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Medical Mycology
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Cutaneous hyalohyphomycosis caused by a
Chrysosporium species related to Nannizziopsis
vriesii in two green iguanas (Iguana iguana)
M. L. Abarca ab; J. Martorell c; G. Castellá ab; A. Ramis b; F. J. Cabañes ab
a
Veterinary Mycology Group, Autonomous University of Barcelona, Bellaterra,
Spain
b
Department of Animal Health and Anatomy, Autonomous University of Barcelona,
Bellaterra, Spain
c
Department of Animal Medicine and Surgery, Autonomous University of Barcelona,
Bellaterra, Spain

First Published on: 07 March 2008

To cite this Article: Abarca, M. L., Martorell, J., Castellá, G., Ramis, A. and
Cabañes, F. J. (2008) 'Cutaneous hyalohyphomycosis caused by a Chrysosporium species related to Nannizziopsis
vriesii in two green iguanas (Iguana iguana)', Medical Mycology, 1 - 6
To link to this article: DOI: 10.1080/13693780701851711
URL: http://dx.doi.org/10.1080/13693780701851711

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 Medical Mycology
2008, 16, iFirst article

Case Report
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Cutaneous hyalohyphomycosis caused by a Chrysosporium

species related to Nannizziopsis vriesii in two green
iguanas (Iguana iguana)
M. L. ABARCA*$, J. MARTORELL%, G. CASTELLÁ*$, A. RAMIS$ & F. J. CABAÑES*$
*Veterinary Mycology Group, and $Department of Animal Health and Anatomy and %Department of Animal Medicine and
Surgery, Autonomous University of Barcelona, Bellaterra, Spain

This report describes the first isolation of a Chrysosporium species as the etiological
agent of dermatomycosis in two green iguanas (Iguana iguana). The ITS-5.8S
rRNA gene of the two strains was sequenced and a search on the GenBank
database revealed that the closest match was Nannizziopsis vriesii. Treatment with
oral ketoconazole, in combination with topical 2% chlorhexidine solution and
terbinafine resulted in clinical cure.
Keywords Chrysosporium, iguana, Nannizziopsis vriesii, pet reptiles,
hyalohyphomycosis

Introduction C. keratinophilum, C. tropicum and Chrysosporium sp.

Many different mycotic diseases have been reported in
captive reptiles. Etiological agents of cutaneous and
systemic infections in reptiles are attributed to a wide
variety of filamentous fungi and yeasts, although they
have been often inadequately identified [1]. As a rule,
fungal infection of reptiles has been regarded as
opportunistic, caused by normally saprophytic organ-
isms that invade living tissue strictly under favorable
circumstances for the pathogen. Predisposing factors
such as suboptimal cage temperatures and inappropri-
ate environmental conditions are often involved [13].
The genus Chrysosporium Corda comprises a large
group of ubiquitous, mostly soil-borne and often
keratinolytic anamorphic species which are responsible
for occasional opportunistic infections in humans [4].
In reptiles, Chrysosporium queenslandicum has been
implicated in disseminated infection in a garter snake
[5]. Some other Chrysosoporium species such as
Received 2 August 2007; Accepted 7 December 2007
Correspondence: M. L. Abarca, Grup de Micologia Veterinària,
Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinà-
ria, Universitat Autonoma de Barcelona E-08193 Bellaterra,
Barcelona, Spain. Tel.: 32 93 5811542; fax: 34 93 5812006.
E-mail: Lourdes.Abarca@uab.es
– 2008 ISHAM
have been cited as agents of cutaneous and systemic
mycoses, as reviewed by Paré et al. [6]. Over the last few
years, several reports have implicated the Chrysospor-
ium anamorph of Nannizziopsis vriesii as the cause of
outbreaks of dermatitis in different reptile species such
as chameleons [6], snakes [7,8], salt-water crocodiles [9]
and bearded dragons [10]. Nannizziopsis vriesii has also
been isolated from a nasal granuloma in an ameiva
lizard [11]. In humans, Chrysosporium anamorph of N.
vriesii has been identified as the cause of a mycotic
brain abscess [12] and has been isolated from bronchial
washings [13].
This report describes for the first time two individual
cases of cutaneous hyalohyphomycosis caused by a
Chrysosporium species in two pet green iguanas (Iguana
iguana).
Case reports
Case 1
A 6-month-old, 75 g male green iguana (Iguana iguana)
was presented to the Veterinary Teaching Hospital of
the Autonomous University of Barcelona on Septem-
ber 2006 due to skin thickening over the distal aspect of
the left hind leg (Fig. 1a). The animal was housed in a
DOI: 10.1080/13693780701851711
 2 Abarca et al.
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Fig. 1 Case 1: Green iguana with skin thickening in the left distal leg (a); Potassium hydroxide mount of scales showing hyaline and septate
hypha (b); Culture of scales on Sabouraud at 258C (c).
glass tank, 6030 40 cm, with a broad-spectrum
ultraviolet lighting and coconut bark for substrate. The
temperature was 28308C, achieved with a commercial
heat mat. The diet consisted of commercial dry food for
young iguanas. The lizard was bright and alert, and ate
and drank well. A physical examination revealed that
the lizard was in good body condition. There was focal
skin thickening, with retention of scaly squames, over
the dorsal aspect of the left tarsus. A sample of
hyperplastic skin was removed from the lesion and
submitted for microbiological culture.
Direct microscopic examination of scales in potas-
sium hydroxide preparations revealed hyaline septate
hypha (Fig. 1b). Numerous white fungal colonies were
obtained in pure culture consistent with Chrysosporium
sp. (strain CCFVB CH10) on Sabouraud dextrose agar
(Oxoid Ltd., Basingstoke, England) supplemented with
chloramphenicol and incubated at 258C. Various
bacterial colonies grew on Columbia agar5% sheep
blood (bioMérieux, Marcy l’Etoile, France) and no
growth was observed on MacConkey agar (Oxoid).
Treatment was initiated with ketoconazole (20 mg/kg/
24 h PO), in combination with 2% chlorhexidine
solution and terbinafine administered topically. The
lizard was re-evaluated after one month and new
healthy skin was developing at the site of the previous
lesion, which was improving with each ecdysis.
Case 2
In January 2007, a 3-year-old, 175 g male green iguana
was examined in the Veterinary Teaching Hospital due
– 2008 ISHAM, Medical Mycology
 Chrysosporium sp. hyalohyphomycosis in green iguanas 3

