Tomotaka Sato

Simple PCR-Based DNA Microarray System To Identify Human Pathogenic Fungi in Skin

ABSTRACT Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

Keratomycosis caused by Aspergillus viridinutans: an Aspergillus fumigatus-resembling mold presenting distinct clinical and antifungal susceptibility patterns

We report here a case of fungal keratitis caused by Aspergillus viridinutans, a member of the genus which was found to have clinical and antifungal susceptibility characteristics distinct from the species it most closely resembles, Aspergillus fumigatus. A 26-year-old woman presented with contact lens-associated keratitis, the etiologic agent of which was initially slow growing and found to be resistant to amphotericin B and voriconazole. Therapeutic keratoplasty was performed and an isolate from a corneal scraping was identified as A. viridinutans through partial DNA sequencing of the β-tubulin gene, along with its slow growth 42°C. In contrast with A. fumigatus, A. viridinutans proved to be resistant to a wider variety of antifungal agents. To the best of our knowledge, this is the first case of keratitis caused by A. viridinutans. Clinical isolates of the genus Aspergillus include rarely encountered species such as A. viridinutans. These species may show clinical manifestations distinct from those of A. fumigatus, including clinical resistance to amphotericin B and voriconazole and relatively low susceptibility to other antifungal agents.

A case of phaeohyphomycosis of the face caused by Exophiala oligosperma in an immunocompromised host.

Phaeohyphomycosis encompasses a heterogeneous group of infections caused by dematiaceous (darkly pigmented) fungi, which grow in tissue as hyphae, pseudohyphae or yeast-like forms. Clinical findings have been classified as follows: 1. superficial: (a) cutaneous, (b) otic, (c) ophthalmic, 2. subcutaneous: (a) cystic (phaeohyphomycotic cyst), (b) nodular disseminated, 3. cerebral, and 4. disseminated or systemic [1]. Clinical Letter The etiological agents of phaeohyphomycosis are numerous species belonging to different genera such as Alternaria, Phialophora and Exophiala [2]. In Japan, Fonsecaea pedrosoi, E. dermatitidis and E. jeanselmei are predominant agents of phaeohyphomycosis [3]. We report an unusual case of phaeohyphomycosis of the face caused by the recently recognized species Exophiala oligosperma. An 80-year-old woman who had been treated with oral prednisolone, 15 mg daily, for Takayasu arteritis presented with a 6-month history of a hyperkeratotic, red-brown papule of the right cheek measuring 6 mm in diameter (Figure 1). Actinic keratosis was suspected and an excisional biopsy was performed. Histopathology showed granuloma formation in the superficial with multinucleated giant cells that contained septate hyphae (Figure 2). Tissue specimens and skin scrapings incubated on Sabouraud dextrose agar yielded black colonies that were morphologically identified as belonging to Exophiala species (Figure 3). Microscopic examination of these colonies revealed septate hyphae, annellidic conidiogenous cells and oval conidia. A sequence of the internal transcribed spacer region of the ribosomal RNA gene was determined [4] and analyzed using BLAST (http://www.ncbi. nlm.nih.gov). It showed more than 99 % homology with sequence data of 21 strains identified as E. oligosperma, including the accession number AY163551 from the type strain CBS 725.88 [5]. We performed thermotherapy and administered oral potassium iodide (KI), but the lesion did not respond. Next, we performed total resection and gave oral itraconazole (200 mg/day for 2 weeks), but the lesion recurred after 40 days. We then performed a second total resection, and

Rapid growth of malignant melanoma in pregnancy.

Malignant melanoma during pregnancy is a difficult problem as a variety of risks to both the mother and fetus must be weighed. We describe a rapidly progressive malignant melanoma diagnosed during pregnancy. There are no standarized guidelines for treatment; each case requires an individualized approach. We review the literature and present an algorithm to aid in approaching such patients.

