Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

AIMS: This retrospective case-control study was aimed at identifying potential independent predictors of severe/lethal COVID-19, including the treatment with Angiotensin-Converting Enzyme inhibitors (ACEi) and/or Angiotensin II Receptor Blockers (ARBs). METHODS AND
RESULTS: All adults with SARS-CoV-2 infection in two Italian provinces were followed for a median of 24 days. ARBs and/or ACEi treatments, and hypertension, diabetes, cancer, COPD, renal and major cardiovascular diseases (CVD) were extracted from clinical charts and electronic health records, up to two years before infection. The sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease (154 deaths; mean age 79.3 years; 70.8% hypertensive, 42.2% with CVD). The youngest deceased person aged 44 years. Among hypertensive subjects (n = 543), the proportion of those treated with ARBs or ACEi were 88.4%, 78.7% and 80.6% among patients with mild, severe and very severe/lethal disease, respectively. At multivariate analysis, no association was observed between therapy and disease severity (Adjusted OR for very severe/lethal COVID-19: 0.87; 95% CI: 0.50-1.49). Significant predictors of severe disease were older age (with AORs largely increasing after 70 years of age), male gender (AOR: 1.76; 1.40-2.23), diabetes (AOR: 1.52; 1.05-2.18), CVD (AOR: 1.88; 1.32-2.70) and COPD (AOR: 1.88; 1.11-3.20). Only gender, age and diabetes also predicted very severe/lethal disease.
CONCLUSION: No association was found between COVID-19 severity and treatment with ARBs and/or ACEi, supporting the recommendation to continue medication for all patients unless otherwise advised by their physicians.

Introduction

Novel coronavirus disease (COVID-19) is spreading worldwide, and has caused over 250,000 deaths so far [1]. The mortality rate varies widely by age and across individuals, ranging from 0.2% among healthy, young-adults, to >10% among older persons with pre-existing conditions [1].

Although the pharmacological treatment was not assessed, the first observational studies on patients with severe disease reported a high prevalence of comorbidities that are often treated with angiotensin converting enzyme (ACE) inhibitors, such as cerebrovascular diseases, coronary heart disease, hypertension and diabetes [2-4]. Observing that human pathogenic coronaviruses bind their target cells through angiotensin-converting enzyme 2 (ACE2) [5-8], and that a few studies reported an increase in ACE2 expression mediated by angiotensin II type-I receptor blockers (ARBs) and ACE inhibitors (more consistently on animals than in humans) [9-16], some hypothesized that the increased expression of ACE2 would facilitate infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), thus the hypertension treatment with ACE2-stimulating drugs, as well as ACE2 polymorphisms, might increase the risk of developing severe COVID-19 [17-19]. Consequently, this would lead to a serious conflict regarding treatment, because ACE2 reduces inflammation and has been suggested as a potential new therapy for inflammatory lung diseases, cancer, diabetes, and hypertension [17, 20–23]. In the wake of two preliminary cohort studies reporting a lower [24] or similar [25] COVID-19 mortality among inpatients hypertensive subjects treated with ARBs and ACE inhibitors, the potential predictors of COVID-19 and of disease severity, including anti-hypertensive medications, were recently analyzed by a few observational studies [26-28]. With one exception [27], no increased risk emerged from the use of ARBs or ACE inhibitors; however, the role of other potentially linked predictors, including age and cardiovascular comorbidities [17, 29, 30], differed across the population analyzed, and still requires confirmation. We have performed a case-control study on all SARS-CoV-2 infected subjects diagnosed in two Italian provinces, retrieving admission and pharmacological data up to two years before infection, in order to confirm the potential independent predictors of severe/lethal COVID-19, including treatment with ACE inhibitors and/or ARBs.

Materials and methods

This case-control, retrospective study compared the proportion of subjects treated with ARBs and/or ACE inhibitors among three groups of subjects with SARS-CoV-2 infection:

asymptomatic infection or mild disease, defined as fever or malaise plus at least one of the followings: sore throat, muscle pain, shortness of breath, dry cough, headache, conjunctivitis, and diarrhea [31], with no hospital admission;

severe disease, requiring hospital admission, not in an intensive care unit;

very severe or lethal disease, requiring admission in an intensive care unit and/or causing death.

The sample includes all subjects with diagnosis of infection made in the Province of Ferrara, up to April 2, and the Province of Pescara, Italy, up to April 24, 2020, by the Central Laboratory of the University Hospital of Ferrara or the Central Laboratory of the Pescara Hospital (and confirmed by the National Institute of Health). All diagnoses were made using (real time) reverse transcription polymerase chain reaction (rRT-PCR) on oropharingeal specimens. The assays were those originally proposed by the Charité-Universitätsmedizin Berlin Institute of Virology [32], and then endorsed by the WHO [33].

The data on background pharmacological treatment up to the previous two years (January 1, 2018) were obtained from the National database of drug prescription, and integrated with clinical chart information for hospitalized subjects. Data have been collected on the following drugs: ACE inhibitors (ATC classes: C09A and C09B), ARBs (C09C and C09D), and insulin or other anti-diabetic drugs (A10). Information on age, gender, and pre-existing conditions of all subjects were obtained through data-linkage with hospital discharge abstracts (Italian SDO), which have been queried from the day of the diagnosis until January 1st, 2015. All admission data have been revised manually by two physicians (LM and MEF) and the following conditions have been included in the analyses: malignant tumors, major cardiovascular diseases (heart failure, myocardial infarction and stroke—CVD), type II diabetes, renal disease and chronic obstructive pulmonary diseases (COPD, bronchitis, pneumonia, asthma, and emphysema).

The study complies with the Declaration of Helsinki, the research protocol was approved by the Ethics Committee of the Emilia-Romagna Region (code 287, approved on March 24, 2020), and the requirement for informed consent was waived because of the retrospective and pseudo-anonymized nature of the data.

First, the differences across groups with mild, severe or very severe/lethal disease were evaluated using two-way ANOVA with post-hoc Tukey HSD test for continuous variables, and Mantel-Haenzsel chi-squared test for categorical ones. A sample restricted to hypertensive subjects was used to compare the subjects treated and untreated with ACE inhibitors or ARBs.

