In Vitro Stability of the Biological Activity of Voriconazole against Acanthamoeba castellanii
and Karin Silva Caumo 1,2,*Round 1
Reviewer 1 Report
1. please use more significant publications as literature for AC keratitis: e.g.: -
First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans; Springer; May 2014, Parasitology Research 113(6); DOI: 10.1007/s00436-014-3918-5
and
An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis; Parasitology Research 102(5): 945-50; DOI: 10.1007/s00436-007-0858-32. Three strains are not enough to proclaim results for the whole genus Ac. You shhould use mor strains from different T-groups!
3. Why didn`t you use Alamar Blue technique? Please sxplain to the reader why you used Trypan Blue.
4. Why not using LDH assay? Please explain to the reader.
5. You state "that it was not possible to calculate the IC50 within 24 hours of exposure to the drug. Please explain why not? What was the reason?
6 Please add the literature of the encystment medium. Where did you find it?
7. Please refer to the role of Ac as vectors of endocytobiontes: e.g.
Free-Living Amoebae as Human Parasites and Hosts for Pathogenic Microorganisms; July 2018; DOI: 10.3390/proceedings2110692
8. In the conclusion part you are lacking a proper statement: One viable cyst is sufficient to build another Ac population on the cornea or in the brain. So, what is the result then: Not sufficiant efficiency against trophozoites and very limited efficiency against cysts! Do you really think that Voriconalzol is a candidate against Ac???
Author Response
Response to Reviewers
Reviewer #1:.
- Please use more significant publications as literature for AC keratitis: e.g.: -
First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans; Springer; May 2014, Parasitology Research 113(6); DOI: 10.1007/s00436-014-3918-5
And An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis; Parasitology Research 102(5): 945-50; DOI: 10.1007/s00436-007-0858-3
Response 1: Informations added as suggested.
- Three strains are not enough to proclaim results for the whole genus Ac. You should use mor strains from different T-groups!
Response 2: In the present work, we used two representative strains and a clinical isolate of the T4 genotype, which is the genotype associated with most cases of Acanthamoeba spp infections. because it is the most commonly found in the environment. The use of other genotypes may be included in future studies.
- Why didn`t you use Alamar Blue technique? Please explain to the reader why you used Trypan Blue.
Response 3: The trypan blue exclusion assay is a widely used method to assess cytotoxicity by measuring cell viability by cell membrane integrity. Widely used, although more labor intensive and more likely to fail. However, the experiments were performed in technical and biological triplicates.
- Why not using LDH assay? Please explain to the reader.
Response 4: In the experimental design, we present the assessment of cytotoxicity in rabbit corneal cells (SIRC), which is a strain used worldwide and used for tests of ocular toxicity validated by the OECD.
- You state "that it was not possible to calculate the IC50 within 24 hours of exposure to the drug. Please explain why not? What was the reason?
Response 5: Figure 1 demonstrates the viability of the Neff strain and the AM isolate after 24 hours of exposure to voriconazole. In this same figure, it is possible to observe that the results obtained do not represent a classic dose-response curve. For the curve of the Neff strain, a plateau is observed in the viabilities obtained, and for the AM isolate there is fluctuation in the same values, which makes it impossible to extrapolate for the calculation of IC50.
- Please add the literature of the encystment medium. Where did you find it?
Response 6: Information added as suggested.
- Please refer to the role of Ac as vectors of endocytobiontes: e.g.Free-Living Amoebae as Human Parasites and Hosts for Pathogenic Microorganisms; July 2018; DOI: 10.3390/proceedings2110692
Response 7: Information not included.
- In the conclusion part you are lacking a proper statement: One viable cyst is sufficient to build another Ac population on the cornea or in the brain. So, what is the result then: Not sufficiant efficiency against trophozoites and very limited efficiency against cysts! Do you really think that Voriconalzol is a candidate against Ac???
Response 8: Given the activity of voriconazole on the forms of Acanthamoeba spp., for acting in the inhibition of ergosterol synthesis, weakening the forms, and for having a low cytotoxic potential on corneal cells, it has potential for use in formulations composed with efficient antiamoebic agents but with greater cytotoxic potential, with the proposition of reducing the concentration of the antiamebic agent, improving its tolerance and its use in cases of polymicrobial infections by Acanthamoeba and fungi. Information added as suggested.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Article
The in vitro stability of the biological activity of voriconazole against clinical strains of Acanthamoeba spp. written by Barbara Czerniak Rodrigues et al. is a quite well-designed scientific study, but needs better presentation, especially regarding the photographs, which are completely illegible at this point.
Title: In your study you use one clinical strain, the title mentions strains in plural. Please verify.
Abstract: Please add information about the cytotoxicity performed and possible future studies/limitations of this experiment.
Introduction:
line 30: reference after 1970 is needed
line 34: how many cases is % needed
line 35-37: ring shaped infiltrate occurs in 50% of cases
line 39: in which mechanism?
line 41-44: what are the concentrations?
line 65: one clinical strain or several?
At some point in the introduction it should be added that the infection usually affects one eye
Results:
line 77: the names of the strains and the letter designations should be in the caption. The photo is of very poor quality and should be changed.
line 81: the scale bar should be placed on the photo
line 87: there is no need to keep repeating the strain designations
line 99: an explanation of the * designation should be added
line 106-108: the photo should also be added, after incubation of 48 h
line 111-113: this is a part of materials and methods
line 125: graphs should be larger, photos are completely illegible
line 147: an explanation should be added as to why you are using SIRC cells
Discussion:
line 213: why was cytotoxicity tested only after 24 h? The same in materials and methods and results.
