Digital PCR Discriminates between SARS-CoV-2 Omicron Variants and Immune Escape Mutations.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, mutations arise that will allow the virus to evade immune defenses and therapeutics. Assays that can identify these mutations can be used to guide personalized patient treatment plans. Digital PCR (dPCR) is a fast and reliable complement to whole-genome sequencing that can be used to discriminate single nucleotide polymorphisms (SNPs) in template molecules. Here, we developed a panel of SARS-CoV-2 dPCR assays and demonstrate its applications for typing variant lineages and therapeutic monoclonal antibody resistance. We first designed multiplexed dPCR assays for SNPs located at residue 3395 in the orf1ab gene that differentiate the Delta, Omicron BA.1, and Omicron BA.2 lineages. We demonstrate their effectiveness on 596 clinical saliva specimens that were sequence verified using Illumina whole-genome sequencing. Next, we developed dPCR assays for spike mutations R346T, K444T, N460K, F486V, and F486S, which are associated with host immune evasion and reduced therapeutic monoclonal antibody efficacy. We demonstrate that these assays can be run individually or multiplexed to detect the presence of up to 4 SNPs in a single assay. We perform these dPCR assays on 81 clinical saliva SARS-CoV-2-positive specimens and properly identify mutations in Omicron subvariants BA.2.75.2, BM.1.1, BN.1, BF.7, BQ.1, BQ.1.1, and XBB. Thus, dPCR could serve as a useful tool to determine if clinical specimens contain therapeutically relevant mutations and inform patient treatment. IMPORTANCE Spike mutations in the SARS-CoV-2 genome confer resistance to therapeutic monoclonal antibodies. Authorization for treatment options is typically guided by general trends of variant prevalence. For example, bebtelovimab is no longer authorized for emergency use in the United States due to the increased prevalence of antibody-resistant BQ.1, BQ.1.1, and XBB Omicron subvariants. However, this blanket approach limits access to life-saving treatment options to patients who are otherwise infected with susceptible variants. Digital PCR assays targeting specific mutations can complement whole-genome sequencing approaches to genotype the virus. In this study, we demonstrate the proof of concept that dPCR can be used to type lineage defining and monoclonal antibody resistance-associated mutations in saliva specimens. These findings show that digital PCR could be used as a personalized diagnostic tool to guide individual patient treatment.

Baseline Sequencing Surveillance of Public Clinical Testing, Hospitals, and Community Wastewater Reveals Rapid Emergence of SARS-CoV-2 Omicron Variant of Concern in Arizona, USA.

The adaptive evolution of SARS-CoV-2 variants is driven by selection for increased viral fitness in transmissibility and immune evasion. Understanding the dynamics of how an emergent variant sweeps across populations can better inform public health response preparedness for future variants. Here, we investigated the state-level genomic epidemiology of SARS-CoV-2 through baseline genomic sequencing surveillance of 27,071 public testing specimens and 1,125 hospital inpatient specimens diagnosed between November 1, 2021, and January 31, 2022, in Arizona. We found that the Omicron variant rapidly displaced Delta variant in December 2021, leading to an "Omicron surge" of COVID-19 cases in early 2022. Wastewater sequencing surveillance of 370 samples supported the synchronous sweep of Omicron in the community. Hospital inpatient COVID-19 cases of Omicron variant presented to three major hospitals 10.51 days after its detection from public clinical testing. Nonsynonymous mutations in nsp3, nsp12, and nsp13 genes were significantly associated with Omicron hospital cases compared to community cases. To model SARS-CoV-2 transmissions across the state population, we developed a scalable sequence network methodology and showed that the Omicron variant spread through intracounty and intercounty transmissions. Finally, we demonstrated that the temporal emergence of Omicron BA.1 to become the dominant variant (17.02 days) was 2.3 times faster than the prior Delta variant (40.70 days) or subsequent Omicron sublineages BA.2 (39.65 days) and BA.5 (35.38 days). Our results demonstrate the uniquely rapid sweep of Omicron BA.1. These findings highlight how integrated public health surveillance can be used to enhance preparedness and response to future variants. IMPORTANCE SARS-CoV-2 continues to evolve new variants throughout the pandemic. However, the temporal dynamics of how SARS-CoV-2 variants emerge to become the dominant circulating variant is not precisely known. Genomic sequencing surveillance offers unique insights into how SARS-CoV-2 spreads in communities and the lead-up to hospital cases during a surge. Specifically, baseline sequencing surveillance through random selection of positive diagnostic specimens provides a representative outlook of the virus lineages circulating in a geographic region. Here, we investigated the emergence of the Omicron variant of concern in Arizona by leveraging baseline genomic sequence surveillance of public clinical testing, hospitals, and community wastewater. We tracked the spread and evolution of the Omicron variant as it first emerged in the general public, and its rapid shift in hospital admissions in the state health system. This study demonstrates the timescale of public health preparedness needed to respond to an antigenic shift in SARS-CoV-2.

SARS-CoV-2 Delta Variant N Gene Mutations Reduce Sensitivity to the TaqPath COVID-19 Multiplex Molecular Diagnostic Assay.

As the SARS-CoV-2 virus evolves, mutations may result in diminished sensitivity to qRT-PCR diagnostic assays. We investigated four polymorphisms circulating in the SARS-CoV-2 Delta lineage that result in N gene target failure (NGTF) on the TaqPath COVID-19 Combo Kit. These mutations were detected from the SARS-CoV-2 genome sequences that matched with the diagnostic assay results of saliva specimens. Full length N genes from the samples displaying NGTF were cloned into plasmids and assayed using three SARS-CoV-2 qRT-PCR assays. These constructs resulted in reduced sensitivity to the TaqPath COVID-19 Combo Kit compared to the controls (mean Ct differences of 3.06, 7.70, 12.46, and 14.12), but were detected equivalently on the TaqPath COVID-19 Fast PCR Combo 2.0 or CDC 2019_nCoV_N2 assays. This work highlights the importance of genomic sequencing to monitor circulating mutations and provide guidance in improving diagnostic assays.

University-Associated SARS-CoV-2 Omicron BA.2 Infections, Maricopa County, Arizona, USA, 2022.

We investigated a university-affiliated cohort of SARS-CoV-2 Omicron BA.2 infections in Arizona, USA. Of 44 cases, 43 were among students; 26 persons were symptomatic, 8 sought medical care, but none were hospitalized. Most (55%) persons had completed a primary vaccine series; 8 received booster vaccines. BA.2 infection was mild in this young cohort.