A Robust, Highly Multiplexed Mass Spectrometry Assay to Identify SARS-CoV-2 Variants.

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.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Striking lineage diversity of severe acute respiratory syndrome coronavirus 2 from non-human sources.

Due to the necessity to control human-to-human spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the overwhelming majority of the generated data on this virus was solely related to the genomic characteristics of strains infecting humans; conversely, this work aimed to recover and analyze the diversity of viral genomes from non-human sources. From a set of 3595 publicly available SARS-CoV-2 genome sequences, 128 lineages were identified in non-human hosts, the majority represented by the variants of concern Delta (n = 1105, 30.7%) and Alpha (n = 466, 12.9%), followed by B.1.1.298 lineage (n = 458, 12.7%). Environment, Neovison vison, Odocoileus virginianus and Felis catus were the non-human sources with the highest number of lineages (14, 12 and 10, respectively). Phylogenomic analyses showed viral clusters from environmental sources, N. vison, O. virginianus, Panthera tigris, and Panthera leo. These clusters were collectively related to human viruses as well as all other non-human sources that were heterogeneously distributed in the phylogenetic tree. Further, the genetic details of viral genomes from bats and pangolins were independently investigated owing to their high divergence, revealing five distinct clusters. Cluster 4 exclusively included bat-sourced genomes and the SARS-CoV-2 reference strain Wuhan-01. In summary, this study identified new genetic landmarks of SARS-CoV-2 evolution. We propose potential interspecies transmission routes of SARS-CoV-2 between animals and humans, which should be considered in order to establish better pathogen surveillance and containment strategies.

Deciphering the introduction and transmission of SARS-CoV-2 in the Colombian Amazon Basin.

BACKGROUND: The SARS-CoV-2 pandemic has forced health authorities across the world to take important decisions to curtail its spread. Genomic epidemiology has emerged as a valuable tool to understand introductions and spread of the virus in a specific geographic location. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the sequences of 59 SARS-CoV-2 samples from inhabitants of the Colombian Amazonas department. The viral genomes were distributed in two robust clusters within the distinct GISAID clades GH and G. Spatial-temporal analyses revealed two independent introductions of SARS-CoV-2 in the region, one around April 1, 2020 associated with a local transmission, and one around April 2, 2020 associated with other South American genomes (Uruguay and Brazil). We also identified ten lineages circulating in the Amazonas department including the P.1 variant of concern (VOC). CONCLUSIONS/SIGNIFICANCE: This study represents the first genomic epidemiology investigation of SARS-CoV-2 in one of the territories with the highest report of indigenous communities of the country. Such findings are essential to decipher viral transmission, inform on global spread and to direct implementation of infection prevention and control measures for these vulnerable populations, especially, due to the recent circulation of one of the variants of concern (P.1) associated with major transmissibility and possible reinfections.