Combined Use of RT-qPCR and NGS for Identification and Surveillance of SARS-CoV-2 Variants of Concern in Residual Clinical Laboratory Samples in Miami-Dade County, Florida.

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

SARS-CoV-2 and Variant Diagnostic Testing Approaches in the United States.

Purpose of Review Given the rapid development of diagnostic approaches to test for and diagnose infection with SARS-CoV-2 and its associated variants including Omicron (B.1.1.529), many options are available to diagnose infection. Multiple established diagnostic companies are now providing testing platforms whereas initially, testing was being performed with simple PCR-based tests using standard laboratory reagents. Recent Findings Additional testing platforms continue to be developed, including those to detect specific variants, but challenges with testing, including obtaining testing reagents and other related supplies, are frequently encountered. With time, the testing supply chain has improved, and more established companies are providing materials to support these testing efforts. In the United States (U.S.), the need for rapid assay development and subsequent approval through the attainment of emergency use authorization (EUA) has superseded the traditional arduous diagnostic testing approval workflow mandated by the FDA. Through these efforts, the U.S. has been able to continue to significantly increase its testing capabilities to address this pandemic; however, challenges still remain due to the diversity of the performance characteristics of tests being utilized and newly discovered viral variants. Summary This review provides an overview of the current diagnostic testing landscape, with pertinent information related to SARS-CoV-2 virology, variants and antibody responses that are available to diagnose infection in the U.S.