The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases.
The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March–May 2020.
Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 98.33% (95% CI, 91.06–99.96%) and 98.73% (95% CI, 97.06–99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients.
The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.
The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide since its first recorded case in the city of Wuhan, China in December 2019. According to the COVID-19 Dashboard on August 31st, 2020 by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, over 25 million people in more than 200 countries have been infected and killed more than 840,000 [1,2,3]. It is expected that these numbers continue to rise, especially in populous countries such as the United States, Brazil, and India. In Thailand, the first documented cases of COVID-19 were two Chinese tourists arriving from the city of Wuhan on January 8th and 13th, 2020, respectively. As of August 31st, 2020, there have been 3,412 confirmed COVID-19 cases with 58 deaths; 2,444 cases were from local transmission [4, 5]. The Thai government mandated a 14-day State Quarantine for all travelers entering Thailand from abroad. Since May 26th, 2020, no new local transmission cases were documented; new confirmed COVID-19 cases were people who have tested positive while in State Quarantine after returning from abroad . SARS-CoV-2 infection causes asymptomatic and mild diseases more than severe pneumonia. Severe cases may develop acute respiratory distress syndrome (ARDS) and death with an average mortality rate of 6% (range 1–14.4%) [1, 3, 6].
The real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, which is the current standard test for laboratory diagnosis of SARS-CoV-2 infection, requires at least four hours of operation performed by skilled technicians. Therefore, rapid and accurate tests for SARS-CoV-2 screening are essential to expedite disease prevention and control, as well as screening during pre-operative management for invasive procedures [7,8,9]. Lateral flow immunoassays using monoclonal anti-SARS-CoV-2 antibodies, which target SARS-CoV-2 antigens, can be the complementary screening tests if their accuracy were comparable to that of the real-time RT-PCR assays [10,11,12,13].
Here, we evaluated a rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea) using 454 respiratory specimens. The performance of this lateral flow immunoassay was compared with the SARS-CoV-2 RT-PCR for viral gene detection assay, Allplex™ 2019-nCoV Assay (Seegene®, Korea). This evaluation is critical before the implementation of the rapid antigen test for screening of SARS-CoV-2 infected individuals.
This study was approved by the Institutional Review Board of the Faculty of Medicine Siriraj Hospital, Mahidol University (SIRB protocol 463/2563(IRB4); COA: Si 503/2020).
Respiratory samples, mainly nasopharyngeal and throat swabs, were collected from 454 suspected COVID-19 cases, including pre-operative patients at Siriraj Hospital, Mahidol University, Bangkok, Thailand, from March to May 2020. Samples were mixed in 2 mL of viral transport media (VTM), consisting of Hanks’ balanced salt, 0.4% fetal bovine serum, HEPES, antibiotic and antifungal agents. Samples were transported at 2–8 °C to the Microbiology laboratory, Siriraj Hospital, for processing within a few hours. All specimens were processed in biosafety level-3 (BSL-3) and biosafety level-2 enhanced (BSL-2 +) facilities with full personal protective equipment.
Viral RNA Extraction
MagLEAD 12gC automated extraction platform (Precision System Science, Chiba, Japan) was used to extract SARS-CoV-2 RNAs from 200 µL of nasopharyngeal and throat swabs. Extraction was performed according to the manufacturer’s instructions. Viral RNA was eluted with 100 µL buffer and used for RT-PCR assay.
SARS-CoV-2 RNA detection using real-time RT-PCR
Allplex™ 2019-nCoV Assay (Seegene, Korea), which targets envelope gene (E) of Sarbecovirus, and RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2, was used for SARS-CoV-2 RNA detection according to the manufacturer’s instructions. Briefly, 8 μL of extracted RNA was added to 5 μL of 5X Real-time One-step Buffer, 5 μL of 2019-nCoV MuDT Oligo Mix (2019-nCoV-MOM), 2 μL of Real-time One-step Enzyme, and 5 μL of RNase free water. The CFX-96 real-time thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for amplification. The conditions consisted of 1 cycle of 20 min at 50 °C, 1 min at 95 °C and followed by 45 cycles of 15 s at 94 °C, 30 s at 58 °C. The result was analysed using Seegene Viewer (Seegene, Korea), in which a cycle threshold value (Ct-value) < 40 for all three target genes was defined as a positive result.
Rapid SARS-CoV-2 antigen detection assay
Standard Q COVID-19 Ag test (SD Biosensor®, Chuncheongbuk-do, Republic of Korea) is a rapid chromatographic immunoassay for the detection of SARS-CoV-2 nucleocapsid (N) antigen in respiratory specimens. This rapid antigen test device has two pre-coated lines on the result window: control (C) and test (T) lines. The control (C) region is coated with mouse monoclonal anti-chicken Igγ antibody; the test (T) region is coated with mouse monoclonal anti-SARS-CoV-2 antibody against SARS-CoV-2 N antigen. Detectors for SARS-CoV-2 N antigen presented in the specimen are mouse monoclonal anti-SARS-CoV-2 antibody conjugated with color particles. The antigen–antibody color particle complex migrates via capillary force and is captured by the mouse monoclonal anti-SARS-CoV-2 antibody coated on the test (T) region. The colored test (T) line’s intensity depends on the amount of SARS-CoV-2 N antigen presented in the sample.
