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Background: Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up-regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC., Objectives: We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092., Methods: The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3-lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively., Results: The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3-lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092., Conclusions: Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum., (© 2021 Wiley-VCH GmbH.)

Penicillium Marneffei: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H + antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2 , and PmMDR3 ) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. Pm MDR1-expressing strain showed resistance to the broadest range of drugs, followed by the Pm MDR3, and Pm MDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3 , may be able to serve as multidrug efflux pumps.

Aspergillus fumigatus is the most prevalent species that causes aspergillosis. A. fumigatus strains with tandem repeats in the cyp51A promoter have emerged in the environment. Aspergillus species other than A. fumigatus have also been recognized as causative agents of aspergillosis; however, they show lower susceptibility to antifungals compared with A. fumigatus. Therefore, it is important to precisely identify Aspergillus species and determine their antifungal susceptibility. Herein, we collected 119 mold strains isolated from clinical specimens collected at a hospital between November 2013 and December 2018. The collected strains were identified by sequencing several regions, including internal transcribed spacers, and determined their susceptibility to the antifungals itraconazole, voriconazole, and amphotericin B. Of 119 strains, 107 were Aspergillus species, which were identified as A. fumigatus (67), Aspergillus section Nigri (21), A. flavus (7), A. terreus (6), and A. nidulans (6). In Aspergillus section Nigri, the number of A. niger was less than the number of A. welwitschiae and A. tubingensis. Two azole-resistant A. fumigatus samples were included among the isolates. Four of the eight A. tubingensis isolates showed less susceptibility to voriconazole; however, all isolates of A. niger and A. welwitschiae were susceptible to itraconazole and voriconazole. Because of lack of susceptibility data for non-fumigatus Aspergillus and an increasing frequency of antifungal resistance among A. fumigatus, our data along with further surveillance may contribute to determining the frequency and susceptibility of Aspergillus spp. clinical isolates in Japan., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We describe a case of a 23-year-old female patient with no apparent underlying diseases. She showed a discoloration of the proximal portion of the left big toenail with paronychia. Direct microscopy revealed septate hyphae with conidiophores, and a periodic acid-Schiff-stained nail specimen revealed septate hyphae branching at angles of approximately 45°. On the basis of phylogenetic analysis, we finally arrived at the diagnosis of ungual aspergillosis caused by Aspergillus subramanianii. After p.o. administration of terbinafine and topical application of 10% efinaconazole solution, the disease resolved in 6 months. A. subramanianii is one of the new species in the genus Aspergillus section Circumdati. Reported clinical isolates have been isolated from lung tissue, wounds and feet. This is the first documented case of onychomycosis caused by A. subramanianii. Onychomycosis due to Aspergillus species is uncommon. We summarized the reported cases of ungual aspergillosis in Japan., (© 2018 Japanese Dermatological Association.)

Cryptic species of Aspergillus fumigatus , including the Aspergillus viridinutans species complex, are increasingly reported to be causes of invasive aspergillosis. Their identification is clinically relevant, as these species frequently have intrinsic resistance to common antifungals. We evaluated the susceptibilities of 90 environmental and clinical isolates from the A. viridinutans species complex, identified by DNA sequencing of the calmodulin gene, to seven antifungals (voriconazole, posaconazole, itraconazole, amphotericin B, anidulafungin, micafungin, and caspofungin) using the reference European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. The majority of species demonstrated elevated MICs of voriconazole (geometric mean [GM] MIC, 4.46 mg/liter) and itraconazole (GM MIC, 9.85 mg/liter) and had variable susceptibility to amphotericin B (GM MIC, 2.5 mg/liter). Overall, the MICs of posaconazole and the minimum effective concentrations of echinocandins were low. The results obtained by the EUCAST method were compared with the results obtained with Sensititre YeastOne (YO) panels. Overall, there was 67% agreement (95% confidence interval [CI], 62 to 72%) between the results obtained by the EUCAST method and those obtained with YO panels when the results were read at 48 h and 82% agreement (95% CI, 78 to 86%) when the results were read at 72 h. There was a significant difference in agreement between antifungals; agreement was high for amphotericin B, voriconazole, and posaconazole (70 to 86% at 48 h and 88 to 93% at 72 h) but was very low for itraconazole (37% at 48 h and 57% at 72 h). The agreement was also variable between species, with the maximum agreement being observed for A. felis isolates (85 and 93% at 48 and 72 h, respectively). Elevated MICs of voriconazole and itraconazole were cross-correlated, but there was no correlation between the other azoles tested., (Copyright © 2018 American Society for Microbiology.)

