JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1988, p. 709-712 Vol. 26, No. 4 0095-1137/88/04709-04$02.00/0 Copyright 0 1988, American Society for Microbiology Subcutaneous Phaeohyphomycosis Caused by Xylohypha emmonsni ARVIND A. PADHYE,l* WARREN B. HELWIG,2 NANCY G. WARREN,3 LIBERO AJELLO,l FRANCIS W. CHANDLER,4 AND MICHAEL R. McGINNIS5 Division of Mycotic Diseases' and Division of Host Factors,4 Center for Infectious Diseases, Centérs for Disease Control, Atlanta, Georgia 30333; Riverside Clinical Laboratories, Newport News, Virginia 236012; Division of Consolidated Laboratory Services, Department of General Services, Commonwealth of Virginia, Richmond, Virginia 232193; and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77550S Received 1 September 1987/Accepted 14 December 1987 The first human phàeohyphomycotic infection caused by Xylohypha emmonsii is described. The patient, an 83-year-old woman, developed a purpuric lesion on her left arm. The pale brown fungal elements observed in biopsy tissue consisted of thin- to thick-walled, oval to spherical, yeastlike cells with single and, occasionally, multiple buds; chains of budding cells; cells with internal septations in one and, rarely, two planes; and septate hyphae. In culture, X. emmonsu grew moderately fast at 25°C, showed minimal growth at 37°C, and failed to grow at 40°C. It produced acropetal chains of one-celled (rarely two-celled) conidia laterally and terminally directly from vegetative hyphal cells. During the past decade, the recognition of phaeohy- phomycotic infections has increased significantly. In 1974, when Ajello et al. (3) proposed the disease name phaeohy- phomycosis, they listed nine fungi as etiologic agents. Four years later, Ajello (1) expanded the definition of phaeohy- phomycosis to include all superficial, cutaneous, subcutane- ous, and systemic infections in humans and lower animals by dematiaceous fungi which, in their invasive form in host tissue, developed as filamentous, dematiaceous mycelial elements. To date, 71 species of dematiaceous fungi, classi- fied in 39 genera, have been described in the literature as etiologic agents of phaeohyphomycosis (2). We describe the first human phaeohyphomycotic infection caused by a newly described species of Xylohypha, namely, Xylohypha em- monsii (13). CASE REPORT The patient, an 83-year-old woman, had a long history (75 years) of chronic osteomyelitis of the right tibia and a 9-year history of rheumatoid arthritis. For the past 1.5 years she had been treated with prednisone in doses varyiiig from 5.0 to 7.5 mg/day. During acute exacerbations of the arthritis, the steroid dosage was increased for short periods. In June 1983, she was hospitalized for à myocdfdial infarct, during which time she scraped her left arm on an X-ray table. During the following weeks, she developed in the area of trauma a reddish-brown, wartlike lesion approx- imately 1.0 cm in diameter. A biopsy of the lesion was done in July 1983, and the lesion was interpreted to represent phaeohyphomycosis. The lesion continued to grow, becom- ing a nonpalpable, somewhat purpuric, intraderniai, 3.0- by 1.5-cm nodule. The nodule was excised in' December 1983. The histologic findings were similar to those of the previous biopsy. Histologic slides and two cultures of fungi isolated from the two biopsy specimens on Sabouraud glucose agar incubated at 30°C were forwarded through the Départment of General Services, Division of Consolidated Laboratory Services, Richmond, Va., for further studies to the Division of Mycotic Diseases, Center for Infectious Diseases, Cen- ters for Disease Control, Atlanta, Ga. The patient was * Corresponding author. treated with flucytosine (Ancobon) orally (1,000 mg 4 times daily). After 4 weeks of therapy, however, the patient discontinued the treatment. When contacted later on, she stated that the purple coloration in the area of the surgical incision had recurred. MATERIALS AND METHODS Histopathology. Portions of the skin biopsy were fixed in 10% neutral buffered Formalin, embedded in paraffin, sec- tioned at 5 ,um, and stained with hematoxylin-eosin (HE) and Gomori methenamine-silver (GMS) for fungi (4). Cultures. The two isolates (CDC B-3875 and CDC B- 3875A) were stored under sterile mineral oil 'on potato