key: cord-0961034-t6q4tjbl
authors: Aoki, Kotaro; Nagasawa, Tatsuya; Ishii, Yoshikazu; Yagi, Shintaro; Okuma, Sadatsugu; Kashiwagi, Katsuhito; Maeda, Tadashi; Miyazaki, Taito; Yoshizawa, Sadako; Tateda, Kazuhiro
title: Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs
date: 2020-12-03
journal: J Infect Chemother
DOI: 10.1016/j.jiac.2020.11.021
sha: 62c29c01a0c9b6d1f6534d2313bab96cfd0cd3b3
doc_id: 961034
cord_uid: t6q4tjbl

Background Expansion of the testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important issue to mitigate the pandemic of coronavirus disease-2019 (COVID-19) caused by this virus. Recently, a sensitive quantitative antigen test (SQT), Lumipulse® SARS-CoV-2 Ag, was developed. It is a fully automated chemiluminescent enzyme immunoassay system for SARS-CoV-2. Methods In this study, the analytical performance of SQT was examined using clinical specimens from nasopharyngeal swabs using reverse transcription polymerase chain reaction (RT-PCR) as a control. Results Receiver operating characteristic analysis of 24 SARS-CoV-2-positive and 524 -negative patients showed an area under the curve of 0.957±0.063. Using a cut-off value of 1.34 pg/ml, the sensitivity was 91.7%, the specificity was 98.5%, and the overall rate of agreement was 98.2%. In the distribution of negative cases, the 99.5 percentile value was 1.03 pg/ml. There was a high correlation between the viral load calculated using the cycle threshold value of RT-PCR and the concentration of antigen. The tendency for the antigen concentration to decrease with time after disease onset correlated with that of the viral load. Conclusions Presented results indicate that SQT is highly concordant with RT-PCR and should be useful for the diagnosis of COVID-19 in any clinical setting. Therefore, this fully automated kit will contribute to the expansion of the testing capability for SARS-CoV-2.

Since December 2019, diseases caused by the novel coronavirus 2019, severe acute respiratory coronavirus 2 (SARS-CoV-2), emerged in China and rapidly spread throughout the world. The World Health Organization stated that the coronavirus disease-2019 (COVID-19) pandemic is not close to ending and is still a serious public health threat [1] . An analysis of viral load kinetics indicated that access to SARS-CoV-2 testing as well as the frequency and rapidity of tests should be prioritized to control the spread of SARS-CoV-2 [2].

Currently, nucleic acid testing (NAT) using reverse transcription polymerase chain reaction (RT-PCR) has been used for the diagnosis of COVID-19. However, its complicated operation, relatively long time to obtain results (several hours to several days), and cost are issues that have prevented the expansion of NAT for the general testing of COVID-19 in several countries. Rapid antigen tests (RAT) are expected to address these issues; however, recent studies have reported their high specificity and poor sensitivity compared with 4] . In addition, RAT is a qualitative test and should not be used to estimate viral loads when monitoring the status of virus replication.

Recently, a sensitive quantitative antigen test (SQT), "Lumipulse® J o u r n a l P r e -p r o o f SARS-CoV-2 Ag", was developed based on a fully automated chemiluminescent enzyme immunoassay system that enables the rapid and multiple testing of SARS-CoV-2 N antigens in nasopharyngeal swabs or saliva.

In this study, we investigated the performance of the SQT and compared it with that of RT-PCR using clinical samples.

Five hundred and forty-eight nasopharyngeal swab specimens were collected from COVID-19 patients or suspected patients with COVID-19 hospitalized in Omori Hospital, who had been diagnosed by RT-PCR. Some samples were collected from the same patients at multiple time points. Informed consent was obtained from all participants in the study. Specimens were stored at 80°C and did not melt during storage (within 2 months). The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Toho University (No. A20028_A20020_A20014_A19099).

RT-PCR analysis was performed according to the "Pathogen Detection (NIID_2019-nCOV_N_F2 and NIID_2019-nCOV_N_R2) and FAM labeled probe (NIID_2019-nCOV_N_P2) were used as they were previously reported to target gene N [5]. The detection limit value of the measurement system was set at 5 copies/reaction. The RNA copy number was calculated using the cycle threshold (Ct) value of the AccuPlex SARS-CoV-2 reference (SeraCare, MA, USA) using known RNA copies.

SARS-CoV-2 antigen was measured using the LUMIPULSE SARS-CoV-2 Ag kit and the LUMIPULSE G1200 automated immunoassay analyzer according to the manufacturer's instructions (Fujirebio Inc., Tokyo, Japan) using nasopharyngeal swab specimens frozen after RT-PCR. To remove viscous materials from the samples, J o u r n a l P r e -p r o o f specimens were centrifuged at 1,300 ×g for 10 min, and the supernatants were used for the analysis. First, 100 μL of the supernatant was added to the treatment solution and consecutively aspirated using a single tip. Then, the mixture was dispensed into an anti-SARS-CoV-2 Ag monoclonal antibody-coated magnetic particle solution and incubated for 10 minutes at 37°C. After the first washing step, alkaline phosphatase-conjugated anti-SARS-CoV-2 Ag monoclonal antibody was added and incubated for 10 minutes at 37°C. After another washing step, the substrate solution was added and incubated for 5 minutes at 37°C. The resulting reaction signals were proportional to the amount of SARS-CoV-2 Ag in the sample, which allowed the quantitative determination of SARS-CoV-2 Ag in the nasopharyngeal swabs. When the antigen levels were greater than the upper range of the measurement (5,000 pg/mL), we tested samples diluted with Lumipulse® sample diluent (Fujirebio Inc.). University, Saitama, Japan), which is a graphical user interface for R and Excel (Microsoft Corporation, Redmond, WA, USA). Receiver operating characteristic (ROC) curve analysis was conducted using R to evaluate the assay performance and to visualize the curves, and areas under the ROC curves (AUC), sensitivity, specificity and the overall percent agreement were calculated. Because the negative group distribution did not follow a normal distribution or have a lognormal distribution, a nonparametric method (99.5 percentile) was used to establish a negative reference value.

The ROC analysis of SQT by RT-PCR showed an AUC of 0.957 ± 0.063 and a cut-off value of 1.34 pg/mL using Youden's Index ( Figure 1A ). The distribution of antigen levels in 510 samples collected from persons without a history of COVID-19, whom had been diagnosed by RT-PCR, is shown in Figure 1B . The 99.5 percentile determined by a nonparametric method was 1.03 pg/mL, and was sufficiently below the cut-off value. Using this cut-off value, the SQT had a sensitivity of 91.7%, a J o u r n a l P r e -p r o o f specificity of 98.5%, and overall rates of agreement of 98.2%.

There were eight RT-PCR-negative and SQT-positive cases (1.5%) (Table 1, Figure 2B ). In all these cases, specimens were collected several times and were from patients with COVID-19 history. We examined the dilution-linearity of five available samples to eliminate the possibility of non-specific interference and variation in the measurements [6]. In the 5-fold dilution measurement, the antigen concentration was decreased approximately in proportion to the degree of dilution in each case ( Table 2) . The corresponding values were obtained when the samples were diluted 5-fold for cases of discrepancy, suggesting that the SQT measures the antigen level accurately.

However, there were two cases with PCR-positive and SQT-negative findings (Table 1, Figure 2B ). These two specimens were collected at 2 and 10 days after the onset of symptoms (initial sample collection); the LAMP method for RNA detection gave a negative result for one of these specimens (data not shown) [7] . because these values differ between testing kits and between facilities. Monitoring antigen levels can provide a more rapid and stable quantitative basis for infection control, treatment progress, and discharge decisions. It might also be possible to quantitatively determine the likelihood of relapse.

The results of this study showed that SQT had a high concordance rate with J o u r n a l P r e -p r o o f the RT-PCR method, and even samples with a low viral load showed positive results with SQT. In addition, the antigen concentration correlated well with the RNA titer.

These data suggest that SQT might replace RT-PCR for the diagnosis of COVID-19.

Using SQT, the antigen concentration can be obtained in approximately 30 minutes using a fully-automated analyzer with a throughput rate of 60 or 120 samples per hour, which will contribute to the expansion of the testing capability for SARS-CoV-2. The cost of SQT is lower than that of NAT, and the insurance points of SQT (600) is less than half of that of NAT (1350 or 1800) in Japan. Because the SQT can provide test results in a short period of time (30 minutes), it is particularly useful in facilities where it is difficult for many people to stay for a long time, such as airports.

Because false-positive results are likely to occur when sputum is used for antigen tests [10] , especially if the viscosity of the sample is high or impurities are present, centrifugation might be an effective method to avoid false-positive results. Several discrepancies were found in this study. Although limitations in the volume and/or concentration of samples hindered further analysis by western blotting or different NAT tests, the dilution-linearity test suggested a specific immunoreaction for measurements. Further analysis of J o u r n a l P r e -p r o o f discrepant specimens will help the manufacturer improve the SQT.

Correlation analysis between the antigen test and RT-PCR showed low variability in the correlation between antigen concentration and RNA titer in samples with high viral loads, and scattered correlation patterns were observed in samples with low viral loads, which were collected from patients at later periods after symptom onset. Virological factors that might affect these observations are not clear; however, we think that the accuracy and precision of the measurements might affect the correlation patterns because the SQT showed high linearity of dilution with a coefficient of variation less than 10% (Table 2) [11]. In addition, differences in the analyte stability in samples might affect the scattered correlation pattern.

Generally, proteins are more resistant to degradation than RNAs [12] [13] [14] [15] . In some cases, intracellular viruses do not replicate as RNA but only produce antigenic proteins [16, 17] . Differences in stability or viral production of the antigen protein

Coronavirus disease (COVID-19) Pandemic: World Health Organization

Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance medRxiv