Dear PhD Santos,

Thank you very much for submitting your manuscript "Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: a proof-of-concept study" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

In addition to observations of the three reviewers, we ask you to pay attention to the following points during revision process:

- probably your are not aware about the commercially available (at least in Europe) LDBio western blot for Chagas disease; reviewer 2 is giving you the reference; please revise the paper taking it into account and add the reference;

- in the introduction and discussion give more notes on the problem of discordance in Chagas disease diagnosis (ref http://link.springer.com/10.1007/s10096-011-1393-9) taking into account the concept of the at least 50% rate of Chagas disease affected individuals among the discordants (see Moure et al DOI:https://doi.org/10.1016/j.cmi.2016.06.001, DOI: 10.1016/j.actatropica.2018.05.010)

- in the conclusions, clearly state that this is a phase I study and a larger sample study is needed to confirm results.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Andrea Angheben

Academic Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************

Dear authors, in addition to observations of the three reviewers, I ask you to pay attention to the following points during revision process:

- probably your are not aware about the commercially available (at least in Europe) LDBio western blot for Chagas disease; reviewer 2 is giving you the reference; please revise the paper taking it into account and add the reference;

- in the introduction and discussion give more notes on the problem of discordance in Chagas disease diagnosis (ref http://link.springer.com/10.1007/s10096-011-1393-9) taking into account the concept of the at least 50% rate of Chagas disease affected individuals among the discordants (see Moure et al DOI:https://doi.org/10.1016/j.cmi.2016.06.001, DOI: 10.1016/j.actatropica.2018.05.010)

- in the conclusions, clearly state that this is a phase I study and a larger sample study is needed to confirm results.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The study was designed and carried out nicely. There is no concern at all on the whole aspect of the study

Reviewer #2: This is an interesting paper about the possible use of four chimeric proteins to have a western-blot reference test for the diagnosis of T. cruzi infection.

The study design is appropriate, the samples clearly described.

The sample size is small but this paper is only a phase I (proof of concept), it is acceptable.

Reviewer #3: The objective of the study were clearly articulated with a clear testable hypothesys stated

The study design is appropriate

The population is clearly described and appropriated

The sample size is enough for this study

I agree with the chisen statistical methods

No concerns about ehic problems or so on

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results were recorded using a non-human reader to prevent bias. The analysis was carried out well using the best statistical calculation.

Reviewer #2: The results are clearly exposed, with figures of sufficient quality

Reviewer #3: The analysis match the analysis plan

Results are shown clearly

OK for figures

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions were based on the study results. The study will fill the gap in the lack of confirmatory chronic Chagas disease

Reviewer #2: The conclusions are supported by the data.

These results need to be conforted by further studies, and the cross reactions studied on a larger sample size.

Reviewer #3: Conclusions are supported by the data presented

Limit of the work are described

It is relevant for the Public Health

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: None

Reviewer #2: The authors say (line 56) : there is no reliable test that could serve as a gold standard.

there are published data (pathogens 2021 Nov 10;10(11):1455) about a commercialised CE marketed western blot for the diagnosis of Chagas disease

maybe it would be interesting to add this paper in the references and to take it in charge in the discussion

Reviewer #3: No suggestion

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: None

Reviewer #2: (No Response)

Reviewer #3: No comment: good work.

--------------------

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: Sukwan Handali

Reviewer #2: No

Reviewer #3: Yes: Romualdo Grande

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

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.

Attachments

Attachment

Submitted filename: Reviewer Comments on chimeric T cruzi PlosNTD 14SEP2022.docx

Dear PhD Santos,

We are pleased to inform you that your manuscript 'Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: a proof-of-concept study' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Andrea Angheben

Academic Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

Dear authors, many thanks for the work you did to comply with reviewers' comments. Moreover we appreciated your will to update the manuscript concerning the already available blots (LDBio and Abbott). This information and the new evidence you generated are very important to imporve diagnosis of chronic Chagas disease.