PONE-D-22-28460Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the AmericasPLOS ONE

Dear Dr. Sanchez,

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 This is an interesting and well-conducted study, with an adequate number of figures and supplemental materials. However, in light of the reviewers' comments, it is necessary to further discuss the limitations of the research, especially the use of monoplicates and the non-adoption of PCR in negative samples.I also suggest that the "diagnostic odds ratios" (DORs) of the panel 1 data and the confidence intervals of the values presented in the abstract be included in the article.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study was well conducted and the data support the results presented. The data is robust, clear and well presented graphically. Supplementary material has been presented and allows you to resolve any doubts you may have.

Reviewer #2: PONE-D-22-28460

The paper describes using two new recombinant antigens K18 and KR95 on VL-ELISA diagnosis. The manuscript is well present and can contribute to the use of the antigens in new variations of immunodiagnostic assays for human visceral leishmaniasis. I recommend the publication after minor revision.

M&M

- please clarify the DAT test used as a standard test (produced by? Which antigen is used in this assay? How about the sensibility and specificity of this assay?).

- Line 165- Why the negative samples were not confirmed by molecular assays like PCR or qPCR? The use of molecular assay is important because the health donors are from endemic regions.

- Line 200- Recombinant antigens-ELISA-Please describe the molecular differences between the recombinant antigens. Are they derived from the same original protein or are each of one are from different background proteins?

Discussion

Lines 624-628 – the authors did not add discussion in this paragraph. I don-t know if the following paragraph is about it. If it is, please unify both paragraphs.

Reviewer #3: The manuscript presents highly relevant public health findings, is very well drafted and meets the standards of the journal. I have a couple of questions:

1. Were the standardization, assessment and validation tests carried out on monoplicate? Were any reproducibility and repeatability tests conducted?

2. Was there a sample blinding plan for carrying out the tests?

3. For the Panel 2: serological tests associated with a compatible clinical are sufficient for the treatment of VL cases in Brazil.However, for use in serological panels, it is important to track these cases and obtain the final diagnostic outcome of these patients, whether due to therapeutic success or notification confirmation. Has this information been verified? If so, I think it's interesting to include.

4. There is significant heterogeneity in Panel 2. I believe that to obtain relevant epidemiological information, a more uniform and representative number of samples from each population would be needed. The tests used to characterize the panels should also be standardized. For VL/AIDS co-infected patients were characterized by the routine diagnostic methods and this could lead to misinterpretation. In this way, I suggest future tests, more homogeneous for validation with this objective. Would it fit as a limitation of the study?

5. Regarding the text, I only noticed a writing error: line 344: FN-false positive

6. I suggest reducing the number of figures.

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Reviewer #1: No

Reviewer #2: Yes: Juliana Lopes Rangel Fietto

Reviewer #3: No

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Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas

PONE-D-22-28460R1

Dear Dr. Sanchez,

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.

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Kind regards,

Vinícius Silva Belo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

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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 #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

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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 #3: Yes: Marcelino, AP

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