PONE-D-21-30161RNA Sequencing in COVID-19 patients identifies neutrophil activation biomarkers as promising diagnostic platform for infections.PLOS ONE

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Reviewer #1: Yes

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Reviewer #1: N/A

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Reviewer #1: Yes

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Reviewer #1: The authors set out to identify biomarkers associated with Covid-19 disease severity. They measured the host response to the infection via blood transcriptome profiling. Their analysis rapidly focused on sets of transcripts associated with neutrophil activation. From there they carried out further work profiling the transcriptome of neutrophils isolated from Covid-19 patients and controls. Downstream assays were also run including DNA staining and the measurement of neutrophil elastase activity, as well as measurement elastase activity in serum. The authors conclude that abundance levels in whole blood of DEFA1 transcripts and neutrophil elastase activity may serve as clinically relevant markers in the context of the management of patients with Covid-19 in particular.

While the study could make a significant contribution to Covid-19 research several limitations should be addressed:

Major

- The work is not well-grounded in the literature. Running the query “Covid-19 AND Netosis” returns 69 articles, which include several which are of direct relevance and are not currently cited [e.g. 34344929]. “Covid-19 AND Neutrophils AND Elastase” returns 36 articles. Some important work investigating the role of neutrophils in viral respiratory infection and association with severity is not discussed either [e.g. 29777224].

- The number of subjects used in comparisons is often very small, which inevitably undermines some of the conclusions. There is probably not much to be done at this stage (unless available public provided opportunities for re-use / independent validation). But it would be preferrable not to subdivide already small study groups (e.g in some instances, n=2, n=4, n=5, n=7 etc…). Just for context, this study, which is not yet publish but seeks to address similar questions comprises > 300 subjects. Given the extent of inter-individual variability in patient cohorts it is the kind of numbers that would permit to bring more convincing answers. In any case this limitation should at least be discussed.

- The transcriptome analysis was very cursory. It seems the authors had decided from the onset to focus on neutrophils markers. The paper would benefit from a more global analysis being performed before deep diving on neutrophil related genes and neutrophils.

- The quality of the figures is generally not very good, and they are not always informative. The DAPI staining images are not very clear and the labels that are overlaid obstruct a good part of the image. The use of an arrow on the line graph on Figure 9 is somewhat unusual for an original research paper. There are currently 9 figures, and the paper would likely benefit from focusing on those that are considered more critical to the message being conveyed.

- Before acceptance should be deposited in GEO and token provided for reviewer access (main point here would be to verify that the level of details provided is sufficient)

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Reviewer #1: No

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RNA Sequencing in COVID-19 patients identifies neutrophil activation biomarkers as promising diagnostic platform for infections.

PONE-D-21-30161R1

Dear Dr. McCaffrey,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Afsheen Raza, PhD

Academic Editor

PLOS ONE

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