Dear Dr. Bosch:

Thank you very much for submitting your manuscript "Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis" (#PNTD-D-19-01624) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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We hope to receive your revised manuscript by Mar 08 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

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Sincerely,

Abdallah M. Samy, PhD

Guest Editor

PLOS Neglected Tropical Diseases

Robert Reiner

Deputy Editor

PLOS Neglected Tropical Diseases

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Editor comments to authors: I invited and received three reviews of your manuscript. All reviews raised some important points to be addressed before considering a revised version of your manuscript. I would kindly ask you to address carefully all of their concerns before submitting your revised manuscript. Thanks, AMS

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: Yes (see comments below)

Reviewer #2: • Are failry clear, concise.

• Nice description of the developed statistical analyses in R

• Human ethics number required?

• L 223: 31 out of how many samples yielded suitable genome sequences?

• Some manufacturer’s details are missing (e.g. Dengue IgM, IgG reagents, antibodies etc)

• Since most statistical results are provided, the authors could include exact p values rather than just general significance.

Reviewer #3: One IRB approval is listed but there are several affiliations of authors. Was IRB approval sought at other study sites?

The visit schedule is not clear in the “Data collection” section of the methods.

For the genome analysis, it is not clear what the mutations were in comparison to? Was a reference sequence used or was this observed temporally during the outbreak? Please deposit the sequences at the time of publication (not only supplemental but in a repository).

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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: Yes

Reviewer #2: • Whilst providing statistical significance e.g. by * p<0.01 is commonly performed, stating exact p values are more suited here (include in figures).

• All figures should include in figure legend or better on y-axes what is depicted, e.g. mean ± 95% CI. Please include this info in all relevant figures. Supp files have often ‘median’ in the figure legend, which is acceptable if no ranges are given.

• Table 1: age sign >=7 should be ≥;

• L290 states 61% were female, whereas Table 1 depicts 8 females in total in the study population. Please clarify.

• Table 2: plateletes = typo, correct to platelets.

• Figure 1: include number of subjects in brackets under each criteria/condition. Change notes * to another symbol to avoid confusion with statistical significance symbol (*).

• Figure 4A needs a description of what total count is depicted: mean? Median? Please include.

• Sup Fig 6A needs to be increased as not legible at 400% zoom

• Abbreviations: check throughout, e.g. use MCP-1, use vWF in figures if used abbreviation in text

• L149-153, why were not some other factors tested that are known in Dengue pathobiology? E.g. MCP-1, TNFa, IFy, etc? Include a comment in results or discussion. It would have been nice to confirm findings from other researchers.

Reviewer #3: The abstract makes a nice distinction between the time points where these factors were significantly increased/decreased (e.g. at defervescence) but this is not clear from the results section.

Lines 312-13 Provide the time point? The abstract indicates at defervescence? Provide the data for sST2 that was not significant?

Figure 2, can plots be provided or absolute values (potentially as a supplemental data set)? The heat map is not specific enough and is difficult to read. Also, the interpretation of differences between significance for the kinetic and intercept are not described adequately or provided context for the broader audience.

Figure 3 legend does not indicate what is meant by Day 0. Fever? Recruitment? Defervescence? Were there other clinical parameters that were collected that were not significant?

For the clinical parameters provided in Fig. 2-3 can the AUC values be provided/discussed?

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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: A shortcoming, which may be attributed to the nature of the study design, is the lack of a control group. Of the 204 patients in the study population, 55 were excluded because they did not present with fever and one or more typical symptom, febrile illness for less than two days and no other obvious infection. It would be interesting, for future studies to also follow the progression of individuals who are asymptomatic but test positive for the presence of dengue RNA. This would enhance the scope of the study to also elucidate which biomarkers are of importance in the symptomatic developments of dengue fever at early timepoints in which individuals are viremic but asymptomatic and their subsequent progression to severe dengue.

Reviewer #2: ok

Reviewer #3: The abstract does not make it clear whether the transitions observed in genomes are reported as a marker of severity or some other association.

Although the intro and discussion adequately discuss the potential of clinical measures to predict SD, there is no acknowledgment of the development of biomarkers for SD prediction.

Line 419- What do the authors consider a “hemostatic factor”. Also the use of the terms “hemodynamically stable” versus “unstable” are used throughout the discussion but how the authors define this is unclear.

Line 463, this data is not included in the results and should be.

The title suggests that new approaches to diagnosis/prognosis are revealed by the data, but that is not fully clear since many of the variables used for this analysis have been considered by other groups and are either not consistent from cohort to cohort or no protocol exists for converting these values into a prognostic strategy.

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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: Minor revisions are suggested, since the nature of the study does not allow for further modifications

Reviewer #2: some figures should be increased in size and font size

figures (graphs) should include what is depicted on the y-axis: mean +/- SD or alike

Accept with minor revisions

Reviewer #3: Specifically Figure 2 doesn't contain any raw data, only a heat map that was generated by the authors and not a statistical program regarding p-values. This needs to be improved and some representation of data itself included.

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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: The manuscript by Vasey presents unique insights into human biomarkers that can support current diagnostic tools in the effort to differentiate between dengue fever and similar febrile illnesses. The main strength of the study is its ability to differentiate dengue fever from other febrile illnesses. In terms of providing predictive approaches for the progression of dengue fever to severe dengue, more characterization would be needed to support the claim that as is, this study could aid as a predictive tool for the progression to severe dengue.

Reviewer #2: It is an interesting study. I would have liked to see some other markers tested in this study to link it to other existing studies ( e.g. TNFa, IFy levels). A major lack is the assessment of other, underlying medical conditions that may/could have masked/increased the symptoms. E.g. a full blood analysis, and viral genome testing by RT-PCR could have been beneficial.

In summary, this manuscript is acceptable for publication upon completion of the suggested edits.

Reviewer #3: Multiple statistical methods were used to identify diagnostic/prognostic factors for dengue (vs. OFI) and mild vs. severe dengue in a cohort of suspected dengue patients from Nicaragua. The study describes a unique cohort that will be informative for the literature. Some of the analysis or results presentation was unclear and additional information are needed before this study is ready for publication.

The abstract does not make it clear whether the transitions observed in genomes are reported as a marker of severity or some other association.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Attachments

Attachment

Submitted filename: PNTD-D-19-01624 Vasey et al review.docx

Dear Dr. Bosch,

We are pleased to inform you that your manuscript 'Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis' 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,

Abdallah M. Samy, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

Robert Reiner

Deputy Editor

PLOS Neglected Tropical Diseases