Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes

PLOS Neglected Tropical Diseases

Dear Dr. Reva,

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,

Ben Pascoe

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

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

Additional Editor Comments:

After careful consideration of two expert reviews, we believe the manuscript can reach publishable quality, but substantial revision is required.

In brief:

• Please articulate an explicit, testable hypothesis and streamline the Introduction to highlight the specific knowledge gap addressed.

• Descriptive SNP/parsimony analyses alone are insufficient. Add a statistically supported phylogeny and at least one population-structure or phylogeographic test linking genotype to ecology/host.

• Explain how strains were chosen from the national collection and acknowledge the inherent sampling imbalance when interpreting ecological patterns.

• Provide an explicit statement of BSL-3 approvals and regulatory compliance for work with Y. pestis.

• Improve figure resolution (vector graphics), split intermixed Results/Discussion text, and enforce consistent terminology for biovars, clades, and strain names.

• Correct Table 1 primer information; supply amplicon sizes, biovar specificity, and software versions/parameters for all tools.

• Add a concise paragraph on how your SNP panel or plasmid findings could aid surveillance and explicitly list analytical limitations (e.g., lack of long-read data, small subgroup sizes).

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1) State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

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If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

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: Are the objectives of the study clearly articulated with a clear, testable hypothesis stated?

Partially.

The manuscript has well-described aims: to characterise Y. pestis isolates from Central Asia and identify genetic polymorphisms associated with ecological adaptation and lineage divergence. However, a clear hypothesis is not explicitly stated, and the narrative blends surveillance goals with exploratory phylogenomic analysis. For a stronger framing, the authors should articulate a central hypothesis (e.g., “Y. pestis genomic variation correlates with host or environmental origin more than with clonal ancestry”) early in the Introduction.

Is the study design appropriate to address the stated objectives?

Yes, broadly.

The use of WGS for 98 Y. pestis isolates, with phenotypic testing and in silico variant analysis, is appropriate for evaluating biovar classification, polymorphisms, and lineage diversity. However, comparative analyses with global strains are missing, which limits broader inference and validation of their findings.

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

Yes.

The manuscript provides detailed ecological and geographic context for the 98 isolates, spanning desert and upland plague foci. The inclusion of multiple hosts (e.g., gerbils, marmots, fleas) is appropriate given the zoonotic ecology of plague. A more structured breakdown of how many isolates came from which host species or region would help clarify population stratification.

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

Generally yes, but with caveats.

For regional genomic surveillance, the sample size is robust (n = 98). However, comparisons across clades or ecological categories (e.g., desert vs upland) sometimes involve much smaller groups (e.g., 4 Talas isolates), which limits statistical power. The authors should acknowledge this limitation, particularly when making ecological inferences.

Were correct statistical analyses used to support conclusions?

Partially.

The authors use custom SNP calling, clustering, and dendrogram construction, but they rely heavily on parsimony-based clustering and descriptive comparisons without formal phylogenetic or population structure modelling. No statistical tests (e.g., Mantel tests, AMOVA, PCA) are used to quantify associations between genotype and geography/host. This weakens the analytical rigour and should be addressed with additional comparative or phylogenetic methods if claims about convergent evolution or ecological adaptation are to be substantiated.

Are there concerns about ethical or regulatory requirements being met?

No major concerns raised, but additional clarity would help.

The authors state that isolates came from a national strain collection and were collected during routine surveillance. However: There is no explicit statement of ethical or biosafety approvals for working with Y. pestis (a BSL-3 agent).

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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: Does the analysis presented match the analysis plan?

Broadly yes, but exploratory in nature.

The study presents genome sequencing, variant detection, plasmid profiling, and phenotypic tests of Y. pestis isolates as planned. The described pipelines and laboratory methods align with the Results section. However, because the manuscript is exploratory rather than hypothesis-driven, there is no formal analysis plan or pre-specified comparisons. The selection of SNPs and construction of polymorphism matrices are well described, but the lack of formal statistical testing makes the structure more observational than analytical.

Are the results clearly and completely presented?

Partially.

The authors provide extensive detail, including strain-level variation, allele states, plasmid content, and CRISPR/transposon profiles. However, the narrative is dense, with long blocks of text that lack synthesis or summarisation.

Some key findings (e.g., upland vs desert divergence, pCKF identification) are buried in technical detail, making them hard to extract.

