When the antigen level could not be measured because it exceeded the detection limit, we tested diluted samples and calculated the antigen level of the original sample based on the dilution factor.

Statistical analysis was performed in R (https://www.r-project.org/) and Excel (Microsoft Corp., Redmond, WA, USA). Receiver operating characteristic (ROC) curve analysis was conducted using Analyse-it (Analyse-it Software, Ltd., Leeds, UK) to evaluate the assay performance and to visualize the curves. Areas under the ROC curves, sensitivity, and specificity were calculated. The median antigen level of the PCR-positive samples was 1.57 pg/mL (range 0.12-194,795 pg/mL) and that of the PCR-negative samples was 0.27 pg/mL (range 0-2.46 pg/mL) (Fig. 1A) . The mean antigen level of the PCRpositive samples was significantly higher than that of the PCR-negative samples (p = 0.02, Student's t-test, Fig. 1A ).

To determine the cutoff antigen level for distinguishing SARS-CoV-2 infection status, we conducted ROC curve analysis. When the cutoff for the antigen level was set to 1.31 pg/mL, the accuracy reached its highest level. ROC analysis yielded an area under the ROC curve (AUC) value of 0.848 ± 0.044, suggesting the antigen test accurately detected SARS-CoV-2 (Fig. 1B) .

The numbers of true-positive, false-positive, true-negative, and false-negative results were 32, 1, 254, and 26, respectively (Fig. 1C) . When the RT-qPCR results were used as a reference, the antigen test diagnosed SARS-CoV-2 infection status with a sensitivity of 55.2% and a specificity of 99.6%. The overall concordance between RT-qPCR and the antigen test was 91.4% (286/313).

The primer/probe set used in RT-qPCR amplified the nucleocapsid gene of SARS-Co-V-2 [19] . The antigen test also detects a portion of the nucleocapsid protein. We next examined the relationship between the SARS-CoV-2 viral loads (as determined by RT-qPCR) and the antigen levels (Fig 2) . The SARS-CoV-2 viral load was positively correlated with the antigen level (R² = 0.768).

To examine the reason underlying the low sensitivity of the antigen test, we investigated the relationship between the number of viral copies in the samples and the positive results obtained with the antigen test.

The antigen test determined samples to be positive with 100% concordance with RT-qPCR when the viral load in the samples was > 100 copies (17/17 samples) and 85% concordance when the viral load was > 10 copies but < 100 copies (23/27 samples) ( Table 1 ). The concordance rate gradually declined with decreasing viral load (60% concordance for samples with 10-100 copies, 33% for samples with 1-10 copies, and 26% for samples with less than 1 copy; Table 1 ).

Therefore, the antigen test was highly accurate when the viral load was J o u r n a l P r e -p r o o f > 100 copies, whereas lower viral loads (< 100 copies) resulted in some samples being missed (i.e., false-negative results).

We performed the antigen test and RT-qPCR on a series of nasopharyngeal swabs from seven infected patients. A total of 82 samples were collected from these patients (range 5-27 samples per patient). Overall, there was a strong correlation between the RT-qPCR and antigen test results (Fig. 3) . During the clinical course of these seven patients, the antigen levels showed a similar declining trend along with viral load as quantitated by RT-qPCR (Fig. 3) .

Of particular interest, there were abrupt positive-to-negative turns and negative-to-positive turns based observed with RT-qPCR, especially when the viral load decreased in the latter phase of the infection (cases #2 and #3, Fig.   3 ), whereas the antigen test rarely showed these abrupt "turns".

In this study, we validated the assay performance of an antigen test based on CLEIA (LUMIPULSE) and compared the results with RT-qPCR. To our knowledge, this is the first report on the clinical validation of the LUMIPULSE SARS-CoV-2 Ag kit. This antigen test is commercially supplied by Fujirebio, Inc.

(Tokyo, Japan) and it was recently approved as an in vitro diagnostic test for COVID-19 on June 19, 2020, in Japan. Compared with the RT-qPCR test, this antigen test can process 60-120 samples in 30 min per run on an automated machine, which greatly shortens the turnaround time. This test could, therefore, be used as a routine high-throughput test in a hospital setting, especially during a pandemic situation.

There are some limitations of the antigen test. The antigen test has low sensitivity compared with RT-qPCR being able to detect a lower SARS-CoV-2 titer by means of the PCR amplification process. However, the antigen test accurately detected SARS-CoV-2 in all samples with > 100 copies/test. Where samples had a viral load of < 100 copies as quantitated by RT-qPCR, the sensitivity of the antigen test decreased. Second, the presence of SARS-CoV-2 antigen does not necessarily mean the presence of viable virus. We should carefully consider whether SARS-CoV-2 antigen-positive patient is infectious to other persons.

The viral load tended to be higher at the onset of infection, which is when human-to-human transmission is at its highest [20, 21] . Epidemiologically, one of the key issues is finding asymptomatic and presymptomatic "super spreaders" to prevent community and nosocomial infection [22] . Super spreaders are more likely to be high viral load carriers. Along with viral loads, super spreader would go out with a lot of contact, be close distance with other people and talk loudly without waring mask. The clusters of COVID-19 were reported at closed environments, which would contribute to secondary transmission and promote super-spreading events [23] . In this context, our results revealed that the antigen test could be used to identify COVID-19-infected individuals who pose a high risk of transmission.

According to the guidelines of the Japanese government, saliva, as well as nasopharyngeal swabs, can be used for testing with the LUMIPULSE SARS-CoV-2 Ag kit. Notably, the self-collection of saliva may decrease the infection risk of healthcare workers [11] . The Japanese government recommends that the antigen test and nucleic acid amplification test of saliva be applied to symptomatic patients within 9 days of onset when viral loads are high.

In the USA, the Sofia SARS Antigen Fluorescent Immunoassay (FIA) Notably, our results for seven patients who were followed from the time of admission to that of hospital discharge suggested that the SARS-CoV-2 antigen levels declined in these consecutively collected samples. This implied that antigen levels could be used to distinguish between the early and late phases of the COVID-19 clinical course. The stable trend in the serial antigen test results contrasted with the abrupt changes observed when using RT-qPCR, which often showed mixed "negative" and "positive" results for the same sample. This may confuse clinicians when they wish to investigate treatment effects or the timing of discharge, for example. Thus, the LUMIPULSE antigen test may offer a wide In summary, both RT-qPCR and LUMIPULSE antigen test quantitatively measure virus RNA and antigen level, respectively. Therefore, we could investigate monitor the clinical condition of COVID-19 patients using these tests.

Furthermore, both tests are expected to identify the asymptomatic or presymptomatic SARS-CoV-2 infected persons who are likely to have high viral loads. Combination assay will help us to estimate the infection phase of COVID-19 patients in routine clinical practice. and all of the medical and ancillary hospital staff and the patients for consenting to participate. We thank Natasha Beeton-Kempen, Ph.D., from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.

YH reports receiving grant support from Fujirebio; SY and SK, being employed by Fujirebio. No other potential conflict of interest relevant to this article was reported.

YH contributed to study design, data collection, data analysis and writingreview (C) Comparison of data obtained with the Ag test and RT-qPCR. An overall agreement of 91.4% was achieved between the two tests, with 55.2% sensitivity and 99.6% specificity obtained with the Ag test.

A positive correlation (R² = 0.768) was observed between the SARS-CoV-2 antigen (Ag) level (log10 pg/mL) and the viral titer (log10 copies/test). J o u r n a l P r e -p r o o f

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This study was supported by a Grant-in-Aid for the Genome Research Project from Yamanashi Prefecture (to M.O. and Y.H.), the Japan Society for the

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.