A study protocol to characterise pathophysiological and molecular markers of rheumatic heart disease and degenerative aortic stenosis using multiparametric cardiovascular imaging and multiomics techniques

Daniel W. Mutithu; Olukayode O. Aremu; Dipolelo Mokaila; Tasnim Bana; Mary Familusi; Laura Taylor; Lorna J. Martin; Laura J. Heathfield; Jennifer A. Kirwan; Lubbe Wiesner; Henry A. Adeola; Evelyn N. Lumngwena; Rodgers Manganyi; Sebastian Skatulla; Richard Naidoo; Ntobeko A. B. Ntusi

doi:10.1371/journal.pone.0303496

Peer Review History

Original SubmissionJanuary 8, 2024
13 Mar 2024 Decision Letter - Mohanad Alkhodari, Editor PONE-D-24-00937A study protocol to characterise pathophysiological and molecular markers of rheumatic heart disease and degenerative aortic stenosis using multiparametric cardiovascular imaging and multi-omics techniquesPLOS ONE Dear Dr. Mutithu, 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 Apr 27 2024 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohanad Alkhodari Academic Editor PLOS ONE Comments from Senior Staff Editor: We note that one or more reviewers has recommended that you cite specific previously published works. As always, we recommend that you please review and evaluate the requested works to determine whether they are relevant and should be cited. It is not a requirement to cite these works. We appreciate your attention to this request. 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. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. 3. We note that Figures 1, 2 and 3 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: 1. You may seek permission from the original copyright holder of Figures 1, 2 and 3 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” 2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses? The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory. Reviewer #1: No Reviewer #2: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable? Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible. Reviewer #1: No Reviewer #2: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 ****** 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 Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the manuscript titled "A Study Protocol to Characterize Pathophysiological and Molecular Markers of Rheumatic Heart Disease and Degenerative Aortic Stenosis Using Multiparametric Cardiovascular Imaging and Multi-omics Techniques," the authors outline a protocol for patient recruitment and the rationale for conducting the study. This initiative is commendable, especially in light of the latest recommendations for open science, which emphasize the importance of publishing study protocols before conducting research and subsequently sharing the results. However, several significant modifications are necessary before publication and, crucially, before initiating the study. 1. Inclusion Criteria: The specified age range of 18-80 years is overly broad, considering the significant physiological differences across younger, middle-aged, and older patients. This criterion should be reassessed, or at the very least, patients should be categorized into distinct age groups as recommended, ensuring adequate representation in each category. 2. Sex Differences: The protocol currently overlooks the critical aspect of sex differences in cardiovascular disease (CVD) pathology. Given the historical underrepresentation of females in CVD research, it is imperative to aim for gender-balanced groups to address this gap effectively. 3. Sample Size: The document does not specify the number of participants for each group, a critical omission. While calculating the power of multi-omics studies may be challenging, it is still necessary to provide power calculations or at least an estimate of the minimum number of subjects required for each omics approach to ensure reliable results. 4. Sample Collection and Processing: Detailed information on blood and tissue sample collection and processing is lacking. This should include the types and brands of collection tubes, the number of tubes for serum/plasma, tube volume, anticoagulants used, centrifugation speed and conditions for serum/plasma separation, time from collection to storage, aliquot numbers, and storage conditions. For transcriptomics, considering the variability in platelet counts among patients, which can significantly affect results, platelet-poor plasma may be preferable for miRNA analysis. Details on RNA preservation and extraction methods for both serum/plasma and tissue samples, including the use of RNA later, Trizol, or snap freezing with liquid nitrogen, should be provided. 5. Open Research Data Initiative and FAIR Principles: The manuscript should also address the Open Research Data Initiative and incorporate the FAIR (Findable, Accessible, Interoperable, and Reusable) principles. Discussing how the study's data will be made available in accordance with these principles is essential. This includes detailing the data management plan, which should outline how data will be stored, shared, and made accessible to ensure that it is discoverable and usable by others in the research community. Highlighting the commitment to these practices not only aligns with the push towards open science but also enhances the transparency, reproducibility, and impact of the research. The considerations and suggestions from the referenced paper (https://doi.org/10.1016/j.molmed.2023.09.004) provide valuable guidance on designing and conducting multi-omic studies. While direct citation is not needed, employing this resource as a framework for study design and execution can greatly enhance the protocol's robustness and scientific contribution. Reviewer #2: The current study represent an interesting topic to study and to conduct. We have few comments: 1- There is a need to further support the current proposed sample size (120 pts). 