Author's response to reviews

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1 Author's response to reviews Title:Isolation and identification of Candida species in patients with orogastric cancer: susceptibility to antifungal drugs, attributes of virulence in vitro and immune response phenotype. Authors: Lourimar V.N.F Sousa Kênia V Santos (keniavaleria@gmail.com) Vera L Santos (verabio@gmail.com) Andrea S Monteiro (andreasmont@gmail.com) Marcus V.D Souza (souzamv@mail.com) Sirelei G Marques (sirleigmarques@gmail.com) Eleine S Faria (fariaspeziali@gmail.com) Simone G Santos (simonegsantos@gmail.com) Elaine A.O Assunção (elainemicrobio@hotmail.com) Rodrigo A Holanda (raholanda@yahoo.com.br) Juan M Zonis (juanzonis@yahoo.com.br) Daniel G Alvarenga (daniel_alvarenga@hotmail.com) Jaqueline G Sousa (jgontijos@yahoo.com.br) Maria Aparecida R Stoianoff (maresend@icb.ufmg.br) Version:6Date:30 December 2015 Author's response to reviews: see over

com) Marcus V.D Souza (souzamv@mail.com) Sirelei G Marques (sirleigmarques@gmail.com) Eleine S Faria (fariaspeziali@gmail.com) Simone G Santos (simonegsantos@gmail.com) Elaine A.

2 Ms. Ref. No.: Title: Isolation and identification of Candida species in patients with orogastric cancer: susceptibility to antifungal drugs, attributes of virulence in vitro and immune response phenotype. Dear Dr George Thompson I am sending a revised version of the manuscript as requested by the reviewers' comments and the list of changes. With kind regards, Ds. Lourimar List of reviewers coments/changes 1 o ) Title page: Please note that no author is indicated to have affiliation k. Please also add the addresses of all authors on the title page. addresses have been added as follows: Lourimar Viana Nascimento F. de Sousa 1,6*, Vera Lúcia Santos 2, Andrea de Souza Monteiro 3, Marcus Vinicíus Dias-Souza 2, Sirlei Garcia Marques 4, Elaine Speziali de Faria 5, Elaine Alves de Oliveira Assunção 6, Simone Gonçalves dos Santos 7, Juan Moises Zonis 8, Daniel Gomes de Alvarenga 9, Rodrigo Assunção de Holanda 1,Jaqueline Gontijo de Sousa 10 Kênia Valéria dos Santos 11, Maria Aparecida de Resende Stoianoff 1 1,6 * Laboratory of Mycology, Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 1,6 lourimar.viana@gmail.com, 1 raholanda@yahoo.com.br, 1 maresend@icb.ufmg.br 2 Laboratory of Applied Microbiology, Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 2 souzamv@gmail.com 2 verabio@gmail.com, 3 Laboratory of Microbiology, CEUMA University, São Luis, MA, Brazil. andreasmont@gmail.com 4 Hospital of Federal University of Maranhão, São Luis, MA, Brazil. sirleigmarques@gmail.com 5 Biomarkers Laboratory Diagnostics and Monitoring, Research Center René Rachou (CPqRR) - FIOCRUZ/MG, Brazil. fariaspeziali@gmail.com 6 Laboratory of Microbiology, Vale do Rio Doce University, Governador Valadares, MG, Brazil. elainemicrobio@hotmail.com

phenotype. Dear Dr George Thompson I am sending a revised version of the manuscript as requested by the reviewers' comments and the list of changes. With kind regards, Ds.

