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.

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.

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.

Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

BACKGROUND: Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. METHODS: The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. RESULTS: The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum) and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. CONCLUSION: It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.

Mycological Microscopic and Culture Examination of 400 Bronchoalveolar Lavage (BAL) Samples.

BACKGROUND: The frequency of invasive opportunistic mycoses has increased significantly over the past decades especially in immunocompromised patients. Invasive aspergillosis (IA) has become a major cause of morbidity and mortality among these patients. As bronchoalveolar lavage (BAL) fluid samples are generally useful specimens in the diagnosis of invasive pulmonary aspergillosis (IPA), this study was designed to evaluate the incidence of fungal elements in at-risk patients by direct microscopy and culture of BAL samples. METHODS: In a 16-month period, 400 BAL samples were obtained from several groups of different patients with pulmonary and respiratory disorders and examined by using both direct microscopy and culture. RESULTS: Of the 400 samples, 16 (4%) were positive direct examination with branching septate hyphae and 46 (11.5%) were positive culture: 25 (54%) Aspergillus flavus, 6 (13%) A. fumigatus, 5 (10.9%) A. niger, 1 (2.2%) A. terreus, 3 (6.5%) Penicillium spp. and 6 (13%) mixed A. flavus/A. niger. A. flavus was the most common cause of Aspergillus infection or colonization. Bone marrow transplant (BMT) recipients were the most susceptible group to fungal infection and/or colonization. CONCLUSION: Among Aspergillus species, A. flavus was the most common isolate in both infections and colonization in Iran. More studies are needed to clarify the epidemiological aspect of aspergillosis in Iran.

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.

Preliminary Identification and Typing of Pathogenic and Toxigenic Fusarium Species Using Restriction Digestion of ITS1-5.8S rDNA-ITS2 Region.

BACKGROUND: Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. METHODS: Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 and ITS4 universal primers followed by sequencing and restriction enzyme digestion of PCR products. RESULTS: The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. CONCLUSION: Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species.

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.

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.

Phylogenetic relationships among medically important yeasts based on sequences of mitochondrial large subunit ribosomal RNA gene.

Sequences of the mitochondrial large subunit ribosomal RNA (mtLsurRNA) gene of medically important yeasts were analysed. Sixteen strains of eight species including two varieties were subjected to sequencing. Sequencing enabled us to recognize the differences between all the species and varieties. Alignment analysis revealed that these sequences consisted of three clusters: the Candida albicans group, the C. glabrata group, and the basidiomycetous group. It is possible, therefore, that the mtLsurRNA gene is one of the targets not only for species identification but also for phylogenetic analysis of closely related yeasts. The dendrogram of each group, obtained from this gene, supports the previous study of yeasts based upon the chromosomal genes.

A case of kerion celsi due to Arthroderma benhamiae identified by DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions.

We describe a case of a 4-year-old boy with a 1-month history of a purulent lesion on his scalp. His hair samples revealed fungal organisms and Trichophyton mentagrophytes was cultured from the sample. We analysed the DNA sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS1) region of the isolated fungus. These sequences were in accordance with T. mentagrophytes animal 4 type. In mating experiments, our strain only responded to the Arthroderma benhamiae Americano-European race (+) mating type tester. We speculate that the patient was infected from contact with his pet guinea pig. This is the first case of a clinical isolate of A. benhamiae being identified by DNA sequences of nuclear ribosomal ITS1 regions.

A case of pulmonary aspergilloma molecular biological identification and typing of the isolates from antemortem sputa and autopsy fungus ball.

Filamentous fungi were isolated from antemortem sputum and an autopsy fungus ball of the lung in a case of aspergilloma. Both of the isolates were analyzed for the sequences of species or strain-specific nuclear ribosomal DNA (partial 28S and ITS1 regions), and were identified as Aspergillus fumigatus. The molecular biological technique saved time and is thought to be a powerful tool in the accurate diagnosis of pulmonary fungal infection to assure effective treatment.

[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.