矢口 貴志

We hereby make the first report of a case of mycosis caused by Purpureocillium lilacinum in CARD9 deficiency. A 40-year-old woman complained of lymph node swellings in the left cervical area. She also had chronic mucocutaneous candidiasis (CMC), and was found to have CARD9 deficiency. Lymphadenitis by P. lilacinum was confirmed. The diagnosis was difficult, as culturing the biopsy specimen at a cautiously selected temperature (25 °C) and genetic analysis were both required. Oral administration of voriconazole improved her lymphadenopathy.

BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

Blastomycosis is a fungal infectious disease that can occur in both immunocompromised and immunocompetent populations endemic in North America, with no previous reports in Japan. A 26-year-old Japanese female patient with no relevant medical history presented intermittent left back pain and an abnormal shadow in the left upper lung field eight months ago at a local clinic. She was referred to our hospital for further evaluation and treatment. The patient currently lives in Japan, but until two years ago had spent several years in New York, Vermont and California. Chest computed tomography revealed a 30 mm mass with a cavity in the left pulmonary apex. The specimens obtained by transbronchial biopsy showed periodic acid-Schiff stain (PAS)-positive and Grocott-positive yeast-like fungi scattered among the granulomas, with no malignant findings, and the initial pathology did not lead to a definitive diagnosis. She was empirically started on fluconazole because of onset of multiple subcutaneous abscesses and was referred to the Medical Mycology Research Center. Although antibody tests could not diagnose the disease, blastomycosis was suspected based on the pathology of the skin and lung tissue at the Medical Mycology Research Center, and Blastomyces dermatitidis was identified by ITS analysis of the rRNA region. Her symptoms and CT findings gradually improved with fluconazole. We reported the first Japanese case of blastomycosis with pulmonary and cutaneous involvement in Japan. As the number of overseas travelers is expected to continue increasing, we would like to emphasize the importance of travel history interviews and information of blastomycosis.

Aspergillus lacticoffeatus WU-2020 is a citric acid hyperproducer that is suitable for solid culture. Here, we present a high-quality draft of its genome sequence (35.9 Mb), which consists of 11 scaffolds and contains 11,490 genes. We also present the mitochondrial genome, which is 31.3 kb in length.

The Aspergillus series Nigri contains biotechnologically and medically important species. They can produce hazardous mycotoxins, which is relevant due to the frequent occurrence of these species on foodstuffs and in the indoor environment. The taxonomy of the series has undergone numerous rearrangements, and currently, there are 14 species accepted in the series, most of which are considered cryptic. Species-level identifications are, however, problematic or impossible for many isolates even when using DNA sequencing or MALDI-TOF mass spectrometry, indicating a possible problem in the definition of species limits or the presence of undescribed species diversity. To re-examine the species boundaries, we collected DNA sequences from three phylogenetic markers (benA, CaM and RPB2) for 276 strains from series Nigri and generated 18 new whole-genome sequences. With the three- gene dataset, we employed phylogenetic methods based on the multispecies coalescence model, including four single-locus methods (GMYC, bGMYC, PTP and bPTP) and one multilocus method (STACEY). From a total of 15 methods and their various settings, 11 supported the recognition of only three species corresponding to the three main phylogenetic lineages: A. niger, A. tubingensis and A. brasiliensis. Similarly, recognition of these three species was supported by the GCPSR approach (Genealogical Concordance Phylogenetic Species Recognition) and analysis in DELINEATE software. We also showed that the phylogeny based on benA, CaM and RPB2 is suboptimal and displays significant differences from a phylogeny constructed using 5 752 single-copy orthologous proteins; therefore, the results of the delimitation methods may be subject to a higher than usual level of uncertainty. To overcome this, we randomly selected 200 genes from these genomes and performed ten independent STACEY analyses, each with 20 genes. All analyses supported the recognition of only one species in the A. niger and A. brasiliensis lineages, while one to four species were inconsistently delimited in the A. tubingensis lineage. After considering all of these results and their practical implications, we propose that the revised series Nigri includes six species: A. brasiliensis, A. eucalypticola, A. luchuensis (syn . A. piperis), A. niger (syn. A. vinaceus and A. welwitschiae), A. tubingensis (syn. A. chiangmaiensis, A. costaricensis, A. neoniger and A. pseudopiperis) and A. vadensis. We also showed that the intraspecific genetic variability in the redefined A. niger and A. tubingensis does not deviate from that commonly found in other aspergilli. We supplemented the study with a list of accepted species, synonyms and unresolved names, some of which may threaten the stability of the current taxonomy.

