Environmental Nematophagous Fungal Control of Soil-Transmitted Helminths in Contaminated Soils Across Latin America.

PLOS Neglected Tropical Diseases

Dear Dr. Mejia,

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,

Robert Adamu SHEY, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Jong-Yil Chai

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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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Rojelio Mejia, Eva Mereles Aranda, Leticia Ojeda, Sandra Ocampos Benedetti, Janitzio J Guzman, Barton Slatko, Cristina Almazan, Melisa Diaz-Fernandez, Ruben Cimino, Marisa Juarez, Natalia Montellano Duran, Estefanía Lorena Mansilla Flores, Paola Andrea Vargas, Amandeep Kaur, Nestor L. Uzcategui, Lucia Estela Mejia, Katherine Keegan, Emilio Rey Mejia, Chiara Cássia Oliveira Amorim, Stefan M. Geiger, Ricardo T Fujiwara, Luz Marina Llangarí-Arizo, Andrea Lopez, Natalia Romero-Sandoval, Irene Guadalupe, Liliana E. Villanueva-Lizama, Julio Vladimir Cruz-Chan, Maritza Dalí Camones Rivera, Eddyson Montalvo Sabino, Carlos Pineda, Eric J. Wetzel, and Philip J Cooper. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: This study clearly states its objectives. The methods section is generally clear, but I have a couple of questions:

Please check that reference 24 is correct.

Could you please provide more details about the sampling sites, i.e., whether they were public areas such as parks or streets; and if there were any specific conditions that facilitated STH contamination?

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The results are clearly presented

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Although the statements on lines 400-406 are correct, please support them with ad hoc references.

The conclusion is appropriate.

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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 Response)

Reviewer #2: (No Response)

Reviewer #3: This manuscript is interesting and presents new knowledge through a little-studied approach on the potential future use of Arthrobotrys oligospora for HTS control.

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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: This manuscript studies the potential role of nematophagous fungi as an environmentally sustainable approach to reduce soil-transmitted helminths (STHs). The combination of laboratory-based experiments and multi-country environmental sampling is ambitious and, in principle, could provide valuable insights into parasite control at the soil level. However, in its current form, the study suffers from substantial conceptual, methodological, and interpretational limitations that critically undermine the validity of its conclusions. Here below are my main concerns:

The manuscript combines controlled in vitro experiments with a large observational soil survey; however, these two components are not mechanistically or causally integrated. The in vitro assays demonstrate fungal–nematode interactions under highly artificial conditions, whereas the environmental study relies solely on cross-sectional DNA detection. No evidence is provided that the mechanisms observed in vitro operate under natural soil conditions, rendering the linkage between the two datasets speculative.

Another major methodological flaw exists in the in vitro soil-based experiments. Commercial garden soil was used, and several control samples were found to naturally contain A. oligospora DNA. As a result, the control condition is not fungus-free, and qPCR cannot distinguish background fungal DNA from experimentally introduced fungus. This fundamental confounding undermines the validity of the reported reduction in Toxocara cati DNA.

In the environmental study, the nematode qPCR assays are acknowledged to lack strict species specificity, yet the results are interpreted at the species level and extrapolated to human health relevance. In addition, only A. oligospora was assayed despite the known diversity of nematophagous fungi (e.g. Duddingtonia flagrans, Monacrosporium spp., Arthrobotrys spp.) in soil, making broad conclusions about fungal-mediated control unjustified.

Finally, the soil data are purely correlational, based on DNA detection that does not indicate organism viability or active killing, and no multivariable analyses were performed to account for major environmental confounders. Despite these limitations, the manuscript repeatedly implies active biological control of STHs in the environment, which is not supported by the presented data.

The central conclusions regarding environmental control of soil-transmitted helminths by Arthrobotrys oligospora substantially exceed what can be supported by the current data. Addressing these limitations would require extensive redesign of both laboratory and field components of the study. Therefore, I do not consider the manuscript suitable for publication in its present form.

Reviewer #2: (No Response)

Reviewer #3: This manuscript is well written and presents novel results. I suggest clarifying a few minor comments before publication.

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Reviewer #2: No

Reviewer #3: No

Figure resubmission:

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Dear Dr Mejia,

We are pleased to inform you that your manuscript 'Distribution of Nematophagous Fungi and Soil-Transmitted Helminths in Outdoor Built Environments Across Latin America' 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,

Robert Adamu SHEY, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Jong-Yil Chai

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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