PONE-D-20-36443

The pulmonary mycobiome - a study of subjects with and without chronic obstructive pulmonary disease

PLOS ONE

Dear Dr. Martinsen,

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 note that there are significant issues raised during the review process focused on the technical aspects of the data acquisition including controls that need to be addressed (please see reviewers comments below). Data availability has to also comply with PLOS policies and our reviewers were not able to access your raw data. Please ensure all of this and he below comments are fully addressed in any revision.

Please submit your revised manuscript by Feb 07 2021 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,

Sanjay Haresh Chotirmall, MD PhD

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. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. ("Parts of the content of this manuscript has been presented at the National Microbiota Conference in Norway, and an abstract was published in an online programme. It was also accepted for an oral presentation at the ERS International Congress in 2019, and published as an abstract in the European Respiratory Journal.") Please clarify whether this conference was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

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

"I have read the journal's policy and the authors of this manuscript have the following competing interests:

EMHM, EOL, IH, GRH, KSK, CD, WS, and APG declare no conflict of interest.

RN reports grants from The endowment of timber merchant A. Delphin and wife (The Norwegian Medical Association) and grants from GlaxoSmithKline during the conduct of the study, and grants and personal fees from AstraZeneca, grants and personal fees from GlaxoSmithKline, grants and personal fees from Boehringer Ingelheim, and grants from Novartis outside the submitted work.

TMLE reports grants from Helse Vest (Western Norway Regional Health Authority) during the conduct of the study, and personal fees from Boehringer Ingelheim outside the submitted work."

Please confirm that this 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 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.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

4. Thank you for stating the following in the Financial Disclosure section:

"The MicroCOPD study was funded by unrestricted grants and fellowships from Helse Vest, GlaxoSmithKline, Bergen Medical Research Foundation, and the Endowment of Timber Merchant A. Delphin and Wife through the Norwegian Medical Association.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that one or more of the authors are employed by a commercial company: 3Sequentia Biotech SL, Carrer D'Àlaba 61, 08005, Barcelona, Spain

(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.

“The funder provided support in the form of salaries for authors [insert relevant initials], 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.

(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.

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.

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

[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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: No

Reviewer #2: No

**********

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: No

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: Martinsen EMH et al; evaluated the oral wash and BAL mycobiome in COPD (n=93) and control subjects (n=100) from a single center in Western Norway. Candida was the dominant genus in all samples with higher abundance in oral compared to BAL samples. No difference in alpha and beta-diversity between study groups and ICS used.

This is the first study using BAL to access the airway mycobiome in COPD, however there were numerous weakness which need to be address

Major comments

• The control subjects were defined as no COPD and other lung diseases, however 22 of the control subjects had FEV/FVC <0.7, is this group of patient smoker? why are their lung function obstructive? Any underlying bronchiectasis or asthma which explained the abnormal lung function? As their spirometry is abnormal is hard to believe that these individuals have no underlying lung diseases, perhaps they should be removed from the control group and reanalyzed.

• The author mention that PBS was obtained as negative control samples, and contamination removed with Decontam, but it seems that negative control samples are also dominated by Candida (Figure S2), what was the abundance of Candida in the negative control samples?

• What is the clinical relevance and impact of this study finding?

• Was the sample collected at stable COPD? how many weeks post exacerbations were patients allow to be recruited for the study?

• Is there any patient on long term oral corticosteroid, antifungal or antibiotics?

• What is the breakdown of patient in each group (Figure 4) how many patients in COPD grade I/II and COPD grade III/IV?

• Please ensure sequencing data is publicly available

• The weakness including cross-sectional nature, interaction between various kingdom and host responses were not access in this study and should be discuss

Reviewer #2: General comments:

The authors present an ITS amplicon sequencing study of involving COPD patients and controls (n = 93 vs n = 100). From each subject, mycobiome profiles were derived for BAL, oral was, and a negative controls. While, the analysis of this data reveals difference between the BAL and Oral rinse samples, control and COPD patients do not significantly differ. No association between the mycobiome and clinical outcome is observed.

I have some specific comments and suggestions below;

1. What DNA yields were achieved in samples (OW and BAL) vs negative extraction blanks? This is a critical point. This data should be plotted and included in the manuscript. Both the DNA and fungal yields need to be quantified in some way to demonstrate that the sampling technique actually works i.e. a significantly higher DNA/fungal/Fungal DNA yield is obtained in samples relative to background contamination. Any samples where this cannot be safely concluded should probably be excluded.

2. What was the total raw sequencing depth and total read count in samples (OW and BAL) vs negative extraction blanks (NCS)? Please include a figure/table. Figure S2 shows only relative abundance i.e. the stacked bar charts are proportionally scaled and there is no way to determine how many reads were detected in the NCS compared to samples. A single figure representing aggregate read numbers for each taxonomic classification (genus level) for OW NCS and BAL would be useful. The multiple graphs in figure S2 are informative but a little difficult to digest at first glance. An accompanying graph summarising the aggregate comparison would help – i.e. Total read counts for NCS, OW and BAL for all reported OTUs.

3. For many subjects, negative controls (NCS) are highly similar to the samples (BAL/OW). Unless there is some other evidence for a signal in these samples (i.e. much higher DNA yield/read count in the samples vs NCS) then these must be excluded from analysis. If NCS profiles match either a BAL or OW sample and have comparable yield in terms of DNA yield or total number of classified reads then there is no way to tell whether this signal is coming from the sample or represents stochastic background contamination signal. Such cases should be systematically identified, excluded and the analysis repeated. This could impact interpretation regarding clinical association.

4. Data and code repository not accessible (https://doi.org/10.5061/dryad.w3r2280nz)

5. Batch effects. The authors assessed batch effects associated with sequencing runs. Assuming the DNA extractions were not all done as a single batch, the same analysis should be performed for DNA extraction batch effects (between-batch effects).

6. Confusing statement in the discussion concerning “medians were dominated by zeros, comparable to the 0.03 found in the CF study”. Please revise and state clearly what is meant by this without requiring the reader to refer to another paper.

7. Final paragraph; “still in its infancy” > “still in their infancy”

**********

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.

The pulmonary mycobiome - a study of subjects with and without chronic obstructive pulmonary disease

PONE-D-20-36443R1

Dear Dr. Martinsen,

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,

Sanjay Haresh Chotirmall, MD PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

Reviewer #2: Yes

**********

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 #2: Yes

**********

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 #2: Yes

**********

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 #2: The authors have provided a detailed and extensive rebuttal that has satisfactorily addressed my concerns in so far as possible. I comment the authors on the thoroughness of the response.

**********

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 #2: No