A Comparative Prospective Study in Evaluating Candida spp. In Vitro Susceptibility through Micronaut-AM and Sensititre Yeast-One

Round 1

Reviewer 1 Report

General comment

The manuscript “A comparative prospective study in evaluating Candida spp. in 2 vitro susceptibility through Micronaut-AM and Sensititre Yeast-3 One", is a work that makes a comparison between the Micronaut-AM and Sensititre Yeast-One methods to define reliable MIC values, in isolates of different Candida species, obtained of positive peripheral blood samples from patients recovered in Internal Medicine, Hematology and Intensive Care units. In general, it is a well-structured and interesting work, since the issue of antifungal resistance is becoming more important every day, particularly for immunocompromised patients, in whom their lives may be at risk. Therefore, as mentioned by the authors, the EUCAST and CLSI guidelines provide well-established and proven reference methods to assess antifungal susceptibility via broth microdilution, however, these methods require long intervals, a lot of manual labor, and a good experience, so the evaluation of commercial methods to test antifungal susceptibility with various species of fungi can be a good alternative. However, I do have some comments that I list below.

Comments

Why was the SY method evaluated against the CLSI reference method, while the M-AM method was evaluated against the EUCAST reference method since these types of comparisons did not allow us to assess which broth microdilution colorimetric method was more reliable.

The authors mention that they used the reference strains of Candida parapsilosis (ATCC22019) and Candida krusei (ATCC6258) as quality control, however, in the results they only mention that the MICs for C. krusei (ATCC 6258) and C. parapsilosis ( ATCC 22019) were in the recommended ranges after running Sensititre Yeast-One and Micronaut-AM. Therefore, it would be advisable to show in the tables the values obtained for each strain, with each of the antifungals.

In the discussion, the authors mention some drawbacks of their work, for example, all the echinocandins showed high CA values, in their tested isolates, except C. krusei, however, they do not discuss this finding further, it would be convenient to expand this discussion.

Likewise, it would be convenient to broaden the discussion of their results of the fluconazole and C. glabrata association, in addition to C. krusei and voriconazole. For example, in the case of C. glabrata, which has been described as a complex of species (C. glabrata sensu stricto, C. bracarensis and C. nivariensis), it would be convenient to discuss whether the molecular identification of these species is important.

It would have been interesting to include molecular confirmation of the possible resistance phenotypes for the different Candida species, in order to verify their results.

Although the authors mention that it is not possible to establish which commercial microdilution method is better than another for defining MICs for all antifungals tested, I suggest clearly including their conclusions from the work in a separate section of the discussion.

Table 1: Change “Pozaconazolo” to “Pozaconazole”

Line 134: Change “Candida” to “Candida”

Line 249: Delete space “experi-mental”

 

I suggest carefully reviewing the references and strictly following the journal format.

Author Response

Comments

1. Comment: Why was the SY method evaluated against the CLSI reference method, while the M-AM method was evaluated against the EUCAST reference method since these types of comparisons did not allow us to assess which broth microdilution colorimetric method was more reliable.

Answer: The manufacturer’s instructions established to evaluate SY according to CLSI guidelines and M-AM following EUCAST rules. We are aware about the impossibility to elect the most valid method, but the main article’s aim was to study a possible agreement between the two techniques.

2. Comment: The authors mention that they used the reference strains of Candida parapsilosis(ATCC22019) and Candida krusei (ATCC6258) as quality control, however, in the results they only mention that the MICs for  krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were in the recommended ranges after running Sensititre Yeast-One and Micronaut-AM. Therefore, it would be advisable to show in the tables the values obtained for each strain, with each of the antifungals.

Answer: We decided to add a table (Table 1). This table describes all the MIC details about the quality control strains.

3. Comment: In the discussion, the authors mention some drawbacks of their work, for example, all the echinocandins showed high CA values, in their tested isolates, except  krusei, however, they do not discuss this finding further, it would be convenient to expand this discussion.

Answer: we added few lines (324-328) to further comment these results.

4. Comment: Likewise, it would be convenient to broaden the discussion of their results of the fluconazole and  glabrata association, in addition to C. krusei and voriconazole. For example, in the case of C. glabrata, which has been described as a complex of species (C. glabrata sensu stricto, C. bracarensis and C. nivariensis), it would be convenient to discuss whether the molecular identification of these species is important.

Answer: We added some comments about C. glabrata/fluconazole and C. krusei/voriconazole, including two further references to expand the detected MIC results (lines 329-331). The possibility to justify high fluconazole MIC values with C. bracarensis or C. nivariensis detection was excluded due to the previous MALDI identification of all the isolates. All the C. glabrata identifications were related to C. glabrata sensu strictu. We detailed this information in the results paragraph.

5. Comment: It would have been interesting to include molecular confirmation of the possible resistance phenotypes for the different Candidaspecies, in order to verify their results.

Answer: Further studies will be performed to investigate about the resistance mechanisms of the resistant isolates which were detected during the study. This type of information is not currently available, because we decided to produce an observation study as a first step. Our study referred to other observational published studies, which led us to submit these initial results. Our research group will evaluate molecular characterization of the resistant isolates during future investigations. We added few lines about this future intention in the conclusion (lines 378-380).

6. Comment: Although the authors mention that it is not possible to establish which commercial microdilution method is better than another for defining MICs for all antifungals tested, I suggest clearly including their conclusions from the work in a separate section of the discussion.

