Review 1: "Clinical Validation of a Novel T-cell Receptor Sequencing Assay for Identification of Recent or Prior SARS-CoV-2 Infection"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

The manuscript titled “Clinical Validation of a Novel T-cell Receptor Sequencing Assay for Identification of Recent or Prior SARS-CoV-2 Infection” by Sudeb C. Dalai and co-authors described the implementation and clinical validation of a novel high-throughput assay, TDetect™ COVID, to determine recent or prior SARS-CoV-2 infection based on T-cell receptor gene sequencing. They utilized >4,000 SARS-CoV-2-associated TCR sequences from 784 cases and 2,447 controls across 5 independent cohorts. They demonstrated high positive and negative percent agreement of this assay to identify or exclude prior SARS-CoV-2 infection in PCR-confirmed SARS-CoV-2 cases across several cohorts and longitudinal timepoints. Thus, the T-Detect COVID Assay comprised immunosequencing and classifier application to yield a qualitative positive or negative result. The manuscript is well-written and clearly presented. The main study claims are very well-justified by the data and analytic methods used. It confirms previous work on the importance of the biology of the T-cell immune response in COVID-19 and their utility for identifying and tracking disease exposure. This T-cell receptor sequencing assay can provide new critical insights on the SARS-CoV-2 immune response, with potential implications for clinical management risk stratification and surveillance. Also, it might help to elucidate the etiology of immune dysregulation in severe COVID-19 and inflammatory sequelae. The authors have adequately discussed ethical concerns. Indeed, all samples were collected pursuant to an Institutional Review Board-approved clinical study protocol. For residual samples collected under prospective study protocols, informed consent was obtained from participants.

There are some minor caveats or limitations, but they would/do not change the major claims of the study. Due to the limited data from pediatric cohorts, the title and abstract of the manuscript should underline that the T assay proposed is for the adult population. Moreover, it remains not clear whether the performance of the T-cell classifier to separate SARS-CoV-2 cases from controls is consistent across different ethnic groups. This assay has been only validated across different age and gender groups. Also, new variants of Sars-Cov-2 are expected because the virus mutates in its genome. Thus, the impact of the new SARS-CoV-2 variants on the test performance should be discussed. Moreover, the authors should more describe the importance of the potential cross-reactivity with other HCoVs.