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Journal of Clinical Microbiology, June 2001, p. 2321-2324, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2321-2324.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Phaeohyphomycotic Cyst Caused by
Colletotrichum crassipes
Luiz Guilherme
Martins Castro,1
Carlos
da Silva
Lacaz,2,3
Josep
Guarro,4,5,*
Josepa
Gené,4,5
Elisabeth
Maria
Heins-Vaccari,2,3
Roseli
Santos
de Freitas
Leite,2,3
Giovana
Letícia
Hernández
Arriagada,2,3
Márcia
Maria
Ozaki Reguera,1
Eunice Miki
Ito,1
Neusa Yuriko Sakai
Valente,1 and
Ricardo
Spina Nunes1
Laboratório de Micologia Médica
do Instituo de Medicina Tropical de São
Paulo,2 Laboratório de
Micologia Médica (LIM-53)3 and
Serviço de Dermatologia1 do
Hospital das Clínicas da Faculdade de Medicina da
Universidade de São Paulo, São Paulo, Brazil, and
Unitat de Microbiologia, Departament de Ciències
Mèdiques Bàsiques, Facultat de Medicina i Ciències de
la Salut, 43201 Reus,4 and Institut
d'Estudis Avançats,5 Universitat Rovira i
Virgili, Tarragona, Spain
Received 12 February 2001/Returned for modification 24 February
2001/Accepted 22 March 2001
 |
ABSTRACT |
A case of phaeohyphomycosis is reported in a male renal transplant
recipient with a nodular lesion in the right leg who was treated
with immunosuppressing drugs. The lesion consisted of a purulent cyst
with thick walls. The cyst was excised surgically, and the
patient did not receive any antifungal therapy. One year later
he remains well. Histological study of the lesion showed a
granulomatous reaction of epithelioid and multinucleate giant cells,
with a central area of necrosis and pus. Fontana-Masson staining
demonstrated the presence of pigmented hyphal elements. The fungus Colletotrichum crassipes was grown in different
cultures from the cyst. The in vitro inhibitory activities of eight
antifungal drugs against the isolate were tested. Clotrimazole and
UR-9825 were the most active drugs. This case represents the first
known reported infection caused by this rare species.
| |
TEXT |
Within the huge number of fungal
species involved in human opportunistic infections, numerous mitosporic
species, which develop their asexual reproductive structures on
cup-shaped (acervuli) or spherical (pycnidia) fruiting bodies
(conidiomata), are frequently being reported. They are classified
within the form class Coelomycetes, and up to now, at least 11 genera
and 22 species have been implicated in human disease (4,
13). Colletotrichum Corda is one of the most common
genera, which is characterized by causing both phaeohyphomycosis
(1, 6, 7) and hyalohyphomycosis (5, 7).
Phaeohyphomycoses comprise a vast array of opportunistic fungal
infections characterized by the presence of different types of
melanized fungal elements in tissue (8). These elements can be clearly detected with the Fontana-Masson stain, because they
become dark, in contrast to the fungal elements present in hyalohyphomycoses, which remain colorless.
The genus Colletotrichum is a typical pathogen of plants,
which has been traditionally included in the coelomycetous order Melanconiales, characterized by the formation of acervular conidiomata when the fungus parasitizes the plant tissue. Species concepts are
based on morphology of the fungi on natural substrate and to a lesser
extent in culture, sometimes combined with host specificity or the lack
of it. In culture, these fungi develop conidiomata, which consist of
conidial masses supported by a superficial cushion-like mass of short
conidiophores, among which erect, unbranched, and darkly pigmented
sterile hyphae (setae) are developed. They also frequently develop
appressoria (flat and dark-pigmented swellings at the end of a hypha),
which they use to attach themselves to the host surface before
penetrating the tissue. These elements are typical of plant-pathogenic
fungi. The presence of these elements is an important diagnostic
feature for genus recognition, and their shape is important for species
separation (12, 14).
Currently, of the several hundred species described, only four species
of Colletotrichum have been associated with human infections (4). These species are C. dematium, C. gloeosporioides, C. coccodes, and C. graminicola.
