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Phaeoacremonium krajdenii, a Cause of White Grain Eumycetoma

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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2006, p. 4619–4622 Vol. 44, No. 12
0095-1137/06/$08.00⫹0 doi:10.1128/JCM.01019-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Phaeoacremonium krajdenii, a Cause of White Grain Eumycetoma䌤

B. M. Hemashettar,1 B. Siddaramappa,2 B. S. Munjunathaswamy,2 A. S. Pangi,3 Jayashree Pattan,4
A. T. Andrade,4 A. A. Padhye,5* Lizel Mostert,6† and R. C. Summerbell6
Department of Microbiology, Basaveshwara Medical College, Chitradurga, India1; Departments of Dermatology, Venereology, and
Leprology2 and Surgery,3 Jawaharlal Nehru Medical College, and Hi-Tech Health Care and Diagnostic Center, Pvt. Ltd.,4
Belgaum, India; Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia5; and
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands6
Received 16 May 2006/Returned for modification 3 July 2006/Accepted 12 September 2006

We describe the first case of white grain pedal eumycetoma caused by Phaeoacremonium krajdenii in a
41-year-old man from Goa, India. Based on histological examination of biopsy tissue showing serpentine
granules, a culture of the granules yielding phaeoid fungal colonies, and morphological characteristics and
sequence comparison of the partial ␤-tubulin gene with the ex-type isolate of P. krajdenii, the causal agent was
identified as P. krajdenii.

CASE REPORT
A 41-year-old patient from Goa, India, visited a dermatol-
ogist in December 2000 with complaints of a single nodular,
painful swelling over the dorsum of his foot that had persisted
for 1 year. About 18 years earlier, he had developed a swelling
and multiple discharging sinuses over the dorsum of his right
foot. At that time, he was treated surgically by a local surgeon,
and based on a histological examination, he was diagnosed as
having a mycetoma of the foot. He was treated with cotrimox-
azole and sulfamethoxazole for a period of 6 months without
improvement. However, following surgery, the swelling of the
foot had subsided. After about 10 years, new nodules and
swellings had developed over the operated scar. At this time,
he consulted a dermatologist and was referred to one of us
(B.M.H.) for mycological examination.
Over the scars of the previous surgery, a single small, nod-
ular lesion measuring 6 by 6 mm was observed. The nodule was
aseptically punctured, and a pale white granule, 0.5 to 2.0 mm
in diameter, was aspirated. Direct examination of one-half of
the granule in potassium hydroxide showed numerous hyalines,
septate hyphae, and a few thick-walled cells. The other half of
the granule was cultured on Sabouraud glucose agar contain-
ing chloramphenicol (Sab⫹C) and incubated at 25 to 30°C.
White to off-white fungal colonies became evident after 8 to 10
days. Colonies slowly became darker and velvety and were
olivaceous grayish brown. A provisional diagnosis of white
grain mycetoma was made. A biopsy could not be done at this
time because of the patient’s refusal of the procedure. He was
advised to take ketoconazole (400 mg/day). However, the pa-
tient did not start the treatment because he was going to a
foreign country on a job assignment. During his stay there for
* Corresponding author. Mailing address: Mycotic Diseases Branch,
Centers for Disease Control and Prevention, Bldg. 17, Room 2027,
G-11, Atlanta, GA 30333. Phone: (404) 639-5476. Fax: (404) 639-3546.
E-mail: aap1@cdc.gov.
† Present address: Department of Plant Pathology, University of
Stellenbosch, Matieland, South Africa.
䌤
Published ahead of print on 27 September 2006.
the next 2 years, new lesions appeared on the plantar aspect of
the foot (Fig. 1A).
After his return in October 2000, a biopsy was performed. A
portion of the biopsy tissue was sent for histopathological ex-
amination, and another portion was cultured. The specimen
yielded the same fungus as had been obtained previously. Both
a biopsy tissue block and a subculture were sent to one of us
(A.A.P.) at the Centers for Disease Control and Prevention
(CDC) for additional studies. The X-ray examination of the
foot had shown no bone involvement but revealed soft-tissue
swelling. Results for routine clinical chemistry, hematological
investigations, and serology for human immunodeficiency virus
were within normal limits. The patient was treated with itra-
conazole tablets (400 mg/day) for 4 months. A surgical rede-
bridement was contemplated after about 4 months of treat-
ment, with the hope that the spread of the infection would be
restricted by then. However, the patient could not be found for
the follow-up.
