Dear Dr. Wu,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Comments to the Authors:

Please note that three reviews are uploaded as attachments.

Reviewers' comments:

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. Although the system achieves high predictive accuracy, it functions as a black box model. However, the features that most strongly predict viability have not been identified. The author can use explainable AI (XAI) tools such as Grad-CAM, SHAP, or LIME to highlight which spatial/temporal features (e.g., worm curvature, contraction frequency) contribute most to predictions. This would provide biological interpretability and improve the trust in the system.

2. The system was validated only for adult worms. However, PZQ is less effective against juveniles, and screening should ideally cover all relevant life stages of schistosomes. It would be advisable to extend the platform to include juvenile schistosomes (e.g., schistosomula or 14-day-old worms), which represent a critical treatment gap for PZQ. This would greatly enhance its translational value.

3. The system evaluates phenotypic changes without integrating the known mechanisms of action (MOAs) of compounds, such as calcium influx or tegument disruption. I recommend the inclusion of parallel mechanistic assays (e.g., confocal imaging, redox state indicators, and calcium signaling) for selected compounds to validate phenotypic predictions and classify responses according to the MOA.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: 1. While the model generalizes to some extent, the test compounds (CPD2, CPD12, and CPD13) may be structurally or functionally similar to the training compounds. It will be good to perform External validation with novel compounds not observed during training would be beneficial, ideally including those with known divergent mechanisms or poor motility impact (e.g., metabolic disruptors). Clarify the compound diversity in the test set.

2. The manuscript reports high Pearson correlation coefficients (PCC); however, the error distribution and misclassification are not thoroughly discussed.

Reviewer #2: (No Response)

Reviewer #3: (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: 1. The authors should clarify the rationale for the selection of the 13 tested molecules with known structural classes and MOAs.

2. In the discussion, the authors should explicitly compare their method with other recent AI-based pipelines, if any

Reviewer #2: (No Response)

Reviewer #3: (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: (No Response)

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: This manuscript presents an innovative and scalable computational platform for assessing Schistosoma mansoni viability using deep learning applied to video data. The integration of advanced tools, such as SAMTrack and VideoMAE, along with augmentation and segmentation strategies, represents a significant step toward automated high-throughput anti-schistosomal drug screening. The model demonstrated robust performance, aligning closely with expert annotations and outperforming previous methods.

However, while the study is methodologically sound and technically advanced, the manuscript could be substantially improved by addressing the following scientific and methodological limitations.

Reviewer #2: Developing an AI-powered scoring system capable of high-throughput and accurate assessment of parasite viability is crucial for anti-parasitic drug discovery, as this system can significantly accelerate the development of novel anti-schistosomal agents. This manuscript is well-written and easy to understand. However, prior to the publication of the article, the authors are request to answer the following questions raised by the reviewer.

1. Why did the authors only use this system to evaluate schistosomes viability after 24 hours of drug exposure? Is this system still applicable for assessing parasite viability after 48 hours, 72 hours, or even longer duration of drug action? To our knowledge, the viability of adult worms will be partially recovered after 48 hours exposure to PZQ.

2. The authors should verify the PZQ concentration used in this study. According to the published data, PZQ at the concentration of 0.5 μM has little effect on the viability of Schistosoma mansoni adult worm. Is 0.5 μM the saturated concentration of PZQ? What are the relevant references?

3. Figure 2 lacks the explanation and description of the PZQ concentration, making it difficult to understand.

4. Line 115-116, Line 128, “Schistosoma mansoni” should be “S. mansoni”. The authors have to check the manuscript carefully.

5. Line 189, “……adapted from” is an incomplete sentence.

Reviewer #3: The development of a high-throughput schistosome viability assessment system based on artificial intelligence technology demonstrates significant innovation and can accelerate the development of novel anti-schistosome drugs. This article is well-written and logically coherent. However, before considering publication, there are still some issues that the author needs to address with more detailed explanations and responses.

1. Why did the authors choose the 24-hour post-treatment period to evaluate the AI assessment system? Why was a longer timeframe not considered to assess the system's accuracy? According to the published data, the viability of schistosomes may recover as the duration of PZQ exposure in vitro increases. Can the AI system monitor such changes in worm viability?

2. The authors emphasize that other researchers can access the 24-hour equivalent PZQ concentration of antischistosomal drugs through the online platform, but what exactly is the role of this "24-hour equivalent PZQ concentration"? This question needs further in-depth discussion.

3. Figure 2 contains numerous images but lacks clear labeling of PZQ concentrations, making it difficult to understand. Additionally, can 0.5μM PZQ induce significant changes in schistosome vibility? The authors need to carefully verify the PZQ concentrations used during the experiments.

4. The citation format for Reference 1 contains an error. “Mutapi F, Maizels R, Fenwick A, Woolhouse M. Human schistosomiasis in the post mass drug administration era. Lancet Infect Dis. 2017;17(2): e42-e8”.

5. Line 189, “……adapted from” is an incomplete sentence. The authors should check the manuscript thoroughly.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Manuscript Number PNTD.docx

Attachment

Submitted filename: renamed_3a62d.docx

Attachment

Submitted filename: reviewer comments.docx

Dear Dr. Wu,

We are pleased to inform you that your manuscript 'An Automated End-to-End System for Schistosome Viability Assessment to Accelerate Anti-Schistosomal Drug Discovery' 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,

Krystyna Cwiklinski, PhD

Section Editor

PLOS Neglected Tropical Diseases

Krystyna Cwiklinski

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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

The authors have addressed the comments raised by the reviewers. The manuscript is now suitable for publication in PLoS NTD.

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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 #2: (No Response)

Reviewer #3: (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 #2: (No Response)

Reviewer #3: (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 #2: (No Response)

Reviewer #3: (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 #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 #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 #2: No

Reviewer #3: No