Answer: Thanks for the suggestion. We decided to create a separate section totally dedicated to our conclusions (paragraph number 5).

7. Comment: Comment: Table 1: Change “Pozaconazolo” to “Pozaconazole”

Answer: We apologize for the typo. The word has been corrected.

8. Comment: Line 134: Change “Candida” to “Candida”

Answer: We apologize for the typo. The word has been corrected.

9. Comment: Line 249: Delete space “experi-mental”

Answer: We apologize for the typo. The word has been corrected.

10. Comment: I suggest carefully reviewing the references and strictly following the journal format.
    Answer: Thanks for the advice. We reviewed the format according to the journal’s instructions for authors.

Reviewer 2 Report

Dear Authors,

This is an original work submitted by Trovato et al. entitled: A comparative prospective study in evaluating Candida spp. in vitro susceptibility through Micronaut-AM and Sensititre Yeast-One.

This manuscript is novelty since it presents a complete study on the comparison between two Candida spp susceptibility test, the well-known Sensititre YeastOne and the alternative Micronaut AM.

Major revisions are suggested before the publication of the manuscript

Deeply discussion must be addressed in a comparison with DOI: 10.3390/jof7050356 since relevant differences are observed between your results and the reported one. For example, Fluconazole susceptibility proportions were comparable in DOI: 10.3390/jof7050356, however, big differences are observed in table for Fluconazole proportions.

Categorical agreement value illustrated in table 3 are considerably different to others reported before, specially in the case of Pozaconazole.

In order to support the test, it would be important to explain the method of characterization of candida strains, eg MALDI-TOF mass spectrometry, microscopic characterization, etc.

Other references with interestingly susceptibility results of Micronaut-AM (DOI 10.5812/jjm.101767) and Sensititre Yeast-One (DOI 10.12982/JAMS.2022.023) could be included and discussed.

Regards

Author Response

1. Comment: Deeply discussion must be addressed in a comparison with DOI: 10.3390/jof7050356 since relevant differences are observed between your results and the reported one. For example, Fluconazole susceptibility proportions were comparable in DOI: 10.3390/jof7050356, however, big differences are observed in table for Fluconazole proportions.
   Answer: The detection of some disagreements compared to previous studies can be justified with the restricted number of isolates. We discussed something about this topic in lines 338-342.
2. Comment: Categorical agreement value illustrated in table 3 are considerably different to others reported before, especially in the case of Pozaconazole.
   Answer: We did not report any categorical agreement for posaconazole, whose EUCAST and CLSI rules are not sufficient to establish clinical breakpoints. We only reported an essential agreement, whose calculation was produced in a species-specific sense.
3. Comment: In order to support the test, it would be important to explain the method of characterization of Candida strains, e.g. MALDI-TOF mass spectrometry, microscopic characterization, etc.
   Answer: We already explained that MALDI-TOF mass spectrometry was used to identify all the yeast grown colonies before the application of the susceptibility testing.
4. Comment: Other references with interestingly susceptibility results of Micronaut-AM (DOI 10.5812/jjm.101767) and Sensititre Yeast-One (DOI 10.12982/JAMS.2022.023) could be included and discussed.
   Answer: We added some comments and references about the two techniques, expanding the discussion (lines 358-369).

 

Reviewer 3 Report

This is a significant study in a very important matter: evaluating Candida spp. susceptibility using  in vitro commercial microdilutions tests Micronaut-AM and Sensititre Yeast One

The purpose of the paper is clear and important; however, some changes should be done in order to make this work proper to be published.

 

Changes that I suggest concerns:

Pag 2, Line 65 where it is said “The University Hospital Policlinico of Catania conducted an 8-months (October 2022-April 2023) prospective study”, it should be “This 8-months (October 2022-April 2023) prospective study was conducted in the University Hospital Policlinico of Catania conducted…”

Pag 8, Results: Some English/scientific changes will increase the document (in pink in the attached PDF). Sometimes it is said 1 C. albicans, other is one. It is always better to say “one”. Please verify this issue throughout the all document.

Page 8, Line 194: The word precisely should be removed.

In my opinion the manuscript should have a conclusion.

 

The subject of the article is very relevant and of immense importance. After the proposed changes, the manuscript should be considered for publication.

Comments for author File: Comments.pdf

Author Response

1. Comment: Pag 2, Line 65 where it is said “The University Hospital Policlinico of Catania conducted an 8-months (October 2022-April 2023) prospective study”, it should be “This 8-months (October 2022-April 2023) prospective study was conducted in the University Hospital Policlinico of Catania conducted…”
   Answer: Thanks for the suggestion. The sentence has been modified (lines 65-66).
2. Comment: Pag 8, Results: Some English/scientific changes will increase the document (in pink in the attached PDF). Sometimes it is said 1  albicans, other is one. It is always better to say “one”. Please verify this issue throughout the all document.
   Answer: The requested changes have been provided. Furthermore, the number “1” referred to strains number has been converted into the word “one” across the all document.
3. Comment: Page 8, Line 194: The word precisely should be removed.
   Answer: the word has been removed.
4. Comment: In my opinion the manuscript should have a conclusion.
   Answer: Thanks for the suggestion. We decided to separate some assumptions from the discussion, creating a dedicated paragraph called “Conclusions”.

Round 2

Reviewer 2 Report

Dear Authors,

Thank you for your answers. In my opinion your manuscript is ready for publication.

Regards