I confirm the following proposed Data Availability statement is accurate and suitable to appear alongside our manuscript.

"Data underlying the study cannot be made publicly available due to ethical concerns about patient confidentiality. Data will be made available to qualified researchers on request to HealthTrends@QuestDiagnostics.com."

Sincerely yours,

Michael F Holick PhD MD

Thank you for submitting your manuscript entitled "SARS-CoV-2 Positivity Rates Associated with Circulating 25-Hydroxyvitamin D Levels" to PLOS ONE. Your manuscript files have been checked in-house but before we can proceed we need you to address the following issues:

(1) Thank you for providing an updated Funding statement. We've made a minor change to meet our requirements, can you please confirm whether the following proposed statement is accurate and suitable to appear alongside your manuscript?

"Quest Diagnostics provided support in the form of salaries for authors JKN, BC, MHK, and HWK and consulting fees for MFH but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

We will update the statement on your behalf with your approval.

Author’s response: Thank you, we approve, please provide middle initial (MHK) for author as well.

(2) We're also requesting the following update to your Competing Interest statement:

"HWK, JKN, MHK, and CB are employees of Quest Diagnostics. HWK, MHK and CB own stock in Quest Diagnostics. MFH is a consultant to Quest Diagnostics and was on the speakers’ bureau for Abbott Inc. and Hyatt Pharmaceutical Industries Company PLC. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials."

If any of this information is incorrect, please clarify, otherwise we will update the statement with your approval.

Author’s Response: Please see the response below for our statement on data sharing. If this conforms to your policies then the updated competing interest statement is approved.

(3) We note that you've stated the following regarding your data: "The data underlying the results presented in the study are available from Quest Diagnostics Clinical Laboratory Database and are stored in the Quest Diagnostics Informatics Data Warehouse.”

PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Fjournals.plos.org%2Fplosone%2Fs%2Fdata-availability%23loc-unacceptable-data-access-restrictions&data=02%7C01%7CHarvey.W.Kaufman%40questdiagnostics.com%7C204b422f20524f74c4bd08d849bc2198%7Cb68c6481b22b46b38c4c0024bb9b9b1f%7C1%7C0%7C637340419657309411&sdata=7dkabK3hbEyGDtegn0XYB5rParh4lkIFSbeny812%2Bsc%3D&reserved=0.

Before we proceed with your manuscript, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bmj.com%2Fcontent%2F340%2Fbmj.c181.long&data=02%7C01%7CHarvey.W.Kaufman%40questdiagnostics.com%7C204b422f20524f74c4bd08d849bc2198%7Cb68c6481b22b46b38c4c0024bb9b9b1f%7C1%7C0%7C637340419657309411&sdata=1tq7CqcqrkL%2F6Ke9QspE%2FQzIGfCmtiNp9rs1tYyFN9I%3D&reserved=0 for guidelines on how to de-identify and prepare clinical data for publication. For a list of recommended repositories, please see https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fjournals.plos.org%2Fplosone%2Fs%2Frecommended-repositories&data=02%7C01%7CHarvey.W.Kaufman%40questdiagnostics.com%7C204b422f20524f74c4bd08d849bc2198%7Cb68c6481b22b46b38c4c0024bb9b9b1f%7C1%7C0%7C637340419657309411&sdata=Og4xLiA%2Fo0ERih%2Byo5fBPwpPWaRkTX3XmJRFeu%2BwJFU%3D&reserved=0. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Authors’ Response: In accordance with Quest Diagnostics company policy on data security we are not able to upload the 191,000 rows of data to a public repository. We cannot violate HIPAA and open our Data Informatics Warehouse to external parties. This legal restriction applies to academic institutions as well as to our commercial organization.

Quest Diagnostics has previously published in PLoSONE:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180840

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118108

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063416

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028201

In all prior circumstances as with this study, we have made data available to researchers upon request. We are eager to have others validate our analysis, suggest new ways to analyze and interpret our data, and explore how we can further understanding of health and disease. In recent months we have worked on other subjects with investigators from many outside organizations including the CDC, Boston University, Penn State University, the University of Alabama, the Alameda Health System, and many others. Requests for data and research collaborations can be sent to HealthTrends@QuestDiagnostics.com

