Antagonistic Interactions in Onychomycosis: Antifungal Activity of Extracts from Pure and Mixed Cultures of Candida parapsilosis and Trichophyton spp.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated whether there is a competitive or antagonistic inhibition of Candida spp against T rubrum/T mentagrophytes and vice versa. This is an old clinical observation that some fungi facilitate the penetration and growth of other fungi in the nails and these '"new fungi" then inhibit the growth of the ones tht enabled them to infect the nail. The authors have now performed a study to put this on a higher scientific level. However, whether this is enough to use double infections as a treatment remains to be proven in clinical routine ("the antifungal activity described here opens possibilities for new therapeutic approaches to manage onychomycosis."). It is also not clear whether the action is really microbicidal; we know this from daily routine that antifungals have fantastic MIC values and there should be a real overkill with the treatment regimen performed and nevertheless the disease does not clear. However, I agree with the authors' conclusion that "... extracts of pure and mixed culture of Candida parapsilosis and dermatophytes present antifungal activity against T. mentagrophytes and T. rubrum, which may influence the laboratory diagnosis of mixed infections. These results can explain cases of mixed fungal infections where only one of the etiologic agents grows in culture."

There are some linguistic issues tht should be corrected.

Comments on the Quality of English Language

The authors presented their work in an acceptable English although some improvements are necessary.

Author Response

The article underwent grammatical review

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article “Antagonistic interactions in onychomycosis: Antifungal activity of extracts from pure and mixed cultures of Candida parapasilosis and Trichophyton spp” is devoted to the important problem of biological interference between the dermatophytes T. mentagrophytes and T. rubrum and Candida. The results indicate that such interference may affect the laboratory diagnosis of mixed infections. Part of the work with the toxicity tests is especially noteworthy, as it allows us to talk about possible new therapeutic approaches to control fungal infections using extracts from pure and mixed cultures of Candida parapasilosis and Trichophyton spp.

At the same time, there are comments on the text of the article:

Lines 63-65: An inoculum suspension in sterile saline (0.85%), with turbidity corresponding to MacFarland Scale 10, was inoculated into 500 mL of Saboraud Dextrose Broth (CSD) and incubated at 35° C for 72 hours.

Comment: What is the volume of an inoculum suspension?

Lines 68-69: Subsequently, the mixture of conidia and hyphae in the suspension was transferred to 500 mL of CSD and incubated at 35° C for 120 hours.

Comment: The same question - What is the volume of transferred suspension?

Lines 72-73: The culture medium containing the inocula was then filtered through a 0.2 μm Millipore membrane and subjected to liquid-liquid extraction with ethyl acetate.

Comment: What is the volume ratio between the culture medium and ethyl acetate?

Line 101: For this assay, CFM values obtained were used.

Comment: CFM? Maybe MFC?

Line 101: 3.1. Susceptibility profile of dermatophyte strains against extracts and antifungal drugs

Comment: It remains unclear why in an article devoted to the activity of extracts from microorganisms a table with the MICs of antifungal drugs should be given, especially since no real comparative analysis of the effect of drugs and extracts was carried out afterwards. Maybe just put an extra row in Table 2 with the MICs of any drug to show that it is much less than the extracts.

Lines 139-141: CFM values followed this trend, with smaller intervals for CPTM (250 and 1000 µg/mL) and CP (500 and 1000 µg/mL). CPTR and TR extracts showed higher CFM values (2000 – 8000 µg/mL).

Comment: CFM? Maybe MFC?

Lines 139-141: The time-kill assay was carried out for 48 hours, exposing the strains TRCBS, TR6185, TMATCC, and TM5094 to the extracts

Comment: An explanation of why these four strains were chosen is desirable.

Line 160: Table 4. The colony forming unit (CFU/mL) of dermatophyte strains exposed to extracts at different times.

Comment: Why are the names of the extracts in the table CP4, CP4TR1, CP4TM3, TR1, TM3 and not CP, CPTR, CPTM, TR, TM?

Line 171: 4. Discussion

Comment: The section lacks a real analysis of the results obtained. It would be desirable to provide, for example, considerations on whether and how the antifungal action of mixed extracts differs from that of pure extracts.

Author Response

Lines 63-65: An inoculum suspension in sterile saline (0.85%), with turbidity corresponding to MacFarland Scale 10, was inoculated into 500 mL of Saboraud Dextrose Broth (CSD) and incubated at 35° C for 72 hours.

Comment: What is the volume of an inoculum suspension?

5mL. Adjusted.

Lines 68-69: Subsequently, the mixture of conidia and hyphae in the suspension was transferred to 500 mL of CSD and incubated at 35° C for 120 hours.

Comment: The same question - What is the volume of transferred suspension?

5mL. Adjusted.

Lines 72-73: The culture medium containing the inocula was then filtered through a 0.2 μm Millipore membrane and subjected to liquid-liquid extraction with ethyl acetate.

Comment: What is the volume ratio between the culture medium and ethyl acetate?

250 mL culture medium/50 mL ethyl acetate. Adjusted

Line 101: For this assay, CFM values obtained were used.

Comment: CFM? Maybe MFC?

MFC. Adjusted

Line 101: 3.1. Susceptibility profile of dermatophyte strains against extracts and antifungal drugs

Comment: It remains unclear why in an article devoted to the activity of extracts from microorganisms a table with the MICs of antifungal drugs should be given, especially since no real comparative analysis of the effect of drugs and extracts was carried out afterwards. Maybe just put an extra row in Table 2 with the MICs of any drug to show that it is much less than the extracts.

The authors decided to place the antifungal susceptibility profile in a separate table for better visualization of the data

Lines 139-141: CFM values followed this trend, with smaller intervals for CPTM (250 and 1000 µg/mL) and CP (500 and 1000 µg/mL). CPTR and TR extracts showed higher CFM values (2000 – 8000 µg/mL).

Comment: CFM? Maybe MFC?

MFC. Adjusted

Lines 139-141: The time-kill assay was carried out for 48 hours, exposing the strains TRCBS, TR6185, TMATCC, and TM5094 to the extracts

Comment: An explanation of why these four strains were chosen is desirable.

Strains resistant to conventional drugs and sensitive strains were selected for comparative inhibitory action.

Line 160: Table 4. The colony forming unit (CFU/mL) of dermatophyte strains exposed to extracts at different times.

Comment: Why are the names of the extracts in the table CP4, CP4TR1, CP4TM3, TR1, TM3 and not CP, CPTR, CPTM, TR, TM?

Adjusted

Line 171: 4. Discussion

Comment: The section lacks a real analysis of the results obtained. It would be desirable to provide, for example, considerations on whether and how the antifungal action of mixed extracts differs from that of pure extracts.

 

Adjusted

Author Response File: Author Response.pdf