Resistance to Antileishmanial Drug Candidates: A Flowchart for an Early Phenotypic Evaluation of Risk

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work starts from the central theme of the risk of drug resistance of anti-Leishmania drugs by outlining a possible evaluation of candidate drugs in a preliminary stage to production. I believe that this tool could become significant in the Research and Development stage.

Author Response

We would like to warmly thank Reviewer 1 who accept the manuscript without an modification.

Reviewer 2 Report

Comments and Suggestions for Authors

1. All species names should be written in italics.
2. The abstract must include the most important findings of the study.
3. Please use “leishmaniasis” not “leishmaniases”
4. Keywords: you can prefer to use “resistance level “
5. Needs reference: “Many factors are involved in the transmission process, for example: the climate, the competence of the insect vector and the immune status of the human and /or animal reservoir. In Southern Europe, visceral leishmaniasis has an important canine reservoir”
6. Which One Health approach indicates that you should be more informative. “The global studies in drug research are now encouraged according to “One Health” approaches with the following requirements”
7. You should focus more on the drug resistance giving data about the frequency of this resistance.
8. The aim sentence at the end of the Introduction is confusing, please be more concise and clearer.
9. IC₅₀ (half maximal inhibitory concentration) change in the text.
10. “(Thermo Scientific™)” give the city, country of the manufacturer.
11. “3.1. Choosing the promastigote forms to select the resistance, differenciating in axenic amastigotes and checking drug resistance phenotype, then evaluating the relevance of drug resistance in the intramacrophage amastigotes” very long for subheading. Please make shorter.
12. A graphical summary of the proposed method would greatly enhance the reader’s understanding of the processes described in the manuscript.
13. Overall, the methodology section was difficult to follow due to the excessive number of subheadings, which could be consolidated, and the presence of overly long sentences that should be split for better clarity

Author Response

• Reply to Reviewer 2

 

We would like to sincerely thank Reviewer 2 of our manuscript for the constructive comments and we appreciate that everyone find our study interesting enough to be reviewed. Please find here the detailed answers to the comments:

 

1. All species names should be written in italics.

As requested by the reviewer, all the species names have been written in italics.

 

2. The abstract must include the most important findings of the study.

This study consists more in describing a sequential method to be used at the in vitro early stage of drug development to get information about the risk of a resistance emergence than in presenting findings and results. Of course, the method proposed is based on experimental results presented in the manuscript. As a consequence, we have added a sentence at the end of the abstract.

 

3. Please use "leishmaniasis" not "leishmaniases".

In the literature, « Leishmaniases » is the plural of « leishmaniasis » since there are several forms of this disease. For this reason, we have maintained the term « leishmaniases » along the text.

 

4. Keywords: you can prefer to use "resistance level".

As suggested by the reviewer, in the keyword list, we have replaced « resistance level evaluation » by « resistance level ».

 

5. Needs reference: "Many factors are involved in the transmission process, for example: the climate, the competence of the insect vector and the immune status of the human and /or animal reservoir. In Southern Europe, visceral leishmaniasis has an important canine reservoir".

As suggested by the reviewer, we have added the following reference to illustrate this statement : Rodriguez-Escolar I et al., Transbound Emerg Dis, 2025 Jul 13:2025:1087533. doi: 10.1155/tbed/1087533.

 

6. Which One Health approach indicates that you should be more informative. "The global studies in drug research are now encouraged according to "One Health" approaches with the following requirements".

Line 41: The sentence has been modified as follows : “The different parameters involved in the leishmaniases transmission should be studied according to a “One-health” approach combining medical, veterinary and environmental parameters in the affected areas in order to get integrative informations for an optimal epidemiological survey”.

Another sentence has been added to illustrate the importance of One Health considerations in drug resistance: “For example, in the North Bihar region of India, the relationship between arsenic in drinking waters and selection of antimony resistance demonstrates how environmental stimuli can influence drug resistance in leishmaniasis (Ponte-Sucre et al., 2017)”.

Line 78: considering the requirements for a One health approach, they are clearly expressed as follows, and the links between the ideas have slightly been re-formulated: “Firstly, green chemistry should be a prioritar main objective of the chemists with a reduced number of synthesis steps leading to get cheaper drugs, and the elimination of toxic solvents that should be replaced by less toxic ones. Secondly, according to that, the use of chemoinformatics tools applied to computer-assisted drug design would help reducing the number of compounds synthesis during pharmacomodulations. Thirdly, the “One Health” approach needs to consider toxicological effects through monitoring the behaviour of drug candidates and their metabolites in the environment”.

