Dear Dr Maillard,

Thank you very much for submitting your manuscript "Early diagnosis of dengue: diagnostic utility of the SD BIOLINE Dengue Duo rapid test in Reunion Island" 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.

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,

Puneet Bhatt, MD

Guest Editor

PLOS Neglected Tropical Diseases

Elvina Viennet

Section Editor

PLOS Neglected Tropical Diseases

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

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: 1. In the Flow chart Fig 1; Information regarding break up of RT-PCR positive in NS1Ag positive and IgM positive samples would be useful for the reason being IgM positive samples by RDT which were RT-PCR negative (being used as Reference standard) would have resulted inputs regarding any cross reactive antibodies to other closely related Flaviviruses such as West Nile or Japanese encephalitis viruses. This could have been resolved by more specific µ-capture IgM ELISA test in such samples.

2. Whether RT-PCR inhibitors were taken care of in the samples which were found negative by RT-PCR but were found positive by RDT for Dengue antibodies?

Reviewer #2: The authors made only patients record review only in this study. Is it possible to say retrospective study?

What is the main topics of this manuscript. According to the title, we understood that the usefulness of RDT kits for diagnosis of dengue infection by comparing gold standard tests. But in this study, the authors did not use the full set of gold standard tests for confirmation of IgM Ab. The authors described that gold standard tests used RT-PCR. It is not full set because the authors also used IgM Ab for diagnosis of acute DENV infection. The authors should also test one gold standard IgM Ab tests which is validated for no cross reactivity with other flaviviruses. This is the biggest weak point of this study and the test performance is decreased due to not include IgM Ab at gold standard.

According to the flowchart Fig-1, it is very difficult to interpret for the readers. The authors described step by step but only 325 samples done for reference tests. So the sample size only 325. For calculation of the sensitivity, specificity, all sample must do reference tests to check your RDT test kit is correct or not? The author need to analyse only 325 samples and the authors must do gold standard IgM Ab ELISA test for publication to meet the standard at the reputed international journal.

Reviewer #3: Sample size calculation not mentioned

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

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: 3. As seen from S3 table “Performance of NS1 and IgM RDT….”, column “4” on IgM RDT; sensitivity percentage on Day 1 being highest (65%) as compared to other subsequent days including more than 5 days (61%) where as my understanding is that Dengue IgM antibody rise in blood is after 3-5 days and can remain detectable till 90 days.

Reviewer #2: At Table-1. The authors described the demographic data and clinical features of study population. Concerning gender, we noted Male” population but cannot find for Female anywhere. Please revise it.

In table 1, RDT was tested on 671 patients. But according to case categories, total number is only 547. What are the discrepancies? And please check it.

As you mentioned that the sensitives were slightly better beyond 5th day, if possible please show the comparison of sensitivities based on day of illness

Reviewer #3: Yes

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

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: NS1 Ag detection by RDT beyond 05 days is indicative of lasting viremia and could have been interesting to see its co-relation with viral load copy number by real time RT-PCR. It is interesting finding which could suggest changing viral dynamics. (For Reference: J Clin. Diagn. Res 2016 Apr; 10 (4):1-4) where authors have found NS1Ag positivity maximum on day 2-5 post onset of illness.

Reviewer #2: The authors described that the standard test as RT-PCR. Please kindly mention that is IT either quantitative or conventional Reverse transcription Polymerase Chain Reaction (RT-PCR)? Moreover, the author should described that it is either one step or two steps? Please also describe the vaidity of this PCR system for cross reactivity of the primers and other flaviviruses and within four serotypes of DENV infection.

In this study, the authors made RT-PCR test Is there any information about the serotype of DENV circulating at this DENV season. It will be interesting to the scientific community and now it is no more new information for the peer group scientific community.

Reviewer #3: No

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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: nil

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

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: 1. In the Flow chart Fig 1; Information regarding break up of RT-PCR positive in NS1Ag positive and IgM positive samples would be useful for the reason being IgM positive samples by RDT which were RT-PCR negative (being used as Reference standard) would have resulted inputs regarding any cross reactive antibodies to other closely related Flaviviruses such as West Nile or Japanese encephalitis viruses. This could have been resolved by more specific µ-capture IgM ELISA test in such samples.

2. Whether RT-PCR inhibitors were taken care of in the samples which were found negative by RT-PCR but were found positive by RDT for Dengue antibodies?

3. As seen from S3 table “Performance of NS1 and IgM RDT….”, column “4” on IgM RDT; sensitivity percentage on Day 1 being highest (65%) as compared to other subsequent days including more than 5 days (61%) where as my understanding is that Dengue IgM antibody rise in blood is after 3-5 days and can remain detectable till 90 days.

4. Similarly, NS1 Ag detection by RDT beyond 05 days is indicative of lasting viremia and could have been interesting to see its co-relation with viral load copy number by real time RT-PCR. It is interesting finding which could suggest changing viral dynamics. (For Reference: J Clin. Diagn.Res 2016 Apr; 10 (4):1-4) where authors have found NS1Ag positivity maximum on day 2-5 post onset of illness.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

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: Ajay Kumar Sahni

Reviewer #2: No

Reviewer #3: Yes: Kundan Tandel

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 Comments.docx

Dear Dr Maillard,

Thank you very much for submitting your manuscript "Early diagnosis of dengue: diagnostic utility of the SD BIOLINE Dengue Duo rapid test in Reunion Island" 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.

Thank you for responding to reviewer's comments.

Please note the few last comments attached.

Thanks so much.

Kind regards,

Elvina Viennet

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,

Elvina Viennet, PhD

Section Editor

PLOS Neglected Tropical Diseases

Elvina Viennet

Section Editor

PLOS Neglected Tropical Diseases

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

Thank you for responding to reviewer's comments.

Please note the few last comments attached.

Thanks so much.

Kind regards,

Elvina Viennet

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: All queries resolved. Accept

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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

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: All queries resolved. Accept

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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

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: All queries resolved. Accept

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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

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: All queries resolved. Accept

Reviewer #2: Accept

Reviewer #4: (No Response)

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

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: All queries resolved. Accept

Reviewer #2: In revision, the manuscript was improved. i have no more comments. I accepted as present version.

Reviewer #4: (No Response)

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

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: Ajay Kumar Sahni

Reviewer #2: No

Reviewer #4: No

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: Early diagnosis of dengue.docx

Dear Dr Maillard,

We are pleased to inform you that your manuscript 'Early diagnosis of dengue: diagnostic utility of the SD BIOLINE Dengue Duo rapid test in Reunion Island' 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,

Puneet Bhatt, MD

Guest Editor

PLOS Neglected Tropical Diseases

Elvina Viennet

Section Editor

PLOS Neglected Tropical Diseases

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