Protocol of a mixed (Retrospective and Prospective) longitudinal observational study of amiodarone for chronic Chagas cardiomyopathy (AMIO-CHAGAS study): Investigating immunomodulatory and trypanocidal effects

Juliana Magalhães Chaves Barbosa; Amanda Faier-Pereira; Alejandro Marcel Hasslocher-Moreno; Andréa Rodrigues Costa; Evelyn Nunes Goulart da Silva Pereira; Fernanda de Souza Nogueira Sardinha Mendes; Gilberto Marcelo Sperandio da Silva; Luiz Henrique Conde Sangenis; Marcelo Teixeira Holanda; Mauro Felippe Felix Mediano; Roberto Magalhães Saraiva; Sérgio Salles Xavier; Otacílio da Cruz Moreira; Anissa Daliry; Henrique Horta Veloso; Kelly Salomão

doi:10.1371/journal.pone.0347305

Peer Review History

Original SubmissionSeptember 1, 2025
10 Sep 2025 Decision Letter - Nicolas Padilla-Raygoza, Editor Dear Dr. Salomão, 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 Oct 25 2025 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.. 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.. 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.. 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. 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. 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Read more information on sharing protocols at . 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, Nicolas Padilla-Raygoza, Doctorate in Sciences 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. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. 3. 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 move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 4. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Additional Editor Comments: I consider that the protocol corresponds to experimental design (the effect of AMIO on CCC in Chagas Disease). This involves registering the protocol on a registry of clinical trials. Also, in the protocol is not include a sample of informed consent to verify if the consent includes the destiny of blood samples after the study. [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 . 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 . 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 . 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.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0347305.r001
Revision 1
15 Sep 2025 Author Response PLOS ONE Decision: Revision required [PONE-D-25-46528] Dear Editor, We thank you for the opportunity to revise and resubmit our manuscript entitled “Protocol of a Mixed (Retrospective and Prospective) Longitudinal Observational study of Amiodarone for Chronic Chagas Cardiomyopathy (AMIO-CHAGAS study): Investigating Immunomodulatory and Trypanocidal Effects ”. In response to the journal’s request: Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The manuscript has been revised in accordance with the PLOS ONE guidelines. 2. Please include your tables as part of your main manuscript and remove the individual files. We have included the table as part of the main manuscript and removed it from the individual file. 3. Your ethics statement should only appear in the Methods section of your manuscript. The ethics statement is included only in the Methods section. 4. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure. We apologize for the mistake; Figure 1 has been included in the manuscript text. Response to the Editor Comments I consider that the protocol corresponds to experimental design (the effect of AMIO on CCC in Chagas Disease). This involves registering the protocol on a registry of clinical trials. Also, in the protocol is not include a sample of informed consent to verify if the consent includes the destiny of blood samples after the study. We appreciate the editor’s comments and the opportunity to clarify these points. We would like to emphasize that the submitted study is an observational clinical study, which includes both retrospective and prospective components. Unlike a clinical trial, our study does not involve the assignment of interventions, randomization of participants, or experimental manipulation of treatment. Instead, it aims to observe and analyze clinical outcomes under routine care conditions. Because of these fundamental differences, the study does not meet the standard definition of a clinical trial, and therefore registration in a clinical trial registry is not applicable. Observational studies of this nature follow patients and collect data according to standard clinical practice rather than testing the efficacy or safety of an intervention under experimental conditions. In summary, the study fully complies with ethical standards and methodological requirements for observational research, while not fitting the criteria for a clinical trial registry. While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. As recommended, we uploaded the figures to PACE during the manuscript revision process. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0347305.r002
25 Feb 2026 Decision Letter - Nicolas Padilla-Raygoza, Editor Dear Dr. Salomão, 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 11 2026 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.. 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.. 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.. 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. A 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 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 . 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 . 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 . 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, Nicolas Padilla-Raygoza, Doctorate in Sciences Academic Editor PLOS One Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: The authors should answer all the comments from reviewers and resubmit a new version higlighted the changes in the manuscript. [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? 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??> Reviewer #1: Yes Reviewer #2: Partly ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable??> Reviewer #1: Yes Reviewer #2: No ****** 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.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: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** 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. Reviewer #1: The AMIO-CHAGAS study protocol is robust, ethically sound, and of high clinical importance. It addresses an underexplored yet highly relevant topic in the management of chronic Chagas cardiomyopathy. The combination of immunological, molecular, and clinical endpoints represents an innovative and comprehensive approach. Recommendations for Minor Improvement Clarify exploratory of confirmatory analyses: Specify which molecular and immunological measures (e.g., cytokines, gene expression, T. cruzi DNA) are confirmatory endpoints linked to the main hypothesis and which are exploratory analyses intended for hypothesis generation. This will improve methodological transparency and interpretability. Describe long-term handling of biological samples: Indicate how serum, plasma, and PBMCs will be managed after analysis—whether stored in a certified biobank, destroyed, or used for future ethically approved studies. Include brief details on storage duration and data governance. Refine language and presentation: A light English revision is suggested to enhance flow and precision: Abstract: simplify and unify tenses. Discussion: clarify links between preclinical findings and the study rationale. Tables/Figures: define all abbreviations in legends. Overall, the protocol is scientifically robust, ethically sound, and of high translational relevance. These minor refinements will improve clarity and publication readiness. Reviewer #2: In its current form, the protocol addresses a relevant and potentially innovative research question; however, it requires substantial methodological clarification, particularly regarding the sample size calculation and the delimitation of the clinical scope of the proposed findings ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: Yes: MSc PhD Rubi Gamboa-LeónMSc PhD Rubi Gamboa-LeónMSc PhD Rubi Gamboa-LeónMSc PhD Rubi Gamboa-León 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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: Review report_ AMIO Chagas_230226.pdf https://doi.org/10.1371/journal.pone.0347305.r003
Revision 2
30 Mar 2026 Author Response Response to Reviewers - PONE-D-25-46528R1 Protocol of a Mixed (Retrospective and Prospective) Longitudinal Observational study of Amiodarone for Chronic Chagas Cardiomyopathy (AMIO-CHAGAS study): Investigating Immunomodulatory and Trypanocidal Effects Reviewer #1: The AMIO-CHAGAS study protocol is robust, ethically sound, and of high clinical importance. It addresses an underexplored yet highly relevant topic in the management of chronic Chagas cardiomyopathy. The combination of immunological, molecular, and clinical endpoints represents an innovative and comprehensive approach. Recommendations for Minor Improvement Clarify exploratory of confirmatory analyses: 1. Specify which molecular and immunological measures (e.g., cytokines, gene expression, T. cruzi DNA) are confirmatory endpoints linked to the main hypothesis and which are exploratory analyses intended for hypothesis generation. This will improve methodological transparency and interpretability. Response: We appreciate this suggestion. In the present study, all molecular and immunological measures—including cytokine levels, gene expression, and T. cruzi DNA—are planned as confirmatory endpoints, as they are directly related to the main hypothesis evaluating the immunomodulatory and trypanocidal effects of amiodarone in patients with CCC. These diverse methodologies were intentionally adopted to broaden the range of parameters analyzed, thereby increasing the robustness and interpretability of the results. No analyses are exploratory, and all are designed to address this central research question. 2. Describe long-term handling of biological samples: Response: Thank you for this important suggestion. The request has been addressed, and the relevant information has been added to the Methods section under the subsection “Blood Collection and Isolation of Plasma, Serum and Peripheral Blood Mononuclear Cells (PBMCs)”. This subsection now describes the procedures for long-term storage, management, and disposal of biological samples. 3. Indicate how serum, plasma, and PBMCs will be managed after analysis—whether stored in a certified biobank, destroyed, or used for future ethically approved studies. Include brief details on storage duration and data governance. Response: Thank you for this comment. As previously indicated, the requested information has been included in the Methods section under the subsection “Blood Collection and Isolation of Plasma, Serum and Peripheral Blood Mononuclear Cells (PBMCs)”. Biological samples will be stored under institutional responsibility for up to five years after study completion and subsequently discarded according to institutional biosafety regulations. During this period, samples may be used for additional subprojects subject to appropriate ethical approval. Refine language and presentation: 4. A light English revision is suggested to enhance flow and precision: Response: Thank you for this suggestion. The manuscript has been carefully revised for English language, grammar, and clarity. All changes have been highlighted in the revised text. 5. Abstract: simplify and unify tenses. Response: Thank you for this helpful suggestion. The abstract has been revised to simplify sentence structure. 6. Discussion: clarify links between preclinical findings and the study rationale. Response: We thank the reviewer for this valuable suggestion. The Discussion has been revised to more clearly highlight how the clinical observations from the BENEFIT study are supported by experimental evidence from murine models, thereby providing robust support for our hypothesis regarding the potential immunomodulatory and trypanocidal effects of amiodarone in patients with CCC. 7. Tables/Figures: define all abbreviations in legends. Response: Thank you for this suggestion. All abbreviations in tables and figure legends have been defined accordingly in the revised manuscript. Overall, the protocol is scientifically robust, ethically sound, and of high translational relevance. These minor refinements will improve clarity and publication readiness. Reviewer #2: In its current form, the protocol addresses a relevant and potentially innovative research question; however, it requires substantial methodological clarification, particularly regarding the sample size calculation and the delimitation of the clinical scope of the proposed findings Scientific Review Report The manuscript presents an ambispective longitudinal protocol (retrospective and prospective), entitled AMIO-CHAGAS, which evaluates the use of amiodarone in patients with chronic Chagas cardiomyopathy treated at a referral center in Brazil. Considering that Chagas disease remains endemic in this country and that Chagas cardiomyopathy represents a significant cause of disability and cardiovascular morbidity and mortality, the topic is clinically relevant. The study population consists of patients with confirmed diagnosis of Chagas disease. The primary objective of the protocol is to evaluate the potential immunomodulatory and trypanocidal effects of amiodarone, exploring whether, in addition to its recognized antiarrhythmic effect, the drug could have an etiological impact on Trypanosoma cruzi infection. 