Two cases of BRAF-mutated, bulbar conjunctival melanoma, and review of the published literature.

We describe two patients with BRAF-mutated melanoma of the epithelioid cell type arising from primary acquired melanosis with severe atypia of the right bulbar conjunctiva. Patient 1 was a 71-year-old Japanese man. After adjuvant cryotherapy and enucleation of the right eyeball, therapy with vemurafenib was administered for a distant metastasis to a lumbar vertebra, accompanied by erythema multiforme and two keratinous tumours. The patient died due to metastases to the liver and multiple vertebrae, despite therapy with nivolumab and combination therapy with dabrafenib plus trametinib. Patient 2 was a 72-year-old Japanese man. After adjuvant cryotherapy, periodic mitomycin C eye drops, and excision of the superficial portion of the right parotid gland and the dissection of cervical lymph nodes, he was treated with adjuvant combination therapy with dabrafenib plus trametinib. Dermatologists should be familiar with BRAF-mutated conjunctival melanoma, which is usually located on the bulbar conjunctiva and associated with more frequent distant metastasis.

Molecular characterization of fungal populations on the tongue dorsum of institutionalized elderly adults.

OBJECTIVES: To characterize the global composition of oral fungal populations in frail elderly adults and to investigate the relationship with their health status. MATERIALS AND METHODS: We investigated the fungal populations on the tongue dorsum in 291 institutionalized elderly adults by molecular PCR-based techniques using internal transcribed spacer regions of nuclear ribosomal DNA. RESULTS: Quantitative PCR analysis showed that fungi were present on the tongue dorsum of 128 subjects at ≥10(4) CFU per sample, and 35 of them exceeded 10(5) CFU per sample. Length heterogeneity-PCR analysis and nucleotide sequence determinations showed that Candida albicans was most frequently detected in those subjects with fungi at ≥10(4) CFU per sample (105 subjects), followed by Candida dubliniensis (78), Malassezia restricta (57), and Candida tropicalis (45). Statistical analysis revealed that those subjects with ≥10(5) CFU of fungi other than C. albicans per sample had an increased risk of fever (≥7 febrile days per 12 months) compared with subjects with <10(5) CFU per sample, after adjustment for other fever-associated confounding factors. CONCLUSIONS: These data demonstrate that the oral cavity of the elderly is inhabited by a diverse array of fungi not limited to typical Candida species and they suggest that the diversity in distribution is associated with health status.

The first case of fingernail onychomycosis due to Neoscytalidium novaehollandiae, molecular identification and antifungal susceptibility.

Onychomycosis is considered a fungal nail infection caused mainly by dermatophytes, yeasts and non-dermatophyte molds including dematiaceous fungi. Onychomycosis caused by non-dermatophyte molds is a health problem in the medical environment as the patients frequently return to outpatient clinics seeking new therapeutic modalities. Here, we report the first case of onychomycosis caused by a black fungus, Neoscytalidium novaehollandiae, in the right hand finger nail of a 52-year-old Iranian female with no history of immunodeficiency and underlying disease. The pattern of nail involvement was recognized as total dystrophic onychomycosis. Examination of nail scrapings with potassium hydroxide revealed brown, septate and branching sub-hyaline to dark-colored hyphae. The black fungus isolated in culture was identified as Neoscytalidium novaehollandiae by molecular analysis. The patient received oral terbinafine plus ciclopirox nail lacquer twice a week and began responding to the treatment three months after initial antifungal therapy. Additional four weeks' use of terbinafine plus ciclopirox nail lacquer completely resolved the clinical manifestations of onychomycosis. After four months, both microscopy and culture were negative.

Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences.

BACKGROUND: Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture. OBJECTIVES: This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method. MATERIALS AND METHODS: The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum, T. mentagrophytes, Aspergillus spp., Scopulariopsis brevicaulis, Fusarium solani, F. oxysporum, F. verticillioides, Candida albicans and C. tropicalis were used after confirmation of their specificity. RESULTS: Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83.0%): T. rubrum in 35 cases (74.5%) and T. mentagrophytes in eight cases (17.0%). Surprisingly, nondermatophytes were detected in 18 cases (38.3%), both dermatophytes and nondermatophytes in 10 cases (21.3%) and nondermatophytes alone in eight cases (17.0%). Aspergillus spp. alone was observed in five cases (10.6%). CONCLUSIONS: This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.

Disseminated aspergillosis following resolution of Pneumocystis pneumonia with sustained elevation of beta-glucan in an Intensive Care Unit: a case report.

Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised patients receiving intensive care. The double-sandwich ELISA for galactomannan is reported to have a high sensitivity (96.5%) for the detection of invasive aspergillosis when a cut-off value of 0.8 ng/ml is used. However, we have experienced a case of lethal disseminated aspergillosis in a patient that presented with a negative galactomannan (GM) test and persistent elevation of beta-D glucan (BG) levels. A 63-year-old female was admitted to our Intensive Care Unit (ICU) in acute respiratory failure and elevated BG. She had been receiving medication for Good-pasture syndrome based on anti-glomerular basement membrane antibodies and myeloperoxidase-antineutrophil cytoplasmic antibodies for 9 months and was receiving long-term prednisolone therapy (20 mg/day). On admission, her trachea was immediately intubated, and a PCR analysis of the bronchoalveolar lavage sample revealed Pneumocystis jiroveci. Trimethoprimsulfamethoxazole therapy was started for Pneumocystis pneumonia. The levels of BG remained elevated (> 100 pg/ml) during the treatment period despite the clinical resolution of Pneumocystis pneumonia, raising concerns of another complicated invasive fungal disease; consequently, fosfluconazole was administered empirically. The serum BG levels, however, did not decrease. Blood cultures did not detect a fungal infection. Serum GM levels measured by a double-sandwich ELISA on the 6th, 11th, and 24th days in the ICU were negative (< 0.2 ng/ml). The patient ultimately died of multiple organ failure on the 45th ICU day. Postmortem examination revealed a disseminated fungal infection with aggressive vascular invasion of the lungs, heart, and brain. In situ hybridization with a 568-bp probe of the alkaline proteinase sequence of Aspergillus fumigatus showed specific positive staining within the fungus present in the infected lung tissue, revealing that this patient may have had a systemic infection by A. fumigatus or A. flavus. This is a case of serum GM-negative disseminated aspergillosis pathologically proven by autopsy. Persistent elevated BG levels (> 100 pg/ml) refractory to trimethoprim-sulfamethoxazole and fosfluconazole may suggest possible Aspergillus infection and should prompt the initiation of empiric anti-aspergillosis therapies in patients at risk for fungal infection.

Methods for identification of Candida auris, the yeast of global public health concern: A review.

Candida auris has recently emerged as a fungus able to cause severe infections, especially bloodstream infections with high mortality rates. This multi-drug-resistant yeast has the capacity of persistence on environmental surfaces, and has been reported to cause hospital-acquired infections. The development of faster and inexpensive tools for identification is critical to controlling, preventing and establishing early diagnosis of this emerging pathogen. Identification of C. auris by use of conventional laboratory methods is challenging, and it is commonly misidentified as other Candida species. Less expensive, reliable DNA-based tests have been used for identifying C. auris in environmental and clinical samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometry is also a useful tool for identification of cultured isolates. This review provides a succinct overview of the available methods for identification of C. auris with particular emphasis on their relative advantages and drawbacks.

Erratum for Mirhendi et al., the first case of onychomycosis in a koala (Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum, a diagnostic challenge.

[This corrects the article DOI: 10.18869/acadpub.cmm.2.2.2.].

Mucormycosis in Iran: A six-year retrospective experience.

Mucormycosis is a devastating infection caused by Mucoralean fungi (Mucormycotina, Mucorales). Data concerning the global epidemiology of mucormycosis are scarce and little is known about the characteristics of mucormycosis in Iran. In this study, we aimed to understand the distribution of this infection in Iran retrospectively and to ascertain whether the patterns of infection are associated with specific host factors or not. A total of 208 cases were included in this study occurring during 2008-2014 and were validated according to (EORTC/MSG) criteria. A rising trend as significant increase from 9.7% in 2008 to 23.7% in 2014 was observed. The majority of patients were female (51.4%) with median age of 50 and the infections were seen mostly in autumn season (39.4%). Diabetes mellitus (75.4%) was the most common underlying condition and sinus involvement (86%) was the mostly affected site of infection. Amphotericin B (AmB) was the drug of choice for the majority of cases. Sixty four isolates did not show any growth in the lab and only 21 cases were evaluated by ITS sequencing, among them; Rhizopus arrhizus var. arrhizus was the dominant species. Considering the high mortality rate of mucormycosis, early and accurate diagnosis, with the aid of molecular methods may provide accurate treatments and improve the survival rate. Therefore, increased monitoring and awareness of this life-threatening disease is critical.

The first case of onychomycosis in a koala (Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum, a diagnostic challenge.

BACKGROUND AND PURPOSE: Superficial mycotic infections have been only poorly described in koalas and there are no reliable mycologically confirmed data regarding clinical isolation of dermatophytes in this animal. We report an 11-year-old female koala, kept in a zoo in Tokyo, Japan, and presenting with hyperkeratotic lesions and scaly plaques on forepaw claws and pads reminiscent of fungal infection. CASE REPORT: Direct microscopy of the scrapings was indicative of a dermatophyte infection. By culture and subsequent repeated subculturing of clinical specimens on Sabouraud dextrose agar, Mycobiotic agar, and potato dextrose agar, two distinct strains with different colony morphotypes (designed as types I and II) were identified. Macroscopic and microscopic characteristics of the strains were suggestive of three different species, i.e. Microsporum canis, M. gypseum, and M. fulvum. However, partial sequencing of internal transcribed spacer (ITS) region of rDNA, translation elongation factor-1α (Tef-1α), and beta-tubulin (BT2) genes confirmed the identity of both isolates as M. gypseum. The animal was treated with a continuous terbinafine regimen (250 mg/kg) once daily for 12 weeks. CONCLUSION: To the best of our knowledge, the present report is the first confirmed case of dermatophytosis in a koala. The genetics underlying a variety of phenotypic traits in most classical dermatophyte species are unknown, and further studies are needed to understand this phenomenon.

