Jpn. J. Med. Mycol. Vol. 37, 135-141, 1996 ISSN 0916-4804

Review Studies on Phaeohyphomycosis and Its Causative Agents in China

Ruoyu Li, Duanli Wang Department of Dermatology, First Hospital of Beijing Medical University, Beijing, China

Abstract Until now, 10 cases of phaeohyphomycosis have been reported in China. Their causative agents were as follows: Exophiala dermatitidis (4 cases), E, spinifera (3), E. jeanselmei (1), Alternaria alternata (1) and Veronaeabotryosa (1). The clinical data will be reviewed in this paper. The pathogenic dematiaceous fungi isolated from the phaeohyphomycosis patients were identified carefully and the conidiogenesis of these organisms was studied by using a scanning electron micro- scope. Besides of these, physiological and other studies were also carried out to aid the correct classi- fication and identification. RAPD-PCR technique was applied to the genomic DNA assay of pathogenic black ' and reveled highly polymorphisms in their genomic fingerprints. The ecological studies were carried out and seven species were isolated from the soil and rotting plant materials. Phialophora aerrucosa was the most dominant species in nature in Shandong, China, followed by Exophiala spinifera and Veronaeabotryosa. The pathogenicity of Yeronaeabotryosa and Exophiala spinifera were studied by using both normal and precompromised mice. The results show that both natural and clinical strains of V. botryosa have potential pathogenicity. E. spinifera is neurotropic and its pathogenicity is specially potential in immunodeficient hosts. Key words: Phaeohyphomycosis, dematiaceous fungi, Exophiala spp., Conidiogenesis, RAPD-PCR

as an opportunistic fungal , and Introduction dematiaceous -like cells, pseudohyphae-like Dematiaceous fungi represent a group of fungi elements, hyphae of various shapes or any combi- that bear melanin-like pigment on the wall of nation of these forms could be found in tissue. hyphae and/or spores. Until now, about 57 genus This concept includes many well-established clinical and 101 species have been found to be pathogenic entities such as , keratitis and so on. to human1). They may produce several types of Phaeohyphomycosis has been diagnosed in clinical patterns such as , China only in the last decade. Before that time, phaeohyphomycosis, mycetoma, , the diseases met the same characteristics of the , black piedra, and keratitis2). definition were diagnosed as chromomycosis. To Chromoblastomycosis is the most frequently the present, 10 cases have been reported in the encountered dematiaceous fungal infection in Chinese literature. Their causative agents were as China. Cladosporiumcarrionii is a dominant pathogen follows: Exophiala dermatitidis (4 cases), Exophiala in northeast China in contrast to Fonsecaeapedrosoi spinifera (3), Exophiala jeanselmei (1), Alternaria sp. in southeast China. (1) and Yeronaea botryosa (1)6-8, 10-12,15-17). This The term phaeohyphomycosis was coined in article aims to review the clinical and 1974 by Dr. Aiello to cover all the infections of the basic researches on their pathogens in recent cutaneous, subcutaneous and systemic nature 10 years in China. caused by hyphomycetous fungi that develop in the host tissues in the form of dark-walled Clinical reports dematiaceous septate mycelial elements 4) The Exophiala dermatitidis infections: The first clinical types of phaeohyphomycosis include five case was reported by Dr. Bai and Zhang in categories, i.e., superficial, cutaneous, subcutaneous, Dalian, China in 19646). The patient was a 19- systemic and allergic. In 1983, Dr. McGinnis 5) year-old girl with lymphadenopathy and low gave a more boundary definition to phaeohypho- fever for four months. Symptoms of the central 136 真 菌誌 第37巻 第3号 平 成8年 nervous system soon appeared and she died. E. dermatitidis (Fonsecaea dermatitidis) was isolated from the lymphonodes, so that she was diagnosed as having chromomycosis. Including this case, a total of four cases of E. dermatitidis infections was reported. Three of them were systemic and two cases of central nervous system infection were fetal',). E. dermatitidis infection in China also has a broad clinical spectrum as was recently summa- rized by Dr. Matsumodo et a19>. Their review found that the fatality rate of these infections was 48%, which indicates that E. dermatitidis is an important neurotropic dematiaceous hyphomycete since it has a marked tendency to invade the central nervous system. E. spinifera infections: The first case of E. spinifera infection was reported in 1987 in Shandong province by Dr. Dai et al 10). The patient was a 9-year-old boy with extensive grey to black, verrucous lesions covering the whole surface of his body for over a year (Fig. IA). X-rays showed multiple cystic degenerations and pathologic fractures of the bone in the upper and lower extremities. The diagnosis was confirmed by repeated fungal cultures and findings of dematiaceous yeast-like cells or their chains in granulomatous lesions (Fig. 1B). Fungal cultures Fig. 1. The first and fetal case of E. spinifera infection were positive in samples of blood, stool, cutaneous found in Shandong in 1987. A) Crusted, verrucous lesions, and lymph nodes. The boy died three lesions on the patient's face. B) Histological findings years after the development of systemic dissemi- showing granulomatous lesion formation and chain of nation. Another case was a 22-year-old girl who brown colored yeast-like cells in the tissue. came from Zhejiang province, China"). The crusted, verrucous lesions could be found on her face and arm. Granulomatous mass also happened in her nasal cavity. Recurrence occurred soon after the surgical removal of the lesions. All of cases of the three E. spinifera infections were associated with disseminated skin lesions, lymph nodes involvement, and skeletal lesions 12). These findings were similar to those reported elsewhere, espe- cially to the first human infection found in India in 19582,13). Another subcutaneous infection caused by E. spinifera found in El ')Salvador in 198314), showing the same clinical -pictures exhibited by our cases except fungal cells were found within multinucleated giant cells. Fig. 2. Histological findings in the first case of phaeohypho- E. jeanselmei infection: IA 1985, Dr. Yu and mycosis caused by Veronaea botryosa. Long septate hyphae his colleagues 15) reported a 23-year-old female could be found located in the microabscess. farmer with swelling and pain of her left eyelid for one year. She didn't show any response to a 5-month Veronaea botryosa infection: The first case of anti-tuberculosis treatment. Histopathological