PNTD-D-26-00096

Molecular and morphologic characterization of Trichuris trichiura infecting free-roaming African vervets (Chlorocebus aethiops sabaeus) on the Caribbean Island of St. Kitts

PLOS Neglected Tropical Diseases

Dear Dr. Richins,

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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We look forward to receiving your revised manuscript.

Kind regards,

Bruce A. Rosa

Academic Editor

PLOS Neglected Tropical Diseases

María Periago

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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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: Study design clearly outlined. Sample size seems appropriate. Methods clearly described. No concerns about ethical or regulatory requirements being met.

Reviewer #2: (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: Figure 1 should be labelled in more detail - specifically, arrows should be added to indicate the features being described as it is not clear from the description for the non-expert reader.

For a non-geneticist, the main part of the results section is hard to follow. Would suggest more word count afforded to make this accessible for general parasitologists - for example what is the significance of the different clades?

Reviewer #2: (No Response)

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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: Conclusions are supported by the data presented and limitations of study are described. Public health relevance clear - vervet monkeys could be a zoonotic source of trichuriasis infection on St Kitts.

Reviewer #2: (No Response)

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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: See comment in results section regarding adding labels to figure 1 to enhance clarity.

Reviewer #2: (No Response)

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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: In general, this article is difficult to follow for non-geneticists. Would recommend some additional text in the results and discussion to describe/discuss the key findings in simple terms. For example, a gentle recap of the findings and their potential impact at the start of the discussion - perhaps move some of the text from the second paragraph into the first. Closing paragraph is strong and provides an excellent summary for non-experts.

Reviewer #2: This study represents an interesting addition to the current body of literature investigating taxonomy and population structure within the Trichuris genus. The St. Kitts vervet population is particularly relevant given its close ecological overlap with humans and the resulting implications for zoonotic transmission. The integration of public genomic datasets is a strength of the manuscript and allows for meaningful phylogenetic contextualization.

Major comments:

C1. Incomplete amplification and marker choice. A substantial proportion of fecal samples (17/34) did not yield sequence data, despite microscopy confirming the presence of Trichuris eggs. While incomplete amplification is expected in fecal-derived DNA, this raises concerns about potential underrepresentation of diversity within this population. I recommend that the authors consider testing additional primer pairs to reduce the risk of missing divergent Trichuris lineages. It has previously been demonstrated that primers designed for T. trichiura may fail to amplify other species, including T. incognita (https://doi.org/10.3201/eid3101.240995). Expanding primer coverage would help ensure that non-trichiura taxa are not overlooked. Furthermore, sequencing of a full-length ITS2 fragment (ideally with a strategy enabling haplotype resolution) would help minimize reconstruction bias and provide an independent nuclear marker for phylogenetic comparison. A strengthened multi-locus approach would considerably reinforce the phylogenetic conclusions presented.

C2. Exclusion of ITS1/ITS2 data. The justification for excluding ITS1 and ITS2 data is not fully convincing. While phasing multi-copy rDNA loci from short-read data can be challenging, this does not preclude analysis of ITS diversity. Amplicon-based ITS analyses in Trichuris, including cases with intra-individual heterogeneity, have been successfully conducted using short-read sequencing (e.g., https://doi.org/10.3201/eid3101.240995). Even if full haplotype phasing at the individual-worm level is not feasible, the authors should at least present the detected ITS1/ITS2 sequence variants and their relative abundances per sample (e.g., as ASVs or clustered variants in supplementary material). Completely omitting these data despite successful sequencing leaves an important component of the dataset unexplored and reduces the overall completeness of the study.

Minor comment:

C1 – Taxonomic status of T. incognita. Line: “noting that the name ‘incognita’ was assigned without a formal taxonomic description and likely represents a nomen nudum [23]”

A formal taxonomic description has now been published (https://doi.org/10.1016/j.lanmic.2025.101264), with further contextual characterization in https://doi.org/10.3201/eid3101.240995. The manuscript should be updated accordingly to reflect the current taxonomic status.

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Reviewer #1: No

Reviewer #2: No

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Dear Mr. Richins,

We are pleased to inform you that your manuscript 'Molecular and morphologic characterization of Trichuris trichiura infecting free-roaming African vervets (Chlorocebus aethiops sabaeus) on the Caribbean Island of St. Kitts' 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.

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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

Maria Victoria Periago

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 reviewers appreciated the revisions to the manuscript and now feel that it is ready for acceptance

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: As outlined in the original review of this manuscript, the study design is clearly outlined. Sample size seems appropriate.

Methods clearly described. No concerns about ethical or regulatory requirements

being met.

Reviewer #2: (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: Yes - thank you to the authors for adding the labels requested for Figure 1.

Reviewer #2: (No Response)

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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: Yes - useful addition of commentary on the relevance of the different clades and limitations of the current study in the discussion.

Reviewer #2: (No Response)

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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: No

Reviewer #2: (No Response)

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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 reviewer's comments have been adequately addressed and I recommend acceptance of the article for publication.

Reviewer #2: Many thanks to the authors for addressing the comments thoroughly. I recommend to accept the manuscript.

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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