Dear Mr. Lim,

Thank you very much for submitting your manuscript "The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Husain Poonawala

Guest Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

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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 fungal isolates tested . It isn't clear how these were all speciated prior to testing. Was this carried out by molecular techniques ( please state how ) - This information should be included in the paper.

It is a pity that some mycetoma agents have not been used for comparison eg Scedosporium apiospermum as other non-pigmented fungi have been included. Was this an oversight ?

In the discussion the authors refer to the in silica assay. Can you expand on this ?

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 but see above 3rd comment

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 these are fine. My second point is relevant to limitations - albeit a minor one

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: It would be helpful if the authors would say where they see the place of reference centres for mycetoma diagnosis. How many are there and do we need more ?

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: See above

Reviewer #2: The manuscript by Lim et al describes the development of a novel PCR assay for the specific detection/identification of cases of eumycetoma caused by Madurella mycetomatis, based on a genome-wide comparative approach.

A series of potential gene targets for PCR were identified in putative coding regions throughout the M. mycetomatis genome, PCR primers were designed and tested against a panel of 60 M. mycetomatis isolates plus a range of sibling species in Madurella, other agents of eumycetoma, and a variety of less related filamentous fungi.

One primer pair was selected due to its high specificity, and was shown to be slightly less sensitive that the previously described spscific PCR (which has since been shown to cross-react with another Madurella species.

A major question that should be addressed is whether this novel PCR is sufficiently sensitive to be used in diagnosis directly from clinical specimens eg fungal grains, or whether it will only be useful when working with pure cultures. I would encourage the authors to address this in the current manuscript as it would substantially strengthen the study.

The English usage is a little poor in places, and grammatical issue should be addressed on lines 35 (were not was); 50 (disappointing? is not clear); 54 (cause not causes); 57 (delete "a"); 57 (suggest: that we recommend"); sentence starting line 78 (developed is repeated)108 (using substituting?);127 (PCR reactions contained); 129 (were as follows); 132 (was this final stage really 10 seconds?); Table 2 (Madurella tropicana and spelling of Trematosphaeria); 154 Primer sets 4, 5 and 12 only amplified ...); 189 (delete "a")

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Reviewer #1: Yes: Roderick J Hay

Reviewer #2: No

Figure Files:

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Dear Mr. Lim,

We are pleased to inform you that your manuscript 'The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach' 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,

Husain Poonawala

Guest Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

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