Multivariate logistic regression was used to investigate the potential independent predictors of severe or very severe/lethal COVID-19. Four models were built, two were restricted to hypertensive subjects (A and B), and two included the total sample (Models C and D) and. Models A and C were fit to predict severe or very severe disease (grouped together), while Models B and D to predict very severe/lethal disease only (and repeated to predict death, with similar results, which have not been shown to avoid redundancy). All recorded variables (age, gender, CVD, diabetes, renal disease, cancer and COPD) were included a priori in all models, with the exception of treatments with ACE inhibitors and ARBs, that were excluded from Models C and D because of multicollinearity with hypertension. Standard diagnostic procedures were adopted to check all models validity: influential observation analysis (Dbeta, change in Pearson chi-square and similar), multicollinearity, interaction terms, Hosmer-Lemeshow test for the goodness of fit and C statistic (area under the Receiving Operator Curve). Statistical significance was defined as a two-sided p-value<0.05, and all analyses were carried out using Stata, version 13.1 (Stata Corp., College Station, TX, 2014).

Power analysis

Our sample of 129 hypertensive subjects with severe/lethal COVID-19, and 414 hypertensive subjects with mild disease or asymptomatic infection, had 80% statistical power to detect a difference of 20% or higher (corresponding to a relative risk ≥1.20) in the risk of severe/lethal disease between users of ARBS or ACE inhibitors (exposed group), and non users (controls).

Results

The sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease (Table 1). The sample of infected subjects, compared with the available estimates of the general population of the two provinces [34], showed higher prevalence of diabetes (12.1% in the sample versus ≅5% in the general population); hypertension (33.9% versus ≅17%), COPD (6.0% versus ≅3%) and CVD (16.1% versus ≅3%). 154 subjects deceased (mean age 79.3 years; 54.6% males); of them, 70.8% were hypertensive, 42.2% were diagnosed a CVD; 27.9% diabetics. Twenty subjects with very severe disease were younger than 60 years; the youngest being a male aged 33 years. Of those deceased, eight were younger than 60 years, and the youngest was a female aged 44 years.

Table 1

Characteristics of the sample.

	Overall sample	Mild	Severe	Very severe/lethal	p A	p B	p C
Variables	(n = 1603)	(n = 957)	(n = 454)	(n = 192)
Mean age in years (SD)	58.0 (20.9)	50.4 (20.2)	66.4 (16.9)	76.2 (12.9)	<0.001	<0.001	<0.001
Male gender, %	47.3	42.5	53.1	57.3	<0.001	<0.001	0.3
Diabetes, %	12.1	6.8	16.5	28.1	<0.001	<0.001	0.001
COPD, %	6.0	2.9	9.2	14.1	<0.001	<0.001	0.070
Cancer, %	7.6	5.1	10.1	14.1	<0.001	<0.001	0.2
Major cardiovascular diseases D, %	16.1	6.9	26.9	36.5	<0.001	<0.001	0.015
Renal disease, %	5.4	2.4	8.8	12.0	<0.001	<0.001	0.2
Hypertension, %	33.9	21.6	45.6	67.2	<0.001	<0.001	<0.001
Antihypertensive treatment with:
• ACE inhibitors, %	15.7	11.2	19.4	29.2	<0.001	<0.001	0.006
•ARBs, %	14.2	9.0	19.8	27.1	<0.001	<0.001	0.042
•ACE inhibitors or ARBs, %	28.1	19.1	35.9	54.2	<0.001	<0.001	<0.001
Sample restricted to hypertensive subjects	(n = 543)	(n = 207)	(n = 207)	(n = 129)
Antihypertensive treatment with:
•ACE inhibitors, %	46.2	51.7	42.5	43.4	0.061	0.14	0.9
•ARBs, %	42.0	41.6	43.5	40.3	0.7	0.8	0.6
•ACE inhibitors or ARBs, %	82.9	88.4	78.7	80.6	0.008	0.049	0.7

A Mild versus severe subjects.

B Mild versus very severe subjects

C Severe versus very severe subjects.

D Congestive heart failure, myocardial infarction, or stroke. COPD = Chronic obstructive pulmonary diseases. ACE = Angiotensin-Converting Enzyme. ARBs = Angiotensin II Receptor Blockers.

At univariate analysis, as compared to the subjects with mild disease, those with severe or very severe/lethal disease were significantly more likely to be older, diabetics, hypertensive, diagnosed with COPD, CVD, and renal disease, and treated with ARBs and/or ACE inhibitors (all p<0.05). Among hypertensive subjects (n = 543), however, the proportion of those treated with ARBs or ACE inhibitors (whose medication was not discontinued during the follow-up) were higher among those with asymptomatic/mild disease (88.4%, versus 78.7% and 80.6% among patients with severe and very severe/lethal disease, respectively—Table 1).

In multivariable analyses restricted to hypertensive subjects (Models A and B, Table 2), the treatment with ARBs and/or ACE inhibitors never increased the likelihood of severe or very severe/lethal disease (all p>0.405).

Table 2

Logistic regression model predicting severe or very severe/lethal COVID-19 syndrome (grouped together, Model A) or very severe/lethal disease only (Model B), in the sample restricted to hypertensive subjects.

	(A) Severe or very severe/lethal	p*	(B) Very severe/lethal	p*
Antihypertensive drugs:	OR (95% CI)		OR (95% CI)
•ACE Inhibitors	0.70 (0.44–1.13)	0.15	0.82 (0.49–1.36)	0.4
•ARBs	0.91 (0.56–1.47)	0.7	0.83 (0.50–1.40)	0.5
•ACE Inhibitors or ARBs *	0.58 (0.34–1.01)	0.056	0.87 (0.50–1.49)	0.6

ACE = Angiotensin-Converting Enzyme. ARBs = Angiotensin II Receptor Blockers. OR = odds ratio. CI = Confidence interval. All estimates have been adjusted for age, gender, diabetes, major cardiovascular diseases, COPD, cancer and renal diseases.

* Models A and B have been repeated including ACE or ARBs treatment, grouped, with no substantial changes for other independent variables.

The significant predictors of severe disease were male gender (Adjusted Odds Ratio—AOR: 1.76; 95% Confidence Interval—CI: 1.40–2.23), diabetes (AOR: 1.52; 1.05–2.18), CVD (AOR: 1.88; 1.32–2.70) and COPD (1.88; 1.11–3.20), and older age, which showed an exponential increase after 70 years: compared with the subjects younger than 50 years, the AORs of those aged 70–79 and ≥80 years were 5.72 (3.81–8.58) and 9.06 (6.04–13.6), respectively (Model C; Table 3). Only male gender, older age and diabetes also predicted very severe/lethal disease (Model D; Table 3).

Table 3

Logistic regression model predicting severe or very severe/lethal COVID-19 syndrome (grouped together, Model C) or very severe/lethal disease only (Model D), in the total sample (n = 1603).

	(C) Severe or very severe/lethal	p*	(D) Very severe/lethal	p*
Variables	OR (95% CI)		OR (95% CI)
Male gender	1.76 (1.40–2.23)	<0.001	1.69 (1.20–2.37)	0.003
Age class, in years
•<50 (Ref. cat.)	1	--	1	--
•50–59.9	2.36 (1.68–3.32)	<0.001	3.92 (1.48–10.3)	0.006
•60–69.9	3.51 (2.43–5.07)	<0.001	11.4 (4.63–28.1)	<0.001
•70–79.9	5.72 (3.81–8.58)	<0.001	16.5 (6.66–40.9)	<0.001
•≥80	9.06 (6.04–13.6)	<0.001	27.1 (11.1–66.3)	<0.001
Diabetes	1.52 (1.05–2.18)	0.025	1.58 (1.06–2.34)	0.023
Hypertension	1.10 (0.82–1.47)	0.5	1.39 (0.94–2.05)	0.097
Major cardiovascular diseases **	1.88 (1.32–2.70)	0.001	1.05 (0.71–1.56)	0.8
Cancer	1.26 (0.81–1.95)	0.3	1.11 (0.67–1.82)	0.7
COPD	1.88 (1.11–3.20)	0.020	1.44 (0.84–2.47)	0.2
Renal disease	1.58 (0.90–2.76)	0.11	1.13 (0.64–1.99)	0.7

COPD = Chronic obstructive pulmonary diseases. OR = odds ratio. CI = Confidence interval. ACE = Angiotensin-Converting Enzyme. ARBs = Angiotensin II Receptor Blockers.

* Treatments with ACE inhibitors and ARBs have not been included in both models because of multicollinearity with hypertension.

** Heart failure, myocardial infarction, or stroke.

Discussion

The main findings of this retrospective, observational study, are the following: first, it is confirmed that, among hypertensive subjects, the use of ACE inhibitors or ARBs up to two years preceding SARS-CoV-2 infection did not affect the severity of COVID-19. Second, older age, male gender, diabetes, and the presence of COPD or CVD were independent predictors of severe disease, with a sharp increase of risk among subjects older than 70 years. Third, only older age, male gender and diabetes were associated with a higher likelihood of very severe/lethal disease.

Several hypotheses have been made on the association between COVID-19 progression and treatment with ACE inhibitors and ARBs [20–22, 35, 36]. On one side, some asked whether the therapy should be discontinued during SARS-Cov-2 pandemic [17, 37] because COVID-19 was strongly associated with hypertension, which is frequently treated with ARBs and ACE inhibitors [2-4]. It was indeed hypothesized that: (a) ACE2 up-regulation mediated by ARBs (and, to a lesser extent, by ACE inhibitors) might increase patients' susceptibility to SARS-CoV-2 entry into host cells and further viral propagation [18, 19], (b) virus binding to ACE2 might reduce its activity, thus leading to increased levels of Angiotensin II and consequent pulmonary vasoconstriction, inflammation and oxidative organ damage, and increased risk of acute lung injury [20]. On the opposite side, other scientists suggested that, other than being harmful, ARBs and ACE inhibitors use in patients with cardiovascular risk factors and known or suspected COVID-19 may even exert a beneficial effect, as ACE2 up-regulation could increase the conversion of Angiotensin II to Angiotensin-(1–7), a peptide with potentially protective anti-inflammatory properties [35, 36].

Recently, a few large observational studies based upon in- and outpatient electronic health records [26-28] examined the association between antihypertensive medications and the risk of COVID-19 and/or a severe/lethal disease: our results are in line with most of the previous findings on an absence of risk with ACE inhibitors and/or ARBs use. In brief, the studies by Mancia et al [26], and Reynolds et al [28] reported no difference in COVID-19 severity or death between treated and untreated subjects, for both ARBs and ACE inhibitors. Instead, the results by Mehta et al [27] were partially discordant: the study found no association between treatment with ARBs and death, but those treated with ACE inhibitors showed a significantly higher risk of ICU admission. In our sample, in order to further investigate the potential beneficial effects of ACE inhibitors, the impact of which might be larger in patients with diabetes, COPD, or cardiovascular diseases, we performed additional analyses, stratified by comorbidities. We found however no significant differences in the risk of severe/lethal COVID-19 among treated and untreated patients with either CVD, or diabetes and COPD (data not shown).

Besides sample size and provenance, there were few differences between this and previous studies: all studies included all infected subjects, hospitalized or not, evaluated disease progression beyond mortality, and retrieved medications and admissions from electronic health records (with the exception of Mehta et al, who assessed the medications exclusively at the time of testing for SARS-CoV-2 [27]).

Overall, the present and previous findings confirm those from preliminary Chinese cohorts [24, 31], and although confirmation from randomized studies is required [38], they strongly support the statements of several experts [39, 40] and scientific societies, including the European Medicines Agency [41], the European Society of Cardiology [42], and the American Heart Association [43], who recommend continuation of ARBs or ACE inhibitors medication for all patients, unless otherwise advised by their physicians.

