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      Multiple Species of Trichosporon Produce Biofilms Highly Resistant to Triazoles and Amphotericin B

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          Abstract

          Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

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          Current knowledge of Trichosporon spp. and Trichosporonosis.

          Trichosporon spp. are basidiomycetous yeast-like fungi found widely in nature. Clinical isolates are generally related to superficial infections. However, this fungus has been recognized as an opportunistic agent of invasive infections, mostly in cancer patients and those exposed to invasive medical procedures. It is possible that the ability of Trichosporon strains to form biofilms on implanted devices, the presence of glucuronoxylomannan in their cell walls, and the ability to produce proteases and lipases are all factors likely related to the virulence of this genus and therefore may account for the progress of invasive trichosporonosis. Disseminated trichosporonosis has been increasingly reported worldwide and represents a challenge for both diagnosis and species identification. Phenotypic identification methods are useful for Trichosporon sp. screening, but only molecular methods, such as IGS region sequencing, allow the complete identification of Trichosporon isolates at the species level. Methods for the diagnosis of invasive trichosporonosis include PCR-based methods, Luminex xMAP technology, and, more recently, proteomics. Treating patients with trichosporonosis remains a challenge because of limited data on the in vitro and in vivo activities of antifungal drugs against clinically relevant species of the genus. Despite the mentioned limitations, the use of antifungal regimens containing triazoles appears to be the best therapeutic approach.
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            Invasive infections caused by Trichosporon species and Geotrichum capitatum in patients with hematological malignancies: a retrospective multicenter study from Italy and review of the literature.

            Trichosporonosis is an uncommon but frequently fatal mycosis in immunocompromised patients. A multicenter retrospective study was conducted to characterize cases of proven or probable invasive trichosporonosis diagnosed over the past 20 years in Italian patients with hematological diseases. Of the 52 cases identified, 17 were classified as Trichosporon sp. infections and 35 were attributed to Geotrichum capitatum. Acute myeloid leukemia accounted for 65.4% of the cases. The incidence rates of Trichosporon sp. and G. capitatum infections in acute leukemia patients were 0.4 and 0.5%, respectively. Overall, 76.9% of cases had positive blood cultures. Pulmonary involvement was documented in 26.9% of cases. Death was reported for 57.1% of G. capitatum infections and for 64.7% of Trichosporon sp. infections. A literature review on trichosporonosis in patients with any underlying disease or condition reveals G. capitatum as a predominantly European pathogen, particularly in certain Mediterranean areas, while Trichosporon sp. infections are seen with similar frequencies on all continents. The majority of published Trichosporon sp. and G. capitatum infections occurred in patients with hematological diseases (62.8 and 91.7%, respectively). Well over half of these were suffering from acute leukemia (68 and 84% of patients with Trichosporon sp. and G. capitatum infections, respectively). Crude mortality rates were 77% for Trichosporon spp. and 55.7% for G. capitatum. The optimal therapy for trichosporonosis has yet to be identified; however, in vitro experiences are providing encouraging evidence of the potential role of the new triazoles, in particular, voriconazole.
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              Biofilm-forming ability of Candida albicans is unlikely to contribute to high levels of oral yeast carriage in cases of human immunodeficiency virus infection.

              An increased prevalence of candidal carriage and oral candidiasis is common in cases of human immunodeficiency virus (HIV) infection, and the reasons for this may include the enhanced ability of colonizing yeasts to produce biofilms on mucosal surfaces. The aim of the present study was therefore to examine the differences, if any, in the biofilm-forming abilities of 26 Candida albicans yeast isolates from HIV-infected individuals and 20 isolates from HIV-free individuals, as this attribute of yeast isolates from patients with HIV disease has not been examined before. Biofilm formation in microtiter plate wells was quantitatively determined by both the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction method and the crystal violet method. Although candidal biofilm formation could be quantitatively evaluated by either technique, the better reproducibility (P < 0.05) of the XTT reduction assay compared with that of the crystal violet method led us to conclude that the former is more reliable. There were no significant quantitative differences in biofilm formation between C. albicans isolates from HIV-infected patients and isolates from HIV-free individuals during in vitro incubation in a multiwell culture system over a period of 66 h. Three of eight host factors in the HIV-infected group were found to be associated with candidal biofilm formation. Thus, yeasts isolated from older individuals and those with higher CD4-cell counts exhibited decreased biofilm formation, while the findings for yeasts from individuals receiving zidovudine showed the reverse (P < 0.05 for all comparison). Our data indicate that attributes other than biofilm formation may contribute to the increased oral yeast carriage rates in cases of HIV infection.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                31 October 2014
                : 9
                : 10
                : e109553
                Affiliations
                [1 ]Laboratório Especial de Micologia, Disciplina de Infectologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
                [2 ]Departamento de Microbiologia e Imunologia, Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas, MG, Brazil
                [3 ]Laboratório de Micologia, Faculdade de Medicina, Universidade Federal do Mato Grosso, Cuiabá, MT, Brazil
                University of Wisconsin Medical School, United States of America
                Author notes

                Competing Interests: Dr. Colombo has received research and educational grants from Astellas, MSD, Pfizer and United Medical in the last two years. All the other authors report no conflicts of interest relevant to this article. The authors alone are responsible for the content and the writing of the paper.

                Conceived and designed the experiments: IAIG ACBP ALC. Performed the experiments: IAIG ACBP FCB. Analyzed the data: IAIG ACBP FCB RCH ALC. Contributed reagents/materials/analysis tools: ACBP FCB RCH ALC. Wrote the paper: IAIG ACBP ALC.

                ¶ These authors are co-first authors on this work.

                Article
                PONE-D-14-20746
                10.1371/journal.pone.0109553
                4215839
                25360765
                393d7c4d-c5e4-4755-bb30-f0083152f507
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 May 2014
                : 30 July 2014
                Page count
                Pages: 10
                Funding
                This study received funds through A.L.C. from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (grant 2007/08575-1) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. I.A.I.G. received a master training fellowship from CNPq; A.C.B.P. received a postdoctoral fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (PNPD 02640-09-0). F.C.B. received a postdoctoral fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (2010/17179-5). R.C.H. received funds from the the Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Microbial Control
                Antimicrobials
                Antifungals
                Mycology
                Fungal Structure
                Medicine and Health Sciences
                Infectious Diseases
                Fungal Diseases
                Opportunistic Infections
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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