Aspergillus niger and its related species, known as Aspergillus section Nigri , are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan, Aspergillus section Nigri is second most often isolated from clinical specimens following Aspergillus fumigatus We determined the species of Aspergillus section Nigri isolated in Japan by DNA sequencing of partial β-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20 Aspergillus niger , 59 Aspergillus welwitschiae , and 39 Aspergillus tubingensis strains. Drug susceptibility testing revealed 30 to 55% of A. niger , 6.8 to 18.6% of A. welwitschiae , and 79.5 to 89.7% of A. tubingensis isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for A. niger previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the cyp51A sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of A. niger and A. tubingensis ; however, none of them were confirmed to be associated with azole resistance. Three nonrelated A. welwitschiae isolates possessed a partial deletion in cyp51A , likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant A. tubingensis isolates showed higher expression of cyp51A than did susceptible strains. Our results show that cyp51A point mutations may have no association with azole resistance but that in some cases the overexpression of cyp51A may lead to the azole resistance in these species., (Copyright © 2017 American Society for Microbiology.)

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu 393 , Phe 397 , Phe 415 , and His 440 ) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes ) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains., (Copyright © 2017 American Society for Microbiology.)

Background: Candida albicans (C. albicans) can become a pathogen causing superficial as well as life-threatening systemic infections, especially in immunocompromised patients. Many phenotypic attributes contribute to its capacity to colonize human organs. In our study, 93 C. albicans isolates from patients of various candidiasis in a hospital of China were surveyed. We aimed to investigate the white-opaque (WO) switching competence, drug sensitivity, and virulence of mating type-like (MTL) a/α isolates., Methods: Internal transcribed spacer (ITS) gene and the MTL configuration were detected in all the isolates by reverse transcription-polymerase chain reaction. White/opaque phenotype and doubling time of cell growth were determined. The minimum inhibitory concentrations of antifungal agent were measured using broth microdilution method., Results: Sixty-four isolates (69.6%) were classified to serotype A, 19 (20.6%) to serotype B, and 9 (9.8%) to serotype C. Moreover, phylogenetic analysis showed that these isolates were divided into four different subgroups of ITS genotypes. Most of our clinical isolates were MTL a/α type, while 6.8% remained MTL a or MTLα type. The frequency of opaque phenotype was 71.0% (66 isolates). Following the guidelines of Clinical and Laboratory Standards Institute M27-A3, all isolates were susceptible to caspofungin and a few (0.6-3.2%) of them showed resistance against amphotericin B, flucytosine, fluconazole, itraconazole, and voriconazole., Conclusions: From these analyses, there were comparatively more C. albicans strains classified into serotype B, and the frequency of opaque phase strains was significant in the clinical isolates from China. Genetic, phenotypic, or drug susceptibility patterns were not significantly different from previous studies. MTL a/α isolates could also undergo WO switching which facilitates their survival.

A 16-year-old boy with chronic granulomatous disease presented with pneumonia and rib osteomyelitis. Emericella nidulans var. echinulata was isolated from his sputum. After starting voriconazole, Rasamsonia piperina was isolated from the rib swelling. A combination therapy of voriconazole and micafungin effectively eradicated this invasive mixed-mold infection. In immunocompromised patients, a precise pathogenic diagnosis is clinically useful for administration of an appropriate treatment regimen.