2- Clarify properly if you recruit the patients from endemic area of RHD or not. The current status of RHD. 3- Justify how your cohort would represent a real world cohort regarding RHD and AS. 4- High light your measures to avoid bias, selection bias. 5- Discuss the expected major limitations or challenges. Regards ****** 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 Reviewer #2: Yes: Rami Riziq Yousef Abumuaileq ******** [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.0303496.r001
Revision 1
11 Apr 2024 Author Response Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field. Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses? The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory. Reviewer #1: No Reviewer #2: Yes Response: Thank you for the feedback. In the revised manuscript we have included the specific descriptions of the cutoff points and the specific experiments to be conducted. ________________________________________ 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable? Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible. Reviewer #1: No Reviewer #2: Yes Response: Thank you for the feedback. In the revised manuscript we have included specifics of the experiments to be conducted and their replications where appropriate. We have also included the accepted power and sample size calculations. ________________________________________ 4. Have the authors described where all data underlying the findings will be made available when the study is complete? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 Response: We have included a data availability statement. Lines 519 – 521 of the revised manuscript. ________________________________________ 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 Reviewer #2: Yes Response: The revised clean manuscript has been reviewed, proofread, and the language edited. ________________________________________ 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the manuscript titled "A Study Protocol to Characterize Pathophysiological and Molecular Markers of Rheumatic Heart Disease and Degenerative Aortic Stenosis Using Multiparametric Cardiovascular Imaging and Multi-omics Techniques," the authors outline a protocol for patient recruitment and the rationale for conducting the study. This initiative is commendable, especially in light of the latest recommendations for open science, which emphasize the importance of publishing study protocols before conducting research and subsequently sharing the results. However, several significant modifications are necessary before publication and, crucially, before initiating the study. Response: Thank you for taking the time to review our manuscript and for your kind and constructive feedback. 1. Inclusion Criteria: The specified age range of 18-80 years is overly broad, considering the significant physiological differences across younger, middle-aged, and older patients. This criterion should be reassessed, or at the very least, patients should be categorized into distinct age groups as recommended, ensuring adequate representation in each category. Responses: Thank you for the feedback. After careful consideration, we have revised the upper age limit to 70 years. The mean age of the patients currently recruited in the study is 44.7±13.7 years for RHD patients and 64.2±12.8 years for degenerative AS patients. We acknowledge that the range is larger than desirable, especially when age is a frequent confounder in such studies. However, degenerative AS is often related to advanced age. Furthermore, at the location where we are recruiting the participants, most of the patients present for valve replacement at the advanced stages of the disease (both RHD and AS) which also tends to make them older than in other populations. Given the larger sample sizes we are now looking to recruit, we believe that there should be sufficient age stratification to enable a confounding factor of age to be appropriately accounted for. 2. Sex Differences: The protocol currently overlooks the critical aspect of sex differences in cardiovascular disease (CVD) pathology. Given the historical underrepresentation of females in CVD research, it is imperative to aim for gender-balanced groups to address this gap effectively. Response: Thank you for highlighting this information. We have added a statement indicating that we shall ensure that there is a balance between males and females in the studied groups. Lines 260-261. 3. Sample Size: The document does not specify the number of participants for each group, a critical omission. While calculating the power of multi-omics studies may be challenging, it is still necessary to provide power calculations or at least an estimate of the minimum number of subjects required for each omics approach to ensure reliable results. Response: Thank you for your suggestions. After reconsideration and analysis, we have increased the sample size to 250. This is based on the information obtained from using available tools for power calculation for metabolomics and genomics studies. In the revised manuscript we have included the justification for using a sample size of 250 VHD participants and an equal number of controls. We have also broken down the sample size per disease group and categories. 