Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model

Javier Fernández; Iván del Valle Fernández; Claudio J. Villar; Felipe Lombó

doi:10.1371/journal.pone.0253979

Peer Review History

Original SubmissionNovember 10, 2020
24 Nov 2020 Decision Letter - Michael R Hamblin, Editor PONE-D-20-35356 Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model PLOS ONE Dear Dr. Lombó, 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. There are several points raised by the reviewers that must be addressed. The most important ones are lack of appropriate controls and lack of statistical analysis. Please submit your revised manuscript by Jan 08 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. 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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 4. Please ensure that you refer to Figure 2 in your text as, if accepted, production will need this reference to link the reader to the figure. [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know 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: 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: No ****** 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 very well organized, the results are promising; however, minor revision is needed before publication. 1. Please add more up-to-date references in the introduction, and cite them appropriately. 2. I cannot see any statistical analysis in this study, please add the statistical analysis for all of your data 3. please compare your results with the literature in the discussion 4. The figures are really poor, and are not sharp. I cannot follow the trends in the figure. please upload high quality images. Minor revision Reviewer #2: The authors propose to use a combination of 405 nm and 630 nm light for the treatment of onychomycosis in vitro and ex vivo. General comments. The authors need to address my comments before considering publication.Important controls are missing from the study – namely, treatments using light alone. In addition, it would be ideal to evaluate effects of each light wavelength alone (with and without ozone) to understand the role (and potential complementation) of each light wavelength. Also, a serious concern I have is how the fungal inhibition was quantified – the results appear to be qualitative rather than quantitative – thus no statistical significance between each group of tests can be evaluated. Specific comments Line 92 – please can you add a citation showing NADPH-oxidase as the target for 405 nm light in fungi? What about porphyrins? Line 100 – the use of ozone appears to come out of the blue, I think it would be better to introduce ozone first rather than just say that studies are required to validate efficacy of laser treatment with or without ozone. Line 102 – what treatments? Also please define ppm when first used in text. Line 113 – is this the irradiance /cm2? If not, what area? Also, this seems awfully high and I would not classify it as LLLT. Have you measured the thermal effects? Line 115: have you measured transmittance of light through the bag? If so, please include. Line 116: please include the emission spectra Line 131: on or inside the bag? I assume they should be placed inside. Also, were the lids placed on top? If so, how does the lid and plastic influence transmittance? Line 133: I would think experiments should be performed at least in triplicate (independently over separate days). Line 137: why were the control samples incubated for only 48 hours and the treated for 5 days? What is the growth rate of each fungal organism? Would you expect these treatments to attenuate growth rather than inhibit/kill. Line 139: what was the total radiant exposure of each light wavelength that was exposed? If the irradiance is 1.8 (J/cm2?) this would be a very high radiant exposure 1080-3240 J/cm2. I would be concerned about thermal effects. Line 151:was potassium hydroxide not used to dissolve nail keratin and observe the infected nail? Line 170: table 1A and B: this method of ‘quantification’ is not easy for the reader to interpret. What does + vs. ++ mean? We know that +++ means indistinguishable from the control but it is not very helpful. I wonder why did the authors not spread the conidia onto the plate to achieve single colonies so that the CFU might be quantified? To me, this seems far too subjective. In addition, How did the authors determine percent inhibition without quantification? Line 184: I am struggling with the fact that the authors only used the combination of 405 nm and 630 nm under in vitro conditions. For an appropriately designed experiment, they should have also evaluated each respective light wavelength alone to better understand the contribution of each wavelength to the inhibition. The role of the 630 nm wavelength (as per the authors suggestion) is for immunomodulation purposes and to increase blood circulation. In vitro or ex vivo, this is not going to occur. However, it is feasible that 630 nm light might influence the fungal organisms themselves to increase the susceptibility to 405 nm light. Some discussion is needed. Line 186: why were only 40 ppm and 60 ppm selected. It seemed from the ozone only results, they were very effective alone. it seems only A. terreus was tolerant to this. Ideally, some controls evaluating the role of each wavelength alone should be included. In addition, it seems like light alone (without ozone) are missing? Line 214: what is know about immune cell trafficking into the keratin? line 240: do the authors hypothesize that light is an adjuvant for ozone or ozone is an adjuvant for light? ****** 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.0253979.r001
Revision 1
5 May 2021 Author Response Dear Editor, Please find enclosed the manuscript entitled “COMBINED LASER AND OZONE THERAPY FOR ONYCHOMYCOSIS IN AN IN VITRO AND EX VIVO MODEL” which is re-submitted to “PLOS ONE” (reference PONE-D-20-35356). This work describes a strong effective novel method for treating onychomycosis infections for the main pathogenic fungi in humans. The developed combined therapy uses two laser wavelengths and ozone gas. We have accomplished all requirements asked by the referees in the previous revision. Answers to referees’ comments: Editor’s comments: 4. Please ensure that you refer to Figure 2 in your text as, if accepted, production will need this reference to link the reader to the figure. -Figures 2 and 3 have been substituted by three new figures, which now include the statistical analysis. Reviewer #1: The manuscript is very well organized, the results are promising; however, minor revision is needed before publication. 1. Please add more up-to-date references in the introduction, and cite them appropriately. -14 references have been updated. 2. I cannot see any statistical analysis in this study, please add the statistical analysis for all of your data -Statistical analyses have been carried out and 4 new figures with these analyses have been included in the new version (replacing 2 figures and Tables 1 and 2 in the former version). 3. please compare your results with the literature in the discussion -Our results have been compared with recent papers in the discussion section. 4. The figures are really poor, and are not sharp. I cannot follow the trends in the figure. please upload high quality images. -Figures quality depends on the PDF conversion generated in the journal webpage during files uploading. We have enclosed high quality TIFF formats in this version, but they will be transformed as well to PDF format. Minor revision Reviewer #2: The authors propose to use a combination of 405 nm and 639 nm light for the treatment of onychomycosis in vitro and ex vivo. The authors need to address my comments before considering publication. Important controls are missing from the study – namely, treatments using light alone. In addition, it would be ideal to evaluate effects of each light wavelength alone (with and without ozone) to understand the role (and potential complementation) of each light wavelength. Also, a serious concern I have is how the fungal inhibition was quantified – the results appear to be qualitative rather than quantitative – thus no statistical significance between each group of tests can be evaluated. -We have included in this new version, new experiments with treatments using each laser alone and together. Statistical analysis has been carried out in this new version. Specific comments Line 92 – please can you add a citation showing NADPH-oxidase as the target for 405 nm light in fungi? What about porphyrins? -We have included a new reference (Huang et al., 2018) and another one for porphyrins effects (den Hollander et al., 2015). Line 100 – the use of ozone appears to come out of the blue, I think it would be better to introduce ozone first rather than just say that studies are required to validate efficacy of laser treatment with or without ozone. -We have changed the position of this paragraph, were ozone is described as antimicrobial, also as onychomycosis treatment. Line 102 – what treatments? Also please define ppm when first used in text. -These have been done. Line 113 – is this the irradiance /cm2? If not, what area? Also, this seems awfully high and I would not classify it as LLLT. Have you measured the thermal effects? -This is the irradiance per 110 cm2 (the surface area). No thermal effects are detected in the chamber: chamber temperature is always below 30 ºC. Line 115: have you measured transmittance of light through the bag? If so, please include. -The transmittance has been measured and added in the M&M section. Line 116: please include the emission spectra -Data for both emission spectra have been included, showing the minimum, typical and maximum wavelength in each case. Line 131: on or inside the bag? I assume they should be placed inside. Also, were the lids placed on top? If so, how does the lid and plastic influence transmittance? -This has been clarified. Inside the bag, without the lids. Line 133: I would think experiments should be performed at least in triplicate (independently over separate days). -This has been clarified: triplicates were carried out (not duplicates). Line 137: why were the control samples incubated for only 48 hours and the treated for 5 days? What is the growth rate of each fungal organism? Would you expect these treatments to attenuate growth rather than inhibit/kill. -Controls were incubated the same days than the experiments: 7 days. At this time point (7 days) all species (control