With regard to the role of the other risk factors that have been suggested for severe COVID-19, including age, male gender, hypertension, diabetes, COPD, and major cardiovascular diseases, it has been correctly argued that, so far, available data were unadjusted, thus the relative importance of underlying health conditions was unclear [21, 29]. In this study, we found support for a potential role of gender, diabetes, COPD and CVD, beyond age, in COVID-19 progression to a severe disease, whereas only gender and diabetes significantly increased the risk of a lethal or very severe outcome. Thus, the present study confirms prior findings on the independent relationship of older age and male gender with death [44], and of COPD with progression towards severe disease [26]. Instead, at least two issues may have influenced the conflicting results on the role of CVD and COPD in predicting very severe/lethal disease (an association showed in some prior populations [44]—but lacking in the present as well as in other recent findings [26]): first, the relatively small sample of the present study; second, a marked difference in the population here enrolled, as compared to previous studies which included randomly selected SARS-CoV2 negative subjects as controls [26]. Given the present scenario, further population-based cohort studies, with longer follow-up are clearly needed [29] to clarify these findings.

In addition to a relatively small sample, a limitation of the present study is the lack of tobacco smoking and body mass index among the variables that have been recorded, because we could not extract such information for half of the deceased subjects, as well as for many of those that were not hospitalized. Other limitations are the lack of an evaluation of the severity of the underlying cardiovascular diseases, and the absence of data on other antihypertensive medications. However, their potential role in altering the relationship between ARBS or ACE inhibitors and risk of severe/lethal COVID-19 remains unclear: none of the previous studies on the topic assessed cardiovascular diseases severity [26-28], and the two studies that included the use of other antihypertensive drugs into multivariable analyses did not find substantial differences between the adjusted and unadjusted relative risks of death [26, 28].

In conclusions, the present study did not find any association between COVID-19 severity and treatment with ARBs, ACE inhibitors, or both, and confirms previous findings in supporting the recommendation of several scientific societies to continue ARBs or ACE inhibitors medication for all patients, unless otherwise advised by their physicians, who should thus be reassured. The adjusted analyses substantially confirm prior reports, indicating that the risk of severe or lethal COVID-19 largely and significantly increases among the elderly, males, diabetics, and those with COPD or major cardiovascular diseases.

Database.

(XLS)

Click here for additional data file.

26 May 2020

PONE-D-20-13065

Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

PLOS ONE

Dear Dr. Manzoli,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Besides novelty, which is not an imoportant scope of our journal, there are some issues to be revised as pointed out by two experts.

Please submit your revised manuscript by Jul 10 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information about the participants included in the analysis . Please ensure you have provided sufficient details to replicate the analyses such as: a)  a description of any inclusion/exclusion criteria that were applied to participant inclusion, b) a statement as to whether your sample can be considered representative of a larger population.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. Please clarify the novelty of this study. Especially, how about discussing a little bit more the strength and shortness of this study compared with a report by Mehra et al.?

2. If the data are available, how about comparing the prevalence of hypertension, CVD, and diabetes between the diseased subjects and the whole residence of Provinces of Ferrara and Pescara or Italy?

3. Table 1. Please use 2-way ANOVA followed by post-hoc analysis for the comparison among 3 groups stratified by the COVID-19 severity.

4. Please clarify each variable included in multivariate logistic regression analysis, instead of the current description: “All recorded variables were included a priori in all models, …”

5. Figure 1 may not be necessary.

6. Lines 170-171. The gist of the description is unclear and misleading.

7. Ref 5,6 were not adequate.

Reviewer #2: The authors conducted retrospective case-control study in Italian citizens trying to identify potential independent predictors of severe/lethal COVID-19, and showed no association between COVID-19 severity and treatment with ARBs and/or ACE inhibitors, supporting the previous recommendation to continue the medication. This is an important topic, but it was unfortunate not to see any new findings.

The diagnosis of COVID-19 should be confirmed by PCR tests. Although they described where the laboratory diagnosis was done, they did not mention PCR tests at all. They should provide detailed information of PCR tests.

The worse symptoms of cardiovascular diseases would increase the opportunity to use ARBs or ACE inhibitors. Normalization of the data with disease severity would be necessary. In addition, treatment with ACE inhibitors or ARBs should be compared with other anti-hypertensive drugs.

The authors compared with or without ARBs / ACE inhibitors in hypertension. Sometimes diseases are overlapped. They should clarify whether diabetes or COPD are overlapped with cardiovascular diseases. RAS inhibitors on diabetes patients might have protective impacts on COVID-19 severity.

It is interesting to see the ACE Inhibitors or ARBs treatment showing a trend for reduced odds ratio (Table 2). Beneficial or adverse effects of the drugs would have to be examined more rigorously. For instance, was prescription or medication continued during the infection for each patient?

For references, they cited recent short review or commentary on COVID-19 but mostly ignored key original papers. This is not acceptable. There are nearly two decades of research on ACE2 and SARS-CoV accumulated, and both basic and clinical papers of origin should be cited. The reference should be extensively revised.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 May 2020

Answers to Referee I comments

I-1. The Referee wrote: “Please clarify the novelty of this study. Especially, how about discussing a little bit more the strength and shortness of this study compared with a report by Mehra et al.?"

We agree that, unfortunately, the results are not novel. Please acknowledge, however, that the evidence on SARS-CoV-2 is emerging so rapidly that a finding becomes old after weeks. Please believe that we are the first to be sorry for that. We also entirely agree that the strengths and weaknesses of the study should have been discussed further, comparing it with Mehra et al. and the other observational studies on the same topic (which now also include Mehta et al., JAMA Cardiol 2020). Accordingly, to comply also with the suggestions of the first reviewer, we entirely revised the Discussion section. In specific, the previous paragraphs:

"Recently, three large observational studies based upon inpatient only [14] or in- and outpatient electronic health records [15, 16] examined the association between antihypertensive medications and the risk of COVID-19 and/or a severe/lethal disease: our results substantially confirm these previous findings and, concordantly, no increased risk emerged with ACE inhibitors and/or ARBs use. Of note, Mehra et al [14], after controlling for age, gender, current smoke and comorbidities, found similar death rates among COVID-19 patients using ARBs, and even lower death rates among ACE inhibitors users. The latter results, however - although in line with a previous observational analysis on Chinese in-hospital COVID-19 subjects [12], are based upon hospitalized patients only, may be due to unmeasured confounding and, in the absence of randomized evidence, should be considered only preliminary [23].

