Response to Reviewers

Hypertension as a sequela in patients of SARS-CoV-2 infection (PONE-D-21-02635)

We are grateful to reviewers and editors for the comments and have made every effort to modify the manuscript accordingly or to address the concerns. Table 1-7 are replaced with the modified ones. Please see our specific responses below.

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Answer: Thanks for this suggestion sincerely. The manuscript has been revised according to the PLOS ONE's style requirements.

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Answer: We are grateful for the visionary suggestion. We have toned down this manuscript.

3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

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Answer: We greatly appreciate your constructive comment. We have included the ethics statement information in Line 115 - 116 Page 4 as “All participants provided written informed consent and agreed to use their medical record for research purpose.”

manuscript:

"The current work was supported by the National Natural Science Foundation

Project of China (Grant No. 81670304 – D.H.)."

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Answer: Thank you for your correction. We have removed any funding-related text from the manuscript and updated our Funding Statement section of the online submission form.

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Answer: We re-make the Data Availability statement as suggested in Line 458 - 462 Page 15 as “Data cannot be shared publicly because of the contents including information that could compromise research participant privacy/consent. Data are available from the Renmin Hospital of Wuhan University Ethics Committee (contact via whdxrmyy@126.com) for researchers who meet the criteria for access to confidential data.”

Reviewer #1:

1.The article is written in a clear manner, however it is unclear at what points the authors use parametric statistics and when they use non-parametric statistics- in this regard they offer no justification for their respective uses.

Answer: The reviewer’s comment is well taken. We have added different symbols represent different test methods in Tables 1 - 7.

2.There are only a few minor grammatical errors and only one instance where there is a reference to a paper but no reference number or citation was supplied in the main test.

Answer: We appreciate the reviewer’s valuable comments. The manuscript has been checked thoroughly again and some grammar errors were corrected. Besides, the reference number has been rearranged in some parts of the manuscript.

3.Most importantly, the authors suggest that cardiac injury results from secondary bacterial injury, which based on the design of the trial and that no supporting evidence is provided to this effect (ie any form of analysis of culture data), this represents a jump in logic that I do not believe is justifiable. The authors appear to make this supposition based on elevated procalcitonin levels alone- there are published studies that have shown a small subset of patients with severe to critical illness will actually have elevated procalcitonin levels without secondary bacterial co-infection. The confounding effect notwithstanding, this represents a conclusion which is not substantiated by the trial presented here.

Answer: Thank you a lot for your important comment again. We agree with the reviewer and have revised the manuscript accordingly as follows.

First, we agree that it is not rigorous enough to draw the conclusion that cardiac injury results from secondary bacterial injury and decide to remove it. we have adjusted the discussion about cardiac injury in Line 245 - 252 Page 11 as “In this study, older patients with diabetes are more likely to suffer from cardiac injury. Our further analysis shows that the level of white blood cells, neutrophils, procalcitonin, C-reactive protein, lactate, and lactic dehydrogenase were positively associated with cardiac injury. Besides, cardiac injury occurred mostly in severe patients. Consequently, we hypothesize that severe type of COVID-19 characterized by acute inflammation response might be more prone to cardiac injury, especially in patients with preexisting cardiovascular disease. ”

Besides, according to your suggestion, we have rearranged our data of renin-angiotensin system (RAS). These results, integrated into Table. 5A&B, suggesting that Ang Ⅱ levels were significantly higher in the elevated blood pressure group. Thus, we discuss in Line 300-308 Page13 as “On the other hand, sixteen patients with elevated blood pressure show significantly higher levels of cTnI than those normal blood pressure patients. Several studies have demonstrated Ang II direct or indirect effects on cardiomyocytes, some of which were related to pro-inflammatory and pro-hypertrophic responses. Especially when the balance between the ACE and ACE2 was disrupted in COVID-19 patients, the increase in Ang II actions could lead to myocardial inflammation, oxidative stress, and myocyte apoptosis. This hypothesis explains why elevated blood pressure could occur in parallel with mild cardiac injury of COVID-19 patients. ”

Finally, we resummarize our conclusions in the Abstract part (Line 57 - 60 Page 2) as “Hypertension, sometimes accompanied by elevated cTnI, may occur in COVID-19 patients and become a sequela. Enhancing Ang II signaling, driven by SARS-CoV-2 infection, might play an important role in renin-angiotensin system and consequently lead to the development of hypertension in COVID-19”

4.Moreover, given the confounding effect of hydroxychloroquine, which was the practice at the time of the study, the authors should make note of whether or not HCQ was used in any of these patients, which may actually provide an additional, interesting variable for their analysis.

