Dear Dr. White,

Thank you very much for submitting your manuscript "Co-infection of the four major Plasmodium species: effects on densities and gametocyte carriage" 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 change P. ovale to P. ovale sp. - you do not differ between P. ovale curtisi and P. ovale wallikeri.

Moreover the shorten forms for parasite species (like Pf) might not be in accordance to the authors guidelines - please check.

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.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[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).

Important additional instructions are given below your reviewer comments.

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,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

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

Please change P. ovale to P. ovale sp. - you do not differ between P. ovale curtisi and P. ovale wallikeri.

Moreover the shorten forms for parasite species (like Pf) might not be in accordance to the authors guidelines - please check.

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: This manuscript written by Holzschuh et al. investigates human Plasmodium species coinfections, its impact on gametocyte production and parasite densities. The manuscript is very clearly written, and well structured.

In detail: Objectives are clearly stated and the methods are well described. Statistical methods are well explained and adequate.

Overall: information given in the main text is rather short, but all of the information can be found in the supplement. I recommend to add more information to the main text, so that the reader does only have to go for the supplement for very detailed information (short information on villages, on sample collection, i.e. filter paper, volume of blood used).

Reviewer #2: The study is clearly articulated and the study design is appropriate with also clear hypothesis.

the sample size is also well sufficient.

Reviewer #3: Methods are sound. There are no concerns about the methods or ethical concerns; the data is thoroughly analyzed and interpreted with caution. The major achievement of the authors, developing sensitive methodologies for gametocyte carriage of all four species is an important one and makes this article of great value.

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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: The results are clearly presented and the figures are of sufficient quality.

One point is confusing: There are 505 pre-treatment samples mentioned, but according to methods only 504 children are included. Please explain the discrepancy.

Many results are only presented in the supplement. If this is not a requirement I suggest to add more results to the main text, e.g. the tables stating the positivity for Plasmodium species (asexual/sexual) for the different analysis

Reviewer #2: The results are missing a clear description or statment of some data: the Plasmodium species blood-stage infections, and Pf and Pv gametocytes (see general comments)

Reviewer #3: Figures 1-3 are clear and highly valuable. The Venn diagram nicely illustrates the richness (and complexity) of the data. Figure 4 is nice but somewhat misleading since all effects, even those that are not statistically significant, are depicted. It is unclear what the grey scare for Po gametocyte density vs Pv asexual density means. I would suggest to only present colors for those associations approaching statistical significance. 0.05 is obviously not sacred and it would be acceptable to only those associations with, for example, p<0.1. Now the light green suggests, for example, a increased density but it is never statistically significant and the effect size is very small. That may lead to wrong interpretations. Alternatively, the number of observations on which the effect size is based may be presented in the cells.

Figure 5 should include number of observations.

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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: The conclusion section is also well written and is supported by the data. Limitations of the study are described. One additional limitation that should be mentioned is the paucity of positive samples for P. ovale, so that it is difficult to draw conclusions. (This might go too far – but - In addition, as it is now widely accepted in the community that P. o wallikeri and curtisi are two distinct species, that are sympatric and not recombining, and therefore one might have to consider them separately, then it is even less samples).

Reviewer #2: The study general conclusion is supported by the data presented but the limitations should be improved with the specification of the co-infections densities and Pv and Pf gametocytes. The age of the samples could also be limiting for an actual application for reflexion of the results.

Reviewer #3: The discussion is balanced. Authors do not over-interpret their findings.

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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: accept

Reviewer #2: see below in summary and general comments.

Reviewer #3: None

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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 presents very well conducted and written work on human Plasmodium species coinfections, its impact on gametocyte production and parasite densities. A novel PCR for detection of Po and Pm gametocytes has been established and is presented. I recommend to publish the manuscript.

Reviewer #2: The paper is presenting data on “Co-infection of the four major Plasmodium species: effects on densities and gametocyte carriage”. As described in the methods section, 504-505 children aged 5-10 years were actively monitored for infection by qPCR and illness for 32 weeks and blood samples were collected every 2 weeks for the first 12 weeks and every 4 week 14-32. While the topic and results are very interesting, there is an important lack of information of the data used, and also discussion of the results using the sampling structure.

These are my comments:

It is not clearly specified in the paper if the Plasmodium species blood-stage infections, and Pf and Pv gametocytes were analysed one more time together with Po and Pm gametocytes using the same RNA extraction. As the study is using more than 10 years old samples, and the same samples being used in different publications, the authors should clearly specify the exact data newly done in the current study and those taken from old studies.

The information in methods section and supplementary data are showing that only Pm and Po gametocytes study were newly done. The authors need to clarify if they extracted again the RNA or they used left over RNA from previous studies.

In case all analysis were repeated again with newly extracted RNA, the obtained co-infections parasitemia data and Pv, Pf gametocytes data should be discussed with the old data from the same authors.

My second main comment is why the authors did not discuss the data using the sampling structure: a 32 weeks retrospective sampling was done with 9 to 10 sample collection per child. It would have been very interesting to analyse and discuss the co-infections pattern of the same sample source during the 32 weeks.

The obtained co-infections of the 4 plasmodium species in PNG are for more than 10 years again. These data should be discussed using data on plasmodium evolution in PNG.

Line153: please check the sample number. 505 is used in the results

Reviewer #3: Appropriate.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. White,

Thank you very much for submitting your manuscript "Co-infection of the four major Plasmodium species: effects on densities and gametocyte carriage" 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.

Dear Authors,

The response to the reviewers comments is missing. I only see the one for the journal editor.

BW

Hans-Peter Fuehrer

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.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[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).

Important additional instructions are given below your reviewer comments.

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,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

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

Dear Authors,

The response to the reviewers comments is missing. I only see the one for the journal editor.

BW

Hans-Peter Fuehrer

Figure Files:

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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. White,

We are pleased to inform you that your manuscript 'Co-infection of the four major Plasmodium species: effects on densities and gametocyte carriage' 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,

Hans-Peter Fuehrer

Section Editor

PLOS Neglected Tropical Diseases

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