However, these infections are very rare and are generally associated
with some form of trauma. They manifest as keratitis or subcutaneous
lesions, although a case of invasive infection has also been reported
(9). The present report describes the first case involving
Colletotrichum crassipes causing a subcutaneous
phaeohyphomycotic cyst.
Case report.
A 34-year-old Brazilian male gardener, resident
of São Paulo, had a renal transplant 2 years earlier due to
chronic and progressive renal failure. The patient was
immunocompromised secondary to cyclosporine, prednisone, azathioprine,
and captopril treatment but otherwise in good condition. One year after
transplantation, he noticed a small area of swelling on the anterior
face of the right leg, which progressed slowly to a painless nodule
with no noticeable antecedent trauma. On examination in September 1998, he showed a subcutaneous nodule (2.5 cm in diameter) which was not
painful, and the overlying skin appeared completely normal, with no
sign of inflammation (Fig. 1A). The
nodule was surgically excised and found to encompass a flesh-colored
cystic lesion. The cyst had a thick wall and was purulent (Fig. 1B).
Part of the nodule was fixed in 10% formalin, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin, Groccott-Gomori
methanamine silver, and Fontana-Masson stains. Three different portions
of the nodule were each homogenized separately in sterile saline with a
sterile mortar and pestle and used for microbiological analysis. Each
homogenized specimen was cultured by standard techniques on routine
media for aerobic or anaerobic bacterial isolation and on Sabouraud
dextrose agar (Oxoid, Basinstoke, England), incorporating chloramphenicol (0.05%) with and without cycloheximide (0.04%), for
fungal isolation. All cultures were incubated at 35 to 37°C.
Examination of hematoxylin- and eosin-stained tissue section of the
excised nodule revealed a granulomatous lesion (Fig.
2A).
The subcutaneous tissue was
fibrotic, and in the areas of fibrosis
there were circumscribed small
foci of mixed granulomatous and
suppurative inflammation. Palisading
epithelioid cells, multinucleate
giant cells, and lymphocytes
surrounded central microabscesses
of polymorphonuclear leukocytes.
Grocott-Gomori stain revealed
the presence of a few distorted hyphal
elements (Fig.
2B), and
Fontana-Masson stain revealed the presence of
melanine in these
hyphae. Routine bacteriological cultures were
negative, while
those for fungi were positive. Multiple dark-colored
colonies,
apparently belonging to the same fungus, grew in all
Sabouraud
petri dishes. For identification purposes, the fungus was
cultured
on potato carrot agar (PCA; 20 g of potato, 20 g of
carrot, 18
g of agar, 1,000 ml of tap water, homemade) and oatmeal
agar (OA;
30 g of oat flakes, 1 g of MgSO
4
· 7H
2O, 1.5 g of KH
2PO
4,
15 g
of agar, 1,000 ml of tap water, homemade) and incubated at
25,
37, 40, and 45°C in the dark.
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FIG. 2.
(A) Hematoxylin and eosin stain of the nodular tissue,
showing a granulomatous lesion. (B) Groccott-Gomori methanamine silver
stain, showing a few hyphal elements. Magnification, ×400.
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|
On PCA and OA at 25°C, similar colonies grew very quickly, covering
the surface of the petri dishes in 10 days. They were
cottony,
olivaceous brown with a black reverse. The best growth
and sporulation
was obtained in PCA. On this medium the fungus
produced abundant stiff,
erect, sterile hyphae (setae) and appressoria
(Fig.
3). The setae were acicular, septate,
brown to dark brown,
thick-walled, and up to 150 µm long. The
appressoria were irregular
and dark brown with crenate or deeply lobed
walls, measuring,
on average, 18 µm long by 10 µm wide. The
conidia, emerging from
slightly clavate conidiogenous cells, were
slimy, hyaline, mostly
cylindrical and measuring 11 to 18 µm long by
6.5 to 8 µm wide.
At 37°C the colonies attained a diameter of 12 to
15 mm in 10
days. The fungus did not grow at 40°C.
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FIG. 3.
C. crassipes FMR 6728. (A) Seta (arrow),
conidiogenous cells and conidia. Magnification, ×640. (B) Conidia and
appressoria (arrows). Magnification by Nomarski optics, ×1,600.
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Based on morphology of the conidiophores and conidia and the presence
of setae and numerous appressoria, the fungus was identified
as a
Colletotrichum sp. We first thought that it was a strain
of
C. gloeosporioides, the most common species of the genus,
which
we recently isolated from a subcutaneous infection in Brazil
(
5).
However, a more detailed study, mainly taking into
account the
morphology of the appressoria with crenate or deeply lobed
margins,
helped us to identify it as
C. crassipes. A
comparison of this
clinical isolate with other strains from different
origins (CBS
159.75 and IMI 302450) confirmed its identity. The isolate
is
maintained in our mycology laboratory at the Medicine Faculty
of the
Rovira i Virigli University in Reus, Spain, as FMR 6728.
Other living
cultures have been deposited in the Centraalbureau
voor
Schimmelcultures of the Netherlands as CBS 109355 and in
the Belgian
Coordinated Collections of Microorganisms/Institute
of Hygiene and
Epidemiology Mycology (BCCM/IHEM).
C. coccodes, C. dematium, C. gloeosporioides, and
C. graminicola, the other opportunistic species of the genus, are
clearly
distinguished from
C. crassipes mainly by the shape
and size of
the conidia.
C. dematium and
C. graminicola have falcate conidia,
while in
C. coccodes
they are fusiform and measure 16 to 22 µm
long by 3 to 4 µm wide.
In
C. gloeosporioides, the closest species
morphologically
to
C. crassipes, the conidia are also cylindrical
but
narrower (3 to 4.5 µm) and generally longer (up to 24 µm).
Furthermore, in
C. gloeosporioides the appressoria show an
irregular
edge but are never deeply lobed.
C. crassipes
might be a composite
species consisting of a number of separate taxa
(
2). Baxter
et al. (
3) described many
variations in the colony colors.
This fungus has been reported as
parasitizing legumes in Malaysia,
Zambia, and India (
2).
Antifungal susceptibility testing of the isolate was accomplished by a
previously described microdilution method (
11) mainly
according to the guidelines of the National Committee for Clinical
Laboratory Standards for molds (
10), using RPMI 1640 medium
buffered to pH 7 with 0.165 M morpholinepropanesulfonic acid
(MOPS),
an inoculum of 10
5 CFU/ml, an incubation
temperature of 30°C, an incubation period
of 72 h, and an
additive drug dilution procedure. MICs were 1
µg/ml for amphotericin
B, >128 µg/ml for 5-fluorocytosine, 2 µg/ml
for itraconazole,
voriconazole, and terbinafine, 4 µg/ml for miconazole,
>16 µg/ml
for fluconazole, 8 µg/ml for ketoconazole, 0.5 µg/ml
for
clotrimazole, and 0.125 µg/ml for UR-9825.
This fungus was less susceptible to antifungals than the other three
opportunistic
Colletotrichum species tested previously
(
5). Their MICs were low, with a few exceptions. In this
study,
eight antifungals displayed MICs higher than 1 µg/ml against
C. crassipes; only clotrimazole and UR-9825, the latter
being a new
potent triazole from Uriach S.A., Barcelona, showed lower
MICs.
The present case, however, was resolved without antifungal
therapy.
| |
ACKNOWLEDGMENTS |
We are indebted to Arvind A. Padhye (Centers for Disease Control
and Prevention, Atlanta, Ga.) for reviewing the manuscript.
This study was supported by CICYT (Ministerio de Educación y
Cultura, Spain) grant PM98-0059.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unitat de
Microbiologia, Departament de Ciències Mèdiques
Bàsiques, Facultat de Medicina i Ciències de la Salut,
Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201-Reus,
Tarragona, Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail:
umb{at}fmcs.urv.es.
| |
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Journal of Clinical Microbiology, June 2001, p. 2321-2324, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2321-2324.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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