Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Mechanisms of antifungal resistance

The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have been reported for variants of Saccharomyces cerevisiae and of Ustilago maydis. Tolerance to morpholines is seldom found. Intrinsic resistance to griseofulvin is due to the absence of a prolonged energy-dependent transport system for this antibiotic. Resistance to azole antifungal agents is known to be exceptional, although it does now appear to be increasing in importance in some groups of patients infected with e.g. Candida spp., Histoplasma capsulatum or Cryptococcus neoformans. For example, resistance to fluconazole is emerging in C. albicans, the major agent of oro-pharyngeal candidosis in AIDS patients, after long-term suppressive therapy. In the majority of cases, primary and secondary resistance to fluconazole and cross-resistance to other azole antifungal agents seems to originate from decreased intracellular accumulation of the azoles, which may result from reduced uptake or increased efflux of the molecules. In most C. albicans isolates the decreased intracellular levels can be correlated with enhanced azole efflux, a phenomenon linked to an increase in the amounts of mRNA of a C. albicans ABC transporter gene CDR1 and of a gene (BEN(r) or CaMDR) coding for a transporter belonging to the class of major facilitator multidrug efflux transporters. Not only fluconazole, ketoconazole and itraconazole are substrates for CDR1, terbinafine and amorolfine have also been established as substrates, BEN(r) overexpression only accounts for fluconazole resistance. Other sources of resistance: changes in membrane sterols and phospholipids, altered or overproduced target enzyme(s) and compensatory mutations in the Delta5,6-desaturase.     

In vitro susceptibility of fungal and yeast clinical isolates to itraconazole and voriconazole

The interest on the in vitro susceptibility to itraconazole has recently increased due the availability of the intravenous formulation. In this study, comparative MICs of this antifungal with voriconazole were carried out in 62 clinical isolates of filamentous fungi and 100 yeasts isolates using the NCCLS microbroth methods described in M38-A and M27-A2 documents. A MIC90 of 0.125 micrograms per ml was observed for itraconazole and voriconazole against Aspergillus fumigatus. Higher susceptibility to itraconazole was found for the filamentous form of Sporotrhix schenckii (p = 0.001). Voriconazole was more effective against Scedosporium apiospermium while Scedosporium prolificans isolates were resistant to both azoles. Some isolates of Rhizopus stolonifer were susceptible to itraconazole and resistant to voriconazole, but without statistical significance. Susceptibility of nine species of Candida was similar for both triazoles used in this study. However, Candida glabrata was more susceptible to voriconazole. Some fluconazole-resistant Candida albicans isolates were susceptible to itraconazole and / or voriconazole. Cryptococcus neoformans was more susceptible to itraconazole than to voriconazole. Itraconazole and voriconazole showed very close in vitro activity against the tested fungal isolated, except against S. schenckii. In spite of this, there were some differences in susceptibility among isolates within the same fungal species.     

Molecular phylogenetics of Candida albicans

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.     

In vitro resistance to fluconazole and itraconazole in clinical isolates of Candida spp and Cryptococcus neoformans

An in vitro susceptibility testing of 181 strains of six species of Candida and 21 strains of Cryptococcus neoformans was carried out in order to investigate the resistance to new antifungal drugs. We have studied clinical isolates from 200 different patients of Hospital del Mar (Barcelona) and Hospital La Inmaculada (Almería). An agar diffusion method (NeoSensitabs, Rosco, Taastrup, Denmark), was employed with fluconazole, itraconazole, and reference drugs amphotericin B, flucytosine, tioconazole and ketoconazole. A high level of susceptibility was found for amphotericin B in C. neoformans strains while 19% of them were resistant to flucytosine. All the strains of C. neoformans and Candida guilliermondii were susceptible to the new azoles derivatives and also Candida parapsilosis and Candida albicans had a great susceptibility to this antifungals. A greater level of resistance was found for Candida krusei, Candida tropicalis and Candida glabrata to fluconazole, itraconazole and ketoconazole, but resistance to fluconazole and itraconazole is not always linked because ten resistant strains for fluconazole were susceptible to itraconazole, and two other resistant to itraconazole were susceptible to fluconazole.     

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida . The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 μg mL⁻¹); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1 , CDR2 , MDR1 , encoding for efflux pumps, and ERG11 , encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly ( P >0.05), probably acting as a Cdrp blocker.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

In vitro susceptibility of isolates of Sporothrix schenckii to amphotericin B, itraconazole, and terbinafine: comparison of yeast and mycelial forms

Forty-three clinical isolates of Sporothrix schenckii derived from humans and animals were evaluated in vitro for their susceptibility to amphotericin B, itraconazole, and terbinafine. MICs were determined by the method of micro dilution in liquid media, using protocols M27-A2 for the yeast form and M38-A for the mycelial form, both standardized by the Clinical Laboratory Standards Institute. In general, higher MICs were found for the mycelial form (intervals of up to two dilutions). In the case of amphotericin B, a significant difference in activity was observed, with higher values (p<0.05) found for the mycelial form. MICs for itraconazole and terbinafine were similar for both yeast and mycelial forms but slightly higher for mycelia. Although data presented here indicate different levels of susceptibility when both growth forms were compared, indicating an intrinsic difference between them, it is still difficult to draw a consensus as to which form correlates better with clinical findings. More studies are necessary to determine the criteria for in vitro tests that will lead to efficient therapeutic choices.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.     

