The coronavirus disease 2019 (COVID-19) pandemic demonstrated the need for accurate diagnostic testing for the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pandemic has ended, accurate assays are still needed to monitor viral spread at national levels and beyond through population and wastewater surveillance. To enhance early detection, SARS-CoV-2 assays should have high diagnostic accuracy and should be validated to assure accurate results. Three distinct SARS-CoV-2 assays were evaluated with clinical samples using the VALCOR (VALidation of SARS-CORona Virus-2 assays) framework, with the TaqPath COVID-19 assay (ThermoFisher Scientific, USA) as a comparator. We evaluated clinical sensitivity, specificity, limit of detection (LOD), and overall concordance between comparator and three index Allplex SARS-CoV-2 assays (Seegene, South Korea): Allplex-SC2, Allplex-SC2Fast (Fast PCR), and Allplex-SC2FabR (SARS-CoV-2/FluA/FluB/respiratory syncytial virus). Analytical performance and LOD of index assays were assessed using a dilution series of three synthetic SARS-CoV-2 sequence reference materials (RMs). Ninety SARS-CoV-2 positives and 90 SARS-CoV-2 negatives were tested. All Allplex assays had 100.0% sensitivity (95%CI = 95.9%-100.0%). Allplex-SC2 and Allplex-SC2Fast assays had 97.8% specificity (95%CI = 92.3%-99.7%) and 98.9% overall concordance [κ = 0.978 (95%CI = 0.947-1.000)]. Allplex-SC2FabR assay showed 100.0% specificity (95%CI = 95.9%-100.0%) and 100.0% overall concordance [κ = 1.000 (95%CI = 1.000-1.000)]. LOD assessment of index assays revealed detection down to 2.61 × 102 copies/mL in clinical samples, while the analytical LOD was 9.00 × 102 copies/mL. In conclusion, the evaluation of the three Seegene Allplex SARS-CoV-2 assays showed high sensitivity and specificity and an overall good assay concordance with the comparator. The assays showed low analytical LOD using RM and even a slightly lower LOD in clinical samples. Non-overlapping target gene sequences between SARS-CoV-2 assays and RMs emphasize the need for aligning targeted sequences of diagnostic assays and RMs.IMPORTANCEThe coronavirus disease 2019 pandemic has a significant impact on global public health, economies, and societies. As shown through the first phases of the pandemic, accurate and timely diagnosis is crucial for disease control, prevention, and monitoring. Though the pandemic phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has concluded, diagnostic assays remain in demand to monitor SARS-CoV-2 at the individual patient level, regionally, and nationally, as well as to remain an infectious disease preparedness instrument to monitor any new SARS-CoV-2 dissemination across borders using population and wastewater surveillance. The anticipation by WHO and central health care policy entities such as the Center for Disease Control, EMA, and multiple national health authorities is that SARS-CoV-2 will reside as an endemic respiratory disease for years to come. The key strategic consideration is hence shifting from combating a pandemic situation with a high number of patients to instead allowing precise diagnostics of suspected patients with the intention of correct management in a low-prevalence setting.
Keywords: Allplex; COVID-19; RT-PCR; SARS-CoV-2; Seegene; VALCOR; diagnostics test accuracy; quality control; reference material; standard; test validation.