study human infection caused by Veronaea botryosa was of the intraorbital tissues and the eyeball revealed found in 1990 in Henan Province. A 24-year-old a fungal granuloma. A black yeast was isolated male farmer complained of a 10-year history of from the biopsy materials. It was the fist time to diffuse, black verrucouse nodules and cyst-like use the term 'phaeohyphomycosis' in the Chinese lesions on the back of his left hand, forearm and literatures. both cheeks. Histological examination showed Jpn. J. Med. Mycol. Vol. 37 (No. 3), 1996 137 groups of brown walled, septate hyphae and thick genic dematiaceous fungi are usually difficult to walled spores in the upper dermis (Fig. 2). A identify correctly. Moreover, there is much con- black was repeatedly isolated from the tissue troversial on speciation of this group of fungi. culture of the patient. This strain was identified Hence, the pathogenic dematiaceous fungi isolated as Veronaeabotryosa by Dr. Nishimura 16). Since this from phaeohyphomycosis patients were carefully is a new pathogenic dematiaceous species, identified, and the conidiogenesis of these organisms its characteristics have been studied extensively was studied under scanning electron microscope by using different methods. ( SEM) . Besides, physiological tests include Many kinds of dematiaceous fungi can be the thermotolerance, gelatin liquefaction, hydrolysis causes of phaeohyphomycosis. The cases reported of urea and starch, assimilation of KNO3, and re- in China demonstrate that some species belonging quirement of Vitamin B1 were also done. (Table 1) to the genus Exophiala play an important role. Exophiala dermatitidis (Kano) de Hoog, 1977: The main type of the conidial ontogeny of Basic studies E. dermatitidis is annellation. Cladosporium type Genus Exophiala, so called black yeasts, contains occasionally can be found. Most of the isolates can several species which are very similar morphologi- grow at 42C but can not utilize KNO3. These cally in the production of annellidec conidiogenous two characteristics are the key points of distin- cells. It is relatively difficult to make an exact guishing E. dermatitidis from E. jeanselmei. Like speciation normally. The taxonomy and classifica- most of the Exophiala sp., E, dermatitidis strains tion of this group of fungi still have problems. In could not hydrolyze urea, gelatin and starch'. recent years, chemical, serological, and molecular E, jeanselmei (Langeron) McGinnis and biological methods have been used successfully in Padhye, 1977: The conidiogenesis of this species this field. Our studies on the pathogens of is similar to that of all Exophiala species. The phaeohyphomycosis also include physiological, bio- conidiogeneous cells are annellides. The maximum logical, ecological, and pathogenicily ones in growth temperature in most strains is 36C, and attempt to understand the characteristics of the in some rare strains 38C. This species can utilize pathogens isolated in our country and to make a KNO3, but can not hydrolyze urea, gelatin and correct identification. starch. Ecological studies: Dematiaceous fungi have E. spinifera (Nielsen et Conant) McGinnis, been found widely distributed in natural environ- 1977: Since this is a relatively rare pathogen, ment through out the world. To understand the more detailed studies have been carried out to get natural distribution of pathogenic dematiaceous more information. In these studies, 20 isolates fungi in different areas of China, we have carried including 2 clinical and 18 natural ones were out a cooperative study with our Japanese col- used. SDA, PDA, CMA, CDA and BHIA media leagues since 198818>.Ninety-seven strains belong- were employed for morphological studies. About ing to seven species of pathogenic dematiaceous half of the strains produced yeast-like colonies fungi were isolated from the soil and rotting plant when growing on CDA. Yeast cells with capsules materials in nature in Shandong Province, where could be found in 75% of the strains. Under the Phialophora aerrucosa is the most dominant species light microscopy, this fungus produced long, followed by Ueronaeabotryosa, A, alternata, Exophiala spine-like brownish black conidiophores, which spinifera, E. jeanselmei, E. dermatitidis and F. pedrosoi. were shown to be annellides under the SEM. The similar results were obtained in another (Fig. 3A, B) With 20-30 annellations around study made in recent years 19), except E. spinifera their tip, the annellides were very long character- has higher natural distribution in South China. istically. Even some yeast cells could also produce These natural strains enable us to compare them annellides (Fig. 3C). Three strains (1 clinical and with clinically isolated strains. 2 natural) were found to produce little small Morphological and physiological studies: phialids on PDA or CMA 4-5 week cultures Because of their polymorphic nature, the patho- (Fig. 3D). This ability was unstable. This finding

