Dear Prof Osei-Safo,

Dear Dr. Dorcas Osei-Safo and collegues.

Thank you very much for submitting your manuscript "Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents" 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.

Whereas the topic is highly important and the helminth community needs to fill up the empty drug development pipe lines by all means, describing tests on herbal remedies without naming the plant sources is an issue. Acknowledging that traditional practitioners are reluctant to release information, it would be an asset for this paper to add this information.

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,

Sabine Specht

Associate Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Deputy Editor

PLOS Neglected Tropical Diseases

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

Dear Dr. Dorcas Osei-Safo and collegues.

Thanks for submitting your manuscript to PLoSNTD. Whereas the topic is highly important and the helminth community needs to fill up the empty drug development pipe lines by all means, describing tests on herbal remedies without naming the plant sources is an issue. Acknowledging that traditional practitioners are reluctant to release information, it would be an asset for this paper to add this information.

With best regards, Sabine Specht

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 methods are standard, but are not adequately described.

The main objection is that the material studied (herbal remedies) is not described.

Reviewer #2: 1) Table 1, number of people successfully treated is not an appropriate heading since you said earlier that ‘According to the practitioners, their efficacy claims are based on the number of people who experience reduction or relief from symptoms as a result of using their products’. So it should be coined as the number of people relieved of symptoms and not successfully treated. Given that no laboratory tests were done to determine parasite loads, it is subjective to conclude that the patients were treated successfully

2) Statistical analysis for comparisons of different extracts and concentrations for onchocerca and schistosoma will enhance the credibility of findings given that you ran the assays in quadruplicates and triplicates repeatedly.This will reveal if the differences observed are statistically significant in different concentrations or at the IC50. A good example is Table 5.

Minor Points

3) We recommend that the quality of traditional medicines be improved (Author summary)

4) Primary screening of adult worms and microfilariae, Secondary screening on adult worms

It should be “screening against adult worms” instead of “screen of adult worms”

5) Correct from GCMSNMR to GC/MS/NMR in the manuscript as they are different techniques.

6) Discussion section, ‘however, is on the rise and possess’ it should be poses

7) Introduction section ‘high cost of bringing new drugs to the market’

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: Some of the figures are illegible, or barely legible.

Reviewer #2: 1) Cytotoxicity assessment of extracts. The 50% cytotoxic concentrations (CC50) of the cells were determined by microscopic examinations.

Looking at Table 4, the cytotoxicity was done at 200 µg/mL only. So the CC50 was not determined as stated in the methodology. The selectivity index (SI) column should be included if the CC50 was determined. Also, consider including this aspect in your abstract

Reviewer #3: The objectives of the study are clearly defined, but without a clear testable hypothesis stated.

An in vitro screening was used to evaluate the efficacy of 15 TM against NTDs. This is an appropriate design for screening the extracts of the various TMs for activity against NTD and evaluating their efficacy. The extracts were also assessed for cytotoxicity against Monkey Kidney Epithelial (LLC-MK2) cells to ensure the safety of the products.

The protocols employed in the evaluation of the activity of the crude extracts of the TMs were appropriate.

Though the statistical analyses were no elaborate, the analyses carried out were sound to provide a good interpretation of the data.

Also, ethical approval was obtained from the appropriate Institutional Review Board

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

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. If anything, more than is necessary.

Reviewer #2: Looking at the results you obtained for the antionchocercal activity of extracts, all the active extracts are dichloromethane extracts. Most filaricidal activities of plants tested come from non-polar extracts. This could be backed up in the discussion section with publications from other authors who had similar findings.

Reviewer #3: The results and data support the conclusions, the discussion was well written and authors clearly indicated how these data can be helpful to advance our understanding of traditional medicine and its benefit the treatment of various diseases including NTDs

The public health relevance of the study is clearly indicated and the authors have emphasized the benefit of traditional medicine and how it could be explored for drug discovery and development for NTDs

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

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: Minor revision

Reviewer #3: Minor revision is required before manuscipt is accepted for publication

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

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: If the authors are able to publish the plant species, and present the Methods and Results correctly, then the financial cost of this project would not be wasted. There is no significant problem with the actual writing of English.

Reviewer #2: Onchocerciasis, schistosomiasis and lymphatic filariasis feature among the most prevalent NTDS and are of top priority to elimination programs. The Expanded Special Programme for Elimination of Neglected Tropical Diseases (ESPEN) recently promotes an integrated approach to eliminating Neglected Tropical Diseases and its strategy is optimisation and expansion of existing drugs to hypo-endemic areas, plus potential integration of alternative strategies.

This manuscript by Twumasi et al fits into the goals of ESPEN and addresses key issues that affect Africa and is of great significance. They evaluated the ‘antischistosomal, antionchocercal, and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents’ of fifteen traditional medicines in vitro. They carried out cytotoxicity assessment of the extracts and bioassay-guided fractionation of the most active antitrypanosomal extract which yielded an oil (eupatoriochromene).

The manuscript is written in good english and the methodology and techniques are appropriate. However there are some issues in the statistical analysis and cytotoxicity data that needs to be adjusted.

Reviewer #3: The authors emphasized the need for the developments of new drugs for Neglected Tropical Diseases (NTDs) to facilitate the elimination and eradication of these diseases. Some current NTD drugs have shown reduced efficacy, serious adverse effects associated treatment, while others are unable to prevent reinfection. However, the development of new drugs for NTDs is very slow because of limited investments and minimal interest by major drug companies. Fortunately, most African countries including Ghana where NTDs are highly prevalent, abounds in indigenous medicinal plant species whose products serve as primary sources of remedies for most affected populations.