PURPOSE: To investigate the clinical characteristics and causative fungi in patients with fungal keratitis in Japan, and to determine factors related to the prognosis. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eligible patients were enrolled from November 2011 to October 2013 at the 1st stage and from April 2015 to March 2016 at the 2nd stage. The corneal foci were scraped, and the scrapings were cultured in potato dextrose agar. The isolated fungi were identified by gene analyses. Data were collected from the clinical records and statistically analyzed by Cox and logistic regression analyses. RESULTS: Ninety-four fungal strains were isolated from 93 cases, including 42 yeast-like fungi and 52 filamentous fungi. The fungi affected the deep layers of the cornea in 23 cases (24.7%) and the peripheral cornea in 29 cases (31.2%). The incidences of lid swelling/redness, ciliary injection, anterior chamber cells/flare, anterior chamber fibrin, and hyphate ulcer in cases of filamentous fungi were significantly higher than in yeast-like fungi. No history of topical steroids, absence of a main lesion in the peripheral cornea, and best-corrected visual acuity (BCVA) of more than 0.04 at the first visit were related to a shorter healing time. No history of ocular surgery, absence of lesion at one-third deep stromal layer and BCVA of more than 0.04 at the first visit were correlated with BCVA at 3 months after the initial examination. CONCLUSION: Fungal keratitis is caused by various species of fungi and can become refractory due to poor prognosis factors.

PURPOSE: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI). RESULTS: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles. CONCLUSIONS: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains.

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA.

We encountered two cases of phaeohyphomycosis caused by Exophiala jeanselmei and E. oligosperma that were treated with fosravuconazole and terbinafine, respectively. Our cases were successfully treated with empiric therapy before the pathogen's species or antifungal sensitivity had been determined. We summarized 32 cases of cutaneous and subcutaneous phaeohyphomycosis caused by Exophiala species in Japan. The patients received antifungals, including itraconazole, terbinafine, voriconazole, and fosravuconazole, and the treatment success rates of these monotherapies were 77% (17/22), 67% (8/12), 100% (5/5), and 50% (1/2), respectively. Although the broad-spectrum azole antifungal itraconazole is the first choice for treatment, terbinafine at 125 mg/day might exert the same efficacy. Fosravuconazole is a novel broad-spectrum azole and a moderate inhibitor of Cyp3A4 that causes fewer drug interactions than itraconazole and voriconazole, indicating a promising drug for this disease.

In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.

Purpureocillium lilacinum has been recently found to contaminate a 20% (200,000 μg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB) . We aimed to elucidate the mechanism underlying the resistance of P. lilacinum to PHMB. First, we induced the PHMB-resistant (IR) strains IFM 67050 (IR) and IFM 65838 (IR) from the type strain P. lilacinum CBS 284.36T via cultivation in a medium containing high concentrations of PHMB. We then analyzed the DNA sequences via Illumina sequencing to evaluate the presence of genetic mutations in IFM 65838 (IR) . Further, we established an IFM 65838 (IR) uridine/uracil auxotrophic strain, and using the orotidine-5'-decarboxylase gene, pyrG as a selection marker, we tried to knockout a mutant gene in IFM 65838 (IR) using the CRISPR-Cas9 genome-editing technique. The growth rates of IFM 67050 (IR) and IFM 65838 (IR) in medium containing PHMB increased, and the minimum inhibitory concentrations (MICs) against PHMB also increased. Based on the DNA sequence analysis, we found a nonsynonymous point mutation in the gene PLI-008146 (G779A) in IFM 67050 (IR) and IFM 65838 (IR) . This point mutation leads to site combinations of splicing changes that cause partial sequences deletion (p.Y251_G281del) in the ΔPLI-008146 locus of IFM 65838 (IR) , and deletion sequences include partial adenosine/AMP deaminase motif (PF00962) orthologous to adenosine deaminase (ADA) (GeneBank: OAQ82383.1) . Furthermore, the mutant gene ΔPLI-008146 was successfully knocked out from the resistanceinduced strain using a novel CRISPR-Cas9 gene transformation method. A considerable reduction in growth rate and MIC against PHMB was observed in the absence of the mutant gene. Therefore, ADA may represent an important resistance factor in PHMB-resistant P. lilacinum.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and also occur in animal intestines; occasionally, infections in humans and animals are observed. In this study, we conducted tests of assimilative abilities and thermotolerance in comparison with morphological characteristics of six opportunistic species (Prototheca blaschkeae, Prototheca bovis, Prototheca ciferrii, Prototheca cutis, Prototheca miyajii, and Prototheca wickerhamii) along with Prototheca paracutis. Five of the seven species could be differentiated by physiological characteristics, but P. wickerhamii and P. cutis had identical profiles. Of the cattle-associated species, only P. bovis was able to grow at 42°C. Both type strains of P. cutis and P. miyajii were most susceptible to ravuconazole compared with the other azoles.

Fludioxonil and iprodione are effective fungicides widely used for crop protection and are essential for controlling plant pathogenic fungi. The emergence of fungicide-resistant strains of targeted pathogens is regularly monitored, and several cases have been reported. Non-targeted fungi may also be exposed to the fungicide residues in agricultural fields. However, there are no comprehensive reports on fungicide-resistant strains of non-targeted fungi. Here, we surveyed 99 strains, representing 12 Penicillium species, that were isolated from a variety of environments, including foods, dead bodies, and clinical samples. Among the Penicillium strains, including non-pathogenic P. chrysogenum and P. camembertii, as well as postharvest pathogens P. expansum and P. digitatum, 14 and 20 showed resistance to fludioxonil and iprodione, respectively, and 6 showed multi-drug resistance to the fungicides. Sequence analyses revealed that some strains of P. chrysogenum and Penicillium oxalicum had mutations in NikA, a group III histidine kinase of the high-osmolarity glycerol pathway, which is the mode of action for fludioxonil and iprodione. The single nucleotide polymorphisms of G693D and T1318P in P. chrysogenum and T960S in P. oxalicum were only present in the fludioxonil- or iprodione-resistant strains. These strains also exhibited resistance to pyrrolnitrin, which is the lead compound in fludioxonil and is naturally produced by some Pseudomonas species. This study demonstrated that non-targeted Penicillium strains distributed throughout the environment possess fungicide resistance.

This study was designed to evaluate the prevalence of antifungal resistance, genetic mechanisms associated with in vitro induction of azole and echinocandin resistance and genotyping of Candida krusei, which is intrinsically resistant to fluconazole and is recovered from clinical and non-clinical sources from different countries. Our results indicated that all the isolates were susceptible or had the wild phenotype (WT) to azoles, amphotericin B, and only 1.27% showed non-WT for flucytosine. Although 70.88% of the isolates were resistant to caspofungin, none of them were categorized as echinocandin-resistant as all were susceptible to micafungin and no FKS1 hotspot 1 (HS1) or HS2 mutations were detected. In vitro induction of azole and echinocandin resistance confirmed the rapid development of resistance at low concentrations of fluconazole (4 μg/ml), voriconazole (0.06 μg/ml) and micafungin (0.03 μg/ml), with no difference between clinical and non-clinical isolates in the resistance development. Overexpression of ABC1 gene and FKS1 HS1 mutations were the major mechanisms responsible for azole and echinocandin resistance, respectively. Genotyping of our 79 isolates coupled with 217 other isolates from different sources and geography confirmed that the isolates belong to two main subpopulations, with isolates from human clinical material and Asia being more predominant in cluster 1, and environmental and animals isolates and those from Europe in cluster 2. Our results are of critical concern, since realizing that the C. krusei resistance mechanisms and their genotyping are crucial for guiding specific therapy and for exploring the potential infection source.

A 69-year-old woman who had undergone renal transplantation and was receiving sulfamethoxazole/trimethoprim (ST) developed pulmonary nocardiosis. To our knowledge, this is the first report of the identification of Nocardia elegans using nanopore sequencing, supported by 16S rDNA capillary sequencing findings. Chest computed tomography performed after ST initiation revealed significant improvement of the pulmonary shadows compared to previous findings. We herein report the value of nanopore sequencing for rapid identification of rare pathogens, such as Nocardia elegans. Furthermore, our findings suggest that Nocardia may infect even patients receiving ST, which is currently the most effective prophylactic drug.

BACKGROUND: Schizophyllum commune is a basidiomycete that lives in the environment and can cause infections, mainly those of the respiratory system. Although S. commune is increasingly reported as a cause of allergic bronchopulmonary mycosis and sinusitis, cases of fungal ball formation are extremely uncommon. Identification of S. commune is difficult using routine mycological diagnostic methods, and in clinically suspicious cases, internal transcribed spacer sequencing should be used for diagnosis. Here, we report a first case of lung cancer with a fungal ball formation of S. commune, confirmed by analyzing the internal transcribed spacer. CASE PRESENTATION: A 76-year-old man with diabetes and hypertension was admitted to the hospital with a chief complaint of hemosputum, which he had for about 19 months. A computed tomography image of the patient's chest showed a cavity and internal nodule in the left upper lobe of his lung. A left upper lobectomy was performed, and histopathological examination revealed squamous cell carcinoma of the lung and a fungal ball. The isolate from the surgical specimen was identified as S. commune by analyzing the internal transcribed spacer. The patient had no recurrence of the infection during 5 months of follow-up. CONCLUSIONS: Only three cases of lung fungal balls caused by S. commune have been previously reported, and this is the first case of lung cancer cavity with a fungal ball formation. In cases of fungal ball formation in the lung, S. commune should be considered a possible causative microorganism.

BACKGROUND: Exophiala (Wangiella) dermatitidis is a clinically relevant black yeast. Although E. dermatitidis rarely causes human infection, it can cause superficial and deep-seated infections, and cutaneous and subcutaneous diseases. Cases of fungemia and central line-associated bloodstream infections due to E. dermatitidis are extremely uncommon, and their clinical manifestations and prognosis are still not well-known. Herein, we report a case of central line-associated bloodstream infections in a patient with cancer. These infections were caused by melanized yeast that was finally identified as E. dermatitidis via internal transcribed spacer sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CASE PRESENTATION: A 75-year-old man with thoracic esophageal cancer and early gastric cancer presented with a 1-day history of fever during his hospitalization at our hospital. A central venous port was placed in the patient for total parenteral nutrition. Two E. dermatitidis isolates were recovered from two blood samples drawn at different times from a peripheral vein and this central venous port. The isolate was identified as E. dermatitidis by internal transcribed spacer sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The central venous port was removed, and the patient was administered micafungin and voriconazole. Although the minimum inhibitory concentrations of E. dermatitidis for voriconazole and minimum effective concentrations for micafungin were 2 μg/mL and 4 μg/m, respectively, the bacteremia was successfully treated. CONCLUSIONS: Although no clear treatment guidelines have been proposed for E. dermatitidis infections, immediate removal of central venous catheters is the key to improving central line-associated bloodstream infections.

Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of <italic>Aspergillus</italic> section <italic>Fumigati</italic> (<italic>A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae</italic>, and <italic>Neosartorya fischeri</italic>). For expanding our knowledge, we newly sequenced genomes of <italic>A. viridinutans</italic> and <italic>A. pseudoviridinutans</italic>, and reassembled and reannotated the previously released genomes of <italic>A. lentulus</italic> and <italic>A. udagawae</italic>. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7–51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using <italic>A. fumigatus, A. lentulus, A. udagawae, A. viridinutans</italic>, and <italic>N. fischeri</italic> was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32–83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

We present a 76-year-old Japanese male with tinea faciei, tinea corporis, and tinea unguium with dermatophytoma. We performed fungal culture and confirmed the causative fungus to be Trichophyton rubrum. We treated the patient using oral fosravuconazole l-lysine ethanolate (F-RVCZ). More than one year has passed since the end of treatment, but there has been no recurrence. This case suggests that F-RVCZ is effective for tinea other than tinea unguium.

Tinea imbricata and tinea pseudoimbricata are variant types of tinea corporis characterized by annual-ring-shaped erythema. Although the skin lesions manifest similar symptoms, these two diseases are classified based on causative fungi. The former is caused by Trichophyton concentricum, an anthropophilic dermatophyte, and the latter is caused by dermatophytes other than T. concentricum, commonly zoophilic fungi such as Trichophyton mentagrophytes complex. Here, we report a 27-year-old Japanese male diagnosed with tinea pseudoimbricata attributed to Trichophyton tonsurans, an anthropophilic dermatophyte. We suspected that application of steroid ointment caused the annular pattern of his skin lesions. After three months use of topical luliconazole cream, treatment was finished. We also summarize the knowledge about tinea pseudoimbricata through previous reports with bibliographical consideration.

A 57-year-old male patient with >10-year history of type 2 diabetes presented with a left big toenail deformity and pain. A physical examination revealed a white and yellow-to-brown patch on the nail as well as thickening and ingrowth of the nail plate. The nail plate was opened using nippers, and a fungal culture revealed Trichophyton interdigitale with yellow yeast. The yeast isolate was identified as Kocuria koreensis, a Gram-positive aerobic coccoid with keratinolytic properties that is part of the normal flora of the skin. We created an ex vivo onychomycosis model of T. interdigitale infection of the human nail by placing a sterilized normal nail on the cultured slant. K. koreensis initially spread over the normal nail, and T. interdigitale then penetrated the nail plate. After one year and six months, a spiral ingrown nail developed. A histopathological examination of the spiral revealed onychomycosis with superficial and deep abscesses of Gram-positive cocci infection. We performed PCR from paraffin-embedded material, and the sequences obtained were identical to those of T. interdigitale and K. koreensis. These results suggest that the development of onychomycosis by T. interdigitale is introduced and accelerated by K. koreensis, and the symbiosis of these microorganisms is suspected in the nail. This ex vivo model has a number of limitations. Therefore, further research on co-infected cases is needed to confirm this hypothesis.

We isolated a fungus from a 20% (= 200,000 µg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB), a widely used antimicrobial and examined its morphology and drug resistance profile. Based on the sequence of the internal transcribed spacer region of ribosomal DNA, the fungus was identified as Purpureocillium lilacinum. Although the P. lilacinum type and resistant strains showed similar morphology, the latter had extremely low PHMB susceptibility and was able to grow in 20% aqueous solution of PHMB, which eliminated the type strain. The minimum inhibitory concentration (MIC) of PHMB for the resistant strain was significantly higher than that of the type strain and other pathogenic filamentous fungi and yeasts. The susceptibility to antimicrobial agents and antifungal agents other than PHMB was similar to that of the type strain, therefore the drug resistance of the isolate was specific to PHMB. Furthermore, we sequenced the genome of the isolate to predict PHMB resistance-related genes. Despite its high resistance to PHMB, no well-known genes homologous to fungal PHMB-resistant genes were detected in the genome of the resistant strain. In summary, P. lilacinum was found to be significantly more resistant to PHMB than previously reported, via an unidentified mechanism of drug resistance.

[This corrects the article DOI: 10.1093/ve/veaa101.].

By identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation, we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42 per cent of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp.

The prominence of seafood in Japan motivates close monitoring of its seas and marine lives for potentially pathogenic fungi. During the treatments of the male Pacific white-sided dolphin (Lagenorhynchus obliquidens) for paracoccidioidomycosis ceti (PCM-C), 5 white and floccose colonies showing identical genotype and morphological characteristics were isolated from two skin biopsy samples of cutaneous granulomatous lesions in 2018. The isolates were identified as Parengyodontium album known as one of fungal species having abilities to produce industrially important proteases, and to become a causative agent for emerging mycosis based on morphological and molecular biological characteristics. These lesions consisted of non-malignant pearl-like structures of hyperplastic keratinocytes. Interestingly, although the isolates could grow at 35 °C, their DNA sequences were phylogenetically located in a cluster consisting of environmental and clinical isolates lacking the ability to grow at 35 °C, based on previous reports. The opportunistic infection we observed in the dolphin might be caused by immune disorder due to PCM-C. Notably, although P. album is recognized as non-harmful, and has significant industrial importance and antitumor activity, it has potential to cause not only superficial but also systemic infection, and presents difficulties in treatment because of its high resistance to antifungal compounds.

<title>Abstract</title> Lesions of adiaspiromycosis, a respiratory disease affecting wild animals, have been found mainly in dead mammals and free-living mammals captured for surveillance. No report has described an investigation of adiaspore formation progress in the lung. After establishing an experimental mouse model of intratracheal adiaspiromycosis infection with the causative agent <italic>Emmonsia crescens</italic>, we observed adiaspore development. The spores grew and reached a plateau of growth at 70 days post-infection. The median adiaspore diameter showed a plateau of around 40 μm. The characteristic three-layer cell-wall structure of adiaspores was observed in the lung at 70 days post-infection. We examined infection with a few spores, which revealed that adiaspores in the mouse lung progressed from intratracheal infection of at least 400 spores. Moreover, we developed adiaspores in vitro by culture in fetal bovine serum. Although most spores broke, some large spores were intact. They reached about 50 μm diameter. Thick cell walls and dense granules were found as common points between in vitro adiaspores and in vivo adiaspores. These models are expected to be useful for additional investigations of <italic>E. crescens</italic> adiaspores and adiaspiromycosis.

Aspergillus tubingensis WU-2223L, belonging to the section Nigri, is a hyperproducer of citric acid. Here, we present the high-quality draft (35 Mb) and mitochondrial (32.4 kb) genome sequences of this strain, which consisted of 16 scaffolds in total. The draft and mitochondrial genome sequences comprised 11,493 and 15 genes, respectively.

BACKGROUND: Subtenon injection of triamcinolone acetonide (STTA) has been widely adopted in the clinical setting of ophthalmology and its infectious complications are rare. However, orbital abscess following STTA has been reported in seven cases. Furthermore, although eye infections due to Exophiala species are uncommon, there have been 19 cases to date. E. jeanselmei, E. phaeomuriformis, E. werneckii, and E. dermatitidis have been reported to cause human eye infections; however, to the best of our knowledge, orbital abscess caused by E. dermatitidis has not yet been reported. We describe the first documented case of fungal orbital abscess caused by E. dermatitidis following STTA. We also review the related literature of orbital abscess following STTA, as well as eye infections caused by the four Exophiala species. CASE PRESENTATION: The patient was a 69-year-old Japanese woman with diabetic mellitus. She had a macular oedema in her right eye, which occurred secondary to branch retinal vein occlusion. An orbital abscess caused by E. dermatitidis occurred 4 months after the second STTA for the macular oedema, which was successfully treated by a surgical debridement and systemic administration of voriconazole. CONCLUSIONS: Our findings in the patient and from our literature survey caution ophthalmologists to the fact that STTA can cause fungal orbital infections, especially in diabetic patients. Furthermore, surgical treatment is one of the most important risk factors.