Our study, likewise Mancia et al [15] and Reynolds at al [16], included all infected subjects (either hospitalized or not), retrieved all medications and admissions from electronic health records, and evaluated the disease progression beyond mortality. Concordantly, all these findings confirm those from preliminary Chinese cohorts [12, 19] and strongly support the statements of several experts [24, 25] and scientific societies, including the European Medicines Agency [26], the European Society of Cardiology [27], and the American Heart Association [28], who recommend continuation of ARBs or ACE inhibitors medication for all patients, unless otherwise advised by their physicians."

have been replaced by the followings:

"Recently, a few large observational studies based upon inpatient only [26] or in- and outpatient electronic health records [27-29] examined the association between antihypertensive medications and the risk of COVID-19 and/or a severe/lethal disease: our results are in line with most of the previous findings on an absence of risk with ACE inhibitors and/or ARBs use. In brief, the studies by Mancia et al [27], and Reynolds et al [28] reported no difference in COVID-19 severity or death between treated and untreated subjects, for both ARBs and Ace inhibitors. Instead, the results by Mehra et al [26] and Mehta et al [29] were partially discordant: both studies found no association between treatment with ARBs and death, but those treated with ACE inhibitors showed a significantly higher risk of ICU admission in the study by Mehta et al [29], and a significantly lower risk of in-hospital death in the study by Mehra et al. [26]. In our sample, in order to further investigate the potential beneficial effects of ACE inhibitors, the impact of which might be larger in patients with diabetes, COPD, or cardiovascular diseases, we performed additional analyses, stratified by comorbidities. We found however no significant differences in the risk of severe/lethal COVID-19 among treated and untreated patients with either CVD, or diabetes and COPD (data not shown).

Besides sample size and provenance, the main differences between this and previous studies pertain the inclusion criteria (we included all subjects, hospitalized or not, such as Mancia et al [27], Reynolds et al [28], Mehta et al [29], and unlike Mehra et al [26]); the retrieval of all medications and admissions from electronic health records (unlike Mehta et al [29]), and the evaluation of disease progression beyond mortality (unlike Mehra et al [26]).

Overall, the present and previous findings confirm those from preliminary Chinese cohorts [24, 32], and although confirmation from randomized studies is required [39], they strongly support the statements of several experts [40, 41] and scientific societies, including the European Medicines Agency [42], the European Society of Cardiology [43], and the American Heart Association [44], who recommend continuation of ARBs or ACE inhibitors medication for all patients, unless otherwise advised by their physicians."

I-2. The Referee wrote: “If the data are available, how about comparing the prevalence of hypertension, CVD, and diabetes between the diseased subjects and the whole residence of Provinces of Ferrara and Pescara or Italy?"

We agree and accordingly added the following sentences in the Results section: "The sample of infected subjects, compared with the available estimates of the general population of the two provinces [34], showed higher prevalence of diabetes (12.8% in the sample versus �5% in the general population); hypertension (33.9% versus �17%), COPD (6.0% versus �3%) and CVD (16.1% versus �3%). Please acknowledge that, although the estimates of the two provinces were quite similar (with the exception of cardiovascular diseases), the overall estimate from the provinces was derived computing a weighted mean of the latest available data (2018) from the Italian Institute of Statistics.

I-3. The Referee wrote: “Table 1. Please use 2-way ANOVA followed by post-hoc analysis for the comparison among 3 groups stratified by the COVID-19 severity".

We agree and we accordingly used two-way ANOVA followed by Tukey honestly significant difference test. Methods and Table footnotes were updated, while the Results did not differ.

I-4. The Referee wrote: “Please clarify each variable included in multivariate logistic regression analysis, instead of the current description: “All recorded variables were included a priori in all models, …”

We agree and we accordingly added that age, gender, CVD, diabetes, renal disease, cancer and COPD were included a priori in all models.

I-5. The Referee wrote: “Figure 1 may not be necessary".

We agree and accordingly cut Figure 1.

I-6. The Referee wrote: “Lines 170-171. The gist of the description is unclear and misleading."

We agree and we accordingly revised the text. The following sentences:

"On one side, some asked whether the therapy should be discontinued during SARS-Cov-2 pandemic [7, 22] because of a strong association between hypertension and disease severity (although the pharmacological treatment were not assessed) [2-4], and based on the hypothesis that (a) ACE2 up-regulation mediated by ARBs (and, to a lesser extent, by ACE inhibitors) might increase patients' susceptibility to SARS-CoV-2 entry into host cells and further viral propagation [20], (b) virus binding to ACE2 might reduce its activity, thus leading to increased levels of Angiotensin II and consequent pulmonary vasoconstriction, inflammation and oxidative organ damage, and increased risk of acute lung injury [8]"

were replaced with

"On one side, some asked whether the therapy should be discontinued during SARS-Cov-2 pandemic [17, 38] because COVID-19 was strongly associated with hypertension, which is frequently treated with ARBs or ACE inhibitors [2-4]. It was indeed hypothesized that (a) ACE2 up-regulation mediated by ARBs (and, to a lesser extent, by ACE inhibitors) might increase patients' susceptibility to SARS-CoV-2 entry into host cells and further viral propagation [18, 19], (b) virus binding to ACE2 might reduce its activity, thus leading to increased levels of Angiotensin II and consequent pulmonary vasoconstriction, inflammation and oxidative organ damage, and increased risk of acute lung injury [20]."

I-7. The Referee wrote: “Ref 5,6 were not adequate."

We agree, and we accordingly replaced references n. 5-6 with the following (now numbered as [5-8]):

5. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARSCoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020;181:271-80.

6. Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med. 2005;11(8):875-9.

7. Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426: 450-4.

8. Xiao X, Chakraborti S, Dimitrov AS, Gramatikoff K, Dimitrov DS. The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun. 2003;312(4):1159-64

Please also acknowledge that the sentences in the Introduction previously reported as:

"Observing that human pathogenic coronaviruses bind their target cells through angiotensin-converting enzyme 2 (ACE2) [5], and that the expression of ACE2 is substantially increased in diabetics and patients who are treated with ACE inhibitors and angiotensin II type-I receptor blockers (ARBs) [5,6], Fang and colleagues hypothesized that the increased expression of ACE2 would facilitate infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), thus the hypertension treatment with ACE2-stimulating drugs, as well as ACE2 polymorphisms, might increase the risk of developing severe COVID-19 [7]"

were replaced as follows:

"Observing that human pathogenic coronaviruses bind their target cells through angiotensin-converting enzyme 2 (ACE2) [5-8], and that a few studies reported an increase in ACE2 expression mediated by angiotensin II type-I receptor blockers (ARBs) and ACE inhibitors (more consistently on animals than in humans) [9-16], some hypothesized that the increased expression of ACE2 would facilitate infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), thus the hypertension treatment with ACE2-stimulating drugs, as well as ACE2 polymorphisms, might increase the risk of developing severe COVID-19 [17-19]."

We accordingly added the following new references:

9. Furuhashi M, Moniwa N, Mita T, Fuseya T, Ishimura S, Ohno K, et al. Urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin II receptor blocker. Am J Hypertens. 2015;28(1):15-21.

10. Luque M, Martin P, Martell N, Fernandez C, Brosnihan KB, Ferrario CM. Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension. J Hypertens. 1996;14(6):799-805.

11. Zisman LS, Keller RS, Weaver B, Lin Q, Speth R, Bristow MR, et al. Increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2. Circulation. 2003;108(14):1707-12.

12. Ferrario CM, Jessup J, Chappell MC, Averill DB, Brosnihan KB, Tallant EA, et al. Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2. Circulation. 2005;111(20):2605-10.

13. Gallagher PE, Ferrario CM, Tallant EA. MAP kinase/phosphatase pathway mediates the regulation of ACE2 by angiotensin peptides. Am J Physiol Cell Physiol. 2008;295(5):C1169-74.

14. Igase M, Strawn WB, Gallagher PE, Geary RL, Ferrario CM. Angiotensin II AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol. 2005;289(3):H1013-9.

15. Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension. 2004;43(5):970-6.

16. Soler MJ, Ye M, Wysocki J, William J, Lloveras J, Batlle D. Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan. Am J Physiol Renal Physiol. 2009;296(2):F398-405.

18. Esler M, Esler D. Can angiotensin receptor-blocking drugs perhaps be harmful in the COVID-19 pandemic? J Hypertens 2020;38:781-2.

19. Sommerstein R, Grani C. Preventing a COVID-19 pandemic: ACE inhibitors as a potential risk factor for fatal COVID-19. BMJ 2020;368: m810.

Answers to Referee II comments

II-1. The Referee wrote: “The authors conducted retrospective case-control study in Italian citizens trying to identify potential independent predictors of severe/lethal COVID-19, and showed no association between COVID-19 severity and treatment with ARBs and/or ACE inhibitors, supporting the previous recommendation to continue the medication. This is an important topic, but it was unfortunate not to see any new findings. The diagnosis of COVID-19 should be confirmed by PCR tests. Although they described where the laboratory diagnosis was done, they did not mention PCR tests at all. They should provide detailed information of PCR tests."

We entirely agree that his information should have been reported, and we are sorry for the oversight. We accordingly added the following sentences in the Methods section: "All diagnoses were made using (real time) reverse transcription polymerase chain reaction (rRT-PCR) on oropharingeal specimens. The assays were those originally proposed by the Charité-Universitätsmedizin Berlin Institute of Virology [33], and then endorsed by the WHO [34]".

The following references were thus added:

33. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, et al. (2020) Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25.

34. World Health Organization (2020) Laboratory testing for coronavirus disease (COVID-19) in suspected human cases - Interim guidance.

II-2. The Referee wrote: “The worse symptoms of cardiovascular diseases would increase the opportunity to use ARBs or ACE inhibitors. Normalization of the data with disease severity would be necessary. In addition, treatment with ACE inhibitors or ARBs should be compared with other anti-hypertensive drugs."

Please acknowledge that this point is conceptually related to points II-3 and II-4, and they have thus been discussed together. We entirely agree that the worse symptoms of cardiovascular diseases may increase the opportunity to use ARBs or ACE inhibitors, thus RAAS inhibitors on diabetes patients might have protective impacts on COVID-19 severity. And we thus agree that beneficial or adverse effects of the drugs should have been examined more rigorously.

Accordingly, in order to investigate whether RAAS inhibitors might have had protective impacts among the patients with diabetes, COPD and CVD, we stratified the analyses on the potential association between RAAS inhibitors and COVID-19 severity by CVD, COPD, and diabetes. Please find below the results on the sample of hypertensive subjects.

