PONE-D-24-53106The Chagas non-endemic cohort (“ChaNoE”): aims and study protocolPLOS ONE

Dear Dr. Bosch-Nicolau,

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 Feb 22 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.

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,

Faham Khamesipour, Ph.D.

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.  Thank you for stating the following financial disclosure:

“This project was funded by “Proyectos de investigación en salud” program of the Instituto Carlos III, Ministry of Science, Innovation and Universities of the Spanish Government. Grant number: PI22/01894.“

 Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This project was funded by “Proyectos de investigación en salud” program of the Instituto Carlos III, Ministry of Science, Innovation and Universities of the Spanish Government. Grant number: PI22/01894.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This project was funded by “Proyectos de investigación en salud” program of the Instituto Carlos III, Ministry of Science, Innovation and Universities of the Spanish Government. Grant number: PI22/01894.“

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Additional Editor Comments:

For your submission "The Chagas non-endemic cohort (“ChaNoE”): aims and study protocol" to PLOS ONE to proceed further in the review process, you will need to make major revisions to your manuscript. Revise it as suggested by the reviewer.

[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

Reviewer #3: 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: No

Reviewer #3: 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: No

Reviewer #3: 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: No

Reviewer #3: 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

Reviewer #3: 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 propose a multi-center study cohort to investigate Chagas disease in a non-endemic country. This is a very important initiative and I congratulate the authors.

Comments:

1) Please, include in the objectives the study of clinical end-points. Hard end-points, such as all-cause mortality and heart transplant, but also secondary end-points, such as, admission due to heart failure, cardiac device implantation, and stroke. As a lot of data will be collected, it is very important to understand how these variables are associated with clinical outcomes.

2) Please, clarify criteria for disease progression. Do you mean only progression from indeterminate to cardiac form (A to B/C or D) ? Or also progression within stages of the cardiac form (B to C or D)? Will also study progression from indeterminate to digestive form?

3) Please, clarify the criteria for cure that will be followed.

4) Please, clarify if a patient will be classified as B if changes are present in the echocardiogram even in the presence of a normal ECG. I ask this because of the following sentence : “CCC will be defined by the presence of segmental or global left ventricle (LV) or right ventricle (RV) systolic function impairment, increased end-diastolic diameter of the LV (>55mm), ventricular aneurysms or cardiac thrombus”. Usually, an abnormal ECG is always necessary for a patient to be diagnosed with CCC.

5) Table 2. A 24-Holter is always necessary in the initial evaluation of all patients with CCC. Specially if the study objective is the evaluation of prognosis1.

6) Please, evaluate the inclusion of biochemistry and blood count in the initial evaluation and follow-up. A poor kidney function, anemia, low sodium are all associated with a dismal outcome in patients with heart failure.

7) Table 2. Echocardiography in stage A (indeterminate) can be at every 5 years. B1 patients should undergo echocardiograms more frequently as they already have changes in ECG: every two years, and B2 and C patients every year1.

8) Table 6. Correct fifth line “5mg/day (bid or tid)”

9) Benznidazole treatment dose recommendation is different in Brazil. “For adults with chronic CD, benznidazole is administered orally at the dosage of 5mg/kg/day divided into two or three doses, for 60 days, with a recommended maximum dosage of 300mg/day. For individuals with acute CD, the dose can be of as much as 10mg/kg/day. For individuals weighing over 60 kg, the therapeutic schedule can be extended to achieve the ideal target dose, maintaining 300 mg as the daily limit to prevent adverse events. The 300mg benznidazole regimen can be used for the number of days equivalent to the individual’s weight, limited to a total of 80 days even for individuals weighing over 80 kg”2. The authors’ table 6 suggests that there is no upper limit for benznidazole daily dose. For example, a patient with 90 kg would be prescribed 450 mg/day but this can increase the risk of adverse effects.

10) Include in all Tables the references the authors used.

11) Statistical analysis. Include how prognosis will be evaluated. Survival curves, logistic regression, non-adjusted or adjusted, ROC curves? Please, clarify

12) Dissemination plan. Is there a plan to make data available in public repositories?

13) Discussion. There are other papers describing guides for the generation of databases or repositories for Chagas disease cohorts. I invite the authors to read those papers3,4.

1 - Saraiva RM, Mediano MFF, Mendes FS, Sperandio da Silva GM, Veloso HH, et al. Chagas heart disease: An overview of diagnosis, manifestations, treatment, and care. World J Cardiol. 2021 Dec 26;13(12):654-675.

