PNTD-D-26-00052Efficacy of oxfendazole treatment against infective larvae of Litomosoides sigmodontisPLOS Neglected Tropical DiseasesDear Dr. Hübner,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. Please submit your revised manuscript by March 14 2026 If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please include the following items when submitting your revised manuscript:* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.'.* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.'.If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.We look forward to receiving your revised manuscript.Kind regards,Bruce A. RosaAcademic EditorPLOS Neglected Tropical DiseasesLucienne TrittenSection EditorPLOS 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

Additional Editor Comments:Overall, the reviewers were positive about the manuscript. They have provided a few suggested revisions, so please carefully consider their feedback.Journal Requirements:

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Reviewers' comments: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: The Methods are well-described and appropriate for the aims of the study.

Reviewer #2: The paper describes that Oxfendazole exhibits a robust efficacy against the L3 larval stage of Litomosoides sigmodontis in mice. This work supports the ongoing clinical development of Oxfendazole as a broad stage filaricide/nematicide. The benzimidazole originates from veterinary medicine and is now repurposed for human use. While the team showed in previous work, that Oxfendazole showed macrofilaricidal efficacy, but no direct effect on microfilariae, only this manuscripts demonstrates that Oxfendazole is active against L3 larvae.

The experimental design is sufficiently described and scientifically sound:

• BALB/cJ mice were naturally infected with L. sigmodontis via exposure to infected mites.

• Drug was administered orally twice daily, starting 1 day post infection, for 3 or 5 days.

• Doses tested: 5 mg/kg and 12.5 mg/kg.

• Worms were recovered and measured 35 days post infection.

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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: The Results are clear and the analyses justified and appropriate.

Reviewer #2: Key Findings

1. Strong reduction in worm burden

• 5 day treatment at 12.5 mg/kg: → 97.6% median reduction in total worms → Female worms reduced 100%, males 95.7%

• 3 day treatment showed dose dependent efficacy: → 12.5 mg/kg for 3 days reduced total worms by 82.6% → 5 mg/kg for 3 days had only modest effect (~30% reduction)

2. Worm development was impaired

• Surviving worms were significantly shorter, especially after 5 day treatment.

• Oxfendazole is highly effective against the infective L3 stage of L. sigmodontis.

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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: The regimen investigated here was clearly highly effective against L3 stage parasites. My concern is the seemingly minor relevance. To be useful, one would either have to maintain constant plasma levels in people living in endemic regions or treat individuals who were briefly exposed to transmission. Activity against L4 and L5 stages might be more clinically relevant (not considering macrofilaricidal activity, which has been reported).

Reviewer #2: I would encourage the authors to extend the background and discussion about animal health as well. To prevent canine heartworm disease, the migrating larvae need to be treated. This could also lead to an outlook on what the treatment against L4 larvae would look like. Furthermore, the authors may want to discuss the dose-response comparing the larvicidal versus the adulticidal effects to a certain degree (particularly as parasite isolate, mouse strain and drug are uniform).

Minor remark: The graphs and abstract do not clearly state that the treatment is 2 times per day.

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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: A minor concern: the authors should stress that the PK properties of the drug are atypical for benzimidazole anthelmintics and enable much higher systemic exposure than attained with albendazole, for example, which is included in combination therapy for LF and has been investigated for other systemic helminth infections, including as a way to reduce microfilaremia in Loa infections. Thus, one would anticipate higher efficacy with oxfendazole, assuming intrinsic potency is similar to other BZs.

Reviewer #2: I would encourage the authors to extend the background and discussion about animal health as well. To prevent canine heartworm disease, the migrating larvae need to be treated. This could also lead to an outlook on what the treatment against L4 larvae would look like. Furthermore, the authors may want to discuss the dose-response comparing the larvicidal versus the adulticidal effects to a certain degree (particularly as parasite isolate, mouse strain and drug are uniform).

Minor remark: The graphs and abstract do not clearly state that the treatment is 2 times per day.

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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 study is properly and carefully done. Really, my only concern is its relevance for therapy.

Reviewer #2: This is a well‑executed and valuable study that provides the first in vivo evidence of Oxfendazole’s efficacy against infective larvae of Litomosoides sigmodontis. The results are clear, the methodology is appropriate, and the findings significantly advance the understanding of Oxfendazoles therapeutic spectrum potential.

With minor revisions to strengthen context and clarity, the manuscript is well suited for publication.

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Reviewer #1: No

Reviewer #2: Yes: Daniel KulkeDaniel Kulke

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Dear Dr Hübner,

We are pleased to inform you that your manuscript 'Efficacy of oxfendazole treatment against infective larvae of Litomosoides sigmodontis' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.' 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.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Bruce A. Rosa

Academic Editor

PLOS Neglected Tropical Diseases

Lucienne Tritten

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

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The authors have sufficiently addressed reviewer concerns and improved the manuscript accordingly.