key: cord-1022649-lwsxwc9g
authors: Aoki, Kotaro; Nagasawa, Tatsuya; Ishii, Yoshikazu; Yagi, Shintaro; Kashiwagi, Katsuhito; Miyazaki, Taito; Tateda, Kazuhiro
title: Evaluation of Clinical Utility of Novel Coronavirus Antigen Detection Reagent, Espline® SARS-CoV-2
date: 2020-12-23
journal: J Infect Chemother
DOI: 10.1016/j.jiac.2020.11.015
sha: 9d3dc6d5772e45cc4e03ef1b96c96d95ed928e44
doc_id: 1022649
cord_uid: lwsxwc9g

Background To prevent the novel coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to perform early identification and isolation of people shedding the infectious virus in biological materials with high viral loads several days prior to symptom onset. Rapid antigen tests for infectious diseases are useful to prevent the pandemic spread in clinical settings. Methods We evaluated a SARS-CoV-2 antigen test, Espline® SARS-CoV-2 reagent, with reverse transcription polymerase chain reaction (RT-PCR) as reference test, using 129 nasopharyngeal swab specimens collected from COVID-19 hospitalized patients or from patients suspected having COVID-19-like symptoms. Out of these, 63 RT-PCR positive and 66 RT-PCR negative specimens were identified. Results Among 63 RT-PCR positive specimens, 25 were positive in the Espline test. Test sensitivity was estimated based on the 532.4 copies/reaction of SARS-CoV-2 RNA obtained through receiver operating characteristic analysis. When the specimens were classified based on time since symptom onset, Espline test sensitivity were 73.3% and 29.2% in specimens collected before day 9 and after day 10, respectively. Conclusion Although the overall sensitivity of the Espline® SARS-CoV-2 reagent compared with RT-PCR is less, this antigen test can be useful in identifying people with high risk of virus transmission with high viral loads in order to prevent the pandemic and is useful for diagnosing COVID-19 within 30 minutes.

To prevent the novel coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to perform early identification and isolation of people shedding the infectious virus in biological materials with high viral loads several days prior to symptom onset. Rapid antigen tests for infectious diseases are useful to prevent the pandemic spread in clinical settings.

We evaluated a SARS-CoV-2 antigen test, Espline ® SARS-CoV-2 reagent, with reverse transcription polymerase chain reaction (RT-PCR) as reference test, using 129 nasopharyngeal swab specimens collected from COVID-19 hospitalized patients or from patients suspected having COVID-19-like symptoms. Out of these, 63

Coronavirus disease (COVID-19) is a respiratory disease caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] [2] [3] .

It became a global pandemic within a short period since the outbreak began in China in early 2020 [4] . It is reported that more than 17 million people have been infected with this virus and that more than 600,000 people had died till end of July 2020 [5, 6] .

To prevent viral spread, early detection and isolation of infected people who may be asymptomatic or presymptomatic is necessary [7] . Reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 genomic RNA is the main procedure used in viral identification [8] [9] [10] .

Though RT-PCR is a highly sensitive and specific assay, highly skilled personnel and special devices are required because it is prone to analyze contamination and degradation. In addition, the total cost and time required by RT-PCR are also issues.

Rapid antigen tests developed on the POCT device have been widely used in diagnosing infectious diseases such as influenza and has been effective in preventing pandemics despite the fact that these tests have less sensitivity compared to PCR [11] [12] [13] [14] .

SARS-CoV-2 infectious viruses shed from patients have been isolated from J o u r n a l P r e -p r o o f respiratory specimens collected from patients with high viral loads, as RT-PCR (RT-PCR) Ct values of these samples were below 25 within several days since symptom onset [15] [16] [17] [18] [19] [20] . It is possible that rapid antigen tests would be convenient in clinical settings, similar to their use in other infectious diseases.

Recently, rapid and easy SARS-CoV-2 antigen tests have been developed on an immunochromatography device [21] [22] [23] [24] [25] . Several reagents have been evaluated, and evaluation data have been published by the Foundation for Innovative New Diagnostics [26] . However, it is difficult to compare the performance of each since there are no standard materials for evaluation.

Espline ® SARS-CoV-2 reagent was recently developed and approved as an in-vitro diagnostic in Japan [23, 27] . Here we report the performance of this reagent using nasopharyngeal swab samples collected from in-patients, and evaluated this reagent in comparison with RT-PCR and in relation to the number of days since symptom onset.

J o u r n a l P r e -p r o o f (SPC) were used as one-step RT-qPCR master mixes. The primer set (NIID_2019-nCOV_N_F2 and NIID_2019-nCOV_N_R2) and FAM labeled probe (NIID_2019-nCOV_N_P2) were used as these were previously reported as targeting gene N [10, 28] . The detection limit value of the measurement system was set at 5 copies/reaction. Using AccuPlex™ SARS-CoV-2 Reference Material Kit (Seracare, U.S.A.) with known RNA copies, we examined the effect of using ETS on RNA extraction, and we estimated that the recovery rate of RNA from the specimen treated by ETS is approximately 30% by comparison with that obtained using the reference material diluted in UVT (data not shown). The RNA copies by RT-PCR were normalized with the reference materials.