(4) In the Methods section of your manuscript, please include the following statement: "HIPAA clearly defines research use of data as analyzed for this and numerous other studies based on the Quest Diagnostics Data Informatics Warehouse (45 CFR 164.501, 164.508, 164.512(i) (See also 45 CFR 164.514(e), 164.528, 164.532) Link: https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.hhs.gov%2Fhipaa%2Ffor-professionals%2Fspecial-topics%2Fresearch%2Findex.html&data=02%7C01%7CHarvey.W.Kaufman%40questdiagnostics.com%7C204b422f20524f74c4bd08d849bc2198%7Cb68c6481b22b46b38c4c0024bb9b9b1f%7C1%7C0%7C637340419657314405&sdata=R9Yjj38kKasss73QqHM0u2z16rLwk64VX3QAImAFrRg%3D&reserved=0 ). Quest Diagnostics takes the additional step of having its process reviewed annual by the Western Institutional Review Board who has determined the process is “deemed exempt."

Authors’ Response: We have added this statement to the methods section.

Your manuscript has been returned to your account. Please log on to PLOS Editorial Manager at https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.editorialmanager.com%2Fpone%2F&data=02%7C01%7CHarvey.W.Kaufman%40questdiagnostics.com%7C204b422f20524f74c4bd08d849bc2198%7Cb68c6481b22b46b38c4c0024bb9b9b1f%7C1%7C0%7C637340419657314405&sdata=%2F2Lb3ZZU82hslYEkPeYh8KeqjCgaz5Yz8r9Olq47s%2BU%3D&reserved=0 to access your manuscript.

Your manuscript can be found in the "Revisions Sent Back to the Author" link under the New Submissions menu. After you have made the changes requested above, please be sure to view and approve the revised PDF after rebuilding the PDF to complete the resubmission process.

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Agnes Magyar

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

Authors’ Reponse: We have made the formatting changes outlined in these documents.

2. In your ethics statement in the Methods section and in the online submission form, please provide additional information about the data used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Authors’ response: HIPAA clearly defines research use of data as analyzed for this and numerous other studies based on the Quest Diagnostics Data Informatics Warehouse (45 CFR 164.501, 164.508, 164.512(i) (See also 45 CFR 164.514(e), 164.528, 164.532) Link: https://www.hhs.gov/hipaa/for-professionals/special-topics/research/index.html ). Quest Diagnostics takes the additional step of having its process reviewed annual by the Western Institutional Review Board who has determined the process is “deemed exempt.” This statement is included in the manuscript text to assure the readers. Finally, patient privacy and adherence to HIPAA is of prime important to Quest Diagnostics to provide patients, healthcare providers, and the public trust in its operations ( https://www.questdiagnostics.com/home/privacy/ ).

3. Thank you for stating the following in the Competing Interests section:

'HWK, JKN, MHK, and CB are employees of Quest Diagnostics. HWK, MHK and CB

own stock in Quest Diagnostics. MFH is a consultant to Quest Diagnostics and was on the speakers’ bureau for Abbott Inc. and Hyatt Pharmaceutical Industries Company

PLC.'

We note that one or more of the authors are employed by a commercial company: Quest Diagnostics.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

Authors’ Response: We added: “The funder provided support in the form of salaries for authors JKN, BC, MK, and HWK and consulting fees for MFH but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Authors’ response: Not applicable.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Authors’ Response: The Competing Interests Statement is in compliance with this request. We will add the following to the data sharing statement: “The data underlying the results presented in the study are available from Quest Diagnostics Clinical Laboratory Database and are stored in the Quest Diagnostics Informatics Data Warehouse.”

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Authors’ Response: We have added these to an updated cover letter.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Additional Editor Comments (if provided):

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Reviewers' comments:

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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: Partly

Reviewer #2: Partly

________________________________________

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

Reviewer #1: No

Reviewer #2: No

________________________________________

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: No

________________________________________

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

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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: In this manuscript, Kaufman, et al. examined the relationship between the SARS-CoV-2 positivity and circulating levels of 25-hydroxyvitamin D (25OHD). They analyzed the data of from over 190,000 patients and found strong inverse correlation between SARS-CoV-2 positivity and 25OHD levels, which persisted across latitudes, races/ethnicities, both sexes and age ranges. This paper may provide a rationale to investigate the role of vitamin D supplementation in reducing the risk of SARS-CoV-2 infection. The reviewer’s specific comments are described below.