The lack of statistical tests or quantitative summaries (e.g., number of unique SNPs per clade, plasmid prevalence by ecotype) weakens the clarity of the results.

Recommendation: Add a concise summary table per major section (e.g., SNP patterns, plasmid presence, phenotypic test outcomes) and integrate clearer narrative transitions.

Are the figures (Tables, Images) of sufficient quality for clarity?

No

Several figures are blurry or low-resolution, particularly Figures 2–5, which are central to the interpretation of genomic variation and lineage structure.

Font sizes are too small in dendrogram labels and SNP context annotations.

Figure 3 (pipeline diagram) is unnecessary and should be removed or moved to Supplementary Material.

Supplementary tables are useful but need better integration into the main narrative.

**********

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: Are the conclusions supported by the data presented?

Partially.

The conclusions - particularly regarding the ecological structuring of Y. pestis genotypes (upland vs desert) and the possibility of convergent evolution - are intriguing and plausibly supported by the data. However, some conclusions (e.g., strong claims about adaptive convergence or plasmid transfer across lineages) go beyond the data, given the limitations of short-read sequencing and lack of phylogenetic inference.

The authors also understate the ambiguity around taxonomic assignment of outlier strains (e.g., Y. pseudotuberculosis-like genomes classified as Y. pestis).

Are the limitations of the analysis clearly described?

The authors do not sufficiently acknowledge several key limitations:

No long-read data for plasmid resolution or structural variation.

Small subgroup sizes for comparisons (e.g., Talas isolates).

Reliance on selected SNPs and parsimony trees rather than robust phylogenetic or population genetic approaches.

No formal statistical or model-based testing of associations between genotype, host, or environment.

Recommendation: A dedicated paragraph in the Discussion should explicitly list these analytical limitations and their implications for interpretation.

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

Yes, in part.

The study contributes a valuable genomic dataset from a poorly sampled geographic region. The authors suggest that their polymorphism panel could be used for biovar identification and ecological surveillance. However, they could better articulate:

How these tools might integrate with global Y. pestis surveillance frameworks, or

How their SNPs or plasmid findings compare to those in outbreak strains from other continents.

Is public health relevance addressed?

Minimally.

The manuscript briefly mentions zoonotic risk, ecological change, and the importance of surveillance in Kazakhstan, but does not connect this meaningfully to:

The risk of human outbreaks, or

How these findings might guide vector control, diagnostics, or cross-border health policy.

Recommendation: Strengthen the final paragraph of the Discussion to explicitly state how the findings could improve public health risk assessment, surveillance tools, or outbreak preparedness.

**********

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: Clarify and streamline the Introduction

While informative, the Introduction could be shortened and better focused. The authors should clearly articulate the gap this study addresses and explicitly state a testable hypothesis (e.g., genomic variation in Y. pestis correlates with ecological origin more than with shared ancestry).

Improve logical flow and paragraphing

The Results and Discussion sections are dense and occasionally read like internal notes. Use clearer topic sentences and summary statements to guide the reader through complex genomic findings.

Correct typographical and language issues

Line 29: "sequnced" > "sequenced"

Line 33: "suitable tool" > "suitable tools"

Line 54: "ware caused" > "were caused"

Consider a light language edit throughout to improve fluency and clarity.

Ensure consistency in terminology

Terms like “biovar,” “clade,” “subvariant,” and “non-main” are used inconsistently. Define and standardise these terms early, especially when linking phenotypic biovars to genomic groupings.

Add ethical and biosafety statement

Given that Y. pestis is a BSL-3 pathogen, include a statement affirming compliance with appropriate ethical and biosafety regulations, including approvals for handling high-risk agents.

Improve figure resolution and legibility

Figures 2–5 are critical to the paper’s conclusions but are currently low-resolution with small or pixelated text. Please:

Re-export or redraw figures using vector formats (PDF, SVG) to ensure clarity.

Increase font sizes for isolate names and branch labels.

Ensure all abbreviations, symbols, and colour keys are defined in the legends.

Remove or relocate Figure 3

The diagram showing the internal SNP-calling pipeline (Figure 3) is not informative to the scientific conclusions and can be removed or moved to supplementary materials if needed for pipeline documentation.