glucose agar (PGA). Subcultures were maintained by regular transfers every 3 weeks on PGA throughout the course of this investigation. Inocula from 10-day-old colonies on PGA were subcultured on Sabouraud glucose agar (Difco Labo- ratories) containing 0.05 mg of chloramphenicol per ml and 0.5 mg of cycloheximide per ml to study the susceptibility of the two isolates to cycloheximide. Subcultures on the latter medium were incubated at 25°C ih the dark for 2 weeks, at which time their rate of growth was compared. The thermo- tolerance of both of the isolates was determined on PGA by incubating the subcultures at 37 and 40°C for 3 weeks. At the end of the 3 weeks, the growth of the colonies was deter- mined by visual observation. Slide cultures were prepared on PGA to study the micro- scopic morphology of each isolate. The slide cultures were incubated in the dark at 25°C for 3 weeks. The resultant growth, on both the cover slip and the slide, was mounted in lactophenol cotton blue, and thé preparations were exam- ined with a Zeiss universal research microscope equipped with bright-field, phase-contrast, and differential-interfer- ence-contrast optics. Physiologic tests. The urease and proteolytic activities of both isolates were determined by the procedures described by Honbo et al. (5) and Padhye et al. (13). RESULTS Histopathologic findings. Sections of the skin lesion re- veaied segmental hyperkeratosis, acanthosis, and pseudo- epitheliomatous hyperplasia of the epidermis. The dermis 709 on July 13, 2018 by guest http://jcm.asm.org/ Downloaded from
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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1988, p. 709-712 Vol. 26, No. 40095-1137/88/04709-04$02.00/0Copyright 0 1988, American Society for Microbiology
Subcutaneous Phaeohyphomycosis Caused by Xylohypha emmonsniARVIND A. PADHYE,l* WARREN B. HELWIG,2 NANCY G. WARREN,3 LIBERO AJELLO,l
FRANCIS W. CHANDLER,4 AND MICHAEL R. McGINNIS5Division ofMycotic Diseases' and Division ofHost Factors,4 Center for Infectious Diseases, Centérs for Disease Control,
Atlanta, Georgia 30333; Riverside Clinical Laboratories, Newport News, Virginia 236012; Division of ConsolidatedLaboratory Services, Department of General Services, Commonwealth of Virginia, Richmond, Virginia 232193; and
Department ofPathology, University of Texas Medical Branch, Galveston, Texas 77550S
Received 1 September 1987/Accepted 14 December 1987
The first human phàeohyphomycotic infection caused by Xylohypha emmonsii is described. The patient, an83-year-old woman, developed a purpuric lesion on her left arm. The pale brown fungal elements observed inbiopsy tissue consisted of thin- to thick-walled, oval to spherical, yeastlike cells with single and, occasionally,multiple buds; chains of budding cells; cells with internal septations in one and, rarely, two planes; and septatehyphae. In culture, X. emmonsu grew moderately fast at 25°C, showed minimal growth at 37°C, and failed togrow at 40°C. It produced acropetal chains of one-celled (rarely two-celled) conidia laterally and terminallydirectly from vegetative hyphal cells.
During the past decade, the recognition of phaeohy-phomycotic infections has increased significantly. In 1974,when Ajello et al. (3) proposed the disease name phaeohy-phomycosis, they listed nine fungi as etiologic agents. Fouryears later, Ajello (1) expanded the definition of phaeohy-phomycosis to include all superficial, cutaneous, subcutane-ous, and systemic infections in humans and lower animals bydematiaceous fungi which, in their invasive form in hosttissue, developed as filamentous, dematiaceous mycelialelements. To date, 71 species of dematiaceous fungi, classi-fied in 39 genera, have been described in the literature asetiologic agents of phaeohyphomycosis (2). We describe thefirst human phaeohyphomycotic infection caused by a newlydescribed species of Xylohypha, namely, Xylohypha em-monsii (13).
CASE REPORT
The patient, an 83-year-old woman, had a long history (75years) of chronic osteomyelitis of the right tibia and a 9-yearhistory of rheumatoid arthritis. For the past 1.5 years shehad been treated with prednisone in doses varyiiig from 5.0to 7.5 mg/day. During acute exacerbations of the arthritis,the steroid dosage was increased for short periods.
In June 1983, she was hospitalized for à myocdfdialinfarct, during which time she scraped her left arm on anX-ray table. During the following weeks, she developed inthe area of trauma a reddish-brown, wartlike lesion approx-imately 1.0 cm in diameter. A biopsy of the lesion was donein July 1983, and the lesion was interpreted to representphaeohyphomycosis. The lesion continued to grow, becom-ing a nonpalpable, somewhat purpuric, intraderniai, 3.0- by1.5-cm nodule. The nodule was excised in' December 1983.The histologic findings were similar to those of the previousbiopsy. Histologic slides and two cultures of fungi isolatedfrom the two biopsy specimens on Sabouraud glucose agarincubated at 30°C were forwarded through the Départmentof General Services, Division of Consolidated LaboratoryServices, Richmond, Va., for further studies to the Divisionof Mycotic Diseases, Center for Infectious Diseases, Cen-ters for Disease Control, Atlanta, Ga. The patient was
* Corresponding author.
treated with flucytosine (Ancobon) orally (1,000 mg 4 timesdaily). After 4 weeks of therapy, however, the patientdiscontinued the treatment. When contacted later on, shestated that the purple coloration in the area of the surgicalincision had recurred.
MATERIALS AND METHODSHistopathology. Portions of the skin biopsy were fixed in
10% neutral buffered Formalin, embedded in paraffin, sec-tioned at 5 ,um, and stained with hematoxylin-eosin (HE) andGomori methenamine-silver (GMS) for fungi (4).
Cultures. The two isolates (CDC B-3875 and CDC B-3875A) were stored under sterile mineral oil 'on potatoglucose agar (PGA). Subcultures were maintained by regulartransfers every 3 weeks on PGA throughout the course ofthis investigation. Inocula from 10-day-old colonies on PGAwere subcultured on Sabouraud glucose agar (Difco Labo-ratories) containing 0.05 mg of chloramphenicol per ml and0.5 mg of cycloheximide per ml to study the susceptibility ofthe two isolates to cycloheximide. Subcultures on the lattermedium were incubated at 25°C ih the dark for 2 weeks, atwhich time their rate of growth was compared. The thermo-tolerance of both of the isolates was determined on PGA byincubating the subcultures at 37 and 40°C for 3 weeks. At theend of the 3 weeks, the growth of the colonies was deter-mined by visual observation.
Slide cultures were prepared on PGA to study the micro-scopic morphology of each isolate. The slide cultures wereincubated in the dark at 25°C for 3 weeks. The resultantgrowth, on both the cover slip and the slide, was mounted inlactophenol cotton blue, and thé preparations were exam-ined with a Zeiss universal research microscope equippedwith bright-field, phase-contrast, and differential-interfer-ence-contrast optics.
Physiologic tests. The urease and proteolytic activities ofboth isolates were determined by the procedures describedby Honbo et al. (5) and Padhye et al. (13).
RESULTS
Histopathologic findings. Sections of the skin lesion re-veaied segmental hyperkeratosis, acanthosis, and pseudo-epitheliomatous hyperplasia of the epidermis. The dermis
mixed suprative andgranulomatousinfiltrat a t with
numerous elements of X. emmonsii in the dermis. HE stain;magnification, x215.
and subcutaneous tissue contained a dispersed inflammatoryinfiltrate composed of neutrophils, lymphocytes, plasmacells, macrophages, and multinucleate giant cells of both theLanghans and foreign-body types (Fig. 1). Occasional micro-abscesses were alsô present, as were irregular foci of ca-seous necrosis surrounded by palisaded epithelioid his-tiocytes and multinucleate giant ceils. Numerousdematiaceous, polymorphous fungal elements were seenthroughout the inflammatory infiltrate (Fig. i and 2), intransepidermal elimination channels, and in the keratinousskin layer. In the dermis and subcutaneous tissue, fungalelements were located predominantly within multinucleategiant celis (Fig. 2) and within microabscesses and caseousfoci (Fig. 3). Many multinucleated giant cells also containedGMS-positive granular material that represented the resid-uum of fungal cell degradation (Fig. 3 and 4).