Thanks for this suggestion sincerely. We have checked carefully and rearranged each patients’ clinical and self-reported data in this study, and all subjects had no medical history of hydroxychloroquine.

5.In the section for processes of patient screening, an appendix would be helpful to list what "chronic heart diseases" they considered for exclusion. In the results section, the wording is at times confusing, though technically correct- further revision of this section may help in further iterations of the submission, but do not in themselves constitute grounds for rejection.

Answer: We are grateful for the visionary suggestion. We have redefined "chronic heart diseases" in the Method part (Line 119 - 120 Page 5) and Figure legends part (Line 452-453 Page 21), as “Chronic heart disease includes ischemic heart disease, arrhythmia, valvular disease, and heart failure.”

6.The very last sentence of the discussion section is too strong of an assertion, perhaps a softer wording of the assertion would be better received. Lastly, the study limitations section does a poor job of discussing the study limitations- heavy revision is advised here- in fact, this section does not discuss any limitations at all and appears to be only a continuation of either the discussion section or the introduction of the conclusions section.

Answer: Thanks for this suggestion sincerely. We have adjusted the last sentence of the discussion section in Line 300 - 308 Page13, as “On the other hand, sixteen patients with elevated blood pressure show significantly higher levels of cTnI than those normal blood pressure patients. Several studies have demonstrated Ang II direct or indirect effects on cardiomyocytes, some of which were related to pro-inflammatory and pro-hypertrophic responses. Especially when the balance between the ACE and ACE2 was disrupted in COVID-19 patients, the increase in Ang II actions could lead to myocardial inflammation, oxidative stress, and myocyte apoptosis. This hypothesis explains why elevated blood pressure could occur in parallel with mild cardiac injury of COVID-19 patients.

We have re-written the part of study limitations in Line 309 - 320 Page 14 as “In the present study, we propose that hypertension is probably a sequela of SARS-CoV-2 infection. Although several studies of COVID-19 have been reported, there are few reports about the sequela of the disease likely due to lack of long-term clinical follow-up, which also applies to our present research. Next, it is difficult to analyze whether the blood pressure of COVID-19 patients with preexisting hypertension is further increased. Consequently, many patients could not be incorporated in the analysis because of history of hypertension, which results in a relatively low sample size. Besides, the present study uncovered rising Ang II as one possible mechanism that might result in hypertension in COVID-19. However, due to a lack of detection about ACE2 levels and other components, therefore, we cannot gain a comprehensive view of the virus-induced imbalance of RAS pathway. ”

Reviewer #2: The study was well planned, conducted, analyzed, and presented. The title is captivating and engaging. The introduction is appropriate and discussion is well written and thorough. The subject matter and idea is novel and well thought-out. The clinical point is very interesting and relevant and the paper should be published pending revisions.

The English and syntax of the paper needs a lot of work. The conclusion that bacterial superinfection may be a cause of the cardiac damage needs further evidence from the literature. Please, see attached file.

Answer: We are grateful for the visionary suggestion. We agree with the reviewer and have revised the manuscript accordingly as follows.