Detection of a wide range of medically important fungi by the polymerase chain reaction.

A polymerase chain reaction (PCR) method was developed that was capable of detecting a wide range of medically important fungi from clinical specimens. The primer pair was designed in conserved sequences of 18S-ribosomal RNA genes shared by most fungi. The lower limit of detection of this PCR technique was 1 pg of Candida albicans genomic DNA by ethidium bromide staining and 100 fg after Southern analysis. A 687-bp product was amplified successfully by PCR from all 78 strains of 25 medically important fungal species studies, including Candida spp., Hansenula spp., Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon beigelii, Malassezia furfur, Pneumocystis carinii, Aspergillus spp., and Penicillium spp., but not from any strains of Mucor spp., Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), calf thymus or human placenta. This specificity was subsequently confirmed by Southern analysis. PCR analysis of blood specimens collected from mice systemically infected with C. albicans and clinical samples including blood, cerebrospinal fluid and sputum appeared to be a more sensitive diagnostic method for invasive fungal infections than a conventional blood culture technique.

Immunohistochemical diagnosis of Cryptococcus neoformans var. gattii infection in chronic meningoencephalitis: the first case in Japan.

Cryptococcus neoformans (C. neoformans) var. gattii infection usually occurs in tropical and subtropical areas, and rarely in the northern hemisphere. We report the first Japanese with cryptococcal meningoencephalitis caused by C. neoformans var. gattii infection that occurred during a trip to Australia. This agent was identified in a cerebellar biopsy specimen by immunohistochemical technique with serotype-specific anti-sera. Because the meningitis caused by it did not respond well to conventional therapy, we used an aggressive therapeutic regimen to successfully treat the patient. Even in areas where C. neoformans var. gattii does not exist, this infection should be considered possible as a travel-related infection.

Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA.

Nucleotide sequences of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA from seven morphologically unidentified dermatophyte isolates were determined. The sequences were compared with those of typical isolates of Trichophyton (T.) mentagrophytes var. interdigitale, T. rubrum, and Epidermophyton floccosum. Two of the isolates were classified as T. rubrum and the other five as T. mentagrophytes var. interdigitale. The results did not conflict with identifications using other molecular techniques, including random amplification of polymorphic DNA (RAPD) analysis and restriction enzyme analysis of mitochondrial DNAs. Thus, the nuclotide sequence of ITS 1 is possibly a good molecular marker for identification of these major anthropophilic dermatophyte species.

Species identification system for dermatophytes based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1.

This describes a new and reliable species identification and classification system for dermatophytes based on the cluster analysis of nuclear ribosomal internal transcribed spacer 1 (ITS1) DNA sequences. In this system, some phenotypically similar species construct a compact monophyleic cluster which seems to be a species. This ITS1 sequence based species is called an ITS1-genospecies. The classification of genospecies is a practical concept for DNA sequence based species identification. It is possible to perform species identification and/or strain typing of 25 major dermatophytes (anamorphic genera Trichophyton, Microsporum, Epidermophyton, and the teleomorphic genus Arthroderma), some of which are hard to identify from their morphological features, by demonstrating their dendrogram using this system.

[Taxonomy of genus Malassezia: progress and problems].

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.

[Specific and rapid identification of Malassezia species based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions].

Species identification of genus Malassezia is important in epidemiological and etiological studies, however, is difficult by the conventional system. A specific and rapid identification system based on sequences of internal transcribed spacer 1 of ribosomal DNA has therefore been developed. Using this system, we could identify two or more species mixed in the clinical samples.

[A Case of central venous catheter-related infection with Malassezia sympodialis].

We report a 63-year-old male with central venous catheter-related infection caused by Malassezia sympodialis after total gastrectomy for a gastric cancer. He had fever and his leukocyte counts and C-reactive protein were elevated 14 days after his operation. After his central venous hyperalimentation catheter was removed, the inflammatory signs immediately disappeared, suggesting an intravenous catheter-related infection. A yeast-like fungus was cultured in brain-heart infection semi-solid agar ten days later, and was diagnosed morphologically as Malassezia sp. This strain was identified as M. sympodialis by Tween assimilation test and was confirmed by whole-sequence of internal transcribed spacer 1 regions (ITS1). This is the first report of catheter-related infection caused by M. sympodialis. This strain grew and was subcultured on CHROMagar Candida, potato dextrose agar and Sabouraud agar. There have been no reports of such a lipid-independent Malassezia sp. except for M. pachydermatis. The mechanism of lipid independence of this strain is undetermined and future work is needed. Malassezia sp. is receiving increased attention as an etiologic pathogen of catheter-related fungemia in clinical microbiology laboratories and infectious disease sections.

[Advances in molecular biology of dermatophytes].

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.