to an ulcer on the right leg. The owner reported the by direct microscopy, enrofloxacin treatment was dis-
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skin lesion had worsened since he had acquired the continued. The iguana was receiving oral ketoconazole,
animal one month ago. Data from the previous owner and topical 2% clorhexidine solution and terbinafine
was unavailable. The animal was housed in a glass tank, for 14 weeks. Five months after presentation, the
80 40 50cm, with an incandescent light, a piece of lesions had regressed fully (Fig. 2b).
artificial grass for substrate, and two tree branches. The
temperature was 23248C, achieved with a commercial Mycological study
heat rock. The diet consisted of commercial orange
The two strains showed moderate growth on Sabour-
juice and vegetables, mainly lettuce. The animal was
aud dextrose agar (Oxoid) (23 cm diam in 14 days) at
bright and alert, with normal appetite and behavior. A
258C and appeared as yellowish-white, felty and dense
physical examination revealed a deep, severe, crusting,
colonies. Growth rate was slower at 378C (12 cm diam
ulcerative dermatitis over the anterior and dorsal
in 14 days), but no growth was observed at 428C.
aspect of the right hind leg, extending from the
Conidia were typically pyriform to clavate with trun-
proximal thigh to mid-tibia (Fig. 2a). The iguana was
cate bases, 1-celled, 3523 mm, sessile and usually
premedicated with 1 mg/kg butorphanol administered
formed on side branches. Both strains were morpholo-
by intramuscular injection into a tail muscle. Anesthe-
gically identified as members of the anamorphic genus
sia was induced with 5% isoflurane delivered via face
Chrysosporium.
mask. After that, the animal was maintained through
The internal transcribed spacers (ITS 1 and ITS 2)
intubation with uncuffed endotracheal tube of 1 mm in
and the 5.8S rRNA gene of the two strains were
diameter. Biopsy was taken and skin samples were
sequenced. Procedures for DNA extraction, amplifica-
submitted for microbiological culture. Histological
tion, and sequencing were according to the protocols
examination confirmed a severe granulomatous ulcera-
outlined by Accensi et al. [14]. The sequences were
tive fungal dermatitis. Focal areas of epidermal necrosis
aligned with Clustal X (1.81) of multiple sequence
with mononuclear (lymphocytes, macrophages) peri-
alignment computer program [15]. Adjustments for
vascular infiltrate were observed in superficial dermis.
improvement were made visually when necessary. Both
In the deep dermis there were multiple coalescing
isolates showed nearly identical ITS1-5.8S-ITS2 se-
granulomas made up mainly of macrophages, multi-
quences (99.80% identity). A search of the GenBank
nucleated giant cells and scattered heterophils. Some
database revealed that the closest match was N. vriesii
of these granulomas presented central necrotic areas
AJ131681. Phylogenetic analysis of the sequences using
(Fig. 2c), in which scattered hyphae were noted through
the Neighbour-joining method [16] (Fig. 3) revealed
the use of the Periodic acid-Schiff reaction (PAS) and
that strains from iguanas form a strongly supported
Grocott’s methenamine silver stain (GMS). The fungal
clade with N. vriesii AJ131681. Our strains and N.
elements were also found associated with macrophages
vriessi are closely related although they showed only an
and multinucleated giant cells (Fig. 2d). Ziehl-Neelsen
81% matched identity. The sequence of the strains
acid fast staining did not reveal the presence of acid fast
examined in this study has been deposited in de
organisms.
GenBank database under accession no EU018451 and
A Chrysosporium species was recovered in pure
EU018452.
culture on Sabouraud dextrose agar (Oxoid) supple-
mented with chloramphenicol which had been incu-
Antifungal susceptibility testing
bated at 258C (strain CCFVB CH11). On Columbia
agar5% sheep blood (bioMérieux), only coagulase- Susceptibility of the strains to ketoconazole (KTZ),
negative Staphylococcus sp., later identified as Staphy- itraconazole (ITZ) and terbinafine (TBF) was per-
loccocus saprophyticus, was isolated and no growth was formed using the agar diffusion method Neo-Sensitabs
observed on MacConkey agar (Oxoid). According to (Rosco Diagnostica A/S, Taastrup, Denmark). After
the antimicrobial susceptibility results of the isolated seven days of incubation, there were broad zones of
bacteria, initial treatment was enrofloxacin (5 mg/kg/ inhibition noted with both strains around antifungal
24 h PO) and ketoconazole (20 mg/kg/24 h PO) in tablets (KTZ: 5054 mm; ITZ: 3032 mm; TBF:
combination with topical 2% chlorhexidine solution 7880 mm).
and terbinafine. The owner was advised to improve
some husbandry practices, such as the kind of light,
Discussion
heat source, and diet. After one month, the lizard was
re-evaluated and the progression of the wound healing This report describes two cases of dermatomycosis
was deemed satisfactory. As no bacteria were observed caused by a Chrysosporium species in green iguanas. To
– 2008 ISHAM, Medical Mycology
 4 Abarca et al.
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Fig. 2 Case 2: Green iguana showing ulcerative dermatitis in the right leg (a); Regression of lesions after treatment (b); Granuloma in deep
dermis (H &E stain) (c); Fragments of hyphae in the central area of a granuloma (PAS stain) (d); Culture of scales on Sabouraud at 258C (e);
Microscopic appearance of the isolate showing fertile hyphae bearing conidia on side branches (f).

