A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are characterized by differences in transmissibility and response to therapeutics. Therefore, discriminating among them is vital for surveillance, infection prevention, and patient care. While whole viral genome sequencing (WGS) is the "gold standard" for variant identification, molecular variant panels have become increasingly available. Most, however, are based on limited targets and have not undergone comprehensive evaluation. We assessed the diagnostic performance of the highly multiplexed Agena MassARRAY ^® SARS-CoV-2 Variant Panel v3 to identify variants in a diverse set of 391 SARS-CoV-2 clinical RNA specimens collected across our health systems in New York City, USA as well as in Bogotá, Colombia (September 2, 2020 - March 2, 2022). We demonstrate almost perfect levels of interrater agreement between this assay and WGS for 9 of 11 variant calls (κ ≥ 0.856) and 25 of 30 targets (κ ≥ 0.820) tested on the panel. The assay had a high diagnostic sensitivity (≥93.67%) for contemporary variants (e.g., Iota, Alpha, Delta, Omicron [BA.1 sublineage]) and a high diagnostic specificity for all 11 variants (≥96.15%) and all 30 targets (≥94.34%) tested. Moreover, we highlight distinct target patterns that can be utilized to identify variants not yet defined on the panel including the Omicron BA.2 and other sublineages. These findings exemplify the power of highly multiplexed diagnostic panels to accurately call variants and the potential for target result signatures to elucidate new ones.

Importance: The continued circulation of SARS-CoV-2 amidst limited surveillance efforts and inconsistent vaccination of populations has resulted in emergence of variants that uniquely impact public health systems. Thus, in conjunction with functional and clinical studies, continuous detection and identification are quintessential to inform diagnostic and public health measures. Furthermore, until WGS becomes more accessible in the clinical microbiology laboratory, the ideal assay for identifying variants must be robust, provide high resolution, and be adaptable to the evolving nature of viruses like SARS-CoV-2. Here, we highlight the diagnostic capabilities of a highly multiplexed commercial assay to identify diverse SARS-CoV-2 lineages that circulated at over September 2, 2020 - March 2, 2022 among patients seeking care at our health systems. This assay demonstrates variant-specific signatures of nucleotide/amino acid polymorphisms and underscores its utility for detection of contemporary and emerging SARS-CoV-2 variants of concern.

FIG 1.. Detection of viral variants by the Agena MassARRAY^® SARS-CoV-2 Variant Panel.

(A) SARS-CoV-2 genome with nucleotide positions from 5’-to-3’ direction depicted above. S gene polymorphisms targeted by the variant panel (lollipops) and corresponding amino acids are depicted below. (B) A color map depicts algorithms of target combinations that define 16 distinct SARS-CoV-2 variants on the panel. Variant results are depicted (left) which include the WHO designation (e.g., Omicron, Delta, etc.) and corresponding PANGO lineage assignments. Note that the B.1.526.1 variant was re-designated as B.1.637 to distinguish it from the Iota variant lineage (https://cov-lineages.org/lineage_list.html, last accessed 4/26/22). The minimum number of targets required to support the corresponding variant result are indicated (right). Target results are depicted as colored cells indicating detectable native (e.g., unchanged from Wuhan-Hu-1 reference) amino acids which do not contribute to the variant target algorithm (grey), detectable native amino acids which do contribute to the algorithm (yellow), detectable amino acid polymorphisms (red), and dropout of the given target polymorphism. (C) Phylogenetic composition of 391 clinical specimen viral RNA recovered for diagnostic evaluation of the variant panel. Numbers of each lineage tested are depicted in brackets.

FIG 2.. Diagnostic sensitivity and specificity of the Agena MassARRAY^® SARS-CoV-2 Variant Panel.

(A) Diagnostic sensitivity and (B) diagnostic specificity of eleven variant calls on the panel are depicted. The number of specimens that correspond with each variant according to WGS are annotated in brackets. Depiction of (C) diagnostic sensitivity and (D) diagnostic specificity of each of thirty distinct panel targets. The number of specimens that correspond with each amino acid polymorphism according to WGS are annotated in brackets for each target. Asterisks (*) indicate targets for which dropout results were excluded from analyses (see Methods). For target N501Y, a separate diagnostic analysis was conducted excluding BA.1 specimens (“N501Y_Excl-BA.1”). Error bars reflect 95% CI in all four panels. ND, not determined.

FIG 3.. Target result patterns of undefined variants on the Agena MassARRAY^® SARS-CoV-2 Variant Panel.

(A) A color map depicts the observed target results for three undefined SARS-CoV-2 variants tested on the panel: Lambda (C.37), Mu (B.1.621), and Omicron (BA.2). Distinct target patterns observed among each of the variant types are depicted. Cells indicate the distinct target results including detectable native amino acid (grey), detection of target polymorphism (red), and target dropout (green). The number of specimens that yielded each of the distinct target result patterns are indicated on the right as well as the output variant ID result generated by the variant panel software. (B) A heatmap depicts the measured prevalence of each variant panel target substitution among publicly-available Omicron sublineage genomes as of May 6, 2022.