2 - Marin-Neto JA, Rassi A Jr, Oliveira GMM, Correia LCL, Ramos Júnior AN, et al. SBC Guideline on the Diagnosis and Treatment of Patients with Cardiomyopathy of Chagas Disease - 2023. Arq Bras Cardiol. 2023 Jun 26;120(6):e20230269.

3-González Martínez A, Losada-Galván I, Gabaldón-Figueira JC, Martínez-Peinado N, Saraiva RM, et al. A standardized clinical database for research in Chagas disease: The NHEPACHA network. PLoS Negl Trop Dis. 2024 Aug 15;18(8):e0012364.

4-Martínez-Peinado N, Gabaldón-Figueira JC, Rodrigues Ferreira R, Carmen Thomas M, López MC, et al. A guide for the generation of repositories of clinical samples for research on Chagas disease. PLoS Negl Trop Dis. 2024 Aug 15;18(8):e0012166.

Reviewer #2: Summary:

This article describes the literature behind, and some of what the Spanish Chagas non-endemic cohort (ChaNoE), a multi-center initiative based in Spain, seeks to accomplish. Their aim is to improve the understanding and management of Chagas disease (CD) in non-endemic regions, and to contribute evidence regarding clinical progression, biomarkers, and overall understanding of Chagas disease. The study focuses on:

They aim to recruit individuals with chronic CD, standardize diagnostic and treatment protocols, monitor disease progression, and create a biobank to facilitate biomarker research for progression and treatment efficacy. A serum biobank is being established to identify and validate biomarkers for disease progression and treatment response. This is a critical need in CD research. The Spanish experience is useful for cohorts elsewhere in non-endemic areas. The authors do a good job reviewing existing literature on CD, including its classification and management. However, the article reads more like a review of the literature rather than a research study protocol. There are many details missing from this description, such as inclusion and exclusion criteria, detailed timelines, where the samples will be taken and stored, which specific serology tests will be used to diagnose CD, who will recruit participants, etc. This is a great idea for a project, and the achievement of the cohort will be very beneficial. However, in order to publish the protocol, it needs to provide much more detail.

Introduction:

• In line 92, it says that treatment is recommended for people in the chronic phase. This is not exactly accurate. The recommendations depend on the age of the individual and their circumstances, as the BENEFIT trial showed that treatment in older individuals with advanced cardiomyopathy did not result in significant improvement. Treatment is more strongly recommended for younger patients. However, guidelines vary by region. Please be specific about the treatment indications for chronic disease in Spain.

• The introduction should typically end with the overall aim of the study/work. In this case, it could be more specific about the particular context in Spain and state that the article will describe the cohort and its intent.

Methods:

• Overall, a large proportion of the paragraphs in the methods section seem more like introductory paragraphs, describing the literature. Please remember that the introduction or background section of an article reviews existing literature, and this often happens again in the discussion section when comparing findings to existing studies. Please reorganize the article to clarify each section. The methods should describe the project itself rather than the literature.

• Line 179: Where is the annual incidence of Chagas cardiomyopathy sourced from? There is a wide range in the literature, so please include more references.

• Line 181: Diagnosis and categorization of CCC also vary. Many newer articles include NYHA severity classes in the definitions, so please adjust to clarify this – you already touch on this in line 195.

• Line 253: Digestive tract involvement is less than 15%. Please correct. As you mentioned, some places in the Southern Cone have a higher proportion of digestive tract involvement, but this is not a generalization for Chagas. The Brazilian article identifies female sex as a predictor; however, this line could mislead readers. It is well known that only some genetic variants of T. cruzi are associated with digestive forms—this is the biggest association, not female sex.

• The methods section reads more like a review of the literature than a description of what the ChaNoE cohort will entail. It is important to be very detailed about your specific cohort, rather than existing literature. I see a review of the current recommendations for Chagas, but it’s not clear how the timeline works, who the team will consist of, how patients will be followed up, and how referrals will be made within the specific hospitals. Are these patients insured? Will they be using free national insurance? How will the team ensure that all follow-up care is completed? Will different sites use either benznidazole or nifurtimox for treatment, and how will that be decided? Based on the broad description, it sounds like these participants will be treated for all possible complications of Chagas disease.

• We don’t know the specific inclusion or exclusion criteria for participants, such as age ranges and eligibility. This is only described briefly in the abstract and not in detail.