This manuscript therefore addresses an interesting and important topic that provides useful evidence for development of Traditional medicine and could potentially lead to policy directions for the development of traditional medicines (TMs). However, these resources are still deployed and practice in Indigenous settings. Thus, TMs are underdeveloped and require significant research and development support to harness the benefits that can be derived.

The authors investigated extracts of fifteen traditional medicines used for treatment of NTDs including schistosomiasis, onchocerciasis and lymphatic filaliasis (LF) and found some extracts to be efficacious against the causative agents of schistosomiasis and onchocerciasis, however, no screening platform was found for LF. The evaluation of the activity of the extracts showed that some TMs were very active against the juvenile and adults of Schistoma mansoni as well as microfilaria and adult male and female of Onchocerca ochengi. Surprisingly, some of the TMs which were not indicated for African trypanosomiasis rather demonstrated the highest activity against Trypanosoma brucei brucei. The authors have demonstrated the need to evaluate for activity of various TMs against other diseases including NTDs to determine the broad-spectrum activity of TM products.

The authors should have explored more options of collaboration to access more screening platforms to test for activity against LF, especially since LF is one of the NTDs in Ghana that have made significant progress towards elimination, but currently facing challenges of advancing towards elimination. LF requires therefore additional tools for eventual elimination and exploring effective TMs will be an advantage.

The findings of this study demonstrates the significant role of traditional medicines and the prospects towards drug discovery and alternate medicine for NTDs and other diseases.

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

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: Yes: Jonathan A. Coles

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 https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Attachments

Attachment

Submitted filename: To Editors.docx

Attachment

Submitted filename: Comments to the Authors.docx

Attachment

Submitted filename: My Review comments PLOS 1.doc

Dear Prof Osei-Safo,

Thank you very much for submitting your manuscript "Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents" 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,

Sabine Specht

Associate Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

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: Yes

Reviewer #3: The objectives of the study were clearly defined with clear hypothesis stated

The methodologies used were appropriate to achieve the objectives of the study. The in vitro assays used to assessed the efficacy of the 15 TMs against NTDs were appropriately designed. Assessing the cytotoxicity of extracts using Monkey Kidney Epithelial (LLC-MK2) cells was an appropriate method to evaluate the safety of the extracts.

Though the statistical analyses were not elaborate, the analyses carried out were sound to provide a good interpretation of the data.

Also, ethical approval was obtained from the appropriate Institutional Review Board

The methodologies used were appropriate to achieve the objectives of the study. The in vitro assays used to assessed the efficacy of the 15 TMs against NTDs were appropriately designed. Assessing the cytotoxicity of extracts using Monkey Kidney Epithelial (LLC-MK2) cells was an appropriate method to evaluate the safety of the extracts.

Though the statistical analyses were not elaborate, the analyses carried out were sound to provide a good interpretation of the data.

Also, ethical approval was obtained from the appropriate Institutional Review Board

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

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

Reviewer #3: The results presented are in line with the methods employed and the analysis carried out by the authors. The evaluation of antischistosomal, antionchocercal and antitrypanosomal activities of extracts showed that some TMs were very active against the juvenile and adults of Schistoma mansoni as well as microfilaria and adult male and female of Onchocerca ochengi, while some of these extracts demonstrated the highest activity against Trypanosoma brucei brucei, the causative agents of African Animal Trypanosomiasis, The authors have demonstrated the need to evaluate for activity of various TMs against other diseases including NTDs to determine the broad-spectrum activity of TM products.

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

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

Reviewer #3: The discussion and conclusions are well written, the data and results support the conclusions. The authors have clearly demonstrated how the data could be helpful in advancing our understanding in traditional medicines and their benefit for treatment of various diseases including NTDs. made by the authors.

The public health relevance of the study is clearly indicated and the authors have emphasized the benefit of traditional medicines in Ghana for the treatment of NTDs and how it could be explored for drug discovery and development for NTDs.

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

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: Accept

Reviewer #3: COMMENTS ON REVISED VERSION OF MANUSCRIPT OR RESUBMISSION

The authors have adequately responded to the comments by all three reviewers and have appropriately revised the first manuscript to meet my expectations.

I therefore RECOMMEND the revised manuscript to be accepted for publication after all minor corrections have been made.

GENERAL CORRECTIONS

1.Pg 4, Line 85 reads … “ ivermectin and albendazole for ONCHO and LF” The sentence should rather read “ ivermectin and albendazole for LF and ivermectin for ONCHO

2.Pg 21 line 388 column 2, “the” should be deleted

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

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: (No Response)

Reviewer #3: The authors addressed an important and interesting subject that provides useful evidence for the development of traditional medicines (TMs) for Neglected Tropical Diseases (NTDs), as well as their potential for policy direction towards harnessing maximum benefit from TMs in Ghana and globally. However, these resources are still deployed and practice in indigenous settings, this emphasizes the need for research and development support for TMs towards the developments of new drugs for the control and elimination of NTDs.

Extracts of fifteen traditional medicines were investigated for treatment of NTDs including schistosomiasis, onchocerciasis and lymphatic filaliasis (LF). Some extracts were found to be efficacious against the causative agents of schistosomiasis and onchocerciasis and animal African Trypanosomiasis (AAT). The evaluation of the activity of the extracts showed that some TMs were very active against some of the disease pathogens while other were not potent. The authors have demonstrated the need to evaluate for activity of various TMs against other diseases including NTDs to determine the broad-spectrum activity of TM products.

The findings of this study demonstrates the significant role of traditional medicines and the prospects towards drug discovery and development of alternate medicine for NTDs

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

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: Yes: Adela Ngwewondo

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 Prof Osei-Safo,

We are pleased to inform you that your manuscript 'Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents' 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,

Sabine Specht

Associate Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Deputy Editor

PLOS Neglected Tropical Diseases

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