High levels of circulating osteopontin in inflammatory lung disease regardless of Sars‐CoV‐2 infection

This study independently confirms increased levels of osteopontin in COVID‐19 patients but also suggests that osteopontin cannot be used as a biomarker of SARS‐CoV‐2 infection, as elevated levels of circulating osteopontin are found in inflammatory lung disease regardless of SARS‐CoV‐2 infection.

(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. Depending on transfer agreements, referee reports obtained elsewhere may or may not be included in this compilation. Referee reports are anonymous unless the Referee chooses to sign their reports.) ***** Reviewer's comments ***** Referee #1 (Remarks for Author): In their correspondence "High levels of circulating osteopontin in inflammatory lung disease regardless of Sars-Cov-2 infection." the authors test osteopontin as a biomarker for SARS-CoV-2 infection. The rationale is based on a report by Gibellini et al. published in 2020 addressing monocyte alterations in SARS-CoV-2 and showing increased osteopontin levels in COVID-19 patients. While Gibellini et al. had compared COVID-19 patients to healthy controls, the authors of this manuscript test whether increased osteopontin is specific for SARS-CoV-2 infection as opposed to other causes of pneumonia, which indeed turned out not to be the case. Even though this might not necessarily be surprising, it is an interesting finding worth reporting. Not least does it demonstrate that findings made with respect to COVID-19 need to be put into context and it needs to be discriminated between what is more general infection-associated inflammation and what is SARS-CoV-2-specific. There are some points to be addressed: 1) As an introduction it is stated that "Gibellini et al. (2020) showed that the bioenergetic alteration of the monocyte compartment affects the pathogenic response to Sars-Cov-2 infection". As such effects on the pathogenic response were not explicitly shown by Gibellini et al. and they rather demonstrated alterations to the monocytic compartment in COVID-19, please revise this introductory statement to more precisely reflect the findings of Gibellini et al.
2) "OPN is a multifaceted molecule involved in the inflammatory response: it modulates leukocyte activation, migration and differentiation and induces cytokine secretion both in acute and chronic inflammation.". Please provide a reference for the effects of OPN.
3) As OPN is affected in a broad array of conditions, the authors might want to indicate their criteria defining healthy controls. 4) Regarding the cohort of Sars-Cov-2 negative patients if possible please indicate whether these were viral, bacterial, any other pneumonia patients or how these characteristics were distributed within the cohort. 5) Even though OPN is not a marker for SARS-CoV-2 infection, could the authors speculate on whether increased OPN might be associated with a certain form or severity of COVID-19. There is quite some heterogeneity in the OPN levels reported in SARS-CoV-2 positive patients. It would be interesting to learn whether e.g. explicitly high levels are associated with severe disease. This might make OPN suitable not as a biomarker for SARS-CoV-2 per se but e.g. for the course of the acute disease or the intensity of inflammation. Q2) "OPN is a multifaceted molecule involved in the inflammatory response: it modulates leukocyte activation, migration and differentiation and induces cytokine secretion both in acute and chronic inflammation.". Please provide a reference for the effects of OPN. R2. As suggested, by the reviewer, we added the missing reference (Clemente et al., 2016).

Response to the reviewer
Q3) As OPN is affected in a broad array of conditions, the authors might want to indicate their criteria defining healthy controls. R3. We thank the reviewer for his/her valuable suggestion. We edited the text to clarify it. Q4) Regarding the cohort of Sars-Cov-2 negative patients if possible please indicate whether these were viral, bacterial, any other pneumonia patients or how these characteristics were distributed within the cohort. R4. We thank the reviewer for his/her comment. We added the following sentences: 'Diagnosis at hospital admission were respiratory failure related to acute or acute-on-chronic heart failure (n=17), sepsis (n=16 of which 25% due to community acquired pneumonia), intracranial bleeding (n=8), trauma (n=5) and others (n=10)''. Q5) Even though OPN is not a marker for SARS-CoV-2 infection, could the authors speculate on whether increased OPN might be associated with a certain form or severity of COVID-19. There is quite some heterogeneity in the OPN levels reported in SARS-CoV-2 positive patients. It would be interesting to learn whether e.g. explicitly high levels are associated with severe disease. This might make OPN suitable not as a biomarker for SARS-CoV-2 per se but e.g. for the course of the acute disease or the intensity of inflammation. R5. The reviewer is right; we added in the text the following sentences: "OPN deregulation may impact on COVID- 19  Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data?

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