• Line 331 says that patients will have easy access to their healthcare provider; however, it is unclear if this provider is part of the study or if it refers to a regular visit, with medical charts being reviewed.

Discussion:

• The discussion reads somewhat broadly about Chagas disease, rather than specifically discussing the cohort study in the context of previous work.

Reviewer #3: Dear Editor and Authors,

Thank you for the opportunity to review the manuscript titled "The Chagas Non-Endemic Cohort (“ChaNoE”): Aims and Study Protocol." This is an intriguing and valuable project. The manuscript effectively describes the establishment of a Spanish cohort designed to identify and monitor chronic cases of Chagas disease. The study employs a modern clinical-laboratory approach and clearly articulates its objectives and rationale in a well-justified manner. The manuscript addresses an important public health challenge: Chagas disease in non-endemic regions, a topic that remains underrepresented in current research. The focus on Spain—a country with a significant population of individuals living with Chagas disease due to migration—highlights the study’s relevance and potential impact. However, before the manuscript is accepted, several questions and concerns need to be addressed, as outlined below:

Major comments

1. The authors mention multiple times that the data obtained from the “ChaNoE” cohort will be compared with other cohorts worldwide. However, the manuscript does not specify these cohorts. Including examples of similar studies, such as Oxente Chagas Bahia, Samitrop, TESEU, and others, would enhance the manuscript by informing readers of relevant ongoing research efforts globally and providing context for the comparative analyses planned in this study.

2. While the protocol is thorough, some methodological aspects, such as the specific strategies for data standardization across centers, are underexplained. A clearer description of quality control for data and biobank management is needed.

3. The study’s reliance on multiple centers for participant recruitment could face logistical hurdles, especially in ensuring consistent diagnostic criteria and follow-up. Comment!

4. Although PCR is mentioned, its limited sensitivity in chronic cases raises concerns. The manuscript could better address how it will account for false negatives or inconsistent results. Explain how the study will mitigate the limitations of current diagnostic tools, such as PCR, and whether alternative or complementary methods will be employed.

5. While the protocol outlines periodic evaluations, the manuscript does not provide sufficient detail on how adherence to follow-up will be ensured, particularly for participants in non-urban areas. Discuss strategies to overcome potential challenges in participant recruitment and retention, including communication methods and incentives.

6. Inclusion criteria requiring diagnosis by two serological tests might exclude some cases, potentially limiting generalizability.

7. Add visual elements, such as flowcharts or timelines, to illustrate the study design, recruitment process, and follow-up schedule.

8. The potential financial burden of implementing such protocols in other settings, including low-resource regions, is not addressed. Include a discussion of how the findings and methodologies could be adapted for low-resource or other non-endemic settings. In addition, provide examples of how the study outcomes will directly influence public health policies or clinical practices in non-endemic areas.

9. Include plans for biobank management and data sharing policies.

Minor comments

1. Line 44: Italicize Trypanosoma cruzi.

2. Line 49: Clarify the term "heterogeneous." As it stands, the meaning is unclear and could benefit from further explanation or rephrasing?

3. Line 81: Remove the word "also." The phrase, as written, might imply that vectorial transmission occurs in non-endemic areas, which could lead to misinterpretation.

4. Line 403: Specify the microbiological tests used for diagnosing Chagas disease. Wouldn't these be more accurately described as parasitological tests?

5. Line 470: Capitalize "Chagas."

**********

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: No

Reviewer #3: Yes:  Fred Luciano Neves Santos

**********

[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.

Attachments

Attachment

Submitted filename: Plos_one_review.pdf

The Chagas non-endemic (ChaNoE) cohort: aims and study protocol

PONE-D-24-53106R1

Dear Dr. Bosch-Nicolau,

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,

Faham Khamesipour, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I recommend the manuscript for publication without further revisions.

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 #3: 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 #3: 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 #3: 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 #3: 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 #3: 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: All questions raised by the reviewer were properly addressed by the authors. No further comments.

Reviewer #3: The authors have incorporated all my suggestions into the manuscript. Following the modifications suggested by all reviewers, the project “ChaNoE” is now clearly presented. Given these strengths, I recommend the manuscript for publication in PLOS ONE without further revisions.

**********

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: Yes:  Roberto Saraiva

Reviewer #3: Yes:  FRED LUCIANO NEVES SANTOS

**********