plates) show good growth rates on the solid media: from 12 mm until 37.5 mm diameter, depending on the fungal species. Line 139: what was the total radiant exposure of each light wavelength that was exposed? If the irradiance is 1.8 (J/cm2?) this would be a very high radiant exposure 1080-3240 J/cm2. I would be concerned about thermal effects. -The actual value is 1.8 W/110.36 cm2, which means 16 mW/cm2. This has been clarified in the manuscript. No thermal effects are detected in the treatment chamber. Line 151: was potassium hydroxide not used to dissolve nail keratin and observe the infected nail? -No, in this work NaOH was not used in the ex vivo model. Line 170: table 1A and B: this method of ‘quantification’ is not easy for the reader to interpret. What does + vs. ++ mean? We know that +++ means indistinguishable from the control but it is not very helpful. I wonder why did the authors not spread the conidia onto the plate to achieve single colonies so that the CFU might be quantified? To me, this seems far too subjective. In addition, How did the authors determine percent inhibition without quantification? -New experiments have been carried out during this time, and Tables 1 and 2 have been replaced for the new 4 figures, which include the statistical analyses. Line 184: I am struggling with the fact that the authors only used the combination of 405 nm and 639 nm under in vitro conditions. For an appropriately designed experiment, they should have also evaluated each respective light wavelength alone to better understand the contribution of each wavelength to the inhibition. The role of the 639 nm wavelength (as per the authors suggestion) is for immunomodulation purposes and to increase blood circulation. In vitro or ex vivo, this is not going to occur. However, it is feasible that 639 nm light might influence the fungal organisms themselves to increase the susceptibility to 405 nm light. Some discussion is needed. -New experiments have been carried out now, with each of the two lasers independently or together. The statistical analyses of these experiments is included in the new version of the manuscript. Line 186: why were only 40 ppm and 60 ppm selected. It seemed from the ozone only results, they were very effective alone. it seems only A. terreus was tolerant to this. Ideally, some controls evaluating the role of each wavelength alone should be included. In addition, it seems like light alone (without ozone) are missing? -The reason is that 20 ppm was poorly effective. 40 and 60 ppm were already quite effective. This allowed to avoid the use of the high dose option (80 ppm), potentially preventing future skin irritations due to the pro-oxidant effect of 80 ppm ozone. Another extra reason is that we wished to detect some potential synergistic effect after including lasers in the ozone treatment. The objective was to select the shorter and less potent treatment option. Line 214: what is known about immune cell trafficking into the keratin? -Blood circulation is enhanced in the subungual bed. line 240: do the authors hypothesize that light is an adjuvant for ozone or ozone is an adjuvant for light? -Our new results indicate that light alone is inactive, but it enhances the ozone effect in the combined treatments. All co-authors have read the report and are in agreement with its content. None of this work has been submitted or published elsewhere. Competing Interest The authors have read the journal’s policy and have the following competing interests: IVF is paid employee of Termosalud SL. 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. All other authors declare that they have no competing interests. Financial Disclosure This study was supported by the Programa Ayudas a Empresas para la Ejecución de Proyectos de I+D+i en el Principado de Asturias en el Periodo 2014-2015 (IE-14-084) and the Programa de Ayudas a Grupos de Investigación del Principado de Asturias (IDI/2018/000120) in the form of grants to FL. IVF received salary from Termosalud SL. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript We thank you for your consideration of this work and we look forward to receiving the new evaluations of this paper. Sincerely, Felipe Lombó PhD. Associate Professor of Microbiology University of Oviedo, SPAIN email: lombofelipe@uniovi.es Attachments Attachment Submitted filename: renamed_7d194.pdf https://doi.org/10.1371/journal.pone.0253979.r002
17 Jun 2021 Decision Letter - Michael R Hamblin, Editor Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model PONE-D-20-35356R1 Dear Dr. Lombó, 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, Michael R Hamblin 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 #1: (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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: (No 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 #1: No https://doi.org/10.1371/journal.pone.0253979.r003
Formally Accepted
21 Jun 2021 Acceptance Letter - Michael R Hamblin, Editor PONE-D-20-35356R1 Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model Dear Dr. Lombó: 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. Michael R Hamblin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0253979.r004

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