Espline ® SARS-CoV-2 (Fujirebio Inc., Japan) is an immunochromatography assay based on sandwich enzyme immunoassay methods and makes use of monoclonal antibodies that recognize SARS-CoV-2 N antigen (N-Ag) [27, 29] . For UVT specimens, To estimate the viral loads required for antigen detection, receiver operating characteristic (ROC) analysis was done with SARS-CoV-2 RNA titers and based on the concordance of the antigen test and RT-PCR test results. The value obtained using the minimum distance method was 532.4 copies/mL (Fig1-B) . Specimens with higher RNA titers than this number were collected from days earlier than J o u r n a l P r e -p r o o f symptom onset compared to those specimens with lower RNA copies. The median duration since symptom onset were 9.5 d, 16 d, and 19 d for specimens with both antigen and RT-PCR positive, those with antigen negative/RT-PCR positive, and those with both antigen and RT-PCR negative, respectively.

We examined the cumulative sensitivities and agreement rates of the antigen test using RT-PCR as the reference along with the collection days since the symptom onset. Both rates are peaking at 8 to 9 d (Fig2-A); therefore, we compared the cumulative sensitivities in specimens collected before 9 d and after 10 d since symptom onset. These rates were 73.3% (11/15) and 29.2% (14/48) in specimens collected before 9 d and after 10 d, respectively (Table 1) . RNA titers were also higher in specimens collected within 9 d since symptom onset compared to those collected after 10 d (Fig2-B) . The maximum and minimum SARS-CoV-2 RNA titers of four RT-PCR positive/antigen-negative specimens collected within the 9 d were 1,320 and 7, respectively (Fig2-C). Antigen test sensitivities of 77.3% (17/22) and 90.9% (10/11) were observed when specimens were classified by their RNA titers at 523.4 copies/mL and when they were classified by both RNA titer and the days of specimen collection before 9 d, respectively (Fig2-C). Espline ® SARS-CoV-2 reagent could be useful for detecting SARS-CoV-2 infected persons efficiently in specimens J o u r n a l P r e -p r o o f collected within 9 d since symptom onset. Viral loads of these specimens were higher than 500 copies/reaction of the SARS-CoV-2 RNA (corresponding to Ct values less than 30).

Espline ® SARS-CoV-2 reagent exhibited approximately 40% overall sensitivity, which was lower than those previously reported [21, 22] . We assume that differences in sensitivity reflect the differences in specimen sets between the clinical studies.

Median Ct values of antigen-positive specimens, antigen-positive/RT-PCR positive specimens, and specimen collection days since symptom onset used in this study were quite different from those in a previous study: most Ct values of the specimens were less than 25, and the previously used specimen collection day was 5 d after symptom onset. As such, it was estimated that the Espline test could give high sensitivity when it was evaluated using the same sample set used in the previous study.

This comparison provides important information: First, the duration since symptom onset greatly affects sensitivity of antigen testing. SARS-CoV-2 viral load is highest several days after symptom onset and decreases rapidly [15, 17, 30] . The J o u r n a l P r e -p r o o f cumulative sensitivity and agreement of the Espline test using RT-PCR as reference test were better before 9 d than after 10 d (Fig2-A) . It also reported that infectious virus could be isolated from specimens with high viral loads collected 8 to 9 d since symptom onset [16, 20, 31, 32] . In addition, asymptomatic carriers with high viral loads would act as the epicenter for viral spread [30] . These findings suggest that the antigen test would be very effective in the rapid identification of virus carriers to prevent future epidemics.

We observed four RT-PCR-positive/antigen-negative cases in patients exhibiting COVID-19 symptoms 9 d since symptom onset (Table 1 ). Viral loads of three of these cases were very low, with less than 100 copies/reaction in the SARS-CoV-2 RNA titers, suggesting that transmission risk from these patients is low; however, using the rapid antigen test should not exclude the use of nucleic acid testing for COVID-19 diagnosis. Although two discrepancies between antigen-positive/RT-PCR negative specimens might be caused by false-positive reaction of the Espline test or by false-negative reaction of RT-PCR, it should be noted that these two discrepancies were collected from COVID-19 patient specimens, of which specimens obtained at previous observation points were RT-PCR positive, indicating that antigen testing gave the correct COVID-19 diagnosis in these cases.

Our study has some important limitations, including limited number of specimens collected before 5 days since symptom onset and bias might have existed in in-house developed RT-PCR test based on the standard protocol established in NIID, which would affect sensitivity and cutoff analysis in this study. We think that the international standards for antigen and RNA, and the panels of specimens would be required for proper evaluation of COVID-19 tests.

In conclusion, the sensitivity of Espline ® SARS-CoV-2 reagent is limited in comparison with RT-PCR. This rapid antigen test is useful as it does not require a special device, it can diagnose COVID-19 within 30 minutes, and it can identify people with a high risk of virus transmission in order to prevent pandemics. Days since symptom onset SARS-CoV-2 RNA titer (Log(RNA copies/reaction)) (C) p=9.70x10 -4 Days since symptom onset Cumulative agreement rates

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