We describe a 66-year-old woman who suffered from fungal keratitis after corneal transplantation. The causative organism was identified as Beauveria bassiana on the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was successfully treated with topical voriconazole (VRCZ) use only. We, hereby, present the first report of a case with B. bassiana fungal keratitis that responded to topical antifungal VRCZ treatment., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Sporotrichosis is a fungal infection caused by the Sporothrix species, which have distinct virulence profiles and geographic distributions. We performed a phylogenetic study in strains morphologically identified as Sporothrix schenckii from clinical specimens in Japan, which were preserved at the Medical Mycology Research Center, Chiba University. In addition, we examined the in vitro antifungal susceptibility and growth rate to evaluate their physiological features. Three hundred strains were examined using sequence analysis of the partial calmodulin gene, or polymerase chain reaction（PCR）method using newly designed species-specific primers; 291 strains were Sporothrix globosa and 9 strains were S. schenckii sensu stricto (in narrow sense, s. s.). S. globosa strains were further clustered into two subclades, and S. schenckii s. s. strains were divided into three subclades. In 38 strains of S. globosa for which antifungal profiles were determined, 4 strains (11%) showed high minimal inhibitory concentration (MIC) value for itraconazole. All tested strains of S. schenckii s. s. and S. globosa showed low sensitivity for amphotericin B. These antifungals are used for treatment of sporotrichosis when infection is severe. S. schenckii s. s. grew better than S. globosa; wherein S. globosa showed restricted growth at 35℃ and did not grow at 37℃. Our molecular data showed that S. globosa is the main causal agent of sporotrichosis in Japan. It is important to determine the antifungal profiles of each case, in addition to accurate species-level identification, to strategize the therapy for sporotrichosis.

5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the S. cerevisiae deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen-particularly arginine-metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for S. cerevisiae as a model system, further studies were conducted in the pathogenic yeast Candida glabrata. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in C. glabrata. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes CgFPS1 and CgFPS2, from C. glabrata, were identified as determinants of 5-flucytosine resistance. CgFPS1 and CgFPS2 were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in C. glabrata cells.

Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Eight eyes of 7 patients with fungal disease received intravenous injections of 150 to 300 mg micafungin, and samples of blood, cornea, retina-choroid, aqueous humor, and vitreous humor were collected. The micafungin levels in all collected samples exceeded the MICs; however, the levels in the vitreous and aqueous humors were lower. Our findings suggest that intravenous micafungin should be given in combination with intravitreal antifungal agents after vitrectomy in severe cases of intraocular fungal diseases.

We report here a case of fungal keratitis caused by Aspergillus viridinutans, a member of the genus which was found to have clinical and antifungal susceptibility characteristics distinct from the species it most closely resembles, Aspergillus fumigatus. A 26-year-old woman presented with contact lens-associated keratitis, the etiologic agent of which was initially slow growing and found to be resistant to amphotericin B and voriconazole. Therapeutic keratoplasty was performed and an isolate from a corneal scraping was identified as A. viridinutans through partial DNA sequencing of the β-tubulin gene, along with its slow growth 42°C. In contrast with A. fumigatus, A. viridinutans proved to be resistant to a wider variety of antifungal agents. To the best of our knowledge, this is the first case of keratitis caused by A. viridinutans. Clinical isolates of the genus Aspergillus include rarely encountered species such as A. viridinutans. These species may show clinical manifestations distinct from those of A. fumigatus, including clinical resistance to amphotericin B and voriconazole and relatively low susceptibility to other antifungal agents.

Two new diketopiperazine metabolites, novoamauromine (1) and ent-cycloechinulin (2) have been isolated from Aspergillus novofumigatus CBS117520. The structures of 1 and 2 were established on the basis of spectroscopic and chemical investigation, including a detailed comparison of the spectroscopic and physico-chemical data of amauromine (3) and cycloechinulin (4).

Antifungal susceptibility tests were performed for 38 Candida albicans strains isolated from oral cavities of 43 Uighurian AIDS patients. Results showed that six isolates were resistant to fluconazole; one showed low susceptibility. We attempted to examine these strains molecular-epidemiologically, but 25S rDNA genotyping was insufficient for their discrimination. To estimate whether the origins of resistant strains were identical, we developed a new combination method of C. albicans tandem repeating units (ALTS)/RFLP and micro-temperature gradient gel electrophoresis (micro-TGGE). This new method was able to distinguish all seven strains. A suspected nosocomial infection was ruled out.

