key: cord-0735323-ti2df6w1 authors: Matsuzaki, Nanako; Orihara, Yuta; Kodana, Masahiro; Kitagawa, Yutaro; Matsuoka, Masaru; Kawamura, Rieko; Takeuchi, Shinichi; Imai, Kazuo; Tarumoto, Norihito; Maesaki, Shigefumi; Maeda, Takuya title: Evaluation of a chemiluminescent enzyme immunoassay‐based high‐throughput SARS‐CoV‐2 antigen assay for the diagnosis of COVID‐19: The VITROS® SARS‐CoV‐2 Antigen Test date: 2021-08-12 journal: J Med Virol DOI: 10.1002/jmv.27153 sha: c51842abf307bfb3002360a47079e77e3d1ea04e doc_id: 735323 cord_uid: ti2df6w1 A high‐throughput, fully automated antigen detection test for SARS‐CoV‐2 is a viable alternative to reverse‐transcription polymerase chain reaction (RT‐qPCR) for mass screening during outbreaks. In this study, we compared RT‐qPCR for viral load and the VITROS® SARS‐CoV‐2 Antigen Test with reference to the results of the LUMIPULSE® SARS‐CoV‐2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS‐CoV‐2, 49 were positive and 79 were negative according to RT‐qPCR. Consistent dose‐dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA‐based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID‐19 patients in the clinical setting. Highlights The performance of the VITROS® SARS‐CoV‐2 Antigen Test was sufficient for the diagnosis of contagious COVID‐19. This test showed high sensitivity and specificity in the detection of SARS‐CoV‐2 in samples with a Ct value of 32 or less. a two-step chemiluminescent enzyme immunoassay (CLEIA) and can be used to perform up to 130 tests per hour for the detection of active SARS-CoV-2 infection. This is a fully automated antigen detection test designed to detect active viral infection and is the first high-throughput COVID-19 antigen test to receive Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA). Recently, Favresse et al. 6 demonstrated that the VITROS ® SARS-CoV-2 Antigen Test completely aligned with RT-qPCR for Ct values up to 33 and found that the test had high specificity (100%). However, evaluation in the clinical setting remains limited, so further studies are needed to confirm the real-world effectiveness of this technology. In this study, we compared RT-qPCR in terms of viral load with two rapid antigen tests, namely, the VITROS ® SARS-CoV-2 Antigen Test and the CLEIA-based LUMIPULSE ® SARS-CoV-2 Ag Test (Fujirebio), to investigate their performance in the analysis of clinical Np swab specimens. 7 Np swab specimens were collected from patients suspected of being infected with SARS-CoV-2 at Saitama Medical University Hospital, Saitama, Japan, between December 22, 2020, and January 7, 2021. Each specimen was well suspended in 1000 μl of phosphate-buffered saline (PBS) and used for the following analysis of COVID-19. Asymptomatic carriers were defined as confirmed COVID-19 patients with no history of clinical signs or symptoms on admission. The day of onset was defined as the first day of symptoms caused by COVID-19 in symptomatic patients or the day of the first positive RT-qPCR result using Np swab specimens in asymptomatic carriers. The definitive diagnosis of COVID-19 was confirmed by RT-qPCR according to the nationally recommended protocol, using RNA extracted from 140 μl of the abovementioned suspension. 8 Positive RNA controls were prepared in 10-fold serial dilutions ranging from 5.0 × 10 5 to 5.0 × 10 0 copies/reaction using Vitro synthesized SARS-CoV-2 RNA, which was kindly provided by the National Institute of Infectious Diseases, Japan. A calibration assay was carried out in parallel to create a calibration curve by RT-qPCR. 8 The VITROS ® SARS-CoV-2 Antigen Test was also performed Table 1 summarizes the antigen detection rates for each quantified viral load from Np swab specimens according to the two tests. The detection rate was 85.5% (34/40) for 50.0 copies/reaction or over, 96.8% (30/31) for 250.0 copies/reaction or over, and 100% (29/29) for 500.0 copies/reaction or over in the VITROS ® SARS-CoV-2 Antigen Test. The detection rate was 97.5% (39/40) for 50.0 copies/reaction or over, 100% (31/31) for 250.0 copies/reaction or over, and 100% (29/29) for 500.0 copies/ reaction or over in the LUMIPULSE ® SASR-CoV-2 Ag Test. To assess the sensitivity of these assays according to the number of days after onset, 49 COVID-19 patients were divided into two groups: 9 days or under after onset and over 10 days after onset. The positivity rate of each period wwas analyzed and the results are shown in Table 2 . The VITROS ® SARS-CoV-2 Antigen Test can detect SASR-CoV-2 within 48 min and is capable of processing up to 130 Np swab specimens per hour. In this study, we demonstrated that the sensitivity of the VITROS ® SARS-CoV-2 Antigen Test was 100% in samples with Ct values below 32.0. It should be noted that, unlike the LUMI-PULSE ® SASR-CoV-2 Ag Test, the VITROS ® SARS-CoV-2 Antigen Test exhibits high specificity without the need for additional pretreatment procedures such as high-speed centrifugation. The VITROS ® SARS-CoV-2 Antigen Test realizes high-throughput and rapid testing, does not require skilled technicians or multistep procedures, and can be performed using equipment already installed in many laboratories. Thus, this test is a viable alternative to RT-qPCR and is suitable for mass screening during outbreaks. Previous reports demonstrated that a "positive" NAA result including RT-qPCR reflects only the detection of viral RNA fragments and does not always indicate the presence of viable virus particles. 9 This is a clinically important point. In a larger cohort, Singanayagam et al. 10 reported that 8% of samples with Ct values above 35.0 were positive for virus culture. In addition, previous reports have also demonstrated that high Ct levels were associated with noninfectious SARS-CoV-2. 9, 11 In our study, the VITROS ® SARS-CoV-2 Antigen Test exhibited 100% sensitivity in Np swab specimens with a viral load above 500 copies/reaction or Ct values below 32.0, as reported previously. 6 However, the CLEIA-based LUMIPULSE ®◻ SASR-CoV-2 Ag Test was able to detect lower viral loads compared with the VITROS ® SARS-CoV-2 Antigen Test, but there was no statistically significant difference between the two tests ( Evaluating the clinical utility and sensitivity of SARS-CoV-2 antigen testing in relation to RT-PCR Ct values Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv Symptomatic reinfection of SARS-CoV-2 with spike protein variant N440K associated with immune escape Phylogenomic evidence of reinfection and persistence of SARS-CoV-2: first report from Colombia Duration of isolation and precautions for adults with COVID-19 Head-to-head comparison of rapid and automated antigen detection tests for the diagnosis of SARS-CoV-2 infection Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients Development of genetic diagnostic methods for novel coronavirus 2019 (nCoV-2019) in Japan Virological assessment of hospitalized patients with COVID-2019 Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19 Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and metaanalysis Effect of remdesivir vs standard care on clinical status at 11 days in patients with moderate COVID-19: a randomized clinical trial Evaluation of a chemiluminescent enzyme immunoassay-based high-throughput SARS-CoV-2 antigen assay for the diagnosis of COVID-19: the VITROS® SARS-CoV-2 Antigen Test Positivity rates of SARS The authors declare that there are no conflict of interests. The study design and protocol were reviewed and approved by the The data that support the findings of this study are available from the corresponding author upon reasonable request. http://orcid.org/0000-0002-8416-7371Takuya Maeda http://orcid.org/0000-0003-0912-4643