Dear Dr Brown,

Thank you very much for submitting your manuscript "Human exposure to zoonotic malaria vectors in village, farm and forest habitats in Sabah, Malaysian Borneo" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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

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

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: Research question: Investigate environmental determinants of Plasmodium knowlesi vector density and infections rates across a wider spatial scale in Sabah.

Key aims were to identify associations with habitat type and test whether results from small-scale sampling in one district (Kudat) are generalizable across the state. In addition, this study tested for associations between entomological variables and human exposure to P. knowlesi as measured in a related sero-prevalence study. The authors say they performed and intensive study to address their research questions.

Methods

A larger area of Sabah was included in the study described (fig 1). Three different habitats (peri-domestic, farm and forest) per village were sampled over 4 nights.

All sites were studied in three months (March to June 2016) the single time frame did not seem to particularly coincide with annual zoonotic malaria prevalence in Sabah. Even-so to attempt sampling 3 habitats, with teams of two per habitat, using only human landing catches (HLC) in 11 villages over a 3-month period would be expected to be limiting.

Sampling was conducted between 6pm and midnight when 6pm to 6am would have been more comprehensive.

The study design would not be expected to produce the information required to address the main research questions.

Reviewer #2: If there are no major new analyses/experiments required prior to publication.

If possible, some kind of power analysis based on detection rates here to determine the length and coverage of sampling that might be required to conclusively evaluate if vector density can be used as a proxy for human infection risk would be useful particularly for the intended audience of PLoS NTDs.

Reviewer #3: (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: While data were analysed using formulae designed to detect vector prevalence the number of vectors collected at each habitat per site were small. Measures of diversity indicated that farming areas and the forest have a greater abundance of Anopheline vectors than the peri-domestic habitats.

Reviewer #2: The analyses match the analyses plan.

Throughout make sure that appropriate statistical test information is reported. For example, on line 369-398. What was the outcome of the GLMM that allows you to state that there is no systemic variation in altitude across site types. On line 433-434, what are the stats that show no significant effect of time on mean predicted number of bites.

It was not clear to me why the outcome variable for vector density was the mean number caught per site per night. If date and site were both included as explanatory variables wouldn't it allow you to directly model number of mosquitoes caught?

Tables and Figures:

Figure 1: I would remove a and include a figure that illustrates the "substantial variation in elevation, the size, and distribution of forest areas, and local agricultural activities". Could you use the Hansen global forest cover 2014 map for this?

Fig 3: I would recommend a box plot for this.

Fig 4: I don't think this figure is needed as long as the lack of statistical significance is fully reported in the text.

Reviewer #3: (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: At best all that can be concluded is that the sampling methods used over short time intervals may not have been sufficient to properly capture the information required to calculate the environmental determinants of vector density and infection rate at the study sites during a single short time interval.

The authors do acknowledge this but yet make statements such as An. balabacensis occurred at the study sites at lower density when compared to a much more intense and prolonged study in the Kudat District of Sabah. Had the two studies been similarly rigorous then indeed a comparison could have been made.

In the absence of rigorous longitudinal sampling the objectives of this study on the environmental determinants of malaria vector density, particularly vectors of zoonotic malaria, would be difficult to test.

Reviewer #2: The authors identify the two key limitations of the study: the low numbers of malaria vectors sampled and the temporal mismatch between the human prevalence data and the vector density data. I thought they did a respectable job of pointing out these issues, but there are a few key points to be considered and pieces of information that I think the reader needs to assess the validity of the conclusions.

1. Are there alternative explanations for the numbers of Anopheles in HLCs? Certainly, one explanation is that there are not many Anopheles. However, only one sampling technique was reported and it maybe that this technique was not appropriate for this particular setting. For example barrier screens have been used in other settings (Pollard et al. 2019, Parasites and Vectors) to collect blood fed Anopheles. Can you offer some kind of assessment of the probability that HLCs were not effective for Anopheles in this environment. Are there any larval surveys or alternative sampling that can support the HLC result? If HLCs are a valid technique (which in many instance they are) then what kind of power would you require to detect an association with serology data. I think this is important for recommendations moving forward. Can you use this data to determine the temporal and geographic spread you might need?

2. What was the impact of El Nino during this period. I can see from the attached Fornace publication that there was drought during the serology study which may have impacted vector densities. Was this also having an effect in 2016? Might this explain the discrepancy between site types in the two studies? During drought the oviposition may have been more likely to occur in peri-domestic habitats where people were storing water?

3. It was not clear to me exactly what the serology data indicates and this seems imperative for helping the reader assess the association between the two data sets. From what I understood serology can be used to detect current and historical infections. On line 554 it is stated that the antigens for P. knowlesi are relatively short lived, but when I looked into the citations provided, I still could not determine how short lived. The vector data is collected in 2016 and the serology in 2015. Over what period is prevalence being captured by these data? Is it anyone exposed within 6 months, a year? We expect vector abundance to fluctuate over time at relatively short time scales compared to host infection rates and it is not clear to me why would expect human prevalence rate from a previous year (potentially a previous year with drastically different environmental factors due to el nino and calculated over a long period of time) to correlate with a snapshot of vector density taken much later and in a different season. I think the authors hit on these issues in the discussion, but they need to be much stronger in their defense of this methodology.

Reviewer #3: (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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (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: Research question: Investigate environmental determinants of Plasmodium knowlesi vector density and infections rates across a wider spatial scale in Sabah.

Key aims were to identify associations with habitat type and test whether results from small-scale sampling in one district (Kudat) are generalizable across the state. In addition, this study tested for associations between entomological variables and human exposure to P. knowlesi as measured in a related sero-prevalence study. The authors say they performed and intensive study to address their research questions.

Methods

A larger area of Sabah was included in the study described (fig 1). Three different habitats (peri-domestic, farm and forest) per village were sampled over 4 nights. Each habitat was assigned to one team of two individuals. Sampling was restricted to human landing catches (HLC) and sampling was conducted between 6pm and midnight. Mosquitoes were collected into ethanol and identified, based on morphological characters, retrospectively. DNA was extracted from the ethanol preserved mosquitos and Plasmodium species were detected by PCR.

It is not possible to comment on the integrity of DNA in the samples following ethanol preservation – however given the small number of Plasmodium vectors collected during the entire study this is perhaps a moot point. All sites were studied in three months (March to June 2016) the single time frame did not seem to particularly coincide with annual zoonotic malaria prevalence in Sabah. Even-so to attempt sampling 3 habitats, a team of two per habitat, using only HLC in 11 villages over a 3-month period is a long shot.

While on the face of it this may appear to be an ‘intensive’ study the protocol and team size assigned for sampling at each habitat per site is, to say the least minimal, and lacks any attempt to include diverse sampling methods with no opportunity to identify differences in vector density over time. Furthermore, the study is unlikely to capture enough data to calculate vector association with seroprevalence data collected six months earlier.

Results

While data were analysed using formulae designed to detect vector prevalence the number of vectors collected at each habitat per site were small. Measures of diversity indicated that farming areas and the forest have a greater abundance of Anopheline vectors than the peri-domestic habitats.

Conclusions

At best all that can be concluded is that the sampling methods used over short time intervals may not have been sufficient to properly capture the information required to calculate the environmental determinants of vector density and infection rate at the study sites during a single short time interval.

Malaria transmission in Southeast Asia, particularly Malaysian Borneo, is under intense control pressure and consequently is relatively low and temporal. The authors attempt to generate information on zoonotic malaria vector density, at best difficult, but they adopt scant, short and single capture methodologies that, in the best of circumstances, would be unlikely to generate the depth, quality or quantity of data required to identify the environmental determinants of Plasmodium knowlesi vector density and infections rates across a wider spatial scale in Sabah.

The authors do acknowledge this but yet make statements such as An. balabacensis occurred at the study sites at lower density when compared to a much more intense and prolonged study in the Kudat District of Sabah. Had the two studies been similarly rigorous then indeed a comparison could have been made.

In the absence of rigorous longitudinal sampling the objectives of this study on the environmental determinants of malaria vector density, particularly zoonotic malaria, would be expected to be difficult.

Reviewer #2: This is a useful data set and data on vector species in this increasingly epidemiologically important region is scarce and notoriously difficult to collect. This makes the data presented here novel and significant to those currently attempting to assess a rapidly evolving transmission landscape. Overall, the vector sampling study is well designed and executed. The data are explained clearly and rigorously assessed. There are a few weaknesses in the study related to the use of serology data, but this weakness is clearly identified. With some additional evaluation of these weakness and discussion of alternative explanations for the lack of correlation observed here I feel the study will be ready for publication.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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.

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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you 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. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Attachments

Attachment

Submitted filename: PNTD-D-19-02061.pdf

Dear Ms Brown,

Thank you very much for submitting your manuscript "Human exposure to zoonotic malaria vectors in village, farm and forest habitats in Sabah, Malaysian Borneo" 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.  

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

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

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 #2: The revisions have improved the clarity of how the methods test the hypotheses. The study design is also clearer with clear and correct use of statistical methods.

Reviewer #3: (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 #2: Yes. Where requested the authors have provided addition data and analyses. The addition of the power test is really good.

Reviewer #3: (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 #2: Yes. The authors conclusions are well-justified by the data presented and they discuss potential limitations. The work certainly advances our understanding of vector ecology in this important area and can be used to inform public health investigation moving forward.

Reviewer #3: (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 #2: No additional comments. The current version reads very clearly.

Reviewer #3: (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 #2: The revision has addressed all of my concerns. The data set remains a rare and important contribution to the literature.

Reviewer #3: Please find in the following my comments on the revised manuscript titled, “Human exposure to zoonotic malaria vectors in village, farm and forest habitats in Sabah, Malaysian Borneo” by Brown et al. The authors revised the manuscript substantially.

Specific comments

1. L41: (2) is it a typographical error?

2. L73: SE should be South East.

3. L110: Please edit km. The meaning is unclear.

4. L132: What does “active” mean?

--------------------

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 #2: No

Reviewer #3: No

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, PLOS recommends that you 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. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Ms Brown,

We are pleased to inform you that your manuscript 'Human exposure to zoonotic malaria vectors in village, farm and forest habitats in Sabah, Malaysian Borneo' 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

Deputy Editor

PLOS Neglected Tropical Diseases

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

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Please check Tab. 1 if gp. is correct (once gp. once in italics).