Antihypertensive treatment with: Mild Severe Very severe/lethal

Variables

Stratified by CVD

Without CVD (n=155) (n=106) (n=69)

- ACE inhibitors, % 54.2 45.1 49.3

- ARBs, % 41.9 46.2 42.0

- ACE inhibitors or ARBs, % 91.0 80.2 88.4

With CVD (n=52) (n=101) (n=60)

- ACE inhibitors, % 44.2 43.6 36.7

- ARBs, % 40.4 40.6 38.3

- ACE inhibitors or ARBs, % 80.8 77.2 71.7

Stratified by diabetes

Without diabetes (n=165) (n=151) (n=88)

- ACE inhibitors, % 55.2 45.7 44.3

- ARBs, % 38.8 39.7 34.1

- ACE inhibitors or ARBs, % 89.1 78.8 76.2

With diabetes (n=42) (n=56) (n=41)

- ACE inhibitors, % 38.1 33.9 41.5

- ARBs, % 52.4 53.6 53.7

- ACE inhibitors or ARBs, % 85.7 78.6 90.2

Stratified by COPD

Without COPD (n=194) (n=179) (n=110)

- ACE inhibitors, % 53.1 44.7 44.6

- ARBs, % 40.7 44.7 42.7

- ACE inhibitors or ARBs, % 88.7 81.6 83.6

With COPD (n=13) (n=28) (n=19)

- ACE inhibitors, % 30.8 28.6 36.8

- ARBs, % 53.9 35.7 26.3

- ACE inhibitors or ARBs, % 84.6 60.7 63.2

Among patients with diabetes, COPD or CVD, the use of RAAS inhibitors was not significantly associated with COVID-19 severity. We accordingly added the following sentences in the Discussion section: " In our sample, in order to further investigate the potential beneficial effects of ARBs and ACE inhibitors, the impact of which might be larger in patients with diabetes, COPD, or cardiovascular diseases, we performed additional analyses, stratified by comorbidities. We found however no significant differences in the risk of severe/lethal COVID-19 among treated and untreated patients with either CVD, or diabetes and COPD (data not shown)".

We also agree that it may be of interest to compare ACE inhibitors or ARBs with other anti-hypertensive drugs. However, unfortunately, we could not have the data, as the original protocol focused only on ARBs or ACE inhibitors, and the data on other anti-hypertensive drugs were therefore not collected.

II-3. The Referee wrote: “The authors compared with or without ARBs / ACE inhibitors in hypertension. Sometimes diseases are overlapped. They should clarify whether diabetes or COPD are overlapped with cardiovascular diseases. RAS inhibitors on diabetes patients might have protective impacts on COVID-19 severity."

We agree that some overlapping between cardiovascular diseases and diabetes or COPD exists. Also, we agree that the potential protective effect of RAAS inhibitors among the patients with diabetes or other comorbidities should have been investigated further (as suggested in the following point). We accordingly performed additional, stratified analyses in order to investigate whether RAAS inhibitors might have had protective impacts among the patients with diabetes, COPD and CVD, separately. Please acknowledge that this is issue is conceptually related to issues II-2 and II-3 and was discussed after point II-2.

Concerning the potential overlapping between CVD and COPD or diabetes, this was certainly the case, as shown below. However, please acknowledge that the overlapping was not as high as to cause multicollinearity. It was therefore possible to perform multivariable analyses, adjusting for the potential influence of CVD on the association between RAAS inhibitors and COVID-19 among the subjects with diabetes and COPD.

| Diabetes

CVD | 0 1 | Total

-----------+----------------------+----------

0 | 1,224 121 | 1,345

1 | 185 73 | 258

-----------+----------------------+----------

Total | 1,409 194 | 1,603

| COPD

CVD | 0 1 | Total

-----------+----------------------+----------

0 | 1,297 48 | 1,345

1 | 209 49 | 258

-----------+----------------------+----------

Total | 1,506 97 | 1,603

II-4. The Referee wrote: “It is interesting to see the ACE Inhibitors or ARBs treatment showing a trend for reduced odds ratio (Table 2). Beneficial or adverse effects of the drugs would have to be examined more rigorously. For instance, was prescription or medication continued during the infection for each patient?"

We entirely agree that we should have clarified this issue, and try to investigate further the potential beneficial effects of the drugs on COVID-19. With regard to medication, we added in the text that the treatment with ARBs or ACE inhibitors was not discontinued for any participant. Concerning the potential beneficial effect of the drugs, we performed additional, stratified analyses in order to investigate whether RAAS inhibitors might have had protective impacts among the patients with diabetes, COPD and CVD, separately. Please acknowledge that this is issue is conceptually related to issues II-2 and II-3 and was discussed after point II-2.

II-5. The Referee wrote: “For references, they cited recent short review or commentary on COVID-19 but mostly ignored key original papers. This is not acceptable. There are nearly two decades of research on ACE2 and SARS-CoV accumulated, and both basic and clinical papers of origin should be cited. The reference should be extensively revised."

We agree and thank the Referee for the suggestion. We accordingly replaced the references previously numbered as 5-6 with the followings (now numbered as [5-8]):

5. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARSCoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020;181:271-80.

6. Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med. 2005;11(8):875-9.

7. Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426: 450-4.

8. Xiao X, Chakraborti S, Dimitrov AS, Gramatikoff K, Dimitrov DS. The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun. 2003;312(4):1159-64

Finally, please acknowledge that we also added the following references:

9. Furuhashi M, Moniwa N, Mita T, Fuseya T, Ishimura S, Ohno K, et al. Urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin II receptor blocker. Am J Hypertens. 2015;28(1):15-21.

10. Luque M, Martin P, Martell N, Fernandez C, Brosnihan KB, Ferrario CM. Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension. J Hypertens. 1996;14(6):799-805.

11. Zisman LS, Keller RS, Weaver B, Lin Q, Speth R, Bristow MR, et al. Increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2. Circulation. 2003;108(14):1707-12.

12. Ferrario CM, Jessup J, Chappell MC, Averill DB, Brosnihan KB, Tallant EA, et al. Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2. Circulation. 2005;111(20):2605-10.

13. Gallagher PE, Ferrario CM, Tallant EA. MAP kinase/phosphatase pathway mediates the regulation of ACE2 by angiotensin peptides. Am J Physiol Cell Physiol. 2008;295(5):C1169-74.

14. Igase M, Strawn WB, Gallagher PE, Geary RL, Ferrario CM. Angiotensin II AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol. 2005;289(3):H1013-9.

15. Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension. 2004;43(5):970-6.

16. Soler MJ, Ye M, Wysocki J, William J, Lloveras J, Batlle D. Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan. Am J Physiol Renal Physiol. 2009;296(2):F398-405.

18. Esler M, Esler D. Can angiotensin receptor-blocking drugs perhaps be harmful in the COVID-19 pandemic? J Hypertens 2020;38:781-2.

