PNTD-D-25-00725

Comparative Performance of a Commercial and In-House Mp1p antigen-detecting Enzyme Immunoassay for the Rapid Diagnosis of Talaromycosis

PLOS Neglected Tropical Diseases

Dear Dr. Brown,

(This is great work Lottie- thank you for choosing Plos NTD. Regarding one point raised in review, I do favor keeping the "neglected" as we have including Talaro as such at this journal! Best, Josh)

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,

Joshua Nosanchuk, MD

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

Journal Requirements:

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: Lottie Brown, Sruthi Venugopalan, Ngo Thi Hoa, Jian-Piao Cai, Kwok-Yung Yuen, Jasper F. W. Chan, and Vo Trieu Ly. 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: Lines 137-139: Consider defining other specimens were considered clinical

Lines 139: Consider referencing another paper that describes clinical syndromes consistent with talaro for those readers who may not be as familiar with the disease

Lines 139-143: Consider describing a bit more about how the controls were randomly selected? Numbering system? Same with the cases.

Lines 186-188: The authors may want to add a bit of reasoning into why likelihood ratios were chosen over the more commonly used predictive values. And a bit more commentary on how to use likelihood ratios.

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: Lines 194-195: Consider removing this sentence and just citing figure after the next sentence for flow of manuscript. Consider doing the same for subsequent sections in the results sections.

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: Lines 270-271: Consider adding reference to end of sentence.

Lines 342-343: Consider removing the designation as an NTD since technically not considered as an NTD by WHO; although, it very likely should be.

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: Figure 1: Consider adding a bit more detail about randomization process.

Figure 4: Will ultimately defer to authors, but the value added from figure 4 may be minimal and could be removed or in supplemental material.

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: - Lines 87-88: While vast majority of talaro occurs in those immunocompromised, disease in immunocompetent hosts have been documented. Consider modifying the end of the sentence to reflect this.

- Lines 93-96: Since this is PLoS NTDs, maybe consider including a sentence after WHO FPPL about how it is absent on WHOs NTD list for advocacy purposes? Will leave up to authors, but just a thought if authors think it will add to their report.

Lines 97-106: May want to include a bit of commentary about other promising point of care tests that the authors have worked on, but not commercially available yet.

Reviewer #2: The original manuscript by Barwatt and colleagues, which presents a comparative analysis of the diagnostic performance between a commercial Wantai EIA and an in-house EIA based on the Mp1p antigen of Talaromyces marneffei, is scientifically written, clear, concise, and focused. However, there remain several aspects that warrant further consideration, as outlined below:

1. Clarification of the Study’s Primary Objective:

It is important for the authors to more clearly articulate the main objective of this experiment. Given that both ELISA systems were developed based primarily on rabbit polyclonal antibodies against Mp1p, the resulting outcomes should reasonably align (as the authors have demonstrated), and significant discrepancies would not be expected. Therefore, the rationale and specific aims of the study should be explicitly stated.

2. Experimental Methodology:

There is some ambiguity regarding the experimental procedures. The methods used for the Wantai EIA and the in-house Mp1p EIA appear to differ slightly in their details. To enhance clarity and accessibility for the reader, the authors are encouraged to provide a schematic diagram or flow chart that visually summarizes the experimental workflow, rather than relying solely on textual descriptions.

3. False Negative and False Positive Characteristics:

Under the section addressing the characteristics of false negatives and false positives, the manuscript would benefit from additional detail. The occurrence of false negatives is not entirely unexpected, given that the mechanisms underlying the secretion of mannoprotein antigens in the pathogenesis of T. marneffei are not yet fully understood. The authors may wish to interpret this within the framework of the damage-response model of T. marneffei pathogenesis. Furthermore, baseline antigen levels within populations residing in endemic areas remain to be thoroughly investigated.

In contrast, the issue of false positives requires a more comprehensive discussion. In particular, potential cross-reactivity involving the rabbit anti-Mp1p antibodies should be considered and addressed in greater depth.

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Reviewer #1: Yes: Dallas Smith

Reviewer #2: No

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Dear Dr Brown,

Thank you, Lottie et al, for your thoughtful, rigorous, and complete answers to the comments on the original version of your work. We are pleased to inform you that your manuscript 'Comparative Performance of a Commercial and In-House Mp1p antigen-detecting Enzyme Immunoassay for the Rapid Diagnosis of Talaromycosis' 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.

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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,

Joshua Nosanchuk, MD

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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