3 7 Laboratory of Oral and Anaerobes Microbiology, Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 8 Oncology Core Experts, Governador Valadares, MG, Brazil. 9 Alvarenga Laboratory, Governador Valadares, MG, Brazil. 10 Laboratory of Basic and Applied Virology, Department of Microbiology, Institute of Biological Sciences, Federal University Minas Gerais, Belo Horizonte, MG, Brazil. 11 Laboratory of General Bacteriology and Clinical, Department of Pathology, Federal University of Espirito Santo, Health Science Center, Vitória, ES, Brazil. 2 o ) Consent: Please confirm in the manuscript that you obtained informed consent from all participants (patients and controls). Please specify if the consent was written or verbal. The consent of the patients and the control group was made in writing through the TIC (Term of Informed Consent) and approved by the ethics committee. Number of the approved protocol in COEP (Ethics in research committee) PQ25/ o ) Missing sections Please add the following sections after Discussion: - Conclusions: this should state clearly the main conclusions and include an explanation of their relevance or importance to the field. You may wish to use the last paragraph of Discussion for that purpose. Conclusion: It was possible to isolate Candida species in 85% and 59% of individuals with COG-C and in the control group, respectively. The species C. albicans was the most prevalent amidst the isolates. As for virulence factors, the isolates obtained from patients with cancer were more virulent than isolates from the control group. The results generated by MIC assessment showed that the isolates were susceptible to all antifungals compounds. The 20 drugs to which the yeast strains presented higher susceptibility were amphotericin B, ketoconazole and isoconazole. Regarding the cytokines evaluated in the serum of individuals with and without cancer, significant differences were observed among individuals with cancer for IL2, IL10, TNF-α, IFN-g and IL17. - List of Abbreviations used. OGC: Orogastric cancer; MALDI-TOF: Matrix Assisted Laser Desorption Ionization Time Of Flight; MS: Mass spectrometry; NaCl: sodium chloride; BEC: Buccal epithelial cells; IL-2: Interleukin 2; IL-10: Interleukin 10; TNF-α: Tumor necrosis factor; IFN-γ: Interferon; IL-17: Interleukin 17; Sap: Aspartyl proteinases; CBA: Cytometric Bead Array; MG: Minas Gerais;

com 10 Laboratory of Basic and Applied Virology, Department of Microbiology, Institute of Biological Sciences, Federal University Minas Gerais, Belo Horizonte, MG, Brazil. jgontijos@yahoo.com.br 11 Laboratory of General Bacteriology and Clinical, Department of Pathology, Federal University of Espirito Santo, Health Science Center, Vitória, ES, Brazil.

4 SDA: Sabouraud dextrose agar; PZ: Phospholipases; HBECs: human buccal cells; MIC: Minimum Inhibitory Concentration; YCa: Yeasts cancer; YCo: Yeasts control; KET: Ketoconazole; FLU: Fluconazole; ISO: Isoconazole; AmpB: Amphotericin B; VOR: Voriconazole. Authors contributions: This should list the individual contributions of all authors. LVNFS and MARS - part of the whole design and study design and analysis and interpretation of data. VLS, ESF, SGS - analysis and interpretation of data. MVDS, SGM, JMZ, DGA, EAOA, RAH and JGS - were involved in data acquisition. ASM, KVS - analysis, interpretation of data and performed statistical analysis. All authors helped to draft the manuscript, read and approved the final version of the manuscript. Please ensure that all authors are explicitly mentioned, using their (unique) initials instead of the full names. LVNFS 1,7 *, VLS 2, ASM 3, MVDS 2, SGM 4, ESF 5, EAOA 6, SGS 7, JMZ 8, DGA 9, RAH 1, JGS 10 KVS 11, MARS 1 Please also confirm here that all authors read and approved the final version of the manuscript. All authors helped to draft the manuscript, read and approved the final version of manuscript. 4. Section titles In order to adhere to the journal style, please rename section "Introduction" to "Background", "Materials and Methods" to "Materials", and "Conflict of Interest" to "Competing Interests". The section was altered to Background, to Materials and to Competing Interests. We would also appreciate if you change the content of the latter section to "The authors declare that they have no competing interests", rather than just writing "None" The section was altered to "The authors declare that they have no competing interests" 5. Figure Please do not include the figure in the main manuscript text document, and do not include figure titles or figure legends in the image file. The figure was removed from the body text and removed the title and legends in the image file. Please note that the figure legend next to the image included in the manuscript text document is not identical to the content of the list of figure and table legends.

LVNFS and MARS - part of the whole design and study design and analysis and interpretation of data. VLS, ESF, SGS - analysis and interpretation of data.

5 The figure legend is now identical to the contents of the list of figure and table legends as suggested by the reviewer. 6. Copyediting: Please ensure that your manuscript is copyedited, and mistakes like "identifiedby" and "suggestan" (in the abstract), The corrections were made in the text as suggested by the reviewer "These patients should be should be healthy" (first paragraph of Methods) are corrected. Please also ensure that all text is in English. The corrections were made in the text as suggested by the reviewer.

The corrections were made in the text as suggested by the reviewer "These patients should be should be healthy" (first paragraph of

Introduction. Materials and methods

Introduction. Materials and methods Acta Medica Mediterranea, 2014, 30: 391 INVESTIGATION OF VIRULENCE FACTORS AND DETERMINATION OF ANTIFUNGAL SUSCEPTIBILITIES OF CANDIDA STRAINS ISOLATED AT SULEYMAN DEMIREL UNIVERSITY HOSPITAL AYSE AYNALI