Evaluation of Four Rapid Antigen Tests for the Detection of SARS-CoV-2 Infection with Nasopharyngeal Swabs.

Owing to the high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, the capacity of testing systems based on the gold standard real-time reverse transcription-polymerase chain reaction (rRT-PCR) is limited. Rapid antigen tests (RATs) can substantially contribute to the prevention of community transmission, but their further assessment is required. Here, using 1503 nasopharyngeal swabs, we compared the diagnostic performance of four RAT kits (Abbott Panbio™ COVID-19 Ag Rapid Test, SD Biosensor Standard™ Q COVID-19 Ag Test, Humasis COVID-19 Ag Test, and SG Medical Acrosis COVID-19 Ag Test) to the cycle threshold (Ct) values obtained from rRT-PCR. The precision values, area under the curve values, SARS-CoV-2 variant detection ability, and non-SARS-CoV-2 specificity of all four kits were similar. An assay using the Acrosis kit had a significantly better positive detection rate with a higher recall value and cut-off value than that using the other three RAT kits. During the current COVID-19 pandemic, the Acrosis kit is an effective tool to prevent the spread of SARS-CoV-2 in communities.

Performance Analysis of Self-Collected Nasal and Oral Swabs for Detection of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic human coronavirus and is rapidly transmitted by infected individuals regardless of their symptoms. During the COVID-19 pandemic, owing to the dearth of skilled healthcare workers (HCWs) to collect samples for early diagnosis, self-collection emerged as a viable alternative. To evaluate the reliability of self-collection, we compared the virus detection rate using 3990 self-collected swabs and HCW-collected swabs, procured from the same individuals and collected immediately after the self-collection. The results of multiplex reverse-transcription quantitative polymerase chain reaction revealed that the viral load in the HCW-collected swabs was marginally (18.4–28.8 times) higher than that in self-collected swabs. Self-collection showed no significant difference in sensitivity and specificity from HCW-collection (κ = 0.87, McNemar’s test;p = 0.19), indicating a comparable performance. These findings suggest that self-collected swabs are acceptable substitutes for HCW-collected swabs, and that their use improved the specimen screening efficiency and reduced the risk of SARS-CoV-2 infection among HCWs during and after the COVID-19 pandemic.

Performance Analysis of Self-Collected Nasal and Oral Swabs for Detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic human coronavirus and is rapidly transmitted by infected individuals regardless of their symptoms. During the COVID-19 pandemic, owing to the dearth of skilled healthcare workers (HCWs) to collect samples for early diagnosis, self-collection emerged as a viable alternative. To evaluate the reliability of self-collection, we compared the virus detection rate using 3990 self-collected swabs and HCW-collected swabs, procured from the same individuals and collected immediately after the self-collection. The results of multiplex reverse-transcription quantitative polymerase chain reaction revealed that the viral load in the HCW-collected swabs was marginally (18.4-28.8 times) higher than that in self-collected swabs. Self-collection showed no significant difference in sensitivity and specificity from HCW-collection (κ = 0.87, McNemar's test; p = 0.19), indicating a comparable performance. These findings suggest that self-collected swabs are acceptable substitutes for HCW-collected swabs, and that their use improved the specimen screening efficiency and reduced the risk of SARS-CoV-2 infection among HCWs during and after the COVID-19 pandemic.

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is still rapidly spreading as of March 2022. An accurate and rapid molecular diagnosis is essential to determine the exact number of confirmed cases. Currently, the viral transport medium (VTM) required for testing is in short supply due to a sharp increase in the laboratory tests performed, and alternative VTMs are needed to alleviate the shortage. Guanidine thiocyanate-based media reportedly inactivate SARS-CoV-2 and are compatible with quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays, but the compatibility and the viral detection capacity have not been fully validated. To evaluate the guanidine thiocyanate-based Gene Transport Medium (GeneTM) as an alternative VTM, we prepared 39 SARS-CoV-2-positive and 7 SARS-CoV-2-negative samples in GeneTM, eNAT™, and phosphate-buffered saline (PBS). The cycle threshold (Ct) values of three SARS-CoV-2 targets (the S, RdRP, and N genes) were analyzed using RT-qPCR testing. The comparison of Ct values from the positive samples showed a high correlation (R 2= 0.95-0.96) between GeneTM and eNAT™, indicating a comparable viral detection capacity. The delta Ct values of the SARS-CoV-2 genes in each transport medium were maintained for 14 days at cold (4°C) or room (25°C) temperatures, suggesting viral samples were stably preserved in the transport media for 14 days. Together, GeneTM is a potential alternative VTM with comparable RT-qPCR performance and stability to those of standard media.

Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations.

Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.

Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37-3.15 × 101, 0.41-3.62 × 101, and 0.33-1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3-100% sensitivity and 100% specificity. Bland-Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

Assay System for Simultaneous Detection of SARS-CoV-2 and Other Respiratory Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers disease with nonspecific symptoms that overlap those of infections caused by other seasonal respiratory viruses (RVs), such as the influenza virus (Flu) or respiratory syncytial virus (RSV). A molecular assay for accurate and rapid detection of RV and SARS-CoV-2 is crucial to manage these infections. Here, we compared the analytical performance and clinical reliability of Allplex™ SARS-CoV-2/FluA/FluB/RSV (SC2FabR; Seegene Inc., Seoul, South Korea) kit with those of four commercially available RV detection kits. Upon testing five target viral strains (SARS-CoV-2, FluA, FluB, RSV A, and RSV B), the analytical performance of SC2FabR was similar to that of the other kits, with no significant difference (p ≥ 0.78) in z-scores. The efficiency of SC2FabR (E-value, 81-104%) enabled reliable SARS-CoV-2 and seasonal RV detection in 888 nasopharyngeal swab specimens processed using a fully automated nucleic acid extraction platform. Bland-Altman analyses revealed an agreement value of 95.4% (SD ± 1.96) for the kits, indicating statistically similar results for all five. In conclusion, SC2FabR is a rapid and accurate diagnostic tool for both SARS-CoV-2 and seasonal RV detection, allowing for high-throughput RV analysis with efficiency comparable to that of commercially available kits. This can be used to help manage respiratory infections in patients during and after the coronavirus disease 2019 pandemic.

Coinfections with Respiratory Pathogens among COVID-19 Patients in Korea.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper and lower respiratory specimens and coinfection with other respiratory pathogens in patients with coronavirus disease 2019 (COVID-19) was investigated. Study subjects (N = 342) were retrospectively enrolled after being confirmed as SARS-CoV-2 positive, and their nasopharyngeal swab (NPS), oropharyngeal swab (OPS), and sputum specimens were restored for SARS-CoV-2 retesting and respiratory pathogen detection. The majority of the subjects (96.5%, N = 330) were confirmed as SARS-CoV-2 positive using NPS/OPS specimens. Among the COVID-19 patients (N = 342), 7.9% (N = 27) and 0.9% (N = 3) were coinfected with respiratory viruses and Mycoplasma pneumoniae, respectively, yielding an 8.8% (N = 30) overall respiratory pathogen coinfection rate. Of the respiratory virus coinfection cases (N = 27), 92.6% (N = 25) were coinfected with a single respiratory virus and 7.4% (N = 2) with two viruses (metapneumovirus/adenovirus and rhinovirus/bocavirus). No triple coinfections of other respiratory viruses or bacteria with SARS-CoV-2 were detected. Respiratory viruses coinfected in the patients with COVID-19 were as follows: rhinovirus (N = 7, 2.1%), respiratory syncytial virus A and B (N = 6, 1.8%), non-SARS-CoV-2 coronaviruses (229E, NL63, and OC43, N = 5, 1.5%), metapneumovirus (N = 4, 1.2%), influenza A (N = 3, 0.9%), adenovirus (N = 3, 0.9%), and bocavirus (N = 1, 0.3%). In conclusion, the diagnostic value of utilizing NPS/OPS specimens is excellent, and, as the first report in Korea, coinfection with respiratory pathogens was detected at a rate of 8.8% in patients with COVID-19.