Dear Dr Lamb,

Thank you very much for submitting your manuscript "The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy - a systematic review and meta-analysis of diagnostic test accuracy." 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.

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,

Jean-Philippe Chippaux, M.D., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Jean-Philippe Chippaux

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

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

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

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

-Were correct statistical analysis used to support conclusions? yes

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

Reviewer #2: (No Response)

Reviewer #3: It is a meta-analysis to meet a clearly defined objective. the methodology is appropriate.

the statistical analysis is properly conducted to meet the objectives.

Compliance with ethical or regulatory requirements is correct.

Reviewer #4: The objectives of the study are clearly stated and study design is quite appropriate to address the stated objectives. Selection criteria of the studies are well described and SROC curves for sensitivity/specificity analysis strongly support the conclusions. There are no concerns about ethical or regulatory issues.

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

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: -Does the analysis presented match the analysis plan? yes

-Are the results clearly and completely presented? yes - but can be improved - see the detailed comments

-Are the figures (Tables, Images) of sufficient quality for clarity? yes - but can be improved - see the detailed comments

Reviewer #2: (No Response)

Reviewer #3: The analysis does presented correspond to the protocol. The results are presented in a clear and appropriate manner.

Figures and tables are of sufficient quality to be understood.

Reviewer #4: Systematic review was said to include 15 studies. However, there was only one study from Nepal [Ref. 30]. In fact, data presented in table is not in accordance with the study published. Navin Bhatt is the first and corresponding author, while Samir Kumar Sharma the last one. Please, review reference in which Sharma was the first author in 2020.

Line 252: paragraph is incomplete.

Correct typing for Thongtonyong, 2020 (ref. 32) in Table 1 and 2.

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

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: -Are the conclusions supported by the data presented? yes

-Are the limitations of analysis clearly described? yes

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

-Is public health relevance addressed? yes - but can be improved - see detailed comments

Reviewer #2: (No Response)

Reviewer #3: The limits of the study are well specified. The conclusions are adapted to the study conducted. The discussion allows a good understanding of the problem and raises important points for the use of the 20WBCT (glass tube, user training). This study confirms the role of the 20WBCT in the management of ophidian envenomations as well as its limitations.

Reviewer #4: Coagulation tests are important in indicating the snakebite patients who should receive antivenom, and in monitoring the recovery of the coagulopathy. Oddly, this review showed that 20WBCT has been performed after antivenom treatment in the minority of the studies, although most of the studies were specifically designed to assess the validity of the test. Please, discuss this apparent inconsistence.

Furthermore, the four studies in which clotting assays were performed hours after antivenom treatment revealed low sensitivity of the 20WBCT, suggesting the inefficiency of test to assess antivenom effectiveness. Would 20WBCT be more effective to detect a “all or nothing” coagulation disturbance, as originally described by Warrell, and less efficient to detect partial coagulation abnormality, as expected few hours after antivenom administration?

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

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: Line 173: “Although subtle variations in methodology have been used, the most common method, as described by the WHO, has been implemented in national snakebite guidelines” : is this a general statement or a statement specific to Myanmar?

Line 252: incomplete

Page 15: Dsilva – please correct the spelling of Daboia

Fig 4b: The unit of fibrinogen value must be indicated on the x-axis label

Line 368: g/dL or mg/dL?

Reviewer #2: (No Response)

Reviewer #3: no suggestion

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: The manuscript titled “The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy – a systematic review and meta-analysis of diagnostic test accuracy.” by Lamb et. al. investigates the accuracy of the 20WBCT to detect coagulopathy (VICC) through a systematic review and meta-analysis. This study addresses a highly important topic related to snake envenoming, particularly relevant to the low-resource settings where an early diagnosis of snake envenoming is a challenge.

I general, the study has been well-designed, follows standard guidelines for methodology, the selection of the cut-off points of comparison tests are justified, analyses the results in adequate depth, presents the results clearly and the conclusions drawn out are supported by the results. Therefore, I have only minor comments.

Although the authors have compared the outcome of WBCT20 with the INR and fibrinogen measurements taken at the same time point, across different studies, the time from the snake bite to the test has not adequately appreciated in analysis. The utility of WBCT20 depends not only on the performance of the test in effectively detecting various severities of VICC. The diagnostic utility of WBCT20 may also depend on how early it detects VICC. This aspect is cannot-be adequately addressed by sub-group analysis of on-admission (admission times could variry greatly across settings) and post-antivenom. WBCT20 This may be very important in envenomings by certain snake species that often leads to the gradual transformation of mild VICC to severe VICC. For example, if a patient bitten by Russell’s viper presents to a hospital two hours after the bite and the WBCT20 becomes negative at an INR of 2.0 (so that the antivenom is not indicated) but six hours later WBCT20 becomes positive at an INR of >10, the diagnostic utility of the test is quite low. Therefore, I suggest the authors address the above issue and include a column to table 1 showing the median and IQR/ range of the time from the bite to test in individual studies, if they have presented the data. In addition, if it is possible, a subgroup analysis for <4hr and >4hr from the bite or even <6hr and >6hr would be quite important.

Reviewer #2: The authors systematically reviewed diagnostic accuracy of the 20-min whole blood clotting test (20WBCT) in snakebite-induced coagulopathy. The pooled specificity was high, but the sensitivity was variable among studies. Limitations and caveats of the tests were also discussed.

1. There are different mechanisms of coagulopathy among snake families. For example, pit vipers mainly consume fibrinogen, while true vipers also affect other clotting factors. I wonder if there are differences in 20WBCT performances for pit vipers vs. other snakes.

2. Line 171: ‘poisoning’ suggests ingestion of toxins. For snakebite, envenoming may be more appropriate.

Reviewer #3: (No Response)

Reviewer #4: This is a comprehensive review of the predicting value of 20WBCT demonstrating its accuracy to the prompt diagnose of venom-induced consumption coagulopathy and antivenom prescription.

As pointed out by the authors, some results do not allow to validate the bedside coagulation test because of the methodological limitations, but truthfully reflect the clinical and laboratory practices in most healthcare services where snakebite patients usually seek medical assistance.

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

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: No

Reviewer #3: 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.

Dear Dr Lamb,

We are pleased to inform you that your manuscript 'The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy - a systematic review and meta-analysis of diagnostic test accuracy.' 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,

Jean-Philippe Chippaux, M.D., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Jean-Philippe Chippaux

Deputy Editor

PLOS Neglected Tropical Diseases

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