PONE-D-21-25422Is the presence of Leptospira in the environment merely random? An epidemiological explanation based on serial analysis of water samplesPLOS ONE

Dear Dr. Agampodi,

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this epidemiologic study the authors collected soil and water samples from several regions (wet, dry and semi-dry) of Sri Lanka based on probable exposure history of confirmed leptospirosis patients and tested it by PCR. There are few leptospirosis epidemiologic studies from Sri Lanka. From the wet area 2/12 water sites were positive. From the dry area 1 well in areas of high human contact were positive. A specific positive site in the dry area was chosen to do serial sample testing from 10 wells surrounding a positive well (nr 9) and they found that at different times 4 to 6 of the 10 wells in that site were positive for Leptospira. The work has merit but as described in this paper is very difficult to follow and the abstract does not capture the essence or importance of the work done. The description of the method to collect the samples including a better flow chart, figures, tables and writing to need to be comprehensively revised.

Other comments:

Methods: If the primers detect nonpathogenic Leptospira species, what is the rationale of detecting virulent species in the water and soil samples?

Study design and presentation of results: Very confusing descriptions of all data.

Throughout the paper (including abstract) avoid using Components 1, 2, 3 and just define the sites as 1-X nr of water and soil samples from Wet area, 2- X nr of Wells in Dry Area, 3 – 10 wells surrounding a positive well in dry area.

Discussion: down the claims of positive Leptospiral transmission routes or correlations with disease hotspots since no clinical data is shown.

It is difficult to design any prophylactic measures based on an epidemiological study. Reservoir based studies or other specific animal study can therefore open such avenues to search for a better alternatives than available Doxycycline.

Reviewer #2: This is a nice manuscript describing findings of Leptospira in the environment, adding to our knowledge of transmission of Leptospira in the environment.

Specific comments:

The authors use "random" to describe Leptospira in the environment, but I'm not sure that's the correct term. Maybe transient? Or variable? Maybe conditions have to be ideal to reach levels above the limit of detection, and with clear correlations of lepto cases in humans and rainy seasons, and as shown in this study, proximity to a lake where feral animals are prevalent, I'm not sure "random" should be used.

Can the authors justify why they tossed the first centrifuged water pellet? It's mentioned in limitations that they may have lost some lepto with the pellet. Also, a DNA blood mini kit was used for environmental water - can the authors justify the extraction kit use, or were they able to determine whether or not PCR inhibitors were present?

Can the authors describe in better detail the definition of PCR positivity? It's not clear to me how many "replicates" were done, and whether the replicates were in the PCR reaction or replicate samples.

What is the difference between an "abundant paddy field" and a paddy field (line 183)? More vegetation? Larger?

It might be helpful in the results section to clarify that the soil sequencing detected both pathogenic and non-pathogenic Leptospira, and that non-pathogenic Leptospira are considered ubiquitous.

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PONE-D-21-25422R1The variable presence of Leptospira in the environment; An epidemiological explanation based on serial analysis of water samplesPLOS ONE

Dear Dr. Agampodi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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.

The requested edits are minor and, if corrected, will likely result in acceptance for publication.  We look forward to receiving your revised manuscript.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

R. Mark Wooten, Ph.D.

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract

Line 23: define the classic triad

Methods:

Line 105 and others: Purposive? What does this word mean?

Discussion

Line 279: How much is “a satisfactory concentration of Leptospira needed in the environment”.

Line 309: This concept of intermittent positivity versus repetitive contamination should be mentioned in the abstract.

Reviewer #2: Thank you, the revision provided clarity and I think the manuscript provides valuable information about Leptospira in the environment in an endemic area. I have only one small comment, to de-capitalize "leptospirosis" in the middle of a sentence.

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Reviewer #1: No

Reviewer #2: No

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The variable presence of Leptospira in the environment; An epidemiological explanation based on serial analysis of water samples

PONE-D-21-25422R2

Dear Dr. Agampodi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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R. Mark Wooten, Ph.D.

Academic Editor

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