Dear Dr Penna,

Thank you very much for submitting your manuscript "Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Your manuscript has been reviewed by two experts, here and in the attachment. While they have found the manuscript interesting, they have raised some issues with the use of this technique. Please address each of their comments, both in an itemized rebuttal and in the manuscript itself. Special consideration should be given to the justification for the use of this technique in leprosy and its clinical applications.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Linda B Adams

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Section Editor

PLOS Neglected Tropical Diseases

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

Your manuscript has been reviewed by two experts, here and in the attachment. While they have found the manuscript interesting, they have raised some issues with the use of this technique. Please address each of their comments, both in an itemized rebuttal and in the manuscript itself. Special consideration should be given to the justification for the use of this technique in leprosy and its clinical applications.

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: Reject

Reviewer #2: The methods are generally satisfactory for a preliminary report. Some details need clarification:

The authors have made a reasonable attempt to assess histopathological findings in the nerves and to correlate these with the PET scans, but they did not (and probably could not) biopsy the same nerve sites that had been assessed by the scans. This is an inherent limitation in studying human peripheral nerves. Since leprosy neuritis and neuropathy may occur intermittently along a nerve, rather than diffusely, it is important that they acknowledge the fact that the sites are not identical and therefore that any correlations must be interpreted very cautiously.

“In short, an axonal lesion was defined as either an isolated reduction in amplitude ≥ 30% of the reference values”. Don’t you mean an isolated reduction in amplitude of < 30% . . .? or a 30% reduction . . .?

Histopathological examination -- “The sensory nerve was biopsied according to clinical or electrophysiological findings” Which sensory nerve?

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

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: (No Response)

Reviewer #2: Most of the results are clear and appropriate. some concerns:

Describing the time of onset of nerve vs dermatological symptoms, only 6 patients are accounted for:

2 (#4 & 7) with nerve onset first

2 (5&6) with nerve and skin onset at the same time

2 (2&8) with nerve onset after skin lesions

What about cases #1 and 3?

In Table 1, ‘Symp on” refers to duration of neural symptoms. What does ‘No” mean ?

In the legend for Table 2, please also include explanations for the terms used in the headings, e.g., “t-SUV, max/bg-SUV, max”

“In patients 7 and 8, PET/CT was performed during a neuritis episode, and 18F-FDG uptake was observed in the peripheral nerves in which the patients had symptoms and signs of neuritis, as well as in other nerves that showed no signs of neuritis on neurological examination, but with neurophysiological changes.” Does the final expression, “ with neurophysiological changes”, refer to the 18F-FDg uptake? If so, it will be less confusing to say so.

Also, if I understand this correctly, this means that some of the nerves with ‘no signs of neuritis’ had increased uptake. Plese comment on this.

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

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: (No Response)

Reviewer #2: The authors appear to have overstated the significance of some of their findings. Specifically, assertions regarding the assessment of Schwann cell activity/ uptake of FDG are overstated, since the methods do not appear to have sufficient sensitivity or resolution to distinguish increased uptake in Schwann cells vs inflammatory infiltrates, i.e., macrophages and lymphocytes.

Discussion

The first paragraph is somewhat confusing and should be edited to more concisely describe the overall findings.

e.g., “ . . . patients with LL present with sensory and motor nerve impairment despite the absence of 18F-FDG uptake in their nerves” – does this mean that they have previously had nerve injury, so they have impairment, but do not have active injury occurring at the time they present to the clinic?

“According to the present and previous studies, neuropathy in LL patients can occur as a result of Schwann cell dedifferentiation . . .” Nothing in this paper presents any evidence regarding Schwann cell dedifferentiation, and the methods do not address such dedifferentiation in any way. Therefore, this statement and reference should be omitted.

“Despite this, the nerve biopsy revealed an inflammatory process, even without clinical neuritis.” There are several possible explanations, but the most obvious is that the biopsy sites do not correspond exactly to the sites imaged by 18F-FDG uptake (see general comments). Therefore, any attempts to correlate these much be stated very cautiously.