our knowledge this is the first report of Chrysosporium
spp. being implicated in a disseminated cutaneous
infection in iguanas. The ITS-5.8S rRNA gene of the
two strains was sequenced and a search on the
GenBank database revealed that the closest match
was Nannizziopsis vriesii. The isolation of the fungi in
pure culture along with the presence of fungal elements
within histological sections of affected tissues, confirms
this fungus as the etiologic agent of the infections in
these reptile species.

– 2008 ISHAM, Medical Mycology


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88
60
100
0.3 0.2
Fig. 3 Neighbour-joining tree based on nucleotide sequences from the ribosomal internal transcribed spacer (ITS) and 5.8S rRNA gene.
Phylogenetic analysis was performed with Kimura 2-parameter model, including transitions and transversions and with pairwise deletion for the
treatment of the handling gaps/missing data. Confidence values for individual branches were determined by bootstrap analyses (1000
replications) and maximum parsimony. Bootstrap replication frequencies are indicated in the internodes. Candida albicans was used as outgroup.
It has been suggested that the Chrysosporium ana-
morph of N. vriesii represents a species complex and
that its members may be allied to specific hosts [1,7,10],
but no results have been published. Molecular data of
iguana isolates revealed that they showed nearly
identical ITS1-5.8S-ITS2 sequences and although they
were closely related to N. vriesii, they showed only an
81% of identity. Concerning some physiologic charac-
teristics, most of the Chrysosporium anamorph of N.
vriesii strains isolated from reptiles, except those from
bearded dragons, were unable to grow at 358C [1,7,10].
The isolates recovered in the present cases could
develop at 378C, although the growth rate was slower
than at 258C. Molecular and phenotypic studies of
these strains are in progress.
In the reported outbreaks of reptile mycoses caused
by the Chrysosporium anamorph of N. vriesii, the
sources of the etiologic agents were not determined
[611]. A survey of the skins of healthy squamate
reptiles from zoological and veterinary institutions
revealed that this fungus is a very rare constituent of
the cutaneous mycobiota of reptiles [17]. It has been
recently reported to induce dermatomycosis in veiled
chameleons (Chamaeleo calyptratus) under experimen-
tal conditions [18], supporting its role as primary
pathogen. Dermatomycosis by the Chrysosporium ana-
morph of N. vriesii has been shown to be contagious,
spreading within a reptile collection, either directly
through contact or indirectly via fomites [18]. The
source of the infections in the present iguana cases is
– 2008 ISHAM, Medical Mycology
Chrysosporium sp. hyalohyphomycosis in green iguanas 5
100 Chrysosporium sp. CCFVB CH10 (EU018451)
100 Chrysosporium sp. CCFVB CH11 (EU018452)
Nannizziopsis vriesii (AJ131687)
Chrysosporium lobatum (AJ131688)
Chrysosporium merdarium (AJ390384)
Chrysosporium queenslandicum (AB219228)
Chrysosporium tropicum (AJ131685)
Chrysosporium keratinophilum (AJ131681)
100
Chrysosporium zonatum (AB219229)
Nannizziopsis albicans (AJ271432)
Chrysosporium undulatum (AJ007845)
Candida albicans (AJ551313)
0.1 0.0
not known. Animals had been acquired in pet shops
and owners did not have other reptiles. In both cases
husbandry was suboptimal, especially in case 2 and this
would be a predisposing factor contributing to the
onset of infection.