In the course of searching for new antifungal agents, a new cyclic depsipeptide, eujavanicin A (1), was isolated from Eupenicillium javanicum as an antifungal agent against the human pathogenic filamentous fungus Aspergillus fumigatus. The structure of 1 was established by spectroscopic and chemical investigations. The absolute stereochemistry was elucidated by Marfey's method and by chiral HPLC analysis.

Silver has long been known to have an antimicrobial activity against bacteria and other microorganisms, and has been used as eating utensils, as dental fillings and so on. We developed a novel adduct, GX-95, of silver with nanometer-scale particles to peptidic hydrolysates from collagen. Antifungal activity of the adduct against pathogenic yeasts and filamentous fungi was examined in terms of minimal inhibitory concentrations (MICs). GX-95 was found to possess strong and broad antifungal activities against all fungi examined in the following MICs: 0.25 to 0.31 microg/ml for Candida albicans including resistant strains to fluconazole, itraconazole and flucytosine, 0.05 to 0.2 microg/ml for Cryptococcus neoformans strains, 0.025 to 0.4 microg/ml for Aspergillus fumigatus strains, 0.4 microg/ml for Trichophyton rubrum, and 0.05 microg/ml for Cladophialophora carrionii.

In the course of our research for new antifungal agents, two new meroterpenoids, penisimplicin A (1) and B (2), were isolated from Penicillium simplicissimum. The absolute structures of 1 and 2 were established by spectroscopic and chemical investigation. Penisimplicin A (1) and B (2) are rare examples of D-ring seco-meroterpenoids. Since compounds 1 and 2 showed no antifungal activities, the isolation of the compounds with antifungal activity will be attempted.

Two new decalin derivatives, eujavanoic acids A (1) and B (2), were isolated from Eupenicillium javanicum, along with several compactin derivatives. The structures of 1 and 2 were determined by spectroscopic methods and modified Mosher's method. The side chain (2-methylbutanoyloxy) and acid functionalities of compactin derivatives were necessary to show the antifungal activity.

The dichloromethane extract of Talaromyces convolutus cultivated on barley exhibited antifungal activity against Candida albicans. In the course of a search for the active compounds, four new tetramic acid derivatives, talaroconvolutins A (1), B (2), C (3), and D (4), were isolated along with ZG-1494alpha (5), and mitorubrin derivatives. The structures of talaroconvolutins A-D (1-4) were established on the basis of spectroscopic and chemical investigations and chemical correlations. The antifungal activity of the talaroconvolutins against the pathogenic fungi Aspergillus fumigates, Aspergillus niger, C. albicans, and Cryptococcus neoformans was determined.

Two novel antifungal antibiotics, PF1163A and B, were isolated from the fermentation broth of Penicillium sp. They were purified from the solid cultures of rice media using ethyl acetate extraction, silica gel and Sephadex LH-20 column chromatographies. PF1163A and B showed potent growth inhibitory activity against pathogenic fungal strain Candida albicans but did not show cytotoxic activity against mammalian cells. These compounds inhibited the ergosterol biosynthesis in Candida albicans.

We report the first case of subcutaneous hyalohyphomycosis caused by the genus Cephalotheca, which has not been reported to cause human infection. A 67-year-old immunocompetent farmer presented with a 10-year history of verrucous erythematous plaques on the right foot dorsum, great toe, heel and sole. Histopathology of the lesions revealed chronic granulomatous inflammation with numerous nonpigmented fungal spores in the dermis. Cultures of biopsy specimens on Sabouraud's dextrose agar for 2 weeks developed into yellowish brown, velvety colonies that subsequently turned black after 8 weeks because of the production of black cleistothecia indicating the teleomorph. On the basis of mycological features, scanning electron microscopic morphology and molecular analysis data, a new species of Cephalotheca was identified and designated Cephalotheca foveolata. Because there was no response to the antifungal agents administered, the patient was successfully treated by surgical excision with skin graft. [ABSTRACT FROM AUTHOR]