This rapid Ag test kit was used for the detection of SARS-CoV-2 antigen in respiratory samples in this study. Specimens were processed in biosafety level-3 (BSL-3) and biosafety level-2 enhanced (BSL-2 +) facilities. Five to ten glass beads were added to the samples in VTM tubes. For highly viscous samples, additional VTM was added to reduce the viscosity. Specimens were mixed using a vortex mixer to disrupt thick mucus. The 200 μL of each nasopharyngeal and throat swab specimen was added to the extraction buffer provided in the kit. The filter nozzle cap was pressed tightly onto the extraction tube. Three drops of the extracted sample were applied on a test device, and the test result was read in 15–30 min. For positive COVID-19 antigen result, two colored lines of control (C) and test (T) lines were presented.
Descriptive statistics were used to describe general information of patients. Continuous data were presented in mean, standard deviation (SD), median, and range. Categorical data were presented in numbers, percentages, and 95% confidence interval (95% CI). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) were calculated using an online statistical tool .
Characteristics of Thai COVID-19 cases
Suspected COVID-19 cases and contact individuals were laboratory-confirmed by the gold-standard RT-PCR assay as a national guideline for laboratory diagnosis of COVID-19 . A total of 454 respiratory samples, including 447 nasopharyngeal (NP) and throat swabs, four endotracheal aspirates (tracheal suctions), and three sputum samples, were collected from suspected COVID-19 cases and pre-operative patients at Siriraj Hospital from March to May 2020. These respiratory samples were collected from subjects with the following conditions: (1) asymptomatic and upper respiratory tract infection individuals who had contacted with confirmed cases or were from COVID-19 risk areas, (2) clusters with acute respiratory infections, (3) unknown causative agents of pneumonia, (4) travelers screened at a port of entry and in quarantine places, and (5) pre-operative patients. Of the samples tested for COVID-19 (n = 454) by real-time RT-PCR assay, Allplex™ 2019-nCoV Assay, 13.2% (n = 60) were positive, while 86.8% (n = 394) were negative for SARS-CoV-2 RNA, as shown in Additional file 1: Supplementary Table S1, Additional file 2: Table S2.
The median age of Thai COVID-19 cases (n = 60) was 38.5 years (range 21–72). Male patients were found to be 60% of the infected cases (n = 36). Of the total COVID-19 cases, 75% (n = 45) of patients had direct contact with a variety of confirmed cases in Thailand, such as family members and friends (30%; n = 18), people from karaoke bars and pubs (23.3%; n = 14), people from boxing stadiums (18.3%; n = 11), taxi drivers (1.7%; n = 1), and peers in workplaces (1.7%; n = 1), as shown in Table 1. Most patients showed signs and symptoms of upper respiratory tract infections (61.7%; n = 37). Around 8.3% (n = 5) of COVID-19 cases were presented with pneumonia and were admitted to an intensive care unit (ICU). The median time from onset to laboratory tests for SARS-CoV-2 infection (both RT-PCR and rapid antigen detection assays) was three days (range 0–14), as shown in Table 1 and Additional file 1: Supplementary Table S1.
Real-time RT-PCR and SARS-CoV-2 antigen assays
Real-time RT-PCR (Allplex™ 2019-nCoV Assay), which targets E of Sarbecovirus, and RdRp and N genes of SARS-CoV-2, was used for SARS-CoV-2 RNA detection. The average cycle threshold (Ct) values in COVID-19 positive cases were 22.79 ± 6.69 (min 10.49, max 35.02) for E gene, 24.73 ± 6.55 (min 13.41, max 39.20) for RdRp gene, and 26.09 ± 6.47 (min 12.07, max 37.17) for N gene, as shown in Table 1 and Additional file 1: Supplementary Table S1. The negative RT-PCR results were defined as having a Ct-value higher than 40 for all three target genes (E, RdRp, N).
We evaluated the performance characteristics of SARS-CoV-2 antigen detection (Standard Q COVID-19 Ag test). The results were interpreted as positive when both control (C) and SARS-CoV-2 antigen (T) lines appeared within 30 min, as shown in Fig. 1. Comparing SARS-CoV-2 antigen detection to RNA detection by RT-PCR assay, the sensitivity and specificity of rapid SARS-CoV-2 antigen detection to identify COVID-19 were 98.33% (59/60; 95%CI, 91.06–99.96%) and 98.73% (389/394; 95%CI, 97.06–99.59%), respectively, as shown in Table 2. Of six samples discordant with RT-PCR results, one was false negative, and five were false positive. There were three weakly positive and two positive results. The false negative sample’s Ct-values were 31.18 for E, 39.2 for RdRp, and 35.54 for N genes, as shown in Table 3 and Additional file 2: Supplementary Table S2.