The slides of the biopsy tissue were stained with hematoxylin
and eosin (H&E) and Gomori’s methenamine silver (GMS)
stains. Sections of the skin biopsy sample stained with H&E
showed swelling, draining sinuses, and intradermal mycetoma.
The mycetoma consisted of a necrotic core where eosinophilic
material surrounded the fungal elements in the granules, a
presentation referred to as the Splendore-Hoeppli reaction
(Fig. 1B and C). The granules measured 0.5 to 2.0 mm in
diameter and were irregular in shape. The fungal elements in
the core were not pigmented in H&E-stained sections. Around
the cores of the granules, there were abundant epithelioid
macrophages, plasma cells, and eosinophils as well as occa-
sional neutrophils. Outside the mycetoma, there was scattered
inflammation with an abundance of eosinophils. Epidermal
psoriasiform hyperplasia covered the mycetoma site. The slides
stained with the GMS procedure showed intertwined hyphal
elements consisting of septate, branched hyphae with various
lengths, 2.0- to 3.0-␮m diameters, and thick-walled, vesiculate
chlamydospores (Fig. 1D).
A portion of the biopsy tissue containing granules was cul-
tured on Sab⫹C and Sab⫹C containing cycloheximide

4619
4620 CASE REPORTS J. CLIN. MICROBIOL.

FIG. 1. (A) Mycetoma on the right foot of the patient showing discharging sinuses on the dorsal aspect of the foot. (B) Tissue section showing
a serpentine granule of Phaeoacremonium krajdenii. Stain, H&E. Magnification, ⫻483. (C) Magnified portion of the granule showing a Splendore-
Hoeppli reaction. Stain, H&E. Magnification, ⫻772. (D) Portion of the granule showing intertwined hyphal elements and vesiculate chlamydo-
spores. Stain, GMS. Magnification, ⫻772. (E) Slide culture of P. krajdenii (CDC B-6093 is CBS 110361) showing mono- and polyphialides of
cylindrical to elongate-ampulliform shapes and ellipsoidal to allantoid conidia. Magnification, ⫻772. 1, 2, and 3, first, second, and third types of
phialides as described in the text; arrows, collarettes.
VOL. 44, 2006
(Sab⫹C⫹C) prepared in-house. The morphology of the isolate
was studied on potato dextrose agar (PDA) (Difco Laborato-
ries, Detroit, MI) and malt extract agar containing 2% malt
extract (Oxoid Ltd., England, United Kingdom) and 1.5% agar
(Difco Laboratories, Detroit, MI). The cultures were incu-
bated at 25°C and 37°C in the dark. The initial colonies on
Sab⫹C and PDA were moist, creamy to off-white, and slightly
raised in the center. They became grayish brown to dark gray
after 8 to 10 days at both temperatures. Growth was not in-
hibited by cycloheximide. The colonies after 2 weeks were
velvety, flat, and olivaceous brown and measured 18 to 20 mm
in diameter at 25°C and 10 to 12 mm in diameter at 37°C. No
growth was observed at 40°C.
The slide cultures on PDA after 2 weeks of growth at 25°C
were mounted in lacto-fuchsin medium. Microscopic mounts
from the malt extract agar culture were made after 2 to 3 weeks
of incubation at 25°C from the aerial mycelium as well as from
the agar surface. The hyphae were septate, verrucose, medium
brown, and 2 to 3 ␮m wide. Warts were observed on many
vegetative hyphae. The conidiophores were short, unbranched,
occasionally constricted at the basal septa, 15 to 45 ␮m long,
and 1.5 to 2 ␮m wide. The apical cells of the conidiophores
produced one or two phialides each. The phialides were
monophialidic (i.e., with one fertile opening) at first but often
became polyphialidic (i.e., they developed additional fertile
openings on short, forking side branches). Phialides differed in
shape. Those of the first type were cylindrical, wide at the base,
tapering toward the apex, 4 to 11 ␮m long, and 1 to 2 ␮m wide.
The second type was elongate ampulliform, attenuated at the
base, 7 to 15 ␮m long, and 1.5 to 2 ␮m wide. The third type was
subcylindrical, elongate ampulliform at the base, 13 to 22 ␮m
long, and 1 to 2 ␮m wide. Numerous phialides at maturity gave
rise percurrently to new phialides in a process referred to as
phialide rejuvenation. When this occurred, the older phialides
were often inflated at the base and had one to five septa. The
collarettes at the tips of the phialides were flaring, narrow, 1 to
3 ␮m long, and 1 to 2 ␮m wide. The conidia were subhyaline,
oblong ellipsoid to allantoid (sausage shaped), and 3 to 5 ␮m long
by 1 to 2.5 ␮m wide (Fig. 1E). The phialides on the agar surface
were cylindrical and were 2 to 11 ␮m long and 1 to 2 ␮m wide.
The conidia on the agar surface were allantoid to oblong ellipsoid,
4 to 6.5 ␮m long, and 1 to 1.5 ␮m wide. The isolate hydrolyzed
gelatin. It was studied in more detail at the Centraalbureau voor
Schimmelcultures (CBS), Utrecht, The Netherlands, and was ac-
cessioned in the CDC and CBS collections as CDC B-6093 and
CBS 110361, respectively.
Genomic DNA extraction was performed on CBS 110361 by
using the isolation protocol of Lee and Taylor (10). Approxi-
mately 560 bp at the 5⬘ end of the ␤-tubulin gene was amplified
using primers T1 (14) and Bt2b (5). The PCR amplification
and sequencing were performed as described by Mostert et al.
(13). A consensus sequence was computed from the forward
and reverse sequences with SeqMan from the Lasergene pack-
age (DNAstar, Madison, WI). The sequence obtained was com-
pared with the ␤-tubulin sequence of the ex-type sequence of
Phaeoacremonium krajdenii (CBS 109479, GenBank accession no.
AY579330) with Sequence Alignment Editor version 2.0a11
(16) and the percent DNA similarity calculated. The ␤-tubulin
sequence of CBS 110361 was 99.8% similar (1 base variation)
to the sequence of the ex-type isolate of P. krajdenii over a total
CASE REPORTS 4621
continuous length of 556 bases. The close DNA similarity of
CBS 110361 to the ex-type isolate confirmed this isolate as P.
krajdenii.
At present, there are 28 species known to cause eumycotic
mycetoma. Thirteen of these species cause white grain mycetoma,
and 15 species are known to cause black grain mycetoma (11).
The majority of described human Phaeoacremonium infec-
tions have been subcutaneous abscesses and cysts or chronic or
acute mycotic arthritis (6, 8, 12, 15). Cases are reported with
approximately equal frequencies in immunocompromised and
immunocompetent hosts. Disseminated infections, fungemia,
and endocarditis have been reported only occasionally (7, 18).
In the course of a recent taxonomic revision of the genus
Phaeoacremonium (13), eight species were recognized as caus-
ing human infection. They were Phaeoacremonium alvesii,
Phaeoacremonium amstelodamense, Phaeoacremonium gris-
eorubrum, Phaeoacremonium krajdenii, Phaeoacremonium
parasiticum, Phaeoacremonium rubrigenum, Phaeoacremonium
tardicrescens, and Phaeoacremonium venezuelense. P. parasiti-
cum, the type species of the genus, was originally described as
Phialophora parasitica (3). At present, two of the eight recog-
nized opportunistic Phaeoacremonium species have been re-
ported to cause white grain eumycetoma. In the first reported
mycetoma case involving what later transpired to be a Phae-
oacremonium isolate, a fungus causing white grain eumyce-
toma in a Venezuelan patient was identified as Cephalosporium
serrae (1), an arcane synonym for the fungus now known as
Verticillium nigrescens (17). Crous et al. (3) reidentified the
case isolate, CBS 651.85, as Phaeoacremonium inflatipes. In a
molecular and morphological reexamination, the isolate was
shown not to be a member of P. inflatipes and was redisposed
as the ex-type isolate of the new species P. venezuelense (13).