Specific comments

1. Figures and line 112-113 in the text. To analyze the relationship between circulating 25OHD levels and SARS-CoV-2 positivity, the authors assigned the 25OHD values <20 ng/mL or ≥�60 ng/mL as 19 ng/mL or 60 ng/mL, respectively. However, since many subjects have 25OHD levels lower than 20 ng/mL in the United States, the authors should use the raw data of 25OHD for the analyses.

Authors’ Response: We chose the cutoff of <20 ng/mL to be more clinically relevant as it is in keeping with the definition of vitamin D deficiency. Given the small number of patients with extreme values, and our message that compares those with vitamin D deficiency to patients where the end of increasing benefits exists at 55 ng/mL we feel these cutoffs are appropriate. However, we acknowledge that most of the patients with vitamin D deficiency have values between 16-20 ng/mL, enough to be statistically relevant. If we were to analyze the data with the bottom bin at 15 ng/mL the following would happen:

The R-squared value in Figure 1 would increase from 0.96 to 0.97. The unadjusted odds ratio for 25(OH) would increase 0.001 from 0.979 (95% CI 0.977-0.980) to 0.980 (95% CI 0.978-0.981). The adjusted odds ratio for 25(OH) would also increase 0.001 from 0.984 (95% CI 0.983-0.986) to 0.985 (95% CI 0.984-0.986). All effect sizes of other factors in the multivariable model remain identical except the predominately black non-Hispanic ZIP codes, which would decrease 0.01. The mean 25(OH) level would fall from 31.7 (SD 11.7) to 31.2 (SD 12.3).

Given the lack of clinically significant impact on the main statistical outcomes, the clinical relevance gained by grouping all vitamin D deficient patients, and the urgent nature of the message this study contains (a major reason we chose to submit to PLOS ONE, expediency) we would greatly prefer to keep the groupings unchanged.

2. Lines 87-91. Four different assays were used as SARS-Cov-2 RNA NAATs. Please provide the information on the sensitivity of each assay.

Authors’ Response: We have added four FDA references for the four tests in question and added the following statement to the methods section: “We combined results from all four tests due to their very similar sensitivity and specificity.[8-11]”

3. Table 1. “Vitamin D” should be changed to “25OHD”.

Authors’ Response: We agree and have made this change. Thank you for catching this.

Reviewer #2: Kaufman et al. implemented retrospective analytic methods to identify an association between vitamin D levels and SARS-CoV-2 infection rate. The results are potentially interesting due to the need for viable SARS-CoV-2 treatments and a greater understanding for SARS-CoV-2 infection pathology. The manuscript is well-written and the figures are clear. However, interpreting these results are difficult due these vitamin D detection methods. Here, vitamin D was measured using two techniques: (1) immunoassay and (2) LC-MS. While LC-MS is considered the gold standard due to both its sensitivity and reliability, it is readily established that vitamin D immunoassays frequently overestimate or underestimate 25(OH)D concentrations (Kocak et al., 2015; Holmes et al., 2013; Farrell et al., 2012). I acknowledge the authors' efforts to consider all potential confounding variables related to ethnicity, geographical location, sex, and age. Nevertheless,the authors do not consider the vitamin D detection method as a limitation. Therefore, the association between SARS-CoV-2 infection rate and vitamin D levels may be confounded by the detection method. The regression analysis in which infection rate was assessed as a function of vitamin D levels appear positively skewed towards extremely low vitamin D levels, which could be due to the method of vitamin D detection.

In order to properly assess SARS-CoV-2 infection rate as a function of vitamin D levels, I suggest the authors to conduct analyses by detection method.

Authors’ Response: 98.8% of specimens were tested using immunoassay. In a company-wide analysis the immunoassay and LC/MS-MS methods obtain nearly identical values, with the immunoassay yielding values 1 to 2 ng/mL less, on average, than the LC/MS-MS methodology. In addition, the difference between the methods is tiny in comparison to the range of results and if it did have an effect, it would only diminish the statistical significance of results, not magnify the effect. In the regression analysis in which infection rate was assessed as a function of 25(OH)D levels the specimens tested by LC/MS-MS had an unadjusted odds ratio of 0.956 (95% CI 0.942-0.970), stronger than the unadjusted odds ratio for immunoassay tests 0.979 (95% CI 0.977-0.980) which is also the results for all specimens combined.

We have added the following sentence to the results: “98.8% of included patients had 25(OH)D levels assessed with immunoassay testing methodology.”

________________________________________

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Reviewer #1: No

Reviewer #2: No

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