Improve integration of supplementary tables

The manuscript frequently references Suppl. Tables (e.g., S1–S5), but their use would be clearer if specific examples were summarised in the main text, and/or a “key findings” summary table were added (e.g., SNP markers per lineage, plasmid carriage, or phenotypic traits by clade).

Explicitly list software versions

To ensure full reproducibility, please add version numbers for all software tools used, including:

Trimmomatic

SPAdes

Bowtie2

BCFtools

Prokka

RagTag

BLASTN

Python version for custom scripts

Even if run with default parameters, this should be stated explicitly.

Discuss limitations of short-read data

The inability to resolve plasmid structure, including the circularisation or structural variation of pCKF and other elements, should be clearly stated. The authors should note that long-read sequencing would provide more definitive plasmid resolution.

Add global context

The manuscript would be strengthened by discussing how these Central Asian lineages relate to global Y. pestis diversity, including pandemic-associated clades or modern outbreak strains. A phylogenetic comparison or even literature summary would enhance impact.

Strengthen public health relevance

Currently, the manuscript emphasises ecological and genetic findings but says little about how these data could inform surveillance, risk assessment, or control. Consider adding a concluding paragraph that:

Links the findings to public health surveillance,

Highlights diagnostic implications (e.g., lineage-specific markers),

Discusses the relevance of cryptic plasmids (e.g., pCKF) for virulence monitoring.

**********

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: (No Response)

Reviewer #2: The manuscript offers a rare and detailed look at Y. pestis evolution in Central Asia - a region of historical and current epidemiological importance. The work has the potential to substantially contribute to our understanding of plague ecology and evolution, particularly in the context of environmental adaptation and mobile genetic elements. However, its broader impact is currently limited by presentation, analytical, and contextual shortcomings that should be addressed in revision.

**********

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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

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Attachments

Attachment

Submitted filename: Plos Neglected Yersinia pestis.docx

Please include the following items when submitting your revised manuscript:

Response to ReviewersRevised Manuscript with Track ChangesManuscript

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

Additional Editor Comments:Journal Requirements:

1) State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

2) State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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: Yes.

Reviewer #2: (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 #1: Yes.

Reviewer #2: (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: Yes.

Reviewer #2: (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: Accept.

Reviewer #2: (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 reviewer acknowledges and accepts all responses provided by the authors to the previous comments raised during the review process. No further remarks.

Reviewer #2: This manuscript analyses ~100 Yersinia pestis isolates from Central Asian plague foci using short-read WGS, targeted polymorphism panels, plasmid profiling and classical phenotyping. The dataset is valuable and the revision improves framing, biosafety statements, figure exports, and data availability. However, several core analytical and reporting issues remain.

Major points

Phylogenomic backbone: Provide a core-genome SNP maximum-likelihood tree (with support) for all isolates and use it to anchor ecological interpretations; the current diagnostic-SNP dendrogram is explicitly not a phylogeny.

Genome QC: Add a per-isolate table (genome size, N50/L50, contig count, %GC, mean depth) and state any exclusions.

Statistics: The random-forest classification of desert vs upland needs clearer methods (feature set, CV scheme, class balance) and proper p-value reporting (e.g., p < 0.001, not 0.0). Consider a simple AMOVA/Mantel/PERMANOVA to quantify genotype–ecology association.

Reproducibility: List exact versions/parameters for Trimmomatic, SPAdes, Bowtie2, BCFtools, Prokka, RagTag (and seeds for ML).

Plasmid claims: The pCKF signal is interesting; include read-level evidence in Supplement (coverage plots; discordant pairs). If feasible, note any targeted validation.

Minor points

Tighten language and standardise terminology (biovar/clade/subvariant).

Keep the pipeline graphic in Supplement; ensure all main figures have legible fonts/keys.

Add a brief global context paragraph situating these lineages relative to established Y. pestis diversity.

End with a clearer public-health relevance statement (surveillance utility of markers, implications for risk assessment).

Ethics & data

BSL-3/biosafety statements appear adequate. Data availability is strong; please also provide a pinned, isolate-level metadata CSV.

**********

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

Figure resubmission:

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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:--> -->-->To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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-->?>

Dear Prof. Reva,

We are pleased to inform you that your manuscript 'Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes' 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,

Ben Pascoe

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

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

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

Thanks for engaging with the review process and addressing all reviewer comments.

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