Table 1. Physiological characteristics of phaeohyphomycosis pathogens in China 138 真 菌 誌 第37巻 第3号 平 成8年

Fig. 3. Morphological characteristics of E. spinifera. A) Long, spine-like, brownish black conidiophores could be seen under LM. B) Many annellations could be found around the tip of the annellides. C) annellated tips of the yeast-like cells. D) small phialids produced by old cultures of E. spinifera. was the same as reported by Nielsen and Conant were similar on SDA, PDA and CMA. After in 196813) and it was confirmed by SEM. It sug- growth at 25C for 3 days, a gray-black or gray- gested that E. spinifera, like E. dermatitidis, be also green colony appeared with a villous-like surface. a polymorphic fungus. The diameter of the colony reached 4 cm with an Veronaea botryosa Ciferri et Montemartini, elevated center in 3 weeks. Under the light 1957: Since this species was newly isolated from microscopy, the conidiophores were erected and humanbeings, the morphological structure was elongated. Most of the conidia were elliptical, studied carefully in identification 16). The colonies two-celled and had a sympodial arrangement. Jpn. J. Med. Mycol. Vol. 37 (No. 3), 1996 139

Fig. 4. SEM observation of V. botryosa.A) four-celled conidium. B) secondary conidiogenous cells.

Sometimes, three to four celled conidia could be seen. The secondary conidiogenous cells which produce similar conidia could also be found (Fig. 4A-B). Molecular biological studies: To get more information about characterizing the pathogenic dematiaceous fungi, RAPD-PCR assay was applied to pathogenic black yeasts' and revealed highly polymorphisms in their genomic fingerprints20). In this study, genomic DNA was extracted by benzyl chloride method that had been reported by Zhu Fig. 5. RAPD fingerprints of isolates of E. spinifera and et a121). PCR amplification was programmed for other black yeasts. The fingerprinting patterns were 45 cycles of 1 min at 94C, 1min at 35C, and 2 obtained by amplification of genomic DNA from each isolates using OPI 14 primer. min at 72C by the random primer. The amplifi- Lanes 1-2 : unidentified black yeasts isolated from the cation products were separated by electronphoresis nature; in 1.4% agarose gel and visualized by ethidium Lane 3 : unidentified black yeast isolated from the bromide. Simple and reproducible results were patient; obtained. These products were distinguishable and Lane 4:E. dermatitidis, clinical isolate, IFM 41689; distributed mainly from 200 to 2000 by in length. Lane 5:E. jeanselmei, IFM 40919; The number of DNA fragments varied from 3 to Lanes 6-15: E. spinifera isolates. Lanes 6, 10-12, 15: natural isolates; Lanes 7-9, 13, 14: clinical isolates. 12, and showed high polymorphisms (Fig. 5). The three species of Exophiala could be distin- methods, such as DNA G+C Mol%22), DNA- guished by comparing their polymorphisms in DNA hybridization") and mtDNA analysis24-26) genomic fingerprints. E. dermatitidis strains showed are shown to be useful in this field. Similarly, our homogeneous fingerprints, which were clearly studies are simple and practical in the identifica- different from those of the other two species-E. tion of the 'black yeasts'. Exophiala spp. could be spinifera and E. jeanselmei. Among the latter two differentiated from each other by RAPD assay. species, their fingerprints pattern showing high Pathogenicity studies: Since V. botryosa infec- polymorphisms suggested that they are composed tion is the first case of humanbeing, this fungus of genetically heterogeneous groups. There was no is widely distributed in nature in China, it is obvious difference between the clinical and natural necessary to understand its pathogenicity. The isolates. In recent years, some molecular biology Veronaea botryosa strains isolated from a patient studies were carried out to find more exact, prac- and nature were inoculated into the testis of ddY tical methods for identification of the 'black mice to confirm their potential pathogenicity27). yeasts' which are difficult to differentiate. These Organisms were recovered from the testis of 140 真 菌 誌 第37巻 第3号 平成8年

A

B C

Fig. 6. Pathogenicity studies of E. spinifera. A) The granulomatous lesion and yeast-like cells in normal mouse. B) Histological findings in cortisone treated mouse. C) Histological changes in cyclophosphamide treated mouse. normal and cortisone-treated mice. Granulomatous China in recent 10 years, and some rare patho- inflammatory reactions with fungal elements were genic dematiaceous fungi have been isolated. observed. Polymorphonuclear cells accumulated at Ecological studies found that these pathogens are the center of the lesions. Well developed hyphae widely distributed in nature in China. We found could be found in some cortisone-treated mice. that the understanding of conidiogenesis is very The pathogenicity of one Exophiala spinifera important in the identification of this group of strain isolated from the patient was studied by fungi. Scanning electron microscopy is a very using both normal and pre-compromised mice2B). useful tool for identification the conidiation type. Forty-two 4-week-old ddY mice were divided into Annellation is the main conidiogenous type of three groups. The first group was treated by beta- Exophiala spp. Cladosporium type and phialids can methasone and the second by cyclophosphamide; be found occasionally. Biological studies, especially the third group was used as a control. Each molecular ones, are helpful in differentiating this mouse was injected intravenously with 0.2m1 (1 group of fungi. Studies on the pathogenesis of the X l07cfu) of fungal cell suspension. Tissue culture newly isolated strains revealed that these species and histopathological examination were performed are specially pathogenic in immunocompromised periodically. The results showed that E. spinifera is hosts. neurotropic and its pathogenicity is specially (Co-investigators: Xiaohong Wang, Zhe Wan, potential in immunodeficient hosts. (Fig. 6A, B, C) Dongmei Li, Aiping Wang, Beijing Medical University, China; Wenli Dai, Shandong Province Conclusion Hospital, China; Makoto Miyajii, Kazuko Nishi- Phaeohyphomycosis has been recognized in mura, Ayako Sano, Research Center for Pathogenic Jpn. J. Med. Mycol. Vol. 37 (No. 3), 1996 141

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