Dry heat sterilization as a method to recycle N95 respirator masks: The importance of fit

John G. Yuen; Amy C. Marshilok; Peter Todd Benziger; Shan Yan; Jeronimo Cello; Chavis A. Stackhouse; Kim Kisslinger; David C. Bock; Esther S. Takeuchi; Kenneth J. Takeuchi; Lei Wang; Sruthi Babu; Glen Itzkowitz; David Thanassi; Daniel A. Knopf; Kenneth R. Shroyer

doi:10.1371/journal.pone.0257963

Peer Review History

Original SubmissionAugust 27, 2021
29 Oct 2021 Decision Letter - Swatantra Pratap Singh, Editor PONE-D-21-27839Dry heat sterilization as a method to recycle N95 respirator masks: the importance of fitPLOS ONE Dear Dr. Shroyer, 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 15th Dec 2021. 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. 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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: Yes 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: No 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 manuscript is technically sound and well written . However, in the results section the complete data for thermal stability on N95 Respirators seems to be missing. Thus same can be provided in the supporting information. Apart from this , following are some of the queries which may improve the paper further. Comment 1: Introduction section line 83; “…OSHA compliant fit factors that quantitatively evaluate whether a respirator fits properly…”. OSHA is used for the first time Thus it is advised to write the full form of OSHA over here. Comment 2: Materials and Methods section line 123; “VeroE6 cells were routinely cultured in DMEM containing Glutagro (Corning) and 8%...”. writing the full form of DMEM media is recommended. Comment 3: N95 FFR Quantitative Fit Tests line 157-158; “Quantitative fit testing was performed on the same operator for all mask types and for all conditions: dry heat treated (n = 3), untreated (n = 1), and autoclaved (n = 1) respirators”. As comparison is done between dry heat treated, untreated, and autoclaved respirators taking the value of n 3, 1, and 1 respectively, then why the heterogeneity is there in terms of the sample size n? Comment 4: N95 FFR Quantitative Fit Tests line 159-165; “Quantitative fit testing procedures for N95 respirators were performed according to Occupational Safety…. passes OSHA guidelines”. It would be preferable for the readers if the authors could add the reference in this paragraph. Comment 5: N95 FFR Quantitative Fit Tests line 166. Please check the formula of the Overall Fit factor and correct the running variable (or summation index). Also, it is advised to the author to describe the term n in the text as this will help the reader to understand better. Comment 6: N95 FFR Quantitative Fit Tests line 168. It would be preferable for the readers if the authors could add the reference for Table 1. Comment 7: Estimated Filtration line 285-286. “A fit factor of 100 and 200 corresponds to a filtration efficiency of 99 and 99.5% respectively (both imply passing of the fit test)”. How this relation between fit factor and filtration efficiency is drawn? It would be preferable for the readers if the authors could offer additional detail and explain the reason behind this trend. Comment 8: Estimated Filtration line 290. In table 2 there is no indication of the calculation used to determine estimated filtration (%). Thus, it would be preferable for the readers if the authors could include the information of calculation done. Comment 9: Please check the uniformity of the References. For instance: Few details of reference 11 are missing. Reviewer #2: The work submitted is interesting and can be accepted with minor revisions. Please check the notations throughout the manuscript and keep it uniform. Line 71: Please mention the time as it is a critical parameter for autoclaving Follow the units in the same format throughout Please provide a reference for Table 1 with a source Please improve figure one ****** 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. https://doi.org/10.1371/journal.pone.0257963.r001
Revision 1
24 Nov 2021 Author Response Manuscript PONE-D-21-27839 Response to Reviewers Dear Dr. Singh and Reviewers, Thank you for the feedback and for giving us the opportunity to submit a revised draft of the manuscript “Dry heat sterilization as a method to recycle N95 respirator masks: the importance of fit” for publication in PLOS ONE. We appreciate the time and effort that you and the reviewers dedicated to the review of the manuscript, and we are grateful for the insightful comments. We have incorporated the suggestions made by the reviewers and have outlined them below and within the manuscript. Reviewer #1: The manuscript is technically sound and well written. However, in the results section the complete data for thermal stability on N95 Respirators seems to be missing. Thus same can be provided in the supporting information. Author response: Thank you very much! As for the data for thermal stability, the experiments were done in triplicate, where SARS-CoV-2 virus was recovered by cutting each virus-treated spot, from each N95 mask and submerging the samples in media. Briefly, samples were incubated for 5 minutes at room temperature then rocked gently to recover virus. Plaque assays were performed to quantify the amount of virus recovered by performing serial dilutions of recovered virus and infecting VeroE6 cells for one hour. To quantify the amount of virus recovered, the overlay was removed, and the plaques were visualized by staining VeroE6 cells with 0.5% crystal violet and 0.8% glutaraldehyde in 50% methanol. Total PFU/mL for each condition was calculated by averaging the mean PFU/mL recovered for each biological replicate. Additional details, including the description above, are written in the Methods section “SARS-CoV-2 Thermal Stability/Viral Recovery and Quantification.” Comment 1: Introduction section line 83; “…OSHA compliant fit factors that quantitatively evaluate whether a respirator fits properly…”. OSHA is used for the first time Thus it is advised to write the full form of OSHA over here. Author response: Thank you for pointing this out, we have written out “Occupational Safety and Health Administration” as advised in line 84 of the revised manuscript. Comment 2: Materials and Methods section line 123; “VeroE6 cells were routinely cultured in DMEM containing Glutagro (Corning) and 8%...”