C andida auris is an emerging, multidrug-resistant pathogen associated with a high mortality rate. Since this yeast's first identification and classification by our research group in 2009 (1), there have been several outbreaks linked to this pathogen in health care facilities around the world (2-10). It has been reported that most clinical isolates are resistant to azoles, and about half of the isolates also are resistant to more than one class of antifungal agent, limiting the therapeutic options (2-8, 10). Moreover, the pathogen can persist on environmental surfaces for weeks, resulting in the yeast's spread among patients in health care facilities (11). Therefore, accurate identification of C. auris is critical for controlling this pathogen's prevalence around the globe and preventing further outbreaks. Traditional methods have proven to be unsuitable for accurate identification of C. auris. Automated identification systems popularly used in clinical laboratories, like the Vitek 2 YST card (bioMérieux, Marcy I'Etoile, France) or API20C AUX (bioMérieux), commonly misidentify C. auris as Candida haemulonii or Rhodotorula glutinis, respectively (2, 4-7, 12), and MicroScan misidentifies C. auris as any of several different Candida species (12). On the other hand, specialized methods can provide accurate identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is useful for identifying C. auris, if a proper reference database is available (13-15). Moreover, sequencing of the genes for the D1/D2 region of large subunit ribosomal DNA (rDNA) or of the internal transcribed spacer (ITS) region of rDNA is a reliable option. Real-time PCR assays also are useful for detection of C. auris (16, 17). However, these methods may not be suitable for local or small clinical settings due to financial and technical issues. As shown in the present study, we have successfully devised and assessed the reliability of a loop-mediated isothermal amplification (LAMP)-based identification approach specific to C. auris, enabling distinction of the pathogen from closely related species and other fungi. To design the LAMP primers, the genome sequences of four Candida species, C. auris (PRJNA342691), C. tropicalis (GCF_000006335.2), C. albicans (GCA_000182965.3), and C. lusitaniae (LYUB00000000.2), were aligned and compared using Mauve (version 20150226) (18). An 869-bp DNA fragment of the C. auris genome (accession no. XM_018317007) that encodes a pyruvate:ferredoxin oxidoreductase domain (19) was identified as sharing low similarity with other Candida species. This DNA fragment was amplified using EmeraldAmp PCR master mix (TaKaRa Bio, Inc.

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.

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI-TOF-MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1-month-old infants using this technique. A Malassezia species-specific MALDI-TOF-MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1-month-old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI-TOF-MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI-TOF-MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia.

Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

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