1. Originality and Relevance The protocol addresses a pharmacological aspect that has not yet been fully elucidated in humans: the potential immunomodulatory and trypanocidal effects of amiodarone beyond its conventional antiarrhythmic indication. Given that the available clinical evidence regarding this effect remains limited, the study aims to contribute to a relevant gap in current knowledge. Furthermore, considering the persistent prevalence of Chagas disease and its impact in terms of cardiovascular morbidity and mortality, the exploration of additional effects of drugs already used in this population holds scientific and clinical interest. 2. Discussion and Clinical Applicability Although the protocol proposes an innovative hypothesis regarding the potential etiological effect of amiodarone in humans, it would be advisable for the authors to more thoroughly contextualize the clinical relevance of these findings. In particular, it is important to discuss the drug's safety profile, characterized by known adverse effects, in comparison with currently established etiological therapies for Chagas disease. It is suggested that the clinical scope of the study be more clearly defined, emphasizing the exploratory nature of the research. Response: We thank the reviewer for this valuable suggestion. The manuscript has been updated to address these points, and the relevant information regarding clinical relevance, safety profile, comparison with conventional therapies, and the exploratory nature of the study has been incorporated into the final paragraph of the Discussion. 3. Methodology The methodological design is consistent with the stated objective; however, the proposed sample size (n = 90) may be limited for detecting clinically significant differences, particularly given the exploratory nature of the immunomodulatory and trypanocidal effects being evaluated. Although the authors report having based their estimate on two previous studies and increased the calculated sample size by 20% to account for potential losses, the parameters used for a formal sample size calculation are not described. The absence of this information makes it difficult to determine whether the study will have sufficient statistical power to support robust conclusions. A more detailed description of the sample size calculation procedure would strengthen the methodological rigor of the protocol. Response: We thank the reviewer for this important comment. We have revised the Methods section to provide additional details regarding the sample size calculation. Specifically, we included the significance level (α), statistical power (1 − β), standard deviations, and the minimum differences considered clinically relevant for the primary and secondary endpoints. The sample size of 90 participants (45 per group) was determined based on the scenario requiring the largest sample, with an additional 20% to account for potential losses, ensuring sufficient statistical power to detect meaningful differences in the inflammatory and echocardiographic parameters evaluated. 4. Ethical Aspects The manuscript states that the study was approved by an institutional ethics committee, including the corresponding approval number. It also specifies that all participants provided informed consent prior to inclusion, and the authors declare the absence of conflicts of interest. Overall, the protocol appears to comply with fundamental ethical standards for research involving human participants. 5. Reporting and Data Availability The manuscript does not include an explicit statement regarding data availability. It is recommended that a specific section be incorporated clarifying whether the data will be publicly available, accessible upon reasonable request, or subject to ethical restrictions. Response: We thank the reviewer for this important comment. We have updated the “Data Collection” section of the manuscript to clarify that the data generated in this study will be made available upon reasonable request, subject to ethical and privacy considerations. This revision ensures compliance with PLOS ONE’s data sharing policy and provides transparency regarding data accessibility. 6. Writing and Clarity The manuscript is clearly written and allows adequate understanding of the objective and methodological structure of the protocol. The organization is coherent and facilitates comprehension of the scientific rationale. 7. Final Recommendation In its current form, the protocol addresses a relevant and potentially innovative research question; however, it requires substantial methodological clarification, particularly regarding the sample size calculation and the delimitation of the clinical scope of the proposed findings. Note: I declare that Google Translate was used exclusively for language translation assistance. No artificial intelligence tools were used in the scientific analysis or evaluation of the manuscript. Attachments Attachment Submitted filename: Response to Reviewers - PONE-D-25-46528R1.docx https://doi.org/10.1371/journal.pone.0347305.r004
1 Apr 2026 Decision Letter - Nicolas Padilla-Raygoza, Editor Protocol of a Mixed (Retrospective and Prospective) Longitudinal Observational study of Amiodarone for Chronic Chagas Cardiomyopathy (AMIO-CHAGAS study): Investigating Immunomodulatory and Trypanocidal Effects PONE-D-25-46528R2 Dear Dr. Salomão, 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. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support.... 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, Nicolas Padilla-Raygoza, Doctorate in Sciences Academic Editor PLOS One Additional Editor Comments (optional): The corrections in the manuscript answer all comment from reviewers. Reviewers' comments: https://doi.org/10.1371/journal.pone.0347305.r005
Formally Accepted
Acceptance Letter - Nicolas Padilla-Raygoza, Editor PONE-D-25-46528R2 PLOS One Dear Dr. Salomão, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Nicolas Padilla-Raygoza Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0347305.r006

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