. writing the full form of DMEM media is recommended. Author response: Thank you for pointing this out, we have written out “Dulbecco’s Modified Eagle Medium” as advised in line 140 of the revised manuscript. Comment 3: N95 FFR Quantitative Fit Tests line 157-158; “Quantitative fit testing was performed on the same operator for all mask types and for all conditions: dry heat treated (n = 3), untreated (n = 1), and autoclaved (n = 1) respirators”. As comparison is done between dry heat treated, untreated, and autoclaved respirators taking the value of n 3, 1, and 1 respectively, then why the heterogeneity is there in terms of the sample size n? Author response: Thank you for pointing this out. At the time the experiment was conducted, there was a shortage of N95 respirators, and the supply of respirators were tightly regulated by Environmental Health and Safety at our institution. We therefore decided to take a conservative approach, as we must destroy the N95 respirators in order to perform fit testing by puncturing the masks in order to affix a valve onto the respirator. Similarly, autoclaving a N95 respirator will undoubtedly damage the respirator as reported in the literature (doi: 10.1016/j.jhin.2020.06.030, 10.1371/journal.pone.0243965) and as observed in our experiment by the deformation of the respirator. Comment 4: N95 FFR Quantitative Fit Tests line 159-165; “Quantitative fit testing procedures for N95 respirators were performed according to Occupational Safety…. passes OSHA guidelines”. It would be preferable for the readers if the authors could add the reference in this paragraph. Author response: Thank you for pointing this out. We have added the appropriate citation. Comment 5: N95 FFR Quantitative Fit Tests line 166. Please check the formula of the Overall Fit factor and correct the running variable (or summation index). Also, it is advised to the author to describe the term n in the text as this will help the reader to understand better. Author response: Thank you for pointing this out. We have clarified the definition of n in the text as the number of exercises and corrected the typo in the summation index. Comment 6: N95 FFR Quantitative Fit Tests line 168. It would be preferable for the readers if the authors could add the reference for Table 1. Author response: Thank you for pointing this out. We have added the citation to Table 1. Comment 7: Estimated Filtration line 285-286. “A fit factor of 100 and 200 corresponds to a filtration efficiency of 99 and 99.5% respectively (both imply passing of the fit test)”. How this relation between fit factor and filtration efficiency is drawn? It would be preferable for the readers if the authors could offer additional detail and explain the reason behind this trend. Author response: Thank you for pointing this out. We have clarified that the estimated filtration can be calculated based off the definition of a fit factor (the ratio of the concentration of ambient particles to the concentration of particles inside the respirator), thus is equal to 1 – 1/FitFactor. These changes are reflected in lines 349-351. Comment 8: Estimated Filtration line 290. In table 2 there is no indication of the calculation used to determine estimated filtration (%). Thus, it would be preferable for the readers if the authors could include the information of calculation done. Author response: We think this is an excellent suggestion and have addressed it with the same clarifications we made in response to Comment 7. Comment 9: Please check the uniformity of the References. For instance: Few details of reference 11 are missing. Author response: Thank you for pointing this out. We have double checked the references and added the missing details to references 11 and 2. Reviewer #2: The work submitted is interesting and can be accepted with minor revisions. Author response: Thank you very much! Please check the notations throughout the manuscript and keep it uniform. Author response: Thank you for pointing this out. We have made some revisions on the notation and have clarified the usage of mask, respirator, and FFR according to their most precise and accurate definitions as most commonly used by the CDC, NIOSH, OSHA and the manufacturers. Line 71: Please mention the time as it is a critical parameter for autoclaving Author response: Thank you for pointing this out. The parameters are detailed more thoroughly in the Methods section, where we have stated that the autoclave time is for 30 minutes. The mention of autoclave conditions in line 71 are to point out in the Introduction that autoclaving involves both steam and pressure and typical conditions are given. Follow the units in the same format throughout Author response: Thank you for pointing this out, we have double checked the units and have made revisions in the text. Please provide a reference for Table 1 with a source Author response: Thank you for pointing this out. We have added the appropriate citation. Please improve figure one Author response: Thank you for the suggestion. We have decided that this figure does not add much to the manuscript, and we have therefore removed it. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0257963.r002
14 Dec 2021 Decision Letter - Swatantra Pratap Singh, Editor Dry heat sterilization as a method to recycle N95 respirator masks: the importance of fit PONE-D-21-27839R1 Dear Dr. Shroyer, 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, Swatantra Pratap Singh, Ph.D. Academic Editor PLOS ONE 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 #1: 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 #1: 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 #1: All the comments has been addressed well. However, the suggestion regarding the the complete data for thermal stability on N95 Respirators could have been addressed in a more effective way. ****** 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 https://doi.org/10.1371/journal.pone.0257963.r003
Formally Accepted
16 Dec 2021 Acceptance Letter - Swatantra Pratap Singh, Editor PONE-D-21-27839R1 Dry heat sterilization as a method to recycle N95 respirator masks: the importance of fit Dear Dr. Shroyer: 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. Swatantra Pratap Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257963.r004

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