First, we agree that it is not rigorous enough to draw the conclusion that cardiac injury results from secondary bacterial injury and decide to remove it. we have adjusted the discussion about cardiac injury in Line 245 - 252 Page11 as “In this study, older patients with diabetes are more likely to suffer from cardiac injury. Our further analysis shows that the level of white blood cells, neutrophils, procalcitonin, C-reactive protein, lactate, and lactic dehydrogenase were positively associated with cardiac injury. Besides, cardiac injury occurred mostly in severe patients. Consequently, we hypothesize that severe type of COVID-19 characterized by acute inflammation response might be more prone to cardiac injury, especially in patients with preexisting cardiovascular disease. ”

Besides, according to your suggestion, we have rearranged our data of renin-angiotensin system (RAS). These results, integrated into Table. 5A&B, suggesting that Ang Ⅱ levels were significantly higher in the elevated blood pressure group. Thus, we discuss in Line 300 - 308 Page13 as “On the other hand, sixteen patients with elevated blood pressure show significantly higher levels of cTnI than those normal blood pressure patients. Several studies have demonstrated Ang II direct or indirect effects on cardiomyocytes, some of which were related to pro-inflammatory and pro-hypertrophic responses. Especially when the balance between the ACE and ACE2 was disrupted in COVID-19 patients, the increase in Ang II actions could lead to myocardial inflammation, oxidative stress, and myocyte apoptosis. This hypothesis explains why elevated blood pressure could occur in parallel with mild cardiac injury of COVID-19 patients. ”

Finally, we resummarize our conclusions in the Abstract part (Line 57 - 60 Page 2) as “Hypertension, sometimes accompanied by elevated cTnI, may occur in COVID-19 patients and become a sequela. Enhancing Ang II signaling, driven by SARS-CoV-2 infection, might play an important role in renin-angiotensin system and consequently lead to the development of hypertension in COVID-19”

Reviewer #3: This manuscript seeks to address hypertension as a sequalae of COVID-19 disease. Though the clinical association is important, especially as we see more long haul COVID (PASC) in the world population, the manuscript attempts to relate multiple causation for hypertension, including secondary bacterial infection and mycocardial injury during concurrent SARS-CoV-2 infection. The role of ACE2 and the RAS symptom and their relationship to SARS-CoV-2 infectivity and also their role in hypertension would be a more relevant topic to discuss in this manuscript.

Major issues for this manuscript include disorganized data that appears to suggest causation of multiple factors, independently, related to COVID-19 disease severity. It is suggested that the manuscript focus on direct hypertension risk factors associated with the SARS-CoV-2 infection and not broad measurements of cTnl and procalcitonin levels may be unrelated to the development of chronic hypertension.

Answer: We greatly appreciate your constructive comment. According to your suggestion, we have rearranged our data of renin-angiotensin system (RAS). These results, integrated into Table. 5A&B, suggesting that the increase in Ang II actions may direct or indirect effects on cardiomyocytes.

Thus, we discuss in Line 300 - 308 Page13 as “On the other hand, sixteen patients with elevated blood pressure show significantly higher levels of cTnI than those normal blood pressure patients. Several studies have demonstrated Ang II direct or indirect effects on cardiomyocytes, some of which were related to pro-inflammatory and pro-hypertrophic responses. Especially when the balance between the ACE and ACE2 was disrupted in COVID-19 patients, the increase in Ang II actions could lead to myocardial inflammation, oxidative stress, and myocyte apoptosis. This hypothesis explains why elevated blood pressure could occur in parallel with mild cardiac injury of COVID-19 patients. ”

Besides, we resummarize our conclusions in the Abstract part (Line 57 - 60 Page 2) as “Hypertension, sometimes accompanied by elevated cTnI, may occur in COVID-19 patients and become a sequela. Enhancing Ang II signaling, driven by SARS-CoV-2 infection, might play an important role in renin-angiotensin system and consequently lead to the development of hypertension in COVID-19”

Minor issues of this manuscript include various typographical and grammatical errors. There is also need to expand definitions of mild and moderate severity of disease as well. Please include the National Health Commission of China clinical severity scale and criteria to clarify this in the results section.

Answer: Thank you for your correction. According to your suggestion, we have defined mild and moderate severity of disease in the Method part (Line 90 - 93 Page 4) as “Mild type is defined as mild clinical symptoms and no pneumonia manifestation found in imaging. Moderate cases refer to those who present with fever and respiratory tract symptoms, etc. And have pneumonia manifestations found in imaging.”

Besides, in the Result part (Line 207 Page 9), the division criteria have been supplemented, as “Results of demographic and laboratory findings between the severe and non-severe group, based on guidelines of the National Health Commission of China, are shown in Table 6.”

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