Abstract
Nucleic acid and antibody detection assays have been utilized in COVID-19 laboratory diagnosis. A leader in PCR is Thermo Kingfisher Flex and 3DMed. However, the use of viral antigenic proteins for diagnosis has not been successfully developed. Using viral antigen allows rapid direct viral detection earlier than production of antibodies. The present study was aimed at evaluating the performance of two COVID-19 rapid antigen detection tests, which are BIOCREDIT COVID-19 Ag (RapiGEN Inc., Korea) and Standard Q COVID-19 Ag (SD Biosensor, Korea), in comparison with RT-PCR. These tests were performed on 80 COVID-19 RT-PCR positive respiratory samples and 20 RT-PCR negative control samples. BIOCREDIT COVID-19 Ag and SD Biosensor RAD kits recorded total sensitivities of 52.5% and 68.7% and specificities of 46% and 96%, respectively. In high viral load samples, BIOCREDIT COVID-19 Ag and SD Biosensor RAD kits recorded higher sensitivities of 60% and 77%, compared to 45% and 60% in normal viral load samples, respectively. Sensitivity and specificity of the 2 antigen kits varied significantly with P values of <0.000001 and 0.0135, respectively. The evaluated RAD tests presented promising performance which was relatively better for SD-Biosensor than BIOCREDIT RAD tests, especially in high viral load samples. However, antigen tests are still considered substandard in comparison with RT-PCR in detecting SARS-CoV-2.

BACKGROUND:

The rapid diagnosis of Coronavirus Disease 2019 (COVID-19) patients is essential to reduce the disease spread. Rapid antigen detection (RAD) tests are available, however, there is scanty data on the performance of RAD tests.

OBJECTIVE:

To evaluate the performance of the commercially available BIOCREDIT COVID-19 Ag test and compare it with RT-PCR for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus. Analytical sensitivity for the detection of SARS-CoV-2 virus was determined for the RAD test using viral  culture and RT-PCR as reference methods. The RAD test was further evaluated using respiratory samples collected from confirmed COVID-19 patients. The results were compared with RT-PCR test.

RESULTS:

The detection limits between RAD test, viral culture and RT-PCR varied hugely. RAD was 103 fold less sensitive than viral culture while RAD was 105 fold less sensitive than RT-PCR. The RAD test detected between 11.1 % and 45.7 % of RT-PCR-positive samples from COVID-19 patients.

CONCLUSIONS:

This study demonstrated that the RAD test serves only as adjunct to RT-PCR test because of potential for false-negative results.

General information:

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the name given to the 2019 novel coronavirus. COVID-19 is the name given to the disease associated with the virus. SARS-CoV-2 is a new strain of coronavirus that has not been previously identified in humans. The 2019-Novel Coronavirus belongs to the genus β. It has a capsule, and its particles are round or oval, often polymorphous, with a diameter of 60-140nm. Its genetic characteristics are significantly different from SARS-CoV and MERS-CoV.

BIOCREDIT COVID-19 Ag is a rapid immunochromatographic test for in vitro qualitative detection of coronavirus SARS-CoV-2 antigen from nasopharyngeal samples.

Reagents and materials included:

  1. Each test device sealed in a foil pouch with a desiccant
  2. Assay diluent tubes
  3. Filter caps
  4. Sterilized swabs for nasopharyngeal specimen collection
  5. Instructions for use
Biocredit Antigen
Biocredit Antigen

Introduction

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new type of coronavirus belonging to the genus β. On 11 Mar 2020, World Health Organization (WHO) declared Coronavirus Disease 2019 (COVID-19) as a pandemic [1]. Viral culture and RT-PCR are the gold standards in the diagnosis of SARS-CoV-2 infection [2]. However, it takes hours to detect the nucleic acid and days to isolate the virus. In addition, specialized instrument and expertise are required. For rapid diagnosis of SARS-CoV-2 infection, rapid antigen detection (RAD) tests for qualitative determination of SARS-CoV-2 antigen are available. RAD tests detect viral antigen by the immobilized coated SARS-CoV-2 antibody on the device. The test results of RAD can be interpreted without specialized instrument and available within 30 min. Hence, RAD tests can relieve the workload in diagnostic hospitals and laboratories and improve the turn-around time. However, according to WHO, the role of RAD tests for antigen detection for SARS−COV-2 needs to be evaluated and is not recommended for clinical diagnosis.

The purpose of this evaluation is to assess the diagnostic use of the commercially available BIOCREDIT COVID-19 Ag test. The aim of the first part of the study was to assess the limit of detection (LOD) between RAD test, viral culture and RT-PCR and the second part was to evaluate performance of RAD test in detecting SARS-CoV-2 virus in different types of respiratory samples.