Morphologically, the fungal elements consisted of globoseto oval yeastlike cells, 6 to 15 pm in diameter, with singleand, occasionally, multiple blastoconidia; chains of buddingcelîs; cells with prominent septations in one and, occasion-ally, two planes; short, closely septate hyphae; and thick-walled swollen cells, up to 25 ,um'in. diameter (Fig. 3 and 4).The dark brown pigmentation of the cell walls highlightedthe fungal elements in HE-stained (Fig. 2) and unstained,cleared, and mounted tissue sections. The actual numberand distribution offungal elements were much more evident,however, and their morphology was better demonstratedwith the GMS stain (Fig. 3 and 4). No evidence was found ofangioinvasion or of foreign material within the lesion thatcould have served as the vehicle of infection. The hostreaction evoked by X. emmonsii was more typical of chro-
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FIG. 2. Dematiaceous, thick-walled cells of X. emmonsùi, someof which are septate (arrows) and located within multinucleate giantcells. HE stain; magnification, x340. The inset shows single thick-walled cells of X. emmonsii divided internally by transverse septa.HE stain; magnification, x720.
moblastomycosis than of subcutaneous phaeohyphomycosis(4).Morphologic findings. On Sabouraud glucose agar with
chloramphenicol and Sabouraud glucose agar with chloram-phenicol and cycloheximide, both isolates grew well. OnPGA the colonies were velvety and mousey gray to black,reached 30 to 32 mm in diameter at 25°C and 3 to 3.5 mm at37°C after 2 weeks, and did not grow at 40°C. The slideculture preparations revealed that both isolates were identi-cal. The hyphae were branched, septate, and hyaline orsubhyaline to midbrown and measured 3.5 to 4.5 ,um indiameter. The conidiophores were poorly differentiated fromthe vegetative hyphae. They were often absent. Conidiawere produced laterally and terminally in acropetal chainsfrom vegetative hyphal cells. The young conidia were oftenasymmetrically bent, sigmoid in form, and monoblastic.Mature conidia were one celled (rarely two celled). Theywere ellipsoid, doliiform, subhyaline, and smooth and mea-sured 3 to 10 by 3 to 5 ,um. The conidial chains werebranched or unbranched, consisting of up to 20 conidia (Fig.5). Both isolates were identified as X. emmonsii (13). Theisolates did not liquefy gelatin after 3 weeks of incubation at25°C but hydrolyzed urea in 7 days.
DISCUSSION
The umbrella term phaeohyphomycosis covers a group ofmycoses caused by fungi that develop in the host tissuepredominantly as dark-walled, septate, mycelial elementswith moniliform hyphae, small chains of yeastlike cells,
FIG. 3. Dermal microabscess containing polymorphous fungalelements that consist of spherical to oval yeastlike cells; chains ofbudding cells; short, closely septate hyphae; and large, thick-walledswollen cells. The inset shows cells of X. emmonsùi with septationsin two planes (sharp arrow) and with multiple blastoconidia (bluntarrow). GMS stain; magnification, x215.
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FIG. 4. Large, thick-walled spherical cells of X. emmonsii in adermal granuloma. Two of the fungal cells are septate (arrows). TheGMS-positive granular material within phagocytes represents theresiduum of fungal cell degradation. GMS stain; magnification,x340.
thick-walled swollen cells, and occasional thick-walled cellswith one transverse septum (9) or, rarely, a few thick-walledmuriform cells (6, 12). Phaeohyphomycosis differs by theimpressive polymorphic expression of its etiologic agents inthe host tissue from chromoblastomycosis, which is a dis-tinct clinical and pathologic entity. The causal agents of thelatter develop in host tissue predominantly in the form ofthick-walled muriform cells. Originally, Ajello et al. (3)proposed the term phaeohyphomycosis as a substitute for"phaeo-sporotrichose," a name coined by Mariat et al. (8) in1967 for all mycotic abscesses that involved cutaneous andsubcutaneous tissue and that were caused by fungi whichdeveloped in the host tissue as dark-walled, septate, myce-lial elements. At that time, Ajello et al. (3) listed 10 causalagents of phaeohyphomycosis but anticipated that otherfungi undoubtedly would be encountered as etiologic agentsof phaeohyphomycosis. Four years later, Ajello (1) ex-panded the concept of phaeohyphomycosis to include allsuperficial, cutaneous, subcutaneous, and systemic infec-tions of humans and lower animals by dematiaceous fungiwhose invasive forms developed predominantly as demati-aceous mycelial elements. The list of etiologic agents hadgrown, in the meantime, to 16. In a later review, McGinnis(10) listed 42 causal agents of phaeohyphomycosis andupdated the concept and definition of phaeohyphomycosis.The most recent update by Ajello (2) enumerates 71 speciesof dematiaceous fungi classified in 39 genera as etiologicagents of phaeohyphomycosis. Some of these causal agents,such as Phaeosclera dematioides (12), Bipolaris hawaiiensis
(6), and X. emmonsii, develop in host tissue a few muriformcells in addition to mycelial elements. The finding of a fewmuriform cells in the present case is not unusual in phaeohy-phomycosis and should not be the basis for diagnosing thepatient to have chromoblastomycosis. The relative abun-dance of moniliform hyphae (Fig. 3), yeastlike budding cells,and thick-walled cells (Fig. 4) in the present case is the basisfor its classification as phaeohyphomycosis rather than chro-moblastomycosis.The Xylohypha species now known to cause phaeohy-
phomycosis in humans and lower animals are X. bantiana(11) and X. emmonsii (13). X. bantiana is an important agentof cerebral phaeohyphomycosis. Two feline subcutaneousphaeohyphomycotic infections caused by X. emmonsii areknown. In one of those cases, the causal agent was misiden-tified as X. bantiana (as "Cladosporium bantianum") (7).Our studies showed that in reality it was X. emmonsii (12).The case described in this report represents the first humanphaeohyphomycotic infection caused by X. emmonsii.X. emmonsii is differentiated from X. bantiana by its
inability to grow at 40°C. The maximum growth temperatureof X. emmonsii is 38°C, while that of X. bantiana is 42 to43°C. The conidial chains of X. emmonsii are sparselybranched, arising from poorly differentiated conidiophores,and consist of up to 20 asymmetrically bent conidia. Incontrast, the conidial chains of X. bantiana are long,sparsely branched, and nonfragile and consist of as many as35 or more blastoconidia.
LITERATURE CITED1. Ajello, L. 1978. The black yeasts as disease agents: historical
perspective. Pan Am. Health Organ. Sci. Publ. 356:9-16.2. Ajeilo, L. 1986. Hyalohyphomycosis and phaeohyphomycosis:
two global disease entities of public health importance. Eur. J.Epidemiol. 2:243-251.
3. Ajello, L., L. K. Georg, R. T. Steigbigel, and C. J. K. Wang.1974. A case of phaeohyphomycosis caused by a new species ofPhialophora. Mycologia 66:490-498.
4. Chandler, F. W., W. Kaplan, and L. Ajello. 1980. A colour atlasand textbook of the histopathology of mycotic diseases. WolfeMedical Publications Ltd., London.
5. Honbo, S., A. A. Padhye, and L. Ajello. 1984. The relationship ofCladosporium carrionii to Cladophialophora ajelloi. Sabourau-dia 22:209-218.
6. Koshi, G., V. Anandi, M. Kurien, M. G. Kirubakazan, A. A.Padhye, and L. Ajello. 1987. Nasal phaeohyphomycosis causedby Bipolaris hawaiiensis. J. Med. Vet. Mycol. 25:397-402.
7. Kwon-Chung, K. J., and G. A. de Vries. 1983. Comparativestudy of an isolate resembling Banti's fungus with Cladospo-rium trichoides. Sabouraudia 21:59-72.
8. Mariat, F., G. Segretain, P. Destombes, and H. Darrassee. 1967.Kyste sous-cutané mycosique (phaeo-sporotrichose) a Phia-lophora gougerotii (Matruchot 1910) Borelli 1955, observé auSenegal. Sabouraudia 5:209-219.
9. Matsumoto, T., A. A. Padhye, L. Ajello, P. G. Standard, andM. R. McGinnis. 1984. Critical review of human isolates ofWangiella dermatitidis. Mycologia 76:232-249.
10. McGinnis, M. R. 1983. Chromoblastomycosis and phaeohy-phomycosis: new concepts, diagnosis and mycology. J. Am.Acad. Dermatol. 8:1-16.
11. McGinnis, M. R., D. Borelli, A. A. Padhye, and L. Ajello. 1986.Reclassification of Cladosporium bantianum in the genus Xy-lohypha. J. Clin. Microbiol. 23:1148-1151.
12. McGinnis, M. R., R. A. McKenzie, and M. D. Connole. 1986.Phaeosclera dematioides, a new etiologic agent of phaeohy-phomycosis in cattle. J. Med. Vet. Mycol. 23:133-135.
13. Padhye, A. A., M. R. McGinnis, L. Ajello, and F. W. Chandler.1988. Xylohypha emmonsùi sp. nov., a new agent of phaeohy-phomycosis. J. Clin. Microbiol. 26:702-708.