An earlier reported case of white grain eumycetoma caused
by a Phaeoacremonium isolate involved a 30-year-old Indian
woman living in the United Kingdom who developed myce-
toma in her right foot after visiting India (9). The causal agent
was identified as Phialophora parasitica. The isolate could not
be reexamined because it was nonviable. The CBS collection
contains a P. krajdenii isolate, CBS 633.93, that was stated to be
from a mycetoma from a 31-year-old male examined in Nor-
way. This case, to our knowledge, was not reported in the
scientific literature. The isolate was originally deposited as
Phialophora repens, a name that was frequently misapplied to
isolates of undescribed Phaeoacremonium species in the
years from 1970 to 2000. Genuine P. repens is not involved
in human infection (13).
The present case, then, represents the first reported case of
white grain mycetoma caused by P. krajdenii. An interesting
feature of this case was that the granules in the tissue were
surrounded by eosinophilic material exhibiting a Splendore-
Hoeppli reaction. Such an intensely eosinophilic reaction has
also been reported surrounding white grains of Pseudallesche-
ria boydii (2). Both of these eumycotic agents with melanized
hyphae or conidia produce whitish grains in tissue. Melanin
production in Phaeoacremonium species is facultative, while
melanin pigment is limited to conidia in P. boydii.
The present case is also the first to be reported with the
4622 CASE REPORTS
recently published name P. krajdenii. Two previous cases in-
volving this species have been published with the name P.
repens; for both, the case isolates in CBS were reexamined and
sequenced as described by Mostert et al. (13) and shown to be
P. krajdenii. Both cases were very similar to the present one,
but the lesions examined were tentatively classified as subcu-
taneous phaeohyphomycotic cysts rather than mycetoma, de-
spite the presence of grain-like “microcolonies” or “hyphal
masses” in the lesions. One involved a lesion on the scalp of a
lepromatous-leprosy patient from what is now the Democratic
Republic of Congo (12), while the other involved a lesion on
the hand of a patient in Japan who had no discernible predis-
posing factors other than mild diabetes mellitus (8). In the
Japanese report, Hironaga et al. (8) commented that the le-
sion, which was first noticed by the patient only 2 months prior
to presentation, contained hyphal aggregates that “looked like
the grains of eumycotic mycetomas but did not manifest the
Splendore-Hoeppli phenomenon sometimes seen in [these]
mycetomas.” These authors also commented that draining
sinuses, a feature essential to the classification of a lesion as a
mycetoma, were not seen. The present case, featuring a lesion
that developed for many years, was more typical of a classic
mycetoma, with well-developed grains, a marked Splendore-
Hoeppli effect, and draining sinuses, as can be seen in Fig. 1A.
It would appear, then, that subcutaneous cases caused by P.
krajdenii will at first manifest as phaeohyphomycotic cysts with
organized grain-like hyphal clusters in granulomatous tissue
but will later mature as classic white grain eumycetomas.
The majority of Phaeoacremonium species are plant patho-
gens (3, 4, 13) and endophytes infecting woody plants. Grape
diseases named Petri disease and esca, apoplexy, or black mea-
sles are known to occur in Italy, the United States, and other
countries. Apart from plant-related isolations, some Phae-
oacremonium species have also been isolated from soil (3, 13).
Other sources of inoculum could include dust and indoor water
sources (13). Medically important Phaeoacremonium species
can easily be distinguished from species not involved in human
disease by their abilities to grow at 37° and 40°C. Even though
the identification of medically important Phaeoacremonium
spp. can be very difficult based on cultural and micromorpho-
logical characters alone, the definitive identification of these
species is facilitated by recently developed identification pro-
cedures involving the combined or separate use of ␤-tubulin
sequence data and reevaluated morphological data. As out-
lined by Mostert et al. (13), an online keying system allowing
the use of both morphological and molecular characters can be
accessed via the CBS website by clicking on “The Phaeoacre-
monium database” at http://www.cbs.knaw.nl/databases/index
.htm.
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J. CLIN. MICROBIOL.
Nucleotide sequence accession number. The nucleotide se-
quence for CBS 110361 described in this study was deposited
in GenBank under accession number AY57933.
L.M. thanks Pedro W. Crous (Centraalbureau voor Schimmelcul-
tures, The Netherlands) for the guidance and supervision given during
her Ph.D. study.
L.M. acknowledges the Odo van Vloten Stichting and the Royal
Netherlands Academy of Arts and Sciences for financial support.
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