Severe Acute Respiratory Syndrome Coronavirus 2 Antigen Detection Kit

Background:

Rapid identification and effective isolation are crucial to slowing the spread of coronavirus 2, severe acute respiratory syndrome (SARS-CoV-2). To meet this requirement, rapid antigen detection diagnostic tests (Ag-RDT) are essential.

Methods:

Between February 2020 and August 2020 we conducted a cohort study of confirmed COVID-19 patients. The clinical performance of the Ag Fast Fluorescence Immunoassay (FIA) and Ag Gold was evaluated and compared in parallel with real-time genomic and subgenomic reverse transcription-polymerase chain reaction (RRT-) cell culture-based assays. PCR).

  • Diagnostic criteria and patients

A positive case was defined as a person who tested positive for SARS-CoV-2 infection according to diagnostic measures such as RRT-PCR and serological tests. RRT-PCR was performed by targeting the internally designed N gene as described below and using the SD Biosensors kit (SD Biosensors, South Korea) targeting the E and RdRP genes, according to the manufacturer’s protocol. The serological diagnosis was based on seroconversion or a more than four-fold increase in antibody titers, as previously described (Amanat et al., 2020; Hueston et al., 2020).

From February 2020 to August 2020, we recruited 11 patients who tested positive for SARS-CoV-2 infection, and thereafter we performed tests on serial respiratory samples. The samples were obtained from patients during their stay at Chosun University Hospital. To determine specificity, samples were obtained from healthy individuals without signs or symptoms of SARS-CoV-2 infection. The samples were collected and transported in a sample collection tube containing 3 ml of the viral transport medium (VTM). All samples were stored at -20 ° C and were used for the extraction and culture of viral RNA.

  • Viral RNA extraction

A fully automated instrument (Bio-seam, South Korea) was used to extract viral RNA using a Real-prep viral DNA / RNA kit (BioSewoom, South Korea). Extraction was carried out with 200 µL of all samples according to the manufacturer’s protocol to obtain a final elution of 100 µL. Thereafter, samples were stored at -80 ° C until later use for RT-PCR analysis.

  • Severe acute respiratory syndrome Coronavirus 2 Cell culture and detection of infectious viruses

The 63 RRT-PCRs characterized SARS-CoV-2 respiratory samples incubated in Vero E6 cells (Korean Cell Line Bank, KCLB # 21587), using 24-well cell culture plates with glass coverslips. The infected cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, USA) supplemented with 2% fetal calf serum and 1x penicillin-streptomycin solution (Gibco, Thermo Fisher Scientific Inc., USA. ) and then cultured at 37 ° C in the presence of 5% CO2 for 3-5 days by daily observation of the cytopathic effect (CPE).

The results were characterized as negative if no CPE was observed in 5 days. Furthermore, viral RNA was extracted using the culture supernatant and analyzed by RRT-PCR in two passages to validate the proliferation of SARS-CoV-2. The full trial was conducted at Biosafety Level 3 at the Gwangju City Health and Environment Research Institute.

  • Statistic analysis

Categorical variables were recorded as percentages and counts with Wilson score in 95% confidence intervals (CI), while continuous variables were presented as mean, standard deviation (SD) or median and interquartile range (IQR). Differences between means were compared using two-sample t-tests. McNemar’s test was used to analyze test differences independent groups. Normality was assessed using the Kolmogorov-Smirnov test. In addition, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using two reference standards: (1) RT-PCR (N gene) to confirm clinical samples for diagnosis and (2) SARS- Culture of CoV-2 in the cell line to identify infectivity.

Interrater agreement between RRT-PCR and Ag-RDT was calculated using Cohen’s weighted kappa (K-value) (Cohen, 1968). Interpretations of the K value were characterized as follows: <0.20 as poor; 0.21-0.40 as regular; 0.41-0.60 as moderate; 0.61-0.80 as substantial; and> 0.8 as a near-perfect agreement (Landis and Koch, 1977). To determine the precision of the assay, the receiver operating characteristic (ROC) curve was generated and the area under the ROC curve (AUC) was observed (Hajian-Tilaki, 2013). All data were analyzed using MedCalc statistical software (Ostend, Belgium), and p-values ​​were reported as two tails with <0.05 indicating statistical significance.

  • Study approval

The study was approved by the Institutional Review Board (IRB) of Chosun University Hospital (CHOSUN 2020-04-003-002). Written informed consent was obtained from all participants.

Results:

In total, 150 samples were analyzed. Of these, 63 serial samples were obtained from 11 patients with SARS-CoV-2 and 87 from negative controls. Serial respiratory samples were obtained 2 days before the onset of symptoms (-2) up to 25 days after the onset of symptoms. In general, for samples positive for RRT-PCR (n = 51), the detection sensitivity of Ag rapid FIA and Ag Gold was 74.5% and 53.49%, respectively, with a specificity of 100%; however, for samples with low cycle threshold values ​​(Ct), Ag Rapid FIA and Ag Gold showed a sensitivity of 82.61% (Ct ≤ 30, 5.6 log10 RNA copies/ml) and 80% (Ct ≤25.6.9 log10 RNA copies/ml), respectively.

Despite the low analytical sensitivity, both Ag-RDTs detected 100% infection in positive cell culture samples (n = 15) and were very effective in distinguishing viable samples from those with subgenomic RNA (66.66%). For both Ag-RDT, all samples that yielded discordant results (RRT-PCR + ve / Ag-RDT -ve) were also negative by culture.

Conclusion:

The data suggest that RDT-Ag reliably detect viable SARS-CoV-2; therefore, they can serve as an important tool for the rapid detection of potentially infectious individuals.

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