Development of Azole Resistance in Aspergillus fumigatus during Azole Therapy Associated with Change in Virulence

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata

Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1 / CgCDR2 , but with upregulation of another ATP-binding cassette transporter, CgSNQ2 , which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N -oxide, which is a ScSNQ2 -specific substrate. A putative gain-of-function mutation, P822L, was identified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas introduction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1 . Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the development of CgPDR1 -dependent azole resistance in C. glabrata . The overlapping in function and regulation between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata .     

真菌对唑类抗真菌药物的耐受机制

唑类抗真菌药物广泛用于临床和农业。唑类药物通过与羊毛甾醇14α-去甲基化酶（Erg11p/Cyp51）结合,抑制麦角甾醇合成,同时导致有毒甾醇积累。真菌可快速在转录水平上对唑类药物胁迫作出响应而导致耐药性,尤其是唑类药物外排泵基因和麦角甾醇合成相关基因表达的上调。农业和临床上绝大多数唑类药物耐药菌株的形成都是由麦角甾醇合成基因和唑类药物外排泵表达的变化或是突变所致。一些转录因子（如Pdr1p、Pdr3p、Upc2p、Yap1p、Tac1p、Mrr1p、CCG-8）和信号通路（如cAMP途径、PKC-MAPK途径、HOG MAPK途径、钙调磷酸酶途径）均参与对药物外排泵基因和麦角甾醇合成基因等的调控,影响唑类药物耐药性。针对于这些调控因子设计的抑制剂将有助于提高唑类药物的治疗效果。本文概述了唑类药物的抑菌机制、真菌对唑类药物耐药性形成的原因、真菌对唑类药物适应性响应机理,并对未来此领域的热点和方向进行了展望。     

Can We Achieve Clinical Breakpoints for the Triazoles in Aspergillosis?

The azoles are first-line agents for the treatment of aspergillosis. A number of recent studies have shown increasing rates of resistance in A. fumigatus. Consequently, reliable in vitro susceptibility testing and breakpoints that appropriately classify resistant isolates are of paramount importance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical Laboratory Standards Institute (CLSI) have developed susceptibility testing standards, but so far no breakpoints have been defined. The following aspects are evaluated during the process of developing EUCAST breakpoints: the most common dosage, the definition of the wild-type population and epidemiologic cutoff values, pharmacokinetic and pharmacodynamic properties, and the correlation between the minimum inhibitory concentration (MIC) and clinical outcome. This article reviews the issues to be considered before breakpoints can be established for azole drugs and Aspergillus and describes how MICs can be interpreted until clinical breakpoints have been defined.     

Species Distribution and Antifungal Susceptibility Profile of Oral Candida Isolates from HIV-infected Patients in the Antiretroviral Therapy Era

In this study, we investigated the yeasts colonization of genus Candida, including C. dubliniensis, isolated of HIV-infected patients oral cavities and we accessed in vitro susceptibility pattern of the Candida isolates to four antifungal agents. Out of 99 patients investigated, 62 (62.6%) were colonized with yeasts. C. albicans was the prevailing species (50%). C. dubliniensis isolates were not recovered in our study. We verified that 8.1% of the yeasts isolated were resistant to fluconazole, 8.1% to itraconazole and 3.2% to voriconazole. The isolates demonstrated very low voriconazole MICs, in which 79% (49/62) presented values of 0.015 μg/ml. All Candida isolates were susceptible to amphotericin B. The results reported here showed that although C. albicans continues to be present in one-half of oral Candida carriage of HIV-infected patients, Candida non-albicans species are increasing among these patients. Besides, the findings of resistant isolates endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

Genotyping and Antifungal Drug Susceptibility of Trichosporon asahii Isolated from Chinese Patients

As a major pathogenic agent of trichosporonosis, Trichosporon asahii can cause life-threatening infection in immunocompromised patients. In this study, we analyzed the genotypes of the intergenic spacer (IGS) 1 region of the rRNA gene and the antifungal drug susceptibility of eight T. asahii isolates obtained from Chinese patients. Five genotypes were identified from the eight isolates, including three novel genotypes, three genotype 1, and two genotype 4. The eight T. asahii isolates were resistant to amphotericin B, 5-flucytosine, and terbinafine, but were highly sensitive to fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC). The mean minimum inhibitory concentrations (MICs) of FLC and VRC were significantly lower than those reported in most other countries, while that of ITC was slightly higher. Our results suggest that genotypes of the T. asahii isolated from China are different from those of other countries, and azole drugs appeared to be more effective on the Chinese isolates. These results provide new insights into the epidemiology and antifungal treatment for T. asahii.