4. Sample Collection and Processing: Detailed information on blood and tissue sample collection and processing is lacking. This should include the types and brands of collection tubes, the number of tubes for serum/plasma, tube volume, anticoagulants used, centrifugation speed and conditions for serum/plasma separation, time from collection to storage, aliquot numbers, and storage conditions. For transcriptomics, considering the variability in platelet counts among patients, which can significantly affect results, platelet-poor plasma may be preferable for miRNA analysis. Details on RNA preservation and extraction methods for both serum/plasma and tissue samples, including the use of RNA later, Trizol, or snap freezing with liquid nitrogen, should be provided. Response: In the revised manuscript we have added the detailed information for sample collection and processing. We have also added some detailed information on miRNA extraction and handling. 5. Open Research Data Initiative and FAIR Principles: The manuscript should also address the Open Research Data Initiative and incorporate the FAIR (Findable, Accessible, Interoperable, and Reusable) principles. Discussing how the study's data will be made available in accordance with these principles is essential. This includes detailing the data management plan, which should outline how data will be stored, shared, and made accessible to ensure that it is discoverable and usable by others in the research community. Highlighting the commitment to these practices not only aligns with the push towards open science but also enhances the transparency, reproducibility, and impact of the research. Response: We have added a data availability statement in the revised manuscript. Lines 548-550. The considerations and suggestions from the referenced paper (https://doi.org/10.1016/j.molmed.2023.09.004) provide valuable guidance on designing and conducting multi-omic studies. While direct citation is not needed, employing this resource as a framework for study design and execution can greatly enhance the protocol's robustness and scientific contribution. Response: Thank you for the suggestion. The article was very informative. Reviewer #2: The current study represent an interesting topic to study and to conduct. Response: Thank you for taking your time to review our manuscript and for your kind and positive feedback. We have few comments: 1- There is a need to further support the current proposed sample size (120 pts). Response: Thank you for the feedback. We have increased the sample size to 250 participants, and we have also included a justification for the sample size in the revised manuscript. 2- Clarify properly if you recruit the patients from endemic area of RHD or not. The current status of RHD. Response: We are recruiting in RHD endemic area, the current prevalence of RHD is 29.40/100,000 as at 2017. 3- Justify how your cohort would represent a real-world cohort regarding RHD and AS. Response: We are a little confused by your answer/request. This population represents a real-world cohort in that it is reflective of the patients that we are regularly treating daily in Cape Town hospitals. In this respect, being able to distinguish between AS and RHD where the prevalence of both is high becomes an important clinical question. 4- High light your measures to avoid bias, selection bias. Response: Since this is a case-control study we have defined clear inclusion criteria that will be used to recruit patients into RHD or AS group and the healthy controls. 5- Discuss the expected major limitations or challenges. Response: Thank you for the suggestion. In the revised manuscript we have highlighted some potential limitations and challenges to the study. Lines 524 – 531. Regards ________________________________________ 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 Reviewer #2: Yes: Rami Riziq Yousef Abumuaileq Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0303496.r002
26 Apr 2024 Decision Letter - Mohanad Alkhodari, Editor A study protocol to characterise pathophysiological and molecular markers of rheumatic heart disease and degenerative aortic stenosis using multiparametric cardiovascular imaging and multiomics techniques PONE-D-24-00937R1 Dear Dr. Mutithu, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Mohanad Alkhodari Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses? The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable? Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 ****** 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 Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have modifed manuscript according to reviewers' suggestions. The manuscirpt is ready for publication. Reviewer #2: The authors have addressed our comments and the manuscript has been improved. No further comments. Regards ****** 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 Reviewer #2: Yes: Rami Riziq Yousef Abumuaileq ******** https://doi.org/10.1371/journal.pone.0303496.r003
Formally Accepted
1 May 2024 Acceptance Letter - Mohanad Alkhodari, Editor PONE-D-24-00937R1 PLOS ONE Dear Dr. Mutithu, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Mohanad Alkhodari Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303496.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .