Osteolytic Phaeohyphomycosis in a German Shepherd Dog Caused by Phialemonium obovatum

JOURNAL OF CLINICAL MICROBIOLOGY, May 1986, p. 987-991 Vol. 23, No. 5 0095-1137/86/050987-05$02.00/0 Copyright C) 1986, American Society for Microbiology Osteolytic Phaeohyphomycosis in a German Shepherd Dog Caused by Phialemonium obovatum LARRY G. LOMAX,1t JOHN R. COLE,1 ARVIND A. PADHYE,2* LIBERO AJELLO,2 FRANCIS W. CHANDLER,3 AND BRON R. SMITH4 Veterinary Diagnostic and Investigational Laboratory, University of Georgia, Tifton, Georgia 317931; Division of Mycotic Diseases2 and Division of Host Factors,3 Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333; and Scenic Plaza Animal Clinic, Lawrenceville, Georgia 302454 Received 26 December 1985/Accepted 13 February 1986 Phialemonium obovatum was found to be the cause of osteomyelitis in a German shepherd dog. Histologic examination of the biopsied material from the left tibia revealed septate, irregularly branched hyphae, swollen cells, and ovate-to-spherical cells divided by a transverse septum. The majority of the fungal elements were hyaline, but a few had lightly pigmented cell walls that had a greenish yellow tint. The presence of melanin in the cell walls of the hyphae and especially in their septa was verified by the use of the Fontana-Masson silver stain. P. obovatum formed moist, off-white-to-ochraceous, spreading colonies with a characteristic green pigment on their reverse side. The pigment was more prominent in cultures grown at 37 C than in those grown at 25 C. The isolate also grew at 40 C. The dog isolate produced characteristic adelophialides without conspicuous collarettes and also basal septa from the creeping vegetative hyphae growing on the surface of the medium. The numerous obovate phialoconidia were smooth and one-celled. Phaeohyphomycosis encompasses a distinct, heterogenous, and important group of infections in which the etiologic agents in tissue occur as dematiaceous, yeastlike cells, pseudohyphal-like elements, toruloid hyphae, small chains of cells, distorted to swollen cells, or any combinations of these forms (1, 2, 7). Even though fungal elements in tissue are generally described as dematiaceous, not all elements present in the tissue manifest their pigmented nature. In some instances, several tissue sections have to be carefully examined before the dematiaceous nature of the fungal elements is recognized. In phaeohyphomycotic infections caused by Alternaria alternata, Bipolaris spicifera (syn. Drechslera spicifera) (3, 4), Exophiala jeanselmei (4), or E. spinifera (9), the majority of the fungal elements in tissue may be hyaline. At times, a careful search of unstained or hematoxylin and eosin (H & E)-stained tissue sections is needed to reveal the pigmented nature of the fungal elements present. Currently, 62 species of dematiaceous fungi belonging to 33 genera have been described in the literature under a wide variety of confusing disease names, all of which, in fact, are agents of phaeohyphomycosis. We describe here another agent of phaeohyphomycotic infection, namely, Phialemonium obovatum (6), which infected the tibial bone of a dog. Case report. In November 1982, a 3-year-old female German shepherd dog was admitted to a veterinary clinic with a 2-week history of weakness and pain in the left hind leg. Upon palpation of this limb, pain was elicited from the proximal thigh region to just proximal of the tarsus. There was soft-tissue swelling over the entire length of the tibia. This was most prominent over the mid-tibial region. The dog was in good body condition otherwise and appeared to be alert. Her rectal temperature was 38.9 C. A tentative diag- * Corresponding author. t Present address: Mammalian and Environmental Toxicology Research Laboratory, Dow Chemical Company, Midland, MI 48674. 987 nosis of local soft-tissue and possibly bone inflammation was made. Therapy consisted of 200 mg of butazolidin administered twice a day for 7 days, followed by 100 mg of this drug twice a day for an additional 7 days. The owner was encouraged to return the dog in 14 days for reexamination. Nineteen days later, the dog was readmitted to the clinic because of poor appetite and severe lameness in the left rear leg. Physical examination revealed a painful, firm swelling over the mid-tibial region of this leg. A similar lesion was palpated over the midregion of the left radius, and the entire diaphyseal length of both femurs was firm, painful, and thick. In addition, the dog had a head tilt to the left side. Blood was obtained for hematologic and serum enzyme chemistry determinations. Radiographs of the pelvis (ventrodorsal view), right femur, left tibia, and left radius (lateral and anteroposterior views) were obtained. A punch biopsy from the lesion in the tibia (the site of the original swelling) was also obtained. Portions of the biopsy were placed into 10% neutral buffered Formalin, and the remainder of the specimens was placed in sterile 0.9% saline (NaCl). This material was submitted to the University of Georgia Veterinary Diagnostic Laboratory at Tifton, Ga. The dog died shortly thereafter, but permission for necropsy was denied. Radiographic findings included soft-tissue swelling, irregular periosteal proliferation, lysis of cortical bone, and increased medullary density in the mid-shaft regions of the left tibia (Fig. 1) and the left radius (Fig. 2). There was also a diffuse periosteal proliferation and increased medullary density throughout the diaphyses of both femurs. The periosteal reaction in the femurs extended from the proximal to the distal metaphyses. The coxofemoral, stifle, tarsal, and elbow joints showed no radiologic signs. Additional laboratory findings included neutrophilic leukocytosis with left shift and moderate monocytosis. The serum alkaline phosphatase was elevated (274 U/liter), whereas the other enzyme chemistry values were within normal ranges. Histologic findings. Histologic examination of the biopsy material from the left tibia revealed a diffuse granulomatous

988 NOTES FIG. 1. Lateral view of left tibia and femur. There is soft-tissue swelling, irregular periosteal proliferation, lysis of cortical bone, and increased medullary density in the mid-shaft region of the tibia. The distal metaphysis of the femur has periosteal proliferation and increased medullary density. inflammation characterized by multinodular foci of macrophages and multinucleated giant cells among areas of hemorrhage and necrotic trabecular bone. The centers of most of the granulomas contained microabscesses and necrotic debris. Replicate tissue sections stained with Gomori methenamine silver revealed numerous pleomorphic fungal elements that were either scattered or arranged in loose aggregates within the areas of inflammation and necrosis (Fig. 3 through 5). The fungal elements consisted of septate, irregularly branched hyphae that ranged from 3 to 4.5,um in diameter. They varied considerably in length. The narrow hyphae were frequently constricted at their prominent, thick septations. They sometimes had terminal and intercalary chlamydoconidialike swellings up to 15,um in diameter (Fig. 3). Budding yeastlike cells were also present, along with hyphal elements, single thick-walled cells up to 20,um in diameter, and spherical to oval cells that were transversely septate (Fig. 4 and 5). Most of the fungal elements in H & E-stained sections appeared hyaline, but a few had lightly pigmented greenish yellow cell walls. The Fontana-Masson silver procedure for melanin stained the cell walls and the prominent septa of the fungal elements. It accentuated their light pigmentation (10). Certain other fungal elements that appeared hyaline in replicate H & E-stained sections also gave a positive reaction for melanin (Fig. 5, inset). Mycological findings. Examination of the smears of the biopsy material and KOH preparations by microscope revealed fungal hyphae. The biopsy fluid and tissue were J. CLIN. MICROBIOL. cultured on Sabouraud dextrose agar and Sabouraud dextrose agar containing chloramphenicol and cycloheximide. The cultures were incubated at 25 C for 2 weeks. Glabrous, dirty white to grayish white colonies with dark green pigment grew within 2 weeks of incubation. Based on an initial examination of the colony and examination of teased preparations by microscope, the isolate was tentatively identified as an Acremonium sp. The majority of the fungal elements in the tissue sections were hyaline in the H & E-stained sections, but in their morphology, they closely resembled the agents of phaeohyphomycosis. The green-pigmented colonies were unlike any of the known pathogenic species of Acremonium. Because of the unusual features of the causal agent observed both in vivo and in vitro, a subculture and tissue slides were sent to the Division of Mycotic Diseases, Centers for Disease Control, Atlanta, Ga., for specific identification of the causal agent. There, upon futher mycologic examination, the colonies on Sabouraud dextrose agar were noted to be moist, flat, and spreading. Initially they were white, but they soon became ochraceous and slightly greenish, reaching a diameter of 28 to 30 mm in 2 weeks at 25 C. As the colonies became older, a green diffusible pigment became prominent on their reverse side. In 4-week-old cultures, aerial hyphae FIG. 2. Lateral view of the left radius and ulna showing overlying soft-tissue swelling and periosteal proliferation on both the radius and ulna. The mid-shaft region of the radius also has cortical lysis and increased medullary density.

VOL. 23, 1986 NOTES 989 ~ -..~~~~~~~. PI 9/ WF 0A * 'V _ 0 6 _ 0 ~~~~~Id I* 0p 0' tv -I_ -0. v *.. av, t e~~~~~~~~f *- R le C..d.. Q~~~~ _l S. A':s i* FIG. 3. Scattered and loosely aggregated hyphae of P. obovatum in a section of the tibial lesion stained with Gomori methenamine silver. Arrows, Two hyphae with terminal chlamydoconidiumlike swellings. Magnification, x700. in the central areas of the colonies became evident (Fig. 6). The isolate grew at 37 and 40 C. Examination of the slide culture preparations on potato dextrose agar by microscope after 2 weeks at 25 C revealed adelophialides which developed as small pegs from the sides of the vegetative hyphae. These adelophialides lacked the basal septa and collarettes which are typical of discrete phialides (Fig. 7). Some well-developed discrete phialides with a basal septum were produced, but only rarely. The conidia were hyaline, one-celled, obovate, and smooth. They measured 3.0 to 6.0 plm by 1.4 to 2.0,um. Chlamydoconidia were abundant in older cultures. They were oval to almost cylindrical, thin walled, and hyaline. i *** t. ov They were intercalary or terminal and measured 5 to 8,um by 2.5 to 4.5 plm (Fig. 8). A subculture of the dog isolate was sent to Walter Gams, Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands, who identified it as P. obovatum. It was accessioned at the CBS under CBS number 400.83. It is also deposited in the Centers for Disease Control Division of Mycotic Diseases' culture collection under accession number B-3703. Discussion. The genus Phialemonium was proposed by Gams and McGinnis (6) as being intermediate between the genera Acremonium and Phialophora. It is characterized by the production of adelophialides arising from vegetative b soit 0 I CC) IV~~~~' f 0) %_ # C FIG. 4. Budding yeastlike cells and pseudohyphae in the necrotic center of a granuloma. Arrows, Spherical-to-oval fungal cells, stained with Gomori methenamine silver, with a single, thin septation. Magnification, x700.

990 NOTES J. CLIN. MICROBIOL..4 a go *db 4. 1%, il v 4 '. t irli 4-r- 0 1 4' gah.r.s j".41- V..; "..T 1.,.:.!".k 401il. :.:7; A -,c 4.05. y 7 ("'N40 FIG. 5. Pleomorphic fungal elements of P. obovatum, stained with Gomori methenamine silver, in a tibial abscess. Magnification, x700. Inset: Replicate section shows positive staining of some elements for melanin with the Fontana-Masson silver procedure. Magnification, x700. hyphae close to the agar surface. The term adelophialide, as defined by Gams (5), is applied to a reduced phialide that arises from intercalary hyphal cells as a lateral, phialidic neck. There are no basal septa that separate these phialidic necks from their supporting hyphal cells. Occasionally, discrete phialides, which are well developed and have basal septa, are produced by the species of Phialemonium. The adelophialides lack conspicuous collarettes and inner periclinal wall thickenings. In the species belonging to the genus Phialophora, a continuous series of phialide types that range from discrete phialides with basal septa to adelophilides is observed. But discrete phialides with basal septa predominate in I...-...i iw '.. " q' Is * Phialophora spp. They are considered to be the characteristic conidiogenous cell for the genus. The same type of conidiogenous cell with discrete phialides is also considered typical for members of the genus Acremonium. Some species of Phialophora and Acremonium may produce adelophialides mainly from submerged hyphae in addition to the characteristic discrete phialides which characterize the two genera. Gams and McGinnis (6) described three species of Phialemonium, namely, P. obovatum, P. curvatum, and P. dimorphosporum. In P. obovatum, the conidia are obovate and colonies develop a greenish pigment with age. Phialemonium curvatum is distinguished by its conidia,..wasf 19 FIG. 6. Colony of P. obovatum on Sabouraud dextrose agar after 2 weeks at 25 C. FIG. 7. Photomicrograph showing an adelophialide and conidia of P. obovatum. Lactophenol cotton blue; magnification, x1,560.

VOL. 23, 1986 FIG. 8. Photomicrograph showing conidia and intercalary chlamydoconidia of P. obovatum. Lactophenol cotton blue; magnification, x 1,560. which are uniformly allantoid, and colonies that do not turn vinaceous buff. The conidia of P. dimorphosporum are partly allantoid, partly ellipsoidal, and straight. The colonies of P. dimorphosporum form pale, vinaceous buff pigment on their reverse side. The colonies of P. obovatum and P. curvatum have been isolated from clinical specimens. P. curvatum and P. dimorphosporum have been isolated from soil, sewage, air samples, and water (6). Recently, McGinnis et al. (8) described P. obovatum as an opportunistic pathogen in a 4.5-month-old child that died of thermal burns. In antemortem and postmortem examinations of the skin and subcutaneous tissue from the burn wounds, they observed hyaline, septate, irregularly branched hyphae. Yeastlike cells and pseudohyphae were absent. In the tissues of the present case, P. obovatum was polymorphic. The fungal elements consisted of septate, irregularly branched hyphae, narrow hyphae with terminal or intercalary chlamydoconidialike swellings, budding yeastlike cells, and spherical-to-oval cells that were transversely septate. When NOTES 991 tissue sections were stained by the Fontana-Masson silver procedure (10) for melanin, the light or previously undetectable pigmentation in hyphal cell walls and especially their septations was markedly accentuated. As more species of dematiaceous fungi are reported as etiologic agents of phaeohyphomycosis, it is becoming clear that the innate brown pigmentation of their mycelia may not always be readily apparent in tissue sections. In most instances, a careful search almost always reveals at least some dematiaceous hyphae in unstained (cleared and mounted) or H & E-stained sections. In the case of P. obovatum, a special staining procedure proved useful to confirm the presence of melanin in its tissue cells. We thank Walter Gams, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands, for identifying the dog isolate; Ray Simons, Laboratory Training and Consultation Division, Laboratory Program Office, Centers for Disease Control, for some of the photomicrographs; and the Mycology Section, Georgia Department of Human Resources, Atlanta, for their mycological studies. LITERATURE CITED 1. Ajello, L. 1975. Phaeohyphomycosis: definition and etiology. Pan Am. Health Organ. Sci. Publ. 304:126-130. 2. Ajello, L. 1981. The gamut of human infections caused by dematiaceous fungi. Jpn. J. Med. Mycol. 22:1-5. 3. Alcorn, J. L. 1983. Generic concepts in Drechslera, Bipolaris and Exserohilum. Mycotaxon 17:1-86. 4. Chandler, F. W., W. Kaplan, and L. Ajello. 1980. A colour atlas and textbook of the histopathology of mycotic diseases, p. 92-95 and 253-262. Wolfe Medical Publications Ltd., London, England. 5. Gams, W. 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes), p. 5-10. G. Fischer, Stuttgart, Federal Republic of Germany. 6. Gams, W., and M. R. McGinnis. 1983. Phialemonium, a new anamorph genus intermediate between Phialophora and Acremonium. Mycologia 75:977-987. 7. McGinnis, M. R. 1983. Chromoblastomycosis and phaeohyphomycosis: new concepts, diagnosis and mycology. J. Am. Acad. Dermatol. 8:1-16. 8. McGinnis, M. R., W. Gams, and M. N. Goodwin. 1986. Phialemonium obovatum infection in a burned child. Sabouraudia 24:69-73. 9. Padhye, A. A., W. Kaplan, M. A. Neuman, P. Case, and G. N. Radcliffe. 1984. Subcutaneous phaeohyphomycosis caused by Exophiala spinifera. Sabouraudia 22:493-500. 10. Wood, C., and B. Russel-Bell. 1983. Characterization of pigmented fungi by melanin staining. Am. J. Dermatopathol. 5:77-81.