Airborne aerosol olfactory deposition contributes to anosmia in COVID-19

Alan D. Workman; Aria Jafari; Roy Xiao; Benjamin S. Bleier

doi:10.1371/journal.pone.0244127

Peer Review History

Original SubmissionJuly 16, 2020
27 Oct 2020 Decision Letter - Vladimir Mikheev, Editor PONE-D-20-22099 Airborne Aerosol Olfactory Dispersion Contributes to Anosmia in COVID-19 PLOS ONE Dear Dr. Workman, 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 submit your revised manuscript by November 9, 2020. 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, Vladimir Mikheev 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 and your ethics statement in the online submission form please clarify whether consent was informed consent. 3. Please provide the source and catalog number for the FUL-GLO Fluorescein Sodium used in this study. 4. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information Additional Editor Comments: Dear Dr. Workman, Deep apologies for this long delay. It was really hard to find reviewers but now we have necessary minimum number of reviews. I think this paper is important one but you need to address minor comments suggested by the reviewers. Again sorry for the delay. [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: I Don't Know 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: 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: The objective of this study is to compare aerosol deposition in the OE and RE of nasal passages. Measurements clearly show that small particles are able to reach the olfactory region (mainly by diffusion given that there is very little airflow into the region) while large particles lack diffusive properties and hence travel mainly through the RE as evidenced by its deposition in this region. COVID particles are considered fine and thus can penetrate the OE region. This reviewer has the following comments regarding the manuscript: The Method section is confusing and lacks details. It needs to clearly describe the experimental system for each particle type, choice and reason for the selection of two classes of particles (lines 61 – 63). Droplets are aerosols too. You need to distinguish the aerosols by size and composition. Also, need to explain why you picked large particles versus small. Lines 44 – 46: The statement is partly inaccurate and the process is well described in the aerosol literature. The inhaled air splits at the junction with the majority of the flow (and thus particles regardless of the size) pass through the RE region. However, there is little deposition due to high flow convection in the RE region (thus small gravitational settling). There are far fewer particles reaching the OE region. However, those that reach there deposit by Brownian diffusion and sedimentation. The reason for higher deposition in OE is not due to having more particles but having more deposition. Please revise your hypothesis and clearly explain how you are going to achieve your hypothesis. Line 60: you did not validate your size distribution but measured it. Reviewer #2: The authors should be commended for this study, which hypothesizes that an aerodynamic mechanism may help explain the link between COVID-19 and olfactory dysfunction. Specifically, they show that airborne aerosols differentially deposit in significant concentrations within the olfactory epithelium. This paper, which is brief but methodologically sound, represents a valuable addition to the literature. I would suggest that the authors address several issues prior to potential publication. A primary concern is that there is no mention in the methods/results section of how many subjects were analyzed. The authors should be more clear that only 3 patients (based on the raw data) were analyzed. This small number limits the conclusions one can make from this data and this should be explicitly stated in the discussion section. Furthermore, there is no descriptions of the subjects other than 'healthy.' Further limitations should be added to the discussion section. ****** 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: Yes:** Bahman Asgharian 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. https://doi.org/10.1371/journal.pone.0244127.r001
Revision 1
24 Nov 2020 Author Response Dear Editors: Thank you very much for your consideration and response, as well as for the helpful comments of the reviewers. We have made changes to the manuscript in line with the requests. These are outlined below and are highlighted in the manuscript text. We are confident that the revised work will be of interest to the PLOS ONE readers. Best regards, Alan Workman, MD, MTR Benjamin Bleier, MD, FACS, FARS Associate Editor Comments 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. These style requirements have now been added to the updated manuscript. 2. In your methods section and your ethics statement in the online submission form please clarify whether consent was informed consent. The statement, “…and informed consent was obtained from all participants was added to the first line of the methods. 3. Please provide the source and catalog number for the FUL-GLO Fluorescein Sodium used in this study. The catalog number is now included. The source was Akorn Inc., Lake Forest, IL, USA (written in text as well). 4. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. A separate section on statistical analysis has been added to the methods, with enough detail that utilizing our raw data as well as the statistical tests described our results would be easily reproducible. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. The file has been renamed and the captions have been added. Reviewer 1 Comments 1. The Method section is confusing and lacks details. It needs to clearly describe the experimental system for each particle type, choice and reason for the selection of two classes of particles (lines 61 – 63). Droplets are aerosols too. You need to distinguish the aerosols by size and composition. Also, need to explain why you picked large particles versus small. Lines 44 – 46: The statement is partly inaccurate and the process is well described in the aerosol literature. The inhaled air splits at the junction with the majority of the flow (and thus particles regardless of the size) pass through the RE region. However, there is little deposition due to high flow convection in the RE region (thus small gravitational settling). There are far fewer particles reaching the OE region. However, those that reach there deposit by Brownian diffusion and sedimentation. The reason for higher deposition in OE is not due to having more particles but having more deposition. Please revise your hypothesis and clearly explain how you are going to achieve your hypothesis. The hypothesis has now been revised to be significantly more clear throughout all sections of the manuscript. As you discussed, we have now distinguished between “persistently aerosolized particulate” of smaller size (sub 5-10�m) and larger aerosolized droplets (30-100�m), with the former generated by the nebulizer and the latter generated by the atomizer. This distinction between the size of particulate forms the basis of our hypothesis, in that the smaller particles will not deposit in the RE but do deposit in the OE by diffusion, while larger particulate does indeed settle in the RE. The composition of the particulate remains the same (identical solutions). We have additionally changed the text to highlight that our hypothesis is that small airborne aerosols selectively deposit in the OE, not that disperse to the OE. 2. Line 60: you did not validate your size distribution but measured it. This has been corrected to utilize the word “measure.” Reviewer Two Comments 1. A primary concern is that there is no mention in the methods/results section of how many subjects were analyzed. The authors should be more clear that only 3 patients (based on the raw data) were analyzed. This small number limits the conclusions one can make from this data and this should be explicitly stated in the discussion section. Furthermore, there is no descriptions of the subjects other than 'healthy.' Further limitations should be added to the discussion section. We have now listed “n=3 subjects” in the description of of the raw data and statistics within the results section. Within the methods, we have added the sentences “Participants were healthy subjects between the ages of 25 and 35 without a history of chronic rhinosinusitis, allergic rhinitis, or other rhinologic disease. At the time of data collection, subjects did not have any symptoms of acute sinusitis, rhinorrhea, or subjective nasal obstruction.” In the discussion section, we have now noted additional limitations, and have added the sentence “our study is limited by a small number of subjects as well as uniformity of ambient conditions; temperature and humidity changes in alternate conditions could also affect intranasal diffusion, sedimentation, or other variables contributing to deposition of particulate.” https://doi.org/10.1371/journal.pone.0244127.r002
1 Dec 2020 Decision Letter - Vladimir Mikheev, Editor PONE-D-20-22099R1 Airborne Aerosol Olfactory Deposition Contributes to Anosmia in COVID-19 PLOS ONE Dear Dr. Workman, 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. ============================== Dear Authors, Thank you for revision of your manuscript. I believe you properly addressed Reviewers comments. I think your manuscript is almost ready for publication but I would like you to address couple of my comments as well. Reviewer 1 stated: "There are far fewer particles reaching the OE region. However, those that reach there deposit by Brownian diffusion and sedimentation." Please note that although in general its true (both Brownian diffusion and sedimentation play role) but talking specifically about COVID-19 it has to be noted that single COVID-19 particles (containing only one virion) could be of ~100 nm (60-150 nm) size but peak concentrations found in hospitals were within ~250-500 nm size (please see reference below). Particles of this size range should not deposit by Brownian diffusion (rather particles below 100 nm are subject to Brownian diffusion). Therefore I would strongly suggest to edit your statement made on L56-57 (Caption to Figure 1) "proportion of these aerosols diffusing through Brownian motion into the olfactory epithelium (OE)" to "proportion of these aerosols penetrating into the olfactory epithelium (OE)". Also, L67 - please specify what type of nebulizer was used to generate smaller sized particulate in the sub 5 micron range. Reference: Liu Y, Ning Z, Chen Y, Guo M, Liu Y, Gali NK, et al. Aerodynamic analysis of 193 SARS-CoV-2 in two Wuhan hospitals. Nature. 2020; https://doi.org/10.1038/s41586-194 020-2271-3 ============================== Please submit your revised manuscript by December 14, 2020. 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, Vladimir Mikheev 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. https://doi.org/10.1371/journal.pone.0244127.r003
Revision 2
3 Dec 2020 Author Response Dr. Mikheev, Thank you again for your ongoing consideration of our manuscript. We are happy to address your comments and agree with you that the requested changes make the manuscript clearer and are more harmonious with the existing literature on SARS-CoV-2. The changes have been made in the text (line 56-57 for the first comment) and addition of the nebulizer model to line 67. Best regards, Alan Workman, MD, MTR Benjamin Bleier, MD, FACS, FARS https://doi.org/10.1371/journal.pone.0244127.r004
4 Dec 2020 Decision Letter - Vladimir Mikheev, Editor Airborne Aerosol Olfactory Deposition Contributes to Anosmia in COVID-19 PONE-D-20-22099R2 Dear Dr. Workman, 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, Vladimir Mikheev Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0244127.r005
Formally Accepted
27 Jan 2021 Acceptance Letter - Vladimir Mikheev, Editor PONE-D-20-22099R2 Airborne Aerosol Olfactory Deposition Contributes to Anosmia in COVID-19 Dear Dr. Workman: 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 Dr. Vladimir Mikheev Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244127.r006

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