2. Methods

2.1. Respiratory isolates

The SARS-CoV-2 culture isolate (strain hCoV-19/Hong Kong/VM20001097/2020, the first COVID-19 case detected in Hong Kong) was used to perform a serial tenfold dilution to determine LOD between different assays.

To evaluate the cross-reactivity of the RAD test, 13 non-SARS-CoV-2 respiratory virus isolates were tested. They were influenza A(H1pdm09), influenza A(H3), influenza B, adenovirus, coronavirus type OC43, coronavirus type 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus, rhinovirus and enterovirus.

2.2. Respiratory samples

From February 1, 2020 to April 21, 2020, respiratory samples from individuals confirmed with SARS-CoV-2 infection by RT-PCR targeting the SARS-CoV-2 virus–specific RdRp gene were retrieved for this evaluation. Samples were placed in viral transport media (VTM) or Phosphate-Buffered Saline (PBS) for RNA extraction. The remaining part of the suspension was stored at −70 °C until use in this study. Virus concentrations in samples were estimated from cycle threshold (Ct) value.

The Public Health Laboratory Services Branch (PHLSB) in Hong Kong has been designated as WHO COVID-19 reference laboratory since April 2020 and all confirmed cases in Hong Kong were either diagnosed or confirmed by PHLSB [4]. Total number of 368 confirmed COVID-19 samples collected with sufficient quantity were available for this study in descending order: throat saliva (N = 122), nasopharyngeal swab and throat swab (NPS & TS, N = 103), nasopharyngeal aspirate and throat swab (NPA & TS, N = 81), sputum (N = 62).

2.3. RAD kits for SARS−CoV-2 detection

We evaluated the only commercially available RAD kit in Hong Kong at the time of starting the evaluation, BIOCREDIT COVID-19 Ag for the diagnosis of SARS-CoV-2 infection.

2.4. BIOCREDIT COVID-19 Ag

The intended use for the BIOCREDIT COVID-19 Ag kit is for nasopharyngeal swab sample. As NPA & TS, NPS & TS, sputum and throat saliva had either been eluted in VTM or suspended in PBS, the test was carried out with modified sample processing methods.

2.5. Sample processing by BIOCREDIT COVID-19 Ag

The recommended sample volume by the BIOCREDIT COVID-19 Ag kit was 90–150 μL. To unify the sample volume, 100 μL sample volume was used. We evaluated two sample processing methods based on the nature of the samples.

2.5.1. For less viscous samples

These samples do not need preparation, 100 μL sample was added directly into a sample well of the device.

2.5.2. For viscous samples

The swab provided by the kit was used to collect the samples and the swab was immersed in the provided assay diluent tube. The subsequent procedures were carried out according to the manufacturer’s instructions.

In an effort to compare the performance of these two sample processing methods (i.e. methods for handling less viscous and viscous samples), LOD was determined using a serial tenfold dilution of virus. The results were then compared with viral culture and RT-PCR.

2.6. Viral culture for SARS-CoV-2 virus

Viral culture was conducted by inoculating samples onto Vero E6 cells. When virus-induced cytopathic effect was examined, identification of SARS-CoV-2 virus in culture fluid was confirmed by the RT-PCR.

2.7. RT-PCR for SARS-CoV-2 virus

The in-house developed RT-PCR was used to detect the presence of SARS-CoV-2 virus nucleic acid in all samples. It was conducted using NxtScript Enzyme and Master Mix (Roche Diagnostics GmbH, Germany). Each 10 μL reaction mixture contained 5 μL RNA samples, 2 μL Reaction Mix (5X), 0.06 μL Adpta Taq DNA polymerase (50U/μL), 0.05 μL NxtScript RT Enzyme (85U/μL), 0.9 μL volume of working primer/probe mix and nuclease-free water to obtain a final volume of 10 μL. The working primer/probe mix was prepared by mixing equal volume of forward primer, NCOV-F4: 5′-GTTGGACTGAGACTGACCTTAC-3′ (10 μM); reverse primer, NCOV-R4: 5′−CCCTAGGATTCTTGATGGATCTG-3′ (10 μM); and probe, NCOV-P4: 5′-FAM-ACAGGGTGATGATTATGTGTACCTTCCT-BHQ1−3’ (10 μM). The reverse transcription, amplification was performed in the LC480 System (Roche Diagnostics GmbH, Germany) according to the following program: 1 cycle of 50 °C for 10 min, 1 cycle of 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 56 °C for 30 s; and holding at 4 °C.

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