ABSTRACT
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.
ABSTRACT
BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is generally diagnosed by reverse transcription (RT)-PCR or serological assays. The SARS-CoV-2 viral load decreases a few days after symptom onset. Thus, the RT-PCR sensitivity peaks at three days after symptom onset (approximately 80%). We evaluated the performance of the ARCHITECT® SARS-CoV-2 IgG assay (henceforth termed IgG assay; Abbott Laboratories, Lake County, IL, USA), and the combination of RT-PCR and the IgG assay for COVID-19 diagnosis. METHODS: In this retrospective study, 206 samples from 70 COVID-19 cases at two hospitals in Tokyo that were positive using RT-PCR were used to analyze the diagnostic sensitivity. RT-PCR-negative (N=166), COVID-19-unrelated (N=418), and Japanese Red Cross Society (N=100) samples were used to evaluate specificity. RESULTS: Sensitivity increased daily after symptom onset and exceeded 84.4% after 10 days. Specificity ranged from 98.2% to 100% for samples from the three case groups. Seroconversion was confirmed from 9 to 20 days after symptom onset in 18 out of 32 COVID-19 cases with multiple samples and from another case with a positive result in the IgG assay for the first available sample. CONCLUSIONS: The combination of RT-PCR and IgG assay improves the robustness of laboratory diagnostics by compensating for the limitations of each method.