42 Phialophora

42 Phialophora Dongyou Liu and R.R.M. Paterson contents 42.1 Introduction ..................................................................................................................................................................... 339 42.1.1 Classification, Morphology, and Biology ............................................................................................................ 339 42.1.2 Clinical Features and Pathogenesis ..................................................................................................................... 340 42.1.2.1 Phialophora verrucosa ......................................................................................................................... 340 42.1.2.2 Phialophora richardsiae....................................................................................................................... 341 42.1.3 Laboratory Diagnosis .......................................................................................................................................... 341 42.2 Methods ........................................................................................................................................................................... 341 42.2.1 Sample Preparation .............................................................................................................................................. 341 42.2.2 Detection Procedures ........................................................................................................................................... 342 42.2.2.1 Pan-Fungal Real-Time PCR and Sequencing Analysis ........................................................................ 342 42.2.2.2 Sequencing Analysis of the ITS1-5.8S-ITS2 Region ............................................................................ 342 42.2.2.3 Sequencing Analysis of the D1/ D2 Domains of 28S rRNA Gene ....................................................... 343 42.3 Conclusion ....................................................................................................................................................................... 343 References ................................................................................................................................................................................. 343 42.1 IntroductIon parasiticum), Phialophora pedrosoi (synonym: Fonsecaea pedrosoi), and Phialophora spinifera (synonym: Exophiala 42.1.1 ClassifiCation, Morphology, and Biology spinifera). The genus Phialophora is a dematiaceous (dark-walled) fila- Phialophora is a ubiquitous organism that inhabits the mentous fungus belonging to the mitosporic Magnaporthaceae soil, plants, and decaying food [2,3]. Several Phialophora group, family Magnaporthaceae, order Magnaporthales, species are the causative agents of human infections such class Sordariomycetes, subphylum Pezizomycotina, phylum as chromoblastomycosis, phaeohyphomycosis, and eumy- Ascomycota. The obsolete synonyms of the genus include cetoma. P. verrucosa and P. richardsiae are often involved Cadophora and Margarinomyces, and teleomorphs of this in phaeohyphomycosis, with clinical presentations rang- genus are found in Chaetosphaeria, Gaeumannomyces, and ing from cutaneous infections, subcutaneous cysts, kerati- Mollisia. tis, endocarditis, arthritis, osteomyelitis, cerebral infection, Currently, the genus Phialophora contains 16 recog- fatal hemorrhage, and disseminated infection. Phialophora nized and over 69 unassigned species, the most notable of verrucosa is the principal causative agent of chromoblas- which are Phialophora bubakii, Phialophora europaea, tomycosis in tropical and subtropical areas, particularly Phialophora gregata, Phialophora repens (obsolete synonym in Japan and South America. Phialophora europaea has Cadophora repens), Phialophora reptans, Phialophora been isolated from cutaneous and nail infections in North- richardsiae (obsolete synonyms: Cadophora brunnescens, Western Europe. Cadophora richardsiae, P. brunnescens, and P. calicifor- Phialophora has short conidiophores (sometimes reduced mis), and Phialophora verrucosa (synonym: P. americana; to phialides) and unicellular conidia, although conidia are obsolete synonym: Cadophora Americana; and teleomorph: sometimes absent. Phialophora colonies grow moderately Capronia semiimmersa) [1]. slowly and reach a diameter of 2–3 cm after 7 days at 25°C. A number of former Phialophora species have been reclas- Colonies are gray to olivaceous black and often become dark sified: Phialophora compactum (synonym: Fonsecaea com- gray-green, brown, or black with age. From the reverse, colo- pacta), Phialophora cyanescens (synonym: Cylindrocarpon nies appear iron-gray to black; colony texture is cottony to cyanescens), Phialophora dermatitidis (synonyms: Exophiala velvety and may be heaped and granular. dermatitidis and Wangiella dermatitidis), Phialophora hoff- Phialophora hyphae (up to 5 μm wide) are branched and mannii (synonym: Lecythophora hoffmannii), Phialophora hyaline to brown. If present, condiophores are short and pale jeanselmei (synonym: Exophiala jeanselmei var. jeansel- brown; conidiogenous cells (phialides) have characteristic mei), Phialophora mutabilis (synonym: Lecythophora muta- flask-shaped or cylindrical phialides with distinctive collar- bilis), Phialophora parasitica (synonym: Phaeoacremonium ettes, which are terminally or laterally located on the hyphae; 339 K10626_C042.indd 339 1/20/2011 9:12:14 AM 340 Molecular Detection of Human Fungal Pathogens conidia are one celled, hyaline to olivaceous brown, smooth osteomyelitis, and prosthetic valve endocarditis as well walled, ovoid to cylindrical or allantoid, and often aggregate as disseminated infections [7–13], with P. verrucosa and in slimy heads at the apices of the phialides, giving the appear- P. richardsiae being most commonly reported [9,14–17]. ance of a vase of flowers; phialides may be solitary or in a brush-like arrangement. In tissues infected with Phialophora 42.1.2.1 Phialophora verrucosa AQ1 species, spherical or polyhedral, dark brown, thick-walled A case of fatal hemorrhage due to Phialophora verrucosa sclerotic bodies (muriform cells) and phaeoid hyphae are occurred in a patient with prolonged neutropenia undergo- often present, although absence of muriform cells is noted in ing autologous bone marrow transplant (BMT) for acute cases involving immunosuppressed or debilitated patients. myelogenous leukemia (AML). Autopsy uncovered granula- At the species level, Phialophora ripens produces inter- tion tissue on the tracheal wall, which showed fungal hyphae calary phialides without basal septa or cylindrical to lageni- by histopathological examination and grew P. verrucosa in form phialides (<20 μm in length) with narrow collarettes; culture [18]. Phialophora richardsiae conidia are globose to cylindrical In a case of Phialophora verrucosa-related multifocal and phialides with saucer- or vase-shaped collarettes; and subcutaneous phaeohyphomycosis, an 85 year-old woman Phialophora verrucosa produces flask-shaped phialides with presented with purulent multifocal subcutaneous nodules vase-shaped collarettes; Phialophora europaea—a member on the right forearm and hand. The patient reported a his- of the P. verrucosa complex—shows reduced, flaring phi- tory of gardening and recalled the occurrence of a painless, alidic collarettes [4]. soybean-sized nodule on the dorsal site of the right forearm 3 years earlier, which gradually extended peripherally to the dorsum of the right hand. The pustules and nodules (rang- 42.1.2 CliniCal features and pathogenesis ing from 5 × 5 mm to 20 × 40 mm in size) showed a yellow AQ2 The term dematiaceous (dark-pigmented) fungi refers to a crust on the surface and were surrounded by erythema and large and heterogeneous group of molds belonging to sev- discharged a purulent fluid upon pressure. Examination of eral different genera that are capable of causing a diverse the biopsied specimen revealed a subcutaneous granuloma range of clinical diseases such as phaeohyphomycosis, (containing faint brown septate hyphae with rare branching) chromoblastomycosis, and eumycetoma. The most notable with central abscess and necrosis, consistent with phaeomy- human pathogens in the dematiaceous fungal group include cotic cyst. Culture of the discharged purulent fluid on potato Alternaria, Bipolaris, Cladophialophora, Curvularia, dextrose agar (PDA) at room temperature grew a nearly black Exophiala (Wangiella), Fonsecaea, Madurella, Phialophora, fungus, attaining a diameter of 20 mm in 14 days and 50 mm Rhinocladiella, Scedosporium, and Scytalidium. Being in 35 days. Under light microscope, flask-shaped phialides widely distributed in the environment including soil, wood, (conidiogenous cells) with cup-shaped collarettes and a and decomposing plant debris, these organisms have the bunch of elliptical conidia at the apices of the collarettes capacity to take advantage of weakened defense mecha- were noted along with brown to light-brown hyphae. These nism of human hosts, inducing cutaneous, subcutaneous, morphologic features confirmed the fungus as P. verrucosa, and corneal infections, invasive and allergic sinusitis, arthri- which responded to oral itraconazole treatment [19]. tis, endocarditis, and systemic and disseminated infections. A cutaneous fungal infection due to P. verrucosa was Traumatic implantation, injury, and inhalation represent the reported in a 53 year-old woman who had

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