PONE-D-21-22404In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovisPLOS ONE

Dear Dr. Antonino Pinna,

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.

Please submit your revised manuscript by Oct 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional details regarding the ivermectin used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials.

Additional Editor Comments:

Dear author,

read carefully the comments of the Reviewer 2 and response to all the suggestion/requirement.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. 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

**********

4. 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

**********

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: The results described in the manuscript are preliminary, however they are very interesting and provide the basis for clinical studies. The only negative point, but not an impediment to publication, is that the authors should have performed the in vitro assays with a greater number of repetitions.

Reviewer #2: Re: PLOS One Manuscript PONE-D-21-22404

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

By Giuseppe D’Amico Ricci

General:

The authors have submitted a well prepared manuscript describing laboratory effectiveness of five formulations, containing one or more of three main components, the parasiticide ivermectin, propylene glycol, used mainly as a carrier, but also tested as a sole ingredient as a control, and povidone-iodine in hyaluronic acid vehicle which is used primarily as an antiseptic but with a previous report of insecticidal activity against Demodex mites noted by the authors.

Usually laboratory testing, such as conducted here, is conducted as a preliminary study to in vivo trials to establish whether any insecticidal action against the target organism is present. If so in vivo effectiveness is validated later. Ivermectin often acts systemically and lack of a topical effect in laboratory studies does not necessarily mean that it would not be effective in vivo and provides very little information about optimal formulation.

However, in this instance there is already at least one paper indicating efficacy of ivermectin in treating human ocular myiasis caused by Oestrus ovis larvae (Kumar et al. ), some studies indicating efficacy of ivermectin against O oestrus in human nasal infestations and a significant number of papers indicating in vivo activity against O. ovis in sheep which draws into question the novelty of the work and need for new laboratory studies to demonstrating insecticidal effect against O. ovis. Furthermore, it could perhaps be argued from the results that the polypropylene glycol was more active in the laboratory studies than ivermectin per se, although whether this would be the case in a clinical setting is questionable.

Methods:

More detail is needed to for the reader to accurately interpret the design used for the study. It is noted that “Each Petri dish was seeded with 25 larvae, filled with 1 mL of each study drug” and that all assays were conducted in triplicate. I may have misunderstood, but to me this suggests that 5 treatments + saline control x 25 larvae x 3 replicates = 450 larvae were used. However, it is mentioned in the abstract and in the body of the paper that 893 larvae were tested. How were the other larvae used? This needs to be clarified

It is noted that larvae for the study were collected over a number of days and that in most cases the experiments were conducted on the day of collection, but in some instances when all heads could not be processed, they were held overnight and processed the following day. Often such closely associated parasites die quickly once separated from their host, or their host dies. Thus how the replicates/treatments were established with respect to time of collection needs to be specified to ensure there is no confounding between treatment effects and time of collection.

Results.

Figure 1.

As I understand it Figure 1, Graph A contains a plot of all treatments, Graph B is exactly the same as A but with 3 of the treatments from Graph A omitted and Graphs C and D just contain subsets of Graph A replotted with a different scale on the X axis?. That is, for example the same data 1% ivermectin in BSSS treatment is replotted 3 times in Graphs A, B, and C. It is unusual to replot the same data repeatedly and is potentially confusing for the reader. Consider just using Graph A with statements about which treatments were significantly different in the caption.

References.

As noted above there is at least one previous reported study of the use of ivermectin to treat human ocular myiasis (Kumar et al. 2013 Annals of Tropical Medicine and Public Health 6: 315-316) as well as a quite a number of previous reports of the effectiveness of ivermectin against Oestrus ovis larvae (in human hosts and in sheep). None of these papers have been cited in the reference list. These papers would seem to be very relevant to the current study and at least some of them should be noted.

**********

6. 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 #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-21-22404R1In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovisPLOS ONE

Dear Dr. Antonio Pinna,

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.

==============================

ACADEMIC EDITOR:

Please improve the paper with the suggestion of the Reviewer #2.

==============================

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

Journal Requirements:

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

**********

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

**********

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: Authors respond to reviewers' comments in a satisfactory manner, in this way the manuscript can be accepted as is.

Reviewer #2: Re: PLOS One Manuscript PONE-D-21-22404-R1

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

The manuscript has been revised appropriately and with some minor suggested amendments noted below should be suitable for publication in PLOS 1.

Abstract

Lines 39-41 “Kaplan-Meyer analysis

…disclosed that the survival curves were significantly lower in the samples treated with ivermectin 41 1% solution, ivermectin 1% solution + 0.6% PVP-I, and propylene glycol.” Please state lower than what. Lower than the RSSS control or lower than the other treatments

Introduction

Line 54. In entomological protocol it is most common, though not essential to state both the Order and the Family e.g. [Diptera:Oestridae]

Line 115- The BSS would usually be considered a negative control. A positive control would usually mean a treatment known to be effective in killing larvae. I suggest just delete “Positive”: ie Control Petri dishes received 1 mL of BSSS.

Line 124 – Log rank test, rather than “..long rank test”

Line 147 – Again, as above, significantly lower than what?

Lines 175-177. This is an important point which would benefit from a supporting reference.

Line 193 to 195 Delete “also”. That is: ‘Surprisingly enough, propylene glycol, the solubilizer chosen for ivermectin, had similar larvicidal activity.

Lines 203-204 Suggest “Our results suggest that ivermectin 1% solution in propylene glycol, ivermectin 1% solution in propylene glycol + 0.6% PVP-I, and propylene glycol might be potential candidates for the topical treatment, as ointment and/or eye-drops, for external ophthalmomyiasis caused by O. ovis.”

Line 224 – closing sentence. Suggest ‘… to establish whether or not these formulations may be considered….’ Rather than “…they..”

**********

7. 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 #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

In vitro larvicidal activity of ivermectin and povidone-iodine against Oestrus ovis

PONE-D-21-22404R2

Dear Dr. Antonio Pinna,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE