Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Fabrication of a BOC-Protected 2-Hydroxyethyl Methacrylate Brush and Deprotection of the BOC Group to Control the Surface Hydrophilicity.

Fabrication of functional surfaces with designed patterns of different hydrophilicity has potential applications in active control of water droplets and water harvesting. For practical applications, the fabrication process needs to be applied to a large area in a cost-effective manner. Herein, we report the fabrication of a polymer brush of 2-(tert-butoxycarbonyloxy)ethyl methacrylate having a BOC-protected hydroxy group. The deprotection of the BOC group converts poly(2-(tert-butoxycarbonyloxy)ethyl methacrylate) (PBHEMA) into poly(2-hydroxyethyl methacrylate) (PHEMA) and hence changes the hydrophilicity. The chemical transformation changes the refractive index and thickness of the brush. This simple chemistry enables easy formation of the boundary of different hydrophilicity. Last, we demonstrate that the shape of the water droplet can be manipulated on the designed surface having different hydrophilicity.

Kerion Celsi due to Microsporum canis in an Adult Woman, Treated Successfully with Fosravuconazole.

A 64-year-old woman presented with a fist-sized, severely painful lesion with scales, crusts, pustules, erythema with subcutaneous abscess, and hair loss on the left temporal region. Direct microscopic examination revealed a large number of spores around the hair, which indicated ectothrix hair invasion, and some hyphae were also found. Histopathological examination showed significant inflammatory cell infiltration from the dermis to the subcutaneous tissues and into the hair follicles, destruction of the hair follicles with granulomatous reactions, and fungal masses along the hair within the hair follicles. Microsporum canis was identified based on morphological features via culture method and molecular biological analysis of the internal transcribed spacer region DNA sequence. The patient was diagnosed with kerion celsi caused by M. canis. For treatment of kerion celsi, we chose an oral antifungal agent, fosravuconazole (FRVCZ), which has been available since 2018 only in Japan. Clinical symptoms were cured in 12 weeks without scarring. No side effects were observed during oral administration of FRVCZ. The results of our case and several previous reports suggest that FRVCZ is effective in treating various types of dermatomycoses.

Changes in the mRNA expression of glycolysis-related enzymes of Candida albicans during inhibition of intramitochondrial catabolism under anaerobic condition.

Candida albicans can cause two major types of infections: superficial infection and systemic candidiasis. C. albicans infects diverse host niches, owing to a wide range of virulence factors and attributes, such as morphological transitions and phenotypic switching. C. albicans uses glycolysis, followed by alcoholic fermentation or mitochondrial respiration to rapidly generate ATP under aerobic conditions. In this study, we quantified the mRNA expression of several glycolysis-related enzymes associated with the initial phase of environmental changes using two strains: a type strain, NBRC 1385, and a strain from a patient with auto-brewery syndrome, LSEM 550. Additionally, we analyzed the regulation of a rate-limiting enzyme in glycolysis, phosphofructokinase 1 (PFK1). Our results showed that the mRNA expression of enzymes in the middle and last stages of glycolysis and alcoholic fermentation increased, and that of mitochondrial respiration enzymes decreased under short-term anaerobic conditions. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) administration showed similar results under anaerobic conditions. Moreover, PFK1 maintained its regulatory effect under different conditions; no significant change was observed in its mRNA expression. Our results suggest that C. albicans obtains energy via carbohydrate catabolism in the early phase of environmental change and survives in various parts of the host.

Begerowomyces aurantius sp. nov., a novel yeast isolated from koalas' habitat in a Japanese zoological park.

Koalas are iconic mammals indigenous to Australia. These rare animals and their habitats are occasionally associated with pathogenic fungi, including species of Cryptococcus, and consequently, monitoring the mycobiota of areas inhabited by koalas is of considerable importance. In this report, we describe a novel basidiomycetous yeast isolated from a site in Kanazawa Zoo, Japan, associated with captive koalas. Swab samples were collected from koala breeding environments, from which we isolated a novel unencapsulated yeast characterized by ovoid to ellipsoidal cells (3.2-4.9 × 3.5-5 µm). These cells were observed to undergo polar budding and grow as parent bud pairs, with an optimal growth temperature of 28°C. Colonies grown on yeast extract peptone dextrose agar at 28°C have a characteristic coral pink color. On the basis of physiological, morphological, and molecular characters, the new species was placed in the genus Begerowomyces, and the name Begerowomyces aurantius JCM33898T(LSEM1333T=CBS16241T) is proposed.

Controlling the thermal conductivity of multilayer graphene by strain.

Straintronics is a new concept to enhance electronic device performances by strain for next-generation information sensors and energy-saving technologies. The lattice deformation in graphene can modulate the thermal conductivity because phonons are the main heat carriers. However, the device fabrication process affects graphene's heat transport properties due to its high stretchability. This study experimentally investigates the change in the thermal conductivity when biaxial tensile strain is applied to graphene. To eliminate non-strain factors, two mechanisms are considered: pressure-induced and electrostatic attraction-induced strain. Raman spectroscopy and atomic force microscopy precisely estimate the strain. The thermal conductivity of graphene decreases by approximately 70% with a strain of only 0.1%. Such thermal conductivity controllability paves the way for applying graphene as high-efficiency thermal switches and diodes in future thermal management devices.

Seven years of progress in determining fungal diversity and characterization of fungi isolated from the Japanese Experiment Module KIBO, International Space Station.

The International Space Station (ISS) is a closed facility that orbits the earth carrying not only its crew but also microorganisms. We have participated in microbiota analysis projects for the Japanese Experiment Module KIBO (ISS; operations nomenclature: Microbe-I, II, III, and IV) and were in charge of fungal screening. The interior of KIBO was sampled using swabs and microbe detection sheets (MDSs) for fungal detection. The dominant genera obtained by culture were Aspergillus and Penicillium. DNA analyses of the fungal biota using a clone library showed that KIBO was dominated by Malassezia, a fungal inhabitant of human skin. Three fungal species, Aspergillus sydowii, Penicillium palitans, and Rhodotorula mucilaginosa, which grew under microgravity in KIBO were observed under a field emission-scanning electron microscope on the ground. No novel phenotypic characteristics were noted. The results of antifungal susceptibility testing of all isolates did not differ significantly from previous reports of corresponding fungi. In Microbe-I (August 2009), MDSs were culture negative, while in the next stages the CFU of MDSs were 10 for Microbe-II (February 2011), 24 for Microbe-III (October 2012), and 151 for Microbe-IV (February 2015). These results indicated that fungi inside KIBO are increasing and expanding over time, and therefore continuous surveillance is crucial.

Identification of fungi isolated from astronaut nasal and pharyngeal smears and saliva.

As part of a series of studies regarding the microbiota in manned space environments, we isolated the fungal strains from nasal and pharyngeal smears and saliva of 21 astronauts preflight, in-flight, and postflight. On the ground, 120 strains from 43 genera of environmental fungi were isolated from the astronauts. The dominant fungal genera were Cladosporium, Penicillium, and Aspergillus. Only 18 strains from four genera were isolated from the astronauts inside the International Space Station. These fungi are currently thought to be harmless, but regular screening and cleaning are necessary to prevent fungus-related health disorders.

Investigation of the Physiological, Biochemical and Antifungal Susceptibility Properties of Candida auris.

BACKGROUND: Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences. OBJECTIVES: To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus. METHODS: Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied. RESULTS: The optimal growth temperatures of all strains were 35-37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to > 64 µg/mL) and miconazole (MIC 8 to > 16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1-4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades. CONCLUSION: Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

Isolation and molecular characterization of clinical and environmental dematiaceous fungi and relatives from Iran.

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran. Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA (rDNA) regions were sequenced and analyzed. Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum, Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0-59, 0-22, 0-4, 0-4, 0-3, 0-2, 0-2, 0-2, 0-2, 0-1, and 0-1 nucleotide, respectively. Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Fast calculation method for parabolic-mirror-reflection holographic 3D display using wavefront segmentation.

Convex-parabolic-mirror reflection enables a very wide viewing zone in a holographic three-dimensional (3D) display. In this work, segmentation is introduced to reduce the calculation time of holograms in a convex-parabolic-mirror-reflection holographic 3D display. Wavefront segmentation can practically limit the lateral spread of the wavefront to be considered, which enables the application of geometrical approximation and conventional diffraction theories such as Fresnel diffraction. Thus, diffraction calculation via the convex parabolic mirror can be derived analytically and calculated rapidly using fast Fourier transform (FFT). Our proposed FFT-based method can calculate the diffraction integral 7000 times faster than our previous method, which involved calculating directly the diffraction integral without FFT. In addition, numerical simulation and an optical experiment are presented to verify our proposal.

DNA topoisomerase 2 gene polymorphism in dermatophytes.

BACKGROUND: Dermatophytes are a group of keratinophilic fungi of medical importance. Despite a relatively long history of molecular taxonomic studies, there is still a need for information on genetic polymorphism in wider variety of genomic loci. OBJECTIVES: Our goal was to study partial DNA topoisomerase 2 gene (TOP2) polymorphism in dermatophytes. METHODS: We performed DNA sequencing of TOP2 in 26 dermatophyte species along with ribosomal internal transcribed spacer (ITS) sequencing. RESULTS: The number of polymorphic sites in TOP2 data set was similar to that one in ITS data set. Nannizzia species formed paraphyletic group in TOP2 tree. Trichophyton simii was paraphyletic in concatenated TOP2-ITS tree, one of its two clades contained solely Iranian isolates. CONCLUSIONS: Our results revealed several unresolved problems in the taxonomy of dermatophytes, including probable polyphyly of the genus Nannizzia and the species T simii.

Real-time interactive holographic 3D display with a 360° horizontal viewing zone.

To realize a real-time interactive holographic three-dimensional (3D) display system, we synthesize a set of 24 full high-definition (HD) binary computer-generated holograms (CGHs) based on a 3D fast-Fourier-transform-based approach. These 24 CGHs are streamed into a digital micromirror device (DMD) as a single 24-bit image at 60 Hz: 1440 CGHs are synthesized in less than a second. Continual updates of the CGHs displayed on the DMD and synchronization with a rotating mirror enlarges the horizontal viewing zone to 360° using a time-division approach. We successfully demonstrate interactive manipulation, such as object rotation, rendering mode switching, and threshold value alteration, for a medical dataset of a human head obtained by X-ray computed tomography.

Specific detection of Trichophyton rubrum and Trichophyton interdigitale based on loop-mediated isothermal amplification (LAMP) from onychomycosis specimens.

In diagnosing onychomycosis, diseases with similar features must be excluded by demonstrating the presence of fungal infection and identifying the fungal species. However, fungal culture of onychomycosis-derived samples usually takes many weeks to yield species identification results, and is associated with a low successful culture rate. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and specific molecular biological method that can amplify DNA at a constant temperature, allowing for a simpler testing procedure, shorter detection time and less cost than conventional techniques including quantitative polymerase chain reaction. We have developed a new LAMP method specifically to detect Trichophyton rubrum (T. rubrum) and Trichophyton interdigitale (T. interdigitale), major causative dermatophytes for onychomycosis, and analyzed the correlation between LAMP results and those of the existing fungal culture method for the detection and identification of Trichophyton species from onychomycosis-derived samples. The results showed that all 59 specimens in which T. rubrum or T. interdigitale was identified by fungal culture also tested positive by LAMP, giving a 100% positivity concordance rate between the two methods. Moreover, all 55 and four specimens in which T. rubrum and T. interdigitale were identified by fungal culture, respectively, also tested positive for each species by LAMP, again giving a 100% species-identification concordance rate. The high correlation demonstrated between LAMP and fungal culture results in detection and identification of Trichophyton species from onychomycosis-derived samples suggests high reliability of LAMP as a promising, alternative mycological detection and identification technique which can serve as an alternative to the fungal culture method.

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi, Japan.

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates' partial ß-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

Epidemiological survey of onychomycosis pathogens in Japan by real-time PCR.

In Japan, an epidemiological survey of onychomycosis pathogens was performed using culture methods; however, the positive culture rate was 40% or less. As part of an epidemiological survey of dermatomycoses in Japan, we overcame this low positive rate by employing a real-time polymerase chain reaction (PCR) assay that allowed rapid and accurate detection and identification. In 2011, nail specimens were collected from patients at nine institutes in various prefectures in Japan and diagnosed as onychomycosis. For the detection and identification of the main pathogens causing onychomycosis, we performed real-time PCR using specific TaqMan® MGB probes and primer sets. Of the 496 onychomycosis samples, real-time PCR detected 382 cases (77.0%) caused by Trichophyton rubrum; 74 cases (15.0%) caused by Trichophyton interdigitale; and eight cases (1.6%) caused by Candida albicans. The real-time PCR positive rate was 96.2%. The most frequent pathogen was T. rubrum throughout life, with the number of patients affected peaking in the range of 60 to 69 years of age and no significant differences in the composition of causative pathogens by sex. We were able to detect and identify pathogens from almost all specimens and succeeded in analyzing the pathogens involved in onychomycosis cases in Japan. These data confirmed that our real-time PCR method was effective for detecting and identifying the main fungal pathogens from onychomycosis specimens.

Super-wide viewing-zone holographic 3D display using a convex parabolic mirror.

To enlarge both horizontal (azimuthal) and vertical (zenithal) viewing zones simultaneously, a convex parabolic mirror is placed after passing through the hologram. Viewers perceive a three-dimensional (3D) object inside the parabolic mirror as a virtual image by capturing the wavefront radially reflected from the parabolic mirror. The optical experiment using the convex parabolic mirror has demonstrated an extremely wide viewing zone with an azimuthal range of 180° and zenithal range of 90°. The viewing zone and the shape of the parabolic surface are analyzed. The hologram is designed considering the parabolic mirror reflection, and its diffraction calculation method based on Fermat's principle is also proposed.

In Vitro Antifungal Activity of Novel Triazole Efinaconazole and Five Comparators against Dermatophyte Isolates.

The objective of this study was to assess the in vitro activity of the novel triazole antifungal drug, efinaconazole, and five comparators (luliconazole, lanoconazole, terbinafine, itraconazole, and fluconazole) against a large collection of Trichophyton interdigitale and Trichophyton rubrum clinical isolates. The geometric mean MICs were the lowest for luliconazole (0.0005 µg/ml), followed by lanoconazole (0.002 µg/ml), efinaconazole (0.007 µg/ml), terbinafine (0.011 µg/ml), itraconazole (0.095 µg/ml), and fluconazole (12.77 µg/ml). It appears that efinaconazole, lanoconazole, and luliconazole are promising candidates for the treatment of dermatophytosis due to T. interdigitale and T. rubrum.

Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.