Treatment of fungal infections in reptiles includes
administration, for a minimum of 2 to 4 weeks,
effective antifungal agents and correction of inap-
propriate environmental conditions. As most cases of
mycotic diseases in reptiles are diagnosed at necropsy,
there are relatively few reports which discuss effective
dosages and dosage intervals of antifungal agents [19].
The systemic drugs of choice for use in reptiles
diagnosed with infection caused by filamentous fungi
include ketoconazole and itraconazole [1]. Very little is
known about drug susceptibility patterns of Chrysos-
porium spp. The reported mean MIC values in some
Chrysosporium spp. (C. keratinophilum, C. tropicum and
C. zonatum), ranged from 0.12 to 0.63 for itraconazole
and from 0.13 to 0.5 for ketoconazole [4]. Paré et al.
[20] reported low MICs for amphotericin B, terbina-
fine, itraconazole and voriconazole in isolates of a
Chrysosporium anamorph of N. vriesii. Nevertheless,
antifungal susceptibility tests for filamentous fungi
remain unstandardized. The guidelines of the CLSI
(formerly The National Committee for Clinical La-
boratory Standards) document M38-A [21] are in-
tended for testing some filamentous fungi, but
Chrysosporium spp. are not included. Although the
disk diffusion method used in this study has been
 6 Abarca et al.
commercialized for yeasts, it has been sporadically
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adapted to evaluate antifungal susceptibility of some
mold genera, and has been reported as a good
alternative method for the antifungal susceptibility
testing of dermatophytes in the routine clinical labora-
tory [22,23]. The larger diameter of inhibition values
obtained, greater than interpretative guidelines pro-
vided by manufacturers for susceptible yeasts, allow us
to consider the antifungal agents tested to be highly
active in vitro against the two isolates. As the treatment
regimen initially prescribed had yielded very good
results in both cases, no changes were proposed.
Although mycosis in captive reptiles is less com-
monly encountered than bacterial disease, it does occur
with regularity and it is likely both underestimated and
under diagnosed because fungal lesions and clinical
signs of fungal disease often are indistinguishable from
those of bacterial disease [1]. The key to evaluating skin
lesions of reptiles is collecting and properly evaluating a
good biopsy specimen for both histopathology and
microbial culture [2]. As reptiles continue to gain
popularity as pets, it is very important to increase the
knowledge of the fungi actually involved in reptile
diseases, their habitat, their role as either primary or
secondary pathogens as well as the possibility that these
infections may be transferred to human handlers.
Acknowledgements
Research was supported by grant No 2005 SGR 00684
from the DURSI, Generalitat de Catalunya.
References
1 Paré JA, Sigler L, Rosenthal KL, Mader DR. Microbiology:
fungal and bacterial diseases of reptiles. In: Mader DR (ed.),
Reptile Medicine and Surgery, 2nd ed., St Louis: Saunders
Elsevier, 2006: 217238.
2 Jacobson ER, Cheatwood JL, Maxwell LK. Mycotic diseases of
reptiles. Semin Avian Exot Pet Med 2000; 9: 94101.
3 Kostka VM, Hoffmann L, Balks E, Eskens U, Wimmershof N.
Review of the literature and investigations on the prevalence and
consequences of yeasts in reptiles. Vet Rec 1997; 140: 282287.
4 de Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of Clinical
Fungi, Centraalbureau voor Schimmelcultures, Utrecht, The
Netherlands and Universitat Rovira i Virgili, Reus, Spain, 2000.
5 Vissiennon T, Schüppel KF, Ullrich E, Kuijpers AFA. Case
report. A disseminated infection due to Chrysosporium queen-
slandicum in a garter snake (Thamnophis). Mycoses 1999; 42: 107
110.
6 Paré JA, Sigler L, Hunter DB, et al. Cutaneous mycoses in
chameleons caused by the Chrysosporium anamorph of Nanniz-
ziopsis vriesii (Apinis) Currah. J Zoo Wildlife Med 1997; 28: 443
453.
7 Bertelsen MF, Crawshaw GJ, Sigler L, Smith DA. Fatal cutaneous
mycosis in tentacled snakes (Erpeton tentaculatum) caused by the
Chrysosporium anamorph of Nannizziopsis vriesii. J Zoo Wildlife
Med 2005; 36: 8287.
8 Nichols DK, Weyant RS, Lamirande EW, Sigler L, Mason RT.
Fatal mycotic dermatitis in captive brown tree snakes (Boiga
irregularis). J Zoo Wildlife Med 1999; 30: 111118.
9 Thomas AD, Sigler L, Peucker S, Norton JH, Nielan A.
Chrysosporium anamorph of Nannizziopsis vriesii associated with
fatal cutaneous mycoses in the salt-water crocodile (Crocodylus
porosus). Med Mycol 2002; 40: 143151.
10 Bowman MR, Paré JA, Sigler L, et al. Deep fungal dermatitis in
three inland bearded dragons (Pogona vitticeps) caused by
Chrysosporium anamorph of Nannizziopsis vriesii. Med Mycol
2007; 45: 371376.
11 Martel A, Fonteyne PA, Chiers K, Decostere A, Pasmans F. Nasal
Nannizziopsis vriesii granuloma in an ameiva lizard (Ameiva
chaitzami). Vlaams Diergeneeskundig Tijdschr 2006; 75: 306307.
12 Steininger C, van Lunzen J, Tintelnot K, et al. Mycotic brain
abscess caused by opportunistic reptile pathogen. Emerg Infect Dis
2005; 11: 349350.
13 Brandt ME, Gaunt D, Iqbal N, et al. False-positive Histoplasma
capsulatum gen-probe chemiluminescent test result caused by a
Chrysosporium species. J Clin Microbiol 2005; 43: 14561458.
14 Accensi F, Cano J, Figuera L, Abarca ML, Cabañes FJ. New PCR
method to differentiate species in the Aspergillus niger aggregate.
FEMS Microbiol Lett 1999; 180: 191196.
15 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG. The Clustal X windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools.
Nucleic Acid Res 1997; 24: 48764882.
16 Saitou N, Nei M. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol E 1987; 4: 406425.
17 Paré JA, Sigler L, Rypien KL, Gibas CFC. Cutaneous mycobiota
of captive squamate reptiles with notes on the scarcity of
Chrysosporium anamorph of Nannizziopsis vriesii. J Herpetol
Med Surg 2003; 13: 1015.
18 Paré JA, Coyle KA, Sigler L, Maas AK, Mitchell RL. Patho-
genicity of the Chrysosporium anamorph of Nannizziopsis vriesii
for veiled chameleons (Chamaeleo calyptratus). Med Mycol 2006;
44: 2531.
19 Schumacher J. Fungal diseases of reptiles. Vet Clin North Am Exot
Anim Pract 2003; 6: 327335.
20 Paré JA, Andes DR, Sigler L. In vitro susceptibility of fungal
isolates from reptiles to antifungal drugs. Proc Am Assoc Zoo Vet
2005; 124.
21 Clinical and Laboratory Standards Institute. 2002. Reference
method for broth M38-A. National Committee for Clinical
Laboratory Standards: Wayne, PA.
22 Esteban A, Abarca ML, Cabañes FJ. Comparison of disk
diffusion method and broth microdilution method for antifungal
susceptibility testing of dermatophytes. Med Mycol 2005; 43: 61
66.
23 Karaca N, Koç AN. In vitro susceptibility testing of dermato-
phytes: comparison of disk diffusion and reference broth dilution
methods. Diagn Microbiol Infect Dis 2004; 48: 259264.
– 2008 ISHAM, Medical Mycology