 

7. You should focus more on the drug resistance giving data about the frequency of this resistance.

On line 72, we have added the following sentence: “For example, the unresponsiveness to sodium stibogluconate, an antileishmanial drug, was about 43% in the Indian region of Bihar generating a serious public health problem in Bihar (Das et al., 2005).”

 

8. The aim sentence at the end of the Introduction is confusing, please be more concise and clearer.

The sentence has been simplified as follows : “This article aims to propose an in vitro protocol of sequential experiments to gain information about a risk of drug resistance emergence in experimental conditions without immunological considerations, but that are potentially extrapolable to the field”.

 

9. IC₅₀ (half maximal inhibitory concentration) change in the text.

This point has been changed in the text to be more explicit.

 

10. (Thermo Scientific ^TM) give the city, country of the manufacter.

Line 164 : the city (Villebon sur Yvette) and the country (France) have been added as requested.

 

11. Choosing the promastigote forms to select the resistance, differenciating in axenic amastigotes and checking drug resistance phenotype, then evaluating the relevance of drug resistance in the intramacrophage amastigotes" very long for subheading. Please make shorter.

The new subheading proposed is:

“Selecting resistance and checking drug resistance phenotype”

 

12. A graphical summary of the proposed method would greatly enhance the reader’s understanding of the processes described in the manuscript.

A graphical summary of the proposed method has been added as requested as Figure 2.

 

13. Overall, the methodology section was difficult to follow due to the excessive number of subheadings, which could be consolidated, and the presence of overly long sentences that should be split for better clarity.

We agree with the reviewer and some sentences have been split. However, the subheadings remain necessary for an easy understanding of the text.

 

 

 

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The work entitled " Resistance to antileishmanial drug candidates : A flowchart for an early phenotypic evaluation of the risk " aims to propose an in vitro protocol through a sequencial flowchart to gain rapid information about a possible emergence of drug resistance in experimental conditions to possible antileishmanials medicines, contributing to the process of developing new treatment alternatives for leishmaniasis, and therefore is interesting and may be published in DDC.

 

However, it's still a bit confusing and needs to be better written and explained. Here are some suggestions:

Introduction:

 

Paragraph 1: Remove italics from “sp.”.

 

 

Results

 

3.1.1: Third line: Put Leishmania in italics.

 

In Table 1 it would also be interesting to add the IC50 values of the compounds in WT and resistant promastigotes.

 

3.1.1: last paragraph: it is necessary to make clear in the text what is shown in table 1: the induction of drug resistance was successfully.

 

3.1.2: Difficult to understand, further explanation needed. What characteristics should be compared? What are the characteristics of the selected parasites?

 

The quality of figures 1 and 2 needs to be improved.

 

The legend of Figure 2 is very short and needs to better explain how it was done, treatment concentration, treatment time, how it was determined, and whether it was in promastigotes.

 

3.2.2: It is necessary to explain and make clear what it is “amplitude of drug resistance”.

 

Table 2: Add to the table the unit of measurement (months) of “Drug pressure duration to get CMR”

 

3.2.4: Make the table call in the text.

 

3.2.5. Where are these results? They need to be shown.

 

3.2.6: Where are these results? They need to be shown.

 

 

 

Discussion

 

There is no "Discussion" section, nor a "Results and Discussion" section. The results are presented first without discussion. At the end of "Results," there is a brief discussion.

The results should be discussed in the presentation sequence or at the end in a separate "Discussion" section. However, the discussion also needs to be improved, and the analyzed data better explained and compared with each other, for example: the relationship of RI with CMR, of these with generation time, with cross-resistance, etc.

Author Response

• Reply to Reviewer 3

 

We would like to sincerely thank Reviewer 3 of our manuscript for the constructive comments and we appreciate that everyone find our study interesting enough to be reviewed. Please find here the detailed answers to the comments:

 

1) 3 1 1. Third line, Put Leishmania in italics.

It has been done as requested.

 

2) 3.1.1 : last paragraph : it is necessary to make clear in the text what is shown in Table 1 : the induction of drug resistance was successfully.

The following sentence has been added at the end of paragraph 3.1.1: “Table 1 shows that the selection of drug-resistant parasites was successful for all the studied drugs”.

 

3) 3.1.2: Difficult to understand, further explanation needed. What characteristics should be compared ? What are the charactristics of the selected parasites ?

The following sentence has been added at the end of paragraph 3.1.2.: “All the characteristics of drug resistance are presented below in the paragraph 3.2”. Moreover, in order to clarify what we are waiting as characteristics for a drug candidate, we have added the following sentence in the Conclusion part: “When observing the resistance characteristics of drugs in clinical use, an idealistic drug candidate will positively reply to the following in vitro criteria to be pushed up in drug development:

• the longest time to get concentration for maximal resistance;
• the lowest resistance index value IC₅₀^R/IC₅₀^WT;
• an absence of cross-resistance with other antileishmanial drugs;
• a difficult development of drug resistant parasites in a murine model”.