“Apparently, 18F-FDG PET/CT was not sensitive enough to detect metabolic changes in infected Schwann cells.“ I do not see that the authors have determined that any of the uptake seen in their own study is specifically due to Schwann cells. Is the imaging of sufficient resolution and specificity to distinguish uptake by Schwann cells vs nearby macrophages or lymphocytes?

“We believe that further studies are necessary regarding silent nerve injuries, where the cellular inflammatory process seems to be different from that of acute neuritis . . .” This assumes that an inflammatory process is present in silent nerve injury, but that has not been determined. Alternatively, it could be a degenerative process rather than an inflammatory one.

“In addition, in cases of already diagnosed patients who develop recurrent neuropathy, there is a differential diagnosis between a new episode of neuritis and chronic neuropathy” The meaning and point of this sentence is not clear.

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

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: (No Response)

Reviewer #2: Author summary

Typos and incomplete sentences in the following: “ . . . our question was about how the uptake of this radiopharmaceutical would be in a common silent neuritis in LL patients.d the uptake . . .”

The phrase real extension seems odd and I don’t understand what this means here.: ´. . . Our data demonstrated, for the first time, the real extension of peripheral nerve . . .’ I think you mean something else -- Perhaps you mean illustration of . . . or example of

Introduction

“Medeiros et.al. (2016) evaluated in vitro and in vivo models of Schwann cells . . .” This should be indicated as reference #3 instead of the year.

The expression “real extension” also appears in the last paragraph of the introduction.

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

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: (No Response)

Reviewer #2: This is an interesting report regarding the use of 18FDG-PET as a means to assess physiological activity, in a small series of patients. The addition of this method to other tools for neurological assessment enables measurement of physiological activity in real time, a valuable new approach.

The peripheral nerve status of the patients has been carefully assessed and well documented using a variety of clinical assessments in addition to the PET scans. The finding of increased uptake during active neuritis is not surprising, but the capability to assess physiological activity in vivo during acute neuritis offers new possibilities in understanding this process. The finding of no uptake in patients with neuropathy but not acute neuritis also adds to our knowledge of neuropathy in leprosy.

The findings in acute neuritis do increase the evidence base regarding the inflammation in nerves in leprosy. It may be valuable to consider this in the broader context of the findings of FDG uptake in other inflammatory conditions such as arthritis, e.g., Graham RN, Panagiotidis E. [18F]FDG PET/CT in rheumatoid arthritis. Q J Nucl Med Mol Imaging. 2022 Sep;66(3):234-244.

It would be interesting to know if the authors believe that 18FDG-PET could be a useful tool to measure changes in inflammation during treatment of acute neuritis in leprosy, i.e., scanning before and during prednisone treatment.

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

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: David Scollard, M.D., Ph.D.

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

Attachments

Attachment

Submitted filename: Reviewer 1 Comments.docx

Dear Dr Penna,

Thank you very much for submitting your manuscript "Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The original manuscript was sent to two highly qualified and knowledgeable reviewers. However, some of their comments were not addressed at all in the rebuttal; it is imperative to provide a response to every comment. In addition, each of their concerns must be addressed in the manuscript itself. In the rebuttal, please include the line numbers of the manuscript where each of the specific concerns were edited and clarified. Modifications to address their comments will improve the manuscript and reduce misunderstandings by other readers.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Linda B Adams

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Section Editor

PLOS Neglected Tropical Diseases

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

This manuscript was sent to two highly qualified and knowledgeable reviewers. However, some of their comments were not addressed at all in the rebuttal; it is imperative to provide a response to every comment. In addition, each of their concerns must be addressed in the manuscript itself. In the rebuttal, please include the line numbers of the manuscript where each of the specific concerns were edited and clarified. Modifications to address their comments will improve the manuscript and reduce misunderstandings by other readers.

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

Dear Dr Penna,

We are pleased to inform you that your manuscript 'Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose' 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,

Linda B Adams

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Section Editor

PLOS Neglected Tropical Diseases

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