A case of blastomycosis-like pyoderma caused by mixed infection of staphylococcus epidermidis and trichophyton rubrum

Blastomycosis-like pyoderma (BLP) is a type of chronic pyoderma characterized histologically by specific epidermal changes namely: pseudoepitheliomatous hyperplasia and intraepithelial abscesses. These epidermal changes are also seen in blastomycosis (referred to as deep dermatophytosis in North America). Here, we describe the case of a 53-year-old male with prurigo nodularis, diabetes, and chronic lymphocytic leukemia who presented with multiple yellowish-red colored papules that coalesced to form a vegetating plaque. In addition to the typical features of BLP, spores with budding were seen histopathologically in a biopsy specimen. Cultures of a skin specimen grew Staphylococcus epidermidis and Trichophyton rubrum. Antibiotic therapy was effective but failed to eliminate the lesion until antifungal therapy using terbinafine was administered concurrently. Past reports suggest that BLP is mainly caused by bacterial infection, but our case suggests that fungal infection can also be involved as the causative organism in BLP.

Application of electron microscopic analysis and fluorescent in situ hybridization technique for the successful diagnosis of extraskeletal Ewing's sarcoma

The diagnosis of soft tissue tumors is often challenging. Immunohistochemical investigation, let alone routine histopathological investigation, may not allow definitive diagnosis in some cases. To overcome such difficulties, more advanced techniques need to be adopted. Herein, we report an extremely rare 56‐year‐old Japanese female case of extraskeletal Ewings sarcoma (ES), successfully diagnosed by electron microscopy (EM) using formalin‐fixed sections and fluorescence in situ hybridization (FISH). The patient had a 2‐year history of a tumor growing on the leg. In routine histopathology, invasive proliferation of tumor cells was observed in the dermis. Tumor cells were round and uniform with large hyperchromatic nuclei, which were positively stained for CD56, VS38c, Ki‐67, MIC2 and vimentin, but not for pan‐keratin AE1 + AE3, cytokeratin 20, chromogranin A, synaptophysin and neuron‐specific enolase. As these findings were not conclusive to make the final diagnosis, EM specimens were prepared from formalin‐fixed sections and subjected to investigation. Cell surface projections and dense core granules were detected, suggestive of either Merkel cell carcinoma or extraskeletal ES. Subsequent FISH analysis identified reciprocal translocation of the ESWR1 gene, enabling the final diagnosis of extraskeletal ES. This study provides useful information enabling the diagnosis of this uncommon soft tissue tumor.

A Rhizopus oryzae strain isolated from resected bone and soft tissue specimens from a sinonasal and palatal mucormycosis case. Report of a case and in vitro experiments of yeastlike cell development

We report a histologically and mycologically proven sinonasal mucormycosis case causing palatal necrosis in a nondiabetic patient with renal failure. Mycological examination of Giemsa stained imprinted tissue preparations revealed abundant yeast-like cells besides the typical mucoraceous hyphae. The fungus was isolated from surgical specimens and identified as Rhizopus oryzae by phenotypic and genotypic tests. Laboratory studies were performed to investigate the association of the yeast-like cells observed in tissue specimens and the fungus recovered in culture. In vitro induced yeast-like cell development of the case isolate was found under certain growth conditions and documented by photomicrographs.

Schnelles Wachstum des malignes Melanom während der Schwangerschaft

Zusammenfassung Das maligne Melanom stellt während der Schwangerschaft ein schwieriges Problem dar, da viele Risiken für Mutter und Fötus in die Überlegungen einbezogen werden müssen. Im folgenden Artikel wird ein rasch progredientes malignes Melanom beschrieben, das während der Schwangerschaft diagnostiziert wurde. Es gibt keine standardisierten Therapierichtlinien; jede Patientin muss individuell behandelt werden. Im Folgenden wird eine Literaturübersicht gegeben und es wird ein Schema vorgestellt, das als Hilfestellung zur Therapie bei diesen Patientinnen dienen soll.