19. Sommerstein R, Grani C. Preventing a COVID-19 pandemic: ACE inhibitors as a potential risk factor for fatal COVID-19. BMJ 2020;368: m810.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

4 Jun 2020

PONE-D-20-13065R1

Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

PLOS ONE

Dear Dr. Manzoli,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please provide statistical power analysis as reviewer 2 requested.

Please submit your revised manuscript by Jul 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adequately responded to all queries I raised. The manuscript has been improved very much.

Reviewer #2: Comparison with other anti-hypertensive drugs is essential to precisely determine the effects of ACE inhibitors or ARBs on COVID-19 severity and lethality. In addition to COVID-19, the severity of underlying cardiovascular diseases should be also included. As such, it revealed that number and information of the patients in this study was not enough to reach their conclusion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Jun 2020

Answers to Editor's comments

E-1

The Editor wrote: "Please provide statistical power analysis as reviewer 2 requested".

We agree and we accordingly added the following paragraph in the Methods section: "Our sample of 129 hypertensive subjects with severe/lethal COVID-19, and 414 hypertensive subjects with mild disease or asymptomatic infection, had 80% statistical power to detect a difference of 20% or higher (corresponding to a relative risk ≥1.20) in the risk of severe/lethal death between the users of ARBS or ACE inhibitors (exposed group), and non users (controls)".

Answers to Referee I comments

I-1. The Referee wrote: "The authors have adequately responded to all queries I raised. The manuscript has been improved very much".

We thank the Referee for his comment.

Answers to Referee II comments

II-1. The Referee wrote: "Comparison with other anti-hypertensive drugs is essential to precisely determine the effects of ACE inhibitors or ARBs on COVID-19 severity and lethality. In addition to COVID-19, the severity of underlying cardiovascular diseases should be also included. As such, it revealed that number and information of the patients in this study was not enough to reach their conclusion".

On one side, we agree that, hypothetically, both the severity of cardiovascular diseases and the use of other antihypertensive medications could be important to discern more precisely the relationship between ARBS or ACE inhibitors and COVID-19. On the other side, however, please acknowledge that:

1.

None of the four studies published on the topic measured the severity of underlying cardiovascular diseases (Mehta, JAMA Cardiol 2020; Mancia, N Engl J Med 2020; Reynolds, N Engl J Med 2020; Mehra, N Engl J Med 2020). Thus, this remains a plausible but unverified hypothesis and, as happened for the above studies, it cannot cancel the validity of the present study.

2.

Concerning the use of other antihypertensive drugs, of the four studies published on the topic, one did not report these data (Mehta, JAMA Cardiol 2020), one study reported the information but did not include this in multivariable analyses (Mehra, N Engl J Med 2020), and the other two (Mancia, N Engl J Med 2020; Reynolds, N Engl J Med 2020) collected these data and included them in multivariable analysis. In both cases, however, there were no substantial differences between the adjusted and unadjusted relative risks of death of ARBS or ACE inhibitors (which suggests that the additional covariates of the models, including other antihypertensive medications, did not impact alter noticeably the relationship between severity and use of ARBS or ACE inhibitors). This is also supported by the fact that our results were in line with those of the two studies from Mancia et al., and Reynolds et al. Therefore, also this hypothesis, although plausible, remains unverified.

Since it is not proven that both the severity of underlying cardiovascular diseases and use of other antihypertensive drugs may confound, or even mediate, the relationship between COVID-19 and use of ARBS or ACE inhibitors, the validity of the study cannot be nullified by the absence of an evaluation of these hypotheses. We do agree, however, that both hypotheses are potential limitations, and they should have been acknowledged in the text. Accordingly, the following sentences were added in the limitations section:

"Other limitations are the lack of an evaluation of the severity of the underlying cardiovascular diseases, and the absence of data on other antihypertensive medications. However, their potential role in altering the relationship between ARBS or ACE inhibitors and risk of severe/lethal COVID-19 remains unclear: none of the previous studies on the topic assessed cardiovascular diseases severity [26-29], and the two studies that included the use of other antihypertensive drugs into multivariable analyses did not find substantial differences between the adjusted and unadjusted relative risks of death [27,28]".

II-2. The Referee wrote: "The authors did not made all data underlying the findings in their manuscript fully available".

Please acknowledge that this is incorrect: we uploaded the entire dataset - named Appendix S1 - as online supporting information since the initial submission, to comply with PLOS One data availability requirement. Please acknowledge that, at the end of the manuscript, under the "Access to data" section, we reported the following sentence: "The complete dataset used in this work is available in Appendix S1".

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

10 Jun 2020

PONE-D-20-13065R2

Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

PLOS ONE

Dear Dr. Manzoli,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authors responded adequately, however, the reference 26 is retracted.  Please edit the manuscript without citing this article.  Also the information of reference 27 is not completed.

Please submit your revised manuscript by Jul 25 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Jun 2020

Answers to Editor's comments

E-1. The Editor wrote: "The authors responded adequately, however, the reference 26 is retracted. Please edit the manuscript without citing this article".

We agree and we thank the Editor for the comment. Accordingly, we deleted all the citations to the retracted article throughout the text. Please acknowledge that, in place of the above reference, we added the following new reference for some of the sentences in the Discussion:

44. Williamson E, Walker AJ, Bhaskaran K, Bacon S, Bates C, Morton CE, et al. OpenSAFELY: factors associated with COVID-19-related hospital death in the linked electronic health records of 17 million adult NHS patients. MedRxiv. 2020. doi: https://doi.org/10.1101/2020.05.06.20092999.

E-2. The Editor also wrote: "Also the information of reference 27 is not completed".

We agree and accordingly updated and completed the reference. Please accept our apologies for the oversight.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

12 Jun 2020

Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

PONE-D-20-13065R3

Dear Dr. Manzoli,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tatsuo Shimosawa, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

16 Jun 2020

PONE-D-20-13065R3

Predictors of severe or lethal COVID-19, including Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers, in a sample of infected Italian citizens.

Dear Dr. Manzoli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Tatsuo Shimosawa

Academic Editor

PLOS ONE