Molecular tests are the standard laboratory diagnosis to confirm SARS-CoV-2 infection; RT-PCR assays for SARS-CoV-2 RNA detection in clinical specimens are widely used in COVID-19 diagnostic laboratories. There were 183 clinical laboratories in Thailand, including our laboratory at Siriraj Hospital that passed the external quality control of RT-PCR tests by the Department of Medical Science (DMSC), Ministry of Public Health, and was authorized a COVID-19 diagnostic laboratory [15, 16]. Rapid antigen immunoassays with equivalent sensitivity and specificity to real-time RT-PCR assays will help speed up disease screening. In this study, the commercially available rapid SARS-CoV-2 antigen detection kit (Standard Q COVID-19 Ag test) was compared with the RT-PCR assay (Allplex™ 2019-nCoV Assay) for detection of SARS-CoV-2 infection.
The sensitivity and specificity of the Standard Q COVID-19 Ag test for rapid detection of SARS-CoV-2 antigen reported by the manufacturer (total n = 202; positive n = 32; negative n = 170) were 84.38% (95% CI, 67.21–94.72%) and 100.00% (95% CI, 97.85–100%), respectively. The sensitivity of this test was evaluated at a trial site in Malaysia using 32 RT-PCR-positive nasopharyngeal swabs from symptomatic patients. The specificity of this test was evaluated by the R&D team of SD Biosensor using 170 RT-PCR-negative samples. The monoclonal antibody specific to SARS-CoV-2 N antigen coated on the Standard Q COVID-19 Ag test was produced from WUHAN-01 strain, which is genetically closely related to the SARS-CoV-2 strains detected in Thailand [17, 18]. Our results showed higher sensitivity (98.33% vs. 84.38%) but less specificity (98.73% vs. 100.00%) than the manufacturer’s results. The difference in our test performance from the manufacturer could be due to various factors, including the batch of kit reagents, the sample quality and level of extracted antigen, and sample handling and processing techniques. We reduced the sample viscosity using glass beads and vortexing before adding to the extraction buffer. The filter nozzle cap provided in the kit also minimized the glutinousness of the samples. A negative test result could be due to lower levels of extracted antigen than the test’s detection limit. Our batch of clinical specimens might generally have higher viral loads (low Ct-value) than that of the manufacturer’s trial site, which enhanced the chance of antigen detection in our study.
Of 60 RT-PCR-positive samples in our study, the sole false negative result was from the NP and throat swab of a female patient with pneumonia tested for SARS-CoV-2 antigen seven days after disease onset (RT-PCR-positive case no.39). The RT-PCR result of this sample had relatively high Ct-values: 31.18 for E gene, 39.2 for RdRp gene, and 35.54 for N gene, which may explain the negative result of the Standard Q COVID-19 Ag test. However, the Standard Q COVID-19 Ag test correctly detected SARS-CoV-2 antigen from another female patient who also had relatively high Ct-values: 33.49 for E gene, 36.94 for RdRp gene, and 37.17 for N gene (RT-PCR-positive case no.23). This patient was presented with upper respiratory tract infection (URI) and was tested four days after symptom onset [see Additional file 1]. SARS-CoV-2 viral load in upper respiratory specimens was detected at a higher level soon after the symptom onset ; thus, a higher chance of positive antigen detection at the early phase can be implied. This SARS-CoV-2 antigen detection kit might be recommended for patients at the early time point after symptom onset where higher viral loads are anticipated. As aforementioned, some other factors such as clinical manifestation, duration from disease onset to laboratory test, type of specimens, and how the specimens were collected and processed (sample handling and processing techniques) potentially affect the result interpretation. Of 394 RT-PCR-negative samples from pre-operative cases, five NP and throat swabs were tested positive for SARS-CoV-2 antigen using the Standard Q COVID-19 Ag test. Although it is unclear what caused the discordant result, we observed that thick and highly viscous mucous tended to yield false positive results when tested with the antigen detection kit. For patients with negative SARS-CoV-2 detection by RT-PCR, clinical data (such as underlying diseases or infection with other pathogens) were not included in the study. Therefore, the possibility of cross-reactivity with other antigens cannot be excluded.
Our results showed higher sensitivity of the rapid SARS-CoV-2 antigen test (98.33% by Standard Q COVID-19 Ag test) than other rapid antigen tests previously reported. Previous studies reported a sensitivity of 93.9% (95% CI, 86.5–97.4%) by Fluorescence Immunochromatographic Assay for 2019-nCoV Ag Test (Bioeasy Biotechnology Co., Shenzhen, China), 50.0% by COVID-19 Ag Respi-Strip CORIS®, and 11.1–45.7% by BIOCREDIT COVID-19 Ag (BioVendor Research and Diagnostic Products) [10,11,12]. The positive and negative predictive values (PPV and NPV) of the assay could not be accurately calculated without the present population prevalence of COVID-19. However, there were five false positive samples tested by the Standard Q COVID-19 Ag test. We can estimate that in a low COVID-19 prevalence area, the PPV for this test is low. Hypothetically, in the 10% COVID-19 prevalence rate, the PPV vs NPV of the Standard Q COVID-19 Ag test would be 89.59% (95% CI, 78.27–95.37%) versus 99.81% (95% CI, 98.71–99.97%). While in the 1% COVID-19 prevalence rate, the PPV vs NPV of the Standard Q COVID-19 Ag test would be 43.91% (95% CI, 24.66–65.17%) versus 99.98% (95% CI, 99.88–100.00%). Thus, the Standard Q COVID-19 Ag test might be useful in the high prevalence area.
The advantage of the Standard Q COVID-19 Ag test as a screening for COVID-19 is its simple procedure and quick results with high NPV, but its disadvantage is low PPV in a low prevalence area. Thus, the nucleic acid test (NAT) for SARS-CoV-2 gene detection, which is more sensitive and specific than this lateral flow immunoassay, is still a standard test for COVID-19 diagnosis. Even with its limitations, the rapid SARS-CoV-2 antigen test can benefit all healthcare workers in managing infected individuals in time effectively, especially in rural and outbreak areas. Therefore, a prospective study of the rapid SARS-CoV-2 antigen test in these fields should be performed before the implementation.