Osteolytic Phaeohyphomycosis in a German Shepherd Dog Caused by Phialemonium obovatum
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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
Downloaded from http://jcm.asm.org/ on February 15, 2021 by guest
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, heteroge- nosis of local soft-tissue and possibly bone inflammation was
nous, and important group of infections in which the etio- made. Therapy consisted of 200 mg of butazolidin adminis-
logic agents in tissue occur as dematiaceous, yeastlike cells, tered twice a day for 7 days, followed by 100 mg of this drug
pseudohyphal-like elements, toruloid hyphae, small chains twice a day for an additional 7 days. The owner was
of cells, distorted to swollen cells, or any combinations of encouraged to return the dog in 14 days for reexamination.
these forms (1, 2, 7). Even though fungal elements in tissue Nineteen days later, the dog was readmitted to the clinic
are generally described as dematiaceous, not all elements because of poor appetite and severe lameness in the left rear
present in the tissue manifest their pigmented nature. In leg. Physical examination revealed a painful, firm swelling
some instances, several tissue sections have to be carefully over the mid-tibial region of this leg. A similar lesion was
examined before the dematiaceous nature of the fungal palpated over the midregion of the left radius, and the entire
elements is recognized. In phaeohyphomycotic infections diaphyseal length of both femurs was firm, painful, and
caused by Alternaria alternata, Bipolaris spicifera (syn. thick. In addition, the dog had a head tilt to the left side.
Drechslera spicifera) (3, 4), Exophiala jeanselmei (4), or E. Blood was obtained for hematologic and serum enzyme
spinifera (9), the majority of the fungal elements in tissue chemistry determinations. Radiographs of the pelvis (ventro-
may be hyaline. At times, a careful search of unstained or dorsal view), right femur, left tibia, and left radius (lateral
hematoxylin and eosin (H & E)-stained tissue sections is and anteroposterior views) were obtained. A punch biopsy
needed to reveal the pigmented nature of the fungal elements from the lesion in the tibia (the site of the original swelling)
present. was also obtained. Portions of the biopsy were placed into
Currently, 62 species of dematiaceous fungi belonging to 10% neutral buffered Formalin, and the remainder of the
33 genera have been described in the literature under a wide specimens was placed in sterile 0.9% saline (NaCl). This
variety of confusing disease names, all of which, in fact, are material was submitted to the University of Georgia Veter-
agents of phaeohyphomycosis. We describe here another inary Diagnostic Laboratory at Tifton, Ga. The dog died
agent of phaeohyphomycotic infection, namely, Phiale- shortly thereafter, but permission for necropsy was denied.
monium obovatum (6), which infected the tibial bone of a Radiographic findings included soft-tissue swelling, irreg-
dog. ular periosteal proliferation, lysis of cortical bone, and
Case report. In November 1982, a 3-year-old female Ger- increased medullary density in the mid-shaft regions of the
man shepherd dog was admitted to a veterinary clinic with a left tibia (Fig. 1) and the left radius (Fig. 2). There was also
2-week history of weakness and pain in the left hind leg. a diffuse periosteal proliferation and increased medullary
Upon palpation of this limb, pain was elicited from the density throughout the diaphyses of both femurs. The
proximal thigh region to just proximal of the tarsus. There periosteal reaction in the femurs extended from the proximal
was soft-tissue swelling over the entire length of the tibia. to the distal metaphyses. The coxofemoral, stifle, tarsal, and
This was most prominent over the mid-tibial region. The dog elbow joints showed no radiologic signs.
was in good body condition otherwise and appeared to be Additional laboratory findings included neutrophilic
alert. Her rectal temperature was 38.9°C. A tentative diag- leukocytosis with left shift and moderate monocytosis. The
serum alkaline phosphatase was elevated (274 U/liter),
*
Corresponding author. whereas the other enzyme chemistry values were within
t Present address: Mammalian and Environmental Toxicology normal ranges.
Research Laboratory, Dow Chemical Company, Midland, MI Histologic findings. Histologic examination of the biopsy
48674. material from the left tibia revealed a diffuse granulomatous
987988 NOTES J. CLIN. MICROBIOL.
cultured on Sabouraud dextrose agar and Sabouraud dex-
trose 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 pig-
ment grew within 2 weeks of incubation. Based on an initial
examination of the colony and examination of teased prep-
arations 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
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both in vivo and in vitro, a subculture and tissue slides were
sent to the Division of Mycotic Diseases, Centers for Dis-
ease 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 diam-
eter 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. 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 macro-
phages and multinucleated giant cells among areas of hem-
orrhage and necrotic trabecular bone. The centers of most of
the granulomas contained microabscesses and necrotic de-
bris. 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 reac-
tion for melanin (Fig. 5, inset). FIG. 2. Lateral view of the left radius and ulna showing overly-
Mycological findings. Examination of the smears of the ing soft-tissue swelling and periosteal proliferation on both the
biopsy material and KOH preparations by microscope re- radius and ulna. The mid-shaft region of the radius also has cortical
vealed fungal hyphae. The biopsy fluid and tissue were lysis and increased medullary density.VOL. 23, 1986 NOTES 989
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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). They were intercalary or terminal and measured 5 to 8 ,um by
The isolate grew at 37 and 40°C. 2.5 to 4.5 pLm (Fig. 8).
Examination of the slide culture preparations on potato A subculture of the dog isolate was sent to Walter Gams,
dextrose agar by microscope after 2 weeks at 25°C revealed Centraalbureau voor Schimmelcultures (CBS), Baarn, The
adelophialides which developed as small pegs from the sides Netherlands, who identified it as P. obovatum. It was
of the vegetative hyphae. These adelophialides lacked the accessioned at the CBS under CBS number 400.83. It is also
basal septa and collarettes which are typical of discrete deposited in the Centers for Disease Control Division of
phialides (Fig. 7). Some well-developed discrete phialides Mycotic Diseases' culture collection under accession num-
with a basal septum were produced, but only rarely. The ber B-3703.
conidia were hyaline, one-celled, obovate, and smooth. Discussion. The genus Phialemonium was proposed by
They measured 3.0 to 6.0 pLm by 1.4 to 2.0 ,um. Gams and McGinnis (6) as being intermediate between the
Chlamydoconidia were abundant in older cultures. They genera Acremonium and Phialophora. It is characterized by
were oval to almost cylindrical, thin walled, and hyaline. the production of adelophialides arising from vegetative
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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.
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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 Phialophora spp. They are considered to be the character-
defined by Gams (5), is applied to a reduced phialide that istic conidiogenous cell for the genus. The same type of
arises from intercalary hyphal cells as a lateral, phialidic conidiogenous cell with discrete phialides is also considered
neck. There are no basal septa that separate these phialidic typical for members of the genus Acremonium. Some spe-
necks from their supporting hyphal cells. Occasionally, cies of Phialophora and Acremonium may produce
discrete phialides, which are well developed and have basal adelophialides mainly from submerged hyphae in addition to
septa, are produced by the species of Phialemonium. The the characteristic discrete phialides which characterize the
adelophialides lack conspicuous collarettes and inner two genera.
periclinal wall thickenings. Gams and McGinnis (6) described three species of
In the species belonging to the genus Phialophora, a Phialemonium, namely, P. obovatum, P. curvatum, and P.
continuous series of phialide types that range from discrete dimorphosporum. In P. obovatum, the conidia are obovate
phialides with basal septa to adelophilides is observed. But and colonies develop a greenish pigment with age.
discrete phialides with basal septa predominate in Phialemonium curvatum is distinguished by its conidia,
..Wasf
19
FIG. 6. Colony of P. obovatum on Sabouraud dextrose agar after FIG. 7. Photomicrograph showing an adelophialide and conidia
2 weeks at 25°C. of P. obovatum. Lactophenol cotton blue; magnification, x1,560.VOL. 23, 1986 NOTES 991
tissue sections were stained by the Fontana-Masson silver
procedure (10) for melanin, the light or previously undetect-
able 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,
Downloaded from http://jcm.asm.org/ on February 15, 2021 by guest
Baarn, The Netherlands, for identifying the dog isolate; Ray
Simons, Laboratory Training and Consultation Division, Labora-
tory 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.
FIG. 8. Photomicrograph showing conidia and intercalary 2. Ajello, L. 1981. The gamut of human infections caused by
chlamydoconidia of P. obovatum. Lactophenol cotton blue; magni- dematiaceous fungi. Jpn. J. Med. Mycol. 22:1-5.
fication, x 1,560. 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
which are uniformly allantoid, and colonies that do not turn and textbook of the histopathology of mycotic diseases, p.
vinaceous buff. The conidia of P. dimorphosporum are 92-95 and 253-262. Wolfe Medical Publications Ltd., London,
partly allantoid, partly ellipsoidal, and straight. The colonies England.
of P. dimorphosporum form pale, vinaceous buff pigment on 5. Gams, W. 1971. Cephalosporium-artige Schimmelpilze (Hypho-
their reverse side. The colonies of P. obovatum and P. mycetes), p. 5-10. G. Fischer, Stuttgart, Federal Republic of
curvatum have been isolated from clinical specimens. P.
Germany.
6. Gams, W., and M. R. McGinnis. 1983. Phialemonium, a new
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Recently, McGinnis et al. (8) described P. obovatum as an 7. McGinnis, M. R. 1983. Chromoblastomycosis and phaeohypho-
opportunistic pathogen in a 4.5-month-old child that died of mycosis: new concepts, diagnosis and mycology. J. Am. Acad.
thermal burns. In antemortem and postmortem examinations Dermatol. 8:1-16.
of the skin and subcutaneous tissue from the burn wounds, 8. McGinnis, M. R., W. Gams, and M. N. Goodwin. 1986.
they observed hyaline, septate, irregularly branched hyphae. Phialemonium obovatum infection in a burned child. Sabou-
Yeastlike cells and pseudohyphae were absent. In the tissues raudia 24:69-73.
of the present case, P. obovatum was polymorphic. The 9. Padhye, A. A., W. Kaplan, M. A. Neuman, P. Case, and G. N.
Radcliffe. 1984. Subcutaneous phaeohyphomycosis caused by
fungal elements consisted of septate, irregularly branched Exophiala spinifera. Sabouraudia 22:493-500.
hyphae, narrow hyphae with terminal or intercalary 10. Wood, C., and B. Russel-Bell. 1983. Characterization of pig-
chlamydoconidialike swellings, budding yeastlike cells, and mented fungi by melanin staining. Am. J. Dermatopathol.
spherical-to-oval cells that were transversely septate. When 5:77-81.You can also read