 

4) The quality of figures 1 and 2 needs to be improved.

Figures 1 and 2 are of suitable quality but seemed altered probably because of the pdf version readable by the reviewers after submission.

 

5) The legend of Figure 2 is very short and needs to better explain how it was done, treatment concentration, treatment time, how it was determined, and whether it was in promastigotes.

The legend of Figure 3 (former Figure 2 in the first version) was completed as follows : “The experiments were carried out in 24-well plastic tissue-culture plates in a final volume of 1 ml and the plates were maintained at 37 °C under an atmosphere of 5% CO₂. Axenic amastigotes were initially submitted to a drug pressure corresponding to almost the third of the IC₅₀ value of the soluble formulation of 2-PQ. A stepwise increase of drug concentration was applied as soon as the drug exposed cultures exhibited a growth rate similar to those of the WT line cultures. This procedure was applied until the maximum concentration allowing parasite growth was reached.”

 

6) 3.2.2: It is necessary to explain and make clear what it is “amplitude of drug resistance”.

It is clearly indicated in line 212 that the Resistance Index (RI), defined as the ratio IC₅₀^R / IC₅₀^WT, is the marker of the amplitude of drug resistance. No change in the text is needed.

 

7) Table 2: Add to the table the unit of measurement (months) of “Drug pressure duration to get CMR”.

The unit of measurement in months has been added within Table 2 as requested.

 

8) 3.2.4 : Make the table call in the text.

Table 2 has been cited on line 439 at the end of paragraph 3.2.4.

 

9) 3.2.5. Where are these results ? They need to be shown.

The absence of cross-resistance between all these drugs was confirmed by the IC₅₀ values of the drugs that were not significantly modified between WT and other drug-resistant strains. Such no significant differences do not justify a additive column in a table. As a consequence, we have added the mention: “data not shown”.

 

10) 3.2.6. Where are these results ? They need to be shown.

We have no complete results about the relationship between drug combination and drug resistance. Therefore, we prefer to limit our contribution as a recommendation for the flowchart.

 

11) Discussion: There is no « Discussion » section, nor a « Results and Discussion « section. The results are presented first without discussion. At the end of « Results », there is a brief discussion. The results should be discussed in the presentation sequence or at the end in a separate « Discussion » section. However, the discussion also needs to be improved, and the analyzed data better explained and compared with each other, for example : the relationship of RI with CMR, of these with generation time, with cross-resistance, etc.

 

It seems that the reviewer has got a truncated text. The initial text had a « Discussion » part of two complete pages just before the Conclusion. We hope that this time at resubmission, the reviewer will get the complete text including the discussion.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for revising the manuscript in accordance with my previous comments. However, I have a concern regarding the ethical approval statement for the animal study. The current statement does not include an approval number. Could you please clarify whether the ethical approval information provided is adequate and enough? " Animals were kept in the animal facility of Université Paris-Saclay in accordance with institutional guidelines and French legislation for animals protection (APAFIS), which complies with all relevant European Union and international guidelines for experimental animals.

Author Response

Reviewer 2: Thank you for revising the manuscript in accordance with my previous comments. However, I have a concern regarding the ethical approval statement for the animal study. The current statement does not include an approval number. Could you please clarify whether the ethical approval information provided is adequate and enough? " Animals were kept in the animal facility of Université Paris-Saclay in accordance with institutional guidelines and French legislation for animals protection (APAFIS), which complies with all relevant European Union and international guidelines for experimental animals.

Reply to Reviewer 2,

Dear Reviewer 2,

Thank you for your question regarding the ethical approval information. Please find below the specific reference of the authorization showing that the approval information is adequate and sufficient and a pdf of this approval.

Approval code : #46296-2023121309336649 v9; Approval Date : 17 May 2024 for a 5 year duration

Title of the project: "Evaluation de l'activité antileishmanienne de molécules ou de formulations sur un

modèle murin à Leishmania donovani ou Leishmania infantum".

The approval number has been added within the manuscript in lines 230-231.

Thank you again for the evaluation of our manuscript.

Best regards.

Philippe Loiseau

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the responses and changes.

Author Response

Figures and tables can be improved

Thank you for Reviewer 3 for the comment regarding Figures and Tables.

We have submitted the documents of the best quality we could do. The graphical processing of the journal will release readable documents.