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-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 and Bogotá, Colombia (September 2, 2020 to March 2, 2022). We demonstrated 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, and Omicron [BA.1 sublineage]) and a high diagnostic specificity for all 11 variants (≥96.15%) and all 30 targets (≥94.34%) tested. Moreover, we highlighted distinct target patterns that could be utilized to identify variants not yet defined on the panel, including the Omicron BA.2 and other sublineages. These findings exemplified 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 amid limited surveillance efforts and inconsistent vaccination of populations has resulted in the emergence of variants that uniquely impact public health systems. Thus, in conjunction with functional and clinical studies, continuous detection and identification are quintessential to informing 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 highlighted the diagnostic capabilities of a highly multiplexed commercial assay to identify diverse SARS-CoV-2 lineages that circulated from September 2, 2020 to March 2, 2022 among patients seeking care in our health systems. This assay demonstrated variant-specific signatures of nucleotide/amino acid polymorphisms and underscored its utility for the detection of contemporary and emerging SARS-CoV-2 variants of concern.

Keywords: MALDI-TOF; Omicron; RT-PCR; SARS-CoV-2; multiplex; variant panel.

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 defined 16 distinct SARS-CoV-2 variants on the panel. Variant results are depicted (left) and included the WHO designation (e.g., Omicron, Delta, etc.) and corresponding PANGO lineage assignments. Note that the B.1.526.1 variant was redesignated B.1.637 to distinguish it from the Iota variant lineage (https://cov-lineages.org/lineage_list.html, accessed April 26, 2022). The minimum number of targets required to support the corresponding variant result is indicated (right). Target results are depicted as colored cells indicating amino acid positions that did not contribute to the defined variant identity algorithm (gray). The remaining three colors reflect native amino acids (e.g., unchanged from Wuhan-Hu-1 reference) (yellow), detectable amino acid polymorphisms (red), and drop out of the given target (green), all of which contributed to the variant identity algorithm. (C) Phylogenetic composition of 391 clinical specimen viral RNA recovered for diagnostic evaluation of the variant panel. The 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 11 variant calls on the panel are depicted. The number of specimens that correspond with each variant according to WGS is annotated in brackets. Depiction of (C) diagnostic sensitivity and (D) diagnostic specificity of each of 30 distinct panel targets. The number of specimens that correspond with each amino acid polymorphism according to WGS is annotated in brackets for each target. Asterisks (*) indicate targets for which dropout results were excluded from analyses (see Materials and 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 were observed among each of the variant types are depicted. Cells indicate the distinct target results, including detectable native amino acid (gray), detection of target polymorphism (red), and target dropout (green). The number of specimens that yielded each of the distinct target result patterns is 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.