Materials and Methods:
line 234: what was the concentration of the antibiotic and which antibiotic was it?
line 236: why bold?
lines 251, 261: please remember italics
line 271: italics or not?
Conclusions
The conclusions should be expanded to include more data.
Author Response
Reviewer #2:.
9. The in vitro stability of the biological activity of voriconazole against clinical strains of Acanthamoeba spp. written by Barbara Czerniak Rodrigues et al. is a quite well-designed scientific study, but needs better presentation, especially regarding the photographs, which are completely illegible at this point.
Response 9: The Resolution of each figure in the manuscript was revised and improved according to the journal's rules and as suggested by the reviewers.
- Title: In your study you use one clinical strain, the title mentions strains in plural. Please verify.
Response 10: Revised and modified as suggested.
11. Abstract: Please add information about the cytotoxicity performed and possible future studies/limitations of this experiment.
Response 11: Information added as suggested.
12. Introduction: line 30: reference after 1970 is needed
Response 12: Reference has been included.
- Line 34: how many cases is % needed
Response 13: Information added as suggested.
14. Line 35-37: ring shaped infiltrate occurs in 50% of cases
Response 14: Revised information as suggested.
- Line 39: in which mechanism?
Response 15: Information added as suggested.
- Line 41-44: what are the concentrations?
Response 16: Information added as suggested.
- line 65: one clinical strain or several?
Response 17: Although other isolates were part of the study, the stability study was with the Acanhamoeba castellanni strain (ATCC 50492). Revised information as suggested.
- At some point in the introduction it should be added that the infection usually affects one eye
Response 18: Information added as suggested.
- line 77: the names of the strains and the letter designations should be in the caption. The photo is of very poor quality and should be changed.
Response 19: The figure has been removed.
- line 81: the scale bar should be placed on the photo.
Response 20: The figure has been removed.
- line 87: there is no need to keep repeating the strain designations
Response 21: Information changed as suggested.
- line 99: an explanation of the * designation should be added
Response 22: Information added as suggested.
- line 106-108: the photo should also be added, after incubation of 48 h
Response 23: The figure has been removed.
- line 111-113: this is a part of materials and methods.
Response 24: Information has been removed as suggested.
- line 125: graphs should be larger, photos are completely illegible
Response 25: The figures are with resolution according to the rules of the journal.
- line 147: an explanation should be added as to why you are using SIRC cells.
Response 26: Information added as suggested lines 204 to 207.
- line 213: why was cytotoxicity tested only after 24 h? The same in materials and methods and results.
Response 27: The time of 24 hours was the estimated time for evaluation and that allowed reading the viability from the MTT, not exceeding the reading threshold given the cell proliferation after 24 hours.
- line 234: what was the concentration of the antibiotic and which antibiotic was it?
Response 28: Information added as suggested.
- line 236: why bold?
Response 29: Bold has been removed.
- lines 251, 261: Please remember italics
Response 30: Corrected information.
- line 271: italics or not?
Response 31:Corrected information.
- The conclusions should be expanded to include more data.
Response 32: Information added as suggested.
Reviewer 3 Report
The manuscript entitled “In vitro stability of biological activity of voriconazole against 2 clinical strains of Acanthamoeba spp.” by Rodrigues et al. is an interesting piece of work. This work tends to evaluate the stability of the biological activity of voriconazole against two strains of Acanthamoeba castellanii and one clinical isolate from an infected Acanthamoeba keratitis patient. The study reports the amoebicidal effect of voriconazole against Acanthamoeba spp. in two distinct time points of exposure and the voriconazole solution was stable and retained antiamoebic activity when stored at 4°C for up to 30 days. The topic selected is interesting and it actually addresses a severe problem encountered for the contact lens users. The methods presented in this article are somewhat clear but there are certain issues related to the figures. I have mentioned them specifically in my comments.
Improve the resolution of Figure 1
Improve resolution of Figure 3 (C, D)
Asterisks are placed here and there. Should be appropriately presented.
However, the conclusion is well supported. Reference formatting should be done properly. Many scientific names have not been italicized.
Author Response
- Improve the resolution of Figure 1.
Response 33: Figure was removed.
- Improve resolution of Figure 3 (C, D).
Response 34: Improved resolution as suggested.
- Asterisks are placed here and there. Should be appropriately presented.
Response 35: Revised as suggested. Asterisks were shown in the figures when referring to *p<005. Therefore, they are presented in the graphs according to the representation.
- However, the conclusion is well supported. Reference formatting should be done properly. Many scientific names have not been italicized.
Response 36: Revised as suggested.
Round 2
Reviewer 2 Report
Thank you for your answers and the improvements made. I think that the graphs and pictures (figure 2) should be separated to increase their visibility, at the moment the graphs are too small and the error bars are not visible
Yours sincerely
Author Response
Figure 2 has been modified as suggested by the reviewer and separated into Figures 2A and 2B and 3A and 3B.
Reviewer 3 Report
The manuscript entitled "In vitro stability of biological activity of voriconazole against clinical strains of Acanthamoeba spp" is now in a good shape. Authors have substantially revised the paper as sugggested.
I am happy to accept this paper for publication.
Author Response
We appreciate the comments after the review.
