JOURNAL OF CLINICAL MICROBIOLOGY, June 2008, p.

21412143
0095-1137/08/$08.000 doi:10.1128/JCM.00205-08
Copyright 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 6

Pseudallescheria fusoidea, a New Cause of Osteomyelitis

Mark D. Lindsley,1* Josep Guarro,2 Raed N. Khairy,3 JoAnna Williams,4
Naureen Iqbal,1 and Preeti Pancholi4*
Mycotic Diseases Branch, Division of Foodborne, Bacterial, and Mycotic Diseases, Centers for Disease Control and
Prevention, Atlanta, Georgia1; Unit of Microbiology, Medical School, Rovira i Virgili University, Reus, Spain2; and
Infectious Disease,3 Department of Internal Medicine, Ohio State University Medical Center, and Department of
Pathology,4 Ohio State University Medical Center, Columbus, Ohio
Received 1 February 2008/Returned for modification 17 March 2008/Accepted 15 April 2008

Osteomyelitis resulting from a mold infection often presents as a chronic and indolent disease process.
Described here for the first time is a case of osteomyelitis of the foot caused by the mold Pseudallescheria
fusoidea, which resulted from traumatic implantation after an injury sustained 3 years earlier.

An otherwise healthy 29-year-old male was admitted to the

hospital with progressive pain, intermittent swelling, tenderness, and erythema of the right foot. Three years earlier, the
patient injured his foot while playing baseball, resulting in a
laceration of the skin which was subsequently left untreated.
Physical examination exhibited a diffusely swollen right foot
with tenderness and erythema; no sinus tract or drainage was
observed. The patient denied any fevers, chills, numbness, or
use of medications. He also had no history of travel outside the
United States. Magnetic resonance imaging (MRI) of the right
foot revealed chronic osteomyelitis extending into the metatarsal shafts of the first through the fifth digit. The patient was
initially placed on vancomycin therapy until a subsequent bone
biopsy demonstrated fibrovascular tissue with chronic inflammation, including many plasma cells, and fungal hyphal elements (Fig. 1).
Culture of the bone tissue on potato dextrose agar produced
growth of a white, woolly colony that turned slightly gray with
age. Microscopic morphology revealed a hyaline mold with a
few pyriform conidia resembling Scedosporium sp. The isolate
was sent to the Centers for Disease Control and Prevention
(CDC) fungus reference unit for confirmation. The mold was
subcultured onto pablum agar, which induced abundant pyriform conidia produced on short, annellidic conidiophores (Fig.
2). Optimal growth of the isolate was observed at 30C, with
slower growth at 25C and 37C, restricted growth at 40C, and
no growth at 45C. DNA was extracted from the mold, and
PCR amplification of the internal transcribed spacer (ITS)
region of the ribosomal DNA (rDNA) was performed using 20
M each of the primers ITS5 (5-GGAAGTAAAAGTCGTA
ACAAGG-3) and ITS4 (5-TCCTCCGCTTATTGATATG
C-3) (22), 0.2 mM of each deoxynucleotide triphosphate, and
1.25 U of Taq polymerase diluted in standard PCR buffer

* Corresponding author. Mailing address for Mark D. Lindsley:

1600 Clifton Road, N.E., Mailstop G-11, Atlanta, Georgia, 30333.
Phone: (404) 639-4340. Fax: (404) 639-3546. E-mail: mlindsley@cdc
.gov. Mailing address for Preeti Pancholi: 1492 E. Broad Street,
Columbus, OH 43205. Phone: (614) 257-3488. Fax: (614) 257-2405.
E-mail: preeti.pancholi@osumc.edu.

Published ahead of print on 23 April 2008.

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(Roche Diagnostics Corp., Indianapolis, IN). Two microliters

of the DNA extract was added to the PCR mixture, and the
target DNA was amplified using an Applied Biosystems 9700
thermocycler for 40 cycles of 94C, 51C, and 72C for 1 min
each. The sequence of the amplified product was obtained
using a BigDye Terminator version 3.1 cycle sequencing kit
(Applied Biosystems, Foster City, CA) and evaluated using a
3730 DNA analyzer (Applied Biosystems). The resulting sequence was compared to those deposited in GenBank, using
the BLASTn program. The 569-bp sequence of the ITS region
exhibited a 97 to 98% match to that of Pseudallescheria boydii,
Pseudallescheria angusta, and Pseudallescheria fusoidea. At the
Rovira i Virgili University mycological laboratory in Spain,
further sequence analysis of the TUB region of the -tubulin
gene, using the primers TUB-F/TUB-R (5, 7), identified the
mold as Pseudallescheria fusoidea. The 544-bp sequence displayed a 99% similarity to the sequence of the type strain of P.
fusoidea (CBS 106.53). The isolate was placed into the CDC
culture collection (CDC no. B7315).
Subsequently, antifungal testing of the patient isolate was
performed at the CDC antifungal drugs unit, using the broth
microdilution assay, following the Clinical and Laboratory
Standards Institute (CLSI) M38-A reference method (4). The
culture and incubation conditions recommended by this document for antifungal susceptibility testing of P. boydii were followed, with the following exceptions: (i) the isolate was grown
on pablum agar, as this agar induced sporulation to a greater
degree than the recommended potato dextrose agar, and (ii)
due to the slow rate of sporulation observed with this isolate,
conidia suspensions used in the assay were obtained after 4
weeks of growth at 30C instead of the recommended 7 days at
35C. As recommended by CLSI document M38-A, the MIC
was determined after incubation at 35C for 72 h and defined
as the lowest drug concentration that caused complete (100%)
inhibition of growth. Antifungal susceptibility testing with amphotericin B was determined using the Etest (17). Susceptibility to the echinocandins was interpreted by using the minimum
effective concentration (MEC), which was defined as the lowest concentration of a drug that produced a morphological
change from filamentous growth to microcolonies. The P.
fusoidea isolate displayed elevated MICs against amphotericin
B (32 g/ml), fluconazole (256 g/ml), itraconazole (16 g/

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CASE REPORTS

J. CLIN. MICROBIOL.

laboratory values were within normal limits, except for an

elevated C-reactive protein (50 mg/liter) value. Because the
patient had a history of not being seen for extended periods of
time and the possibility that he had an undetected infection, he
was placed on broad-spectrum antibiotics for 6 weeks. The
patient was seen in the clinic 4 months after antibiotic therapy
was discontinued and continues to show clinical stability, with
no further progression of disease.

FIG. 1. Gomori methenamine silver-stained section of tissue from

the patients initial bone biopsy demonstrating invasion of fungal elements into the tissue (magnification, 400).

FIG. 2. Microscopic morphology of P. fusoidea isolated from the patients bone biopsy sample after culture on pablum agar at 30C for 12 days
(magnification, 400).

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ml), and ketoconazole (16 g/ml). MICs of 1 g/ml were observed with both voriconazole and posaconazole. Of the three
echinocandins tested, anidulafungin displayed the lowest MIC
of 0.015 g/ml, and caspofungin and micafungin displayed
higher MICs of 8 g/ml and 4 g/ml, respectively.
The patient was placed on a 6-week course of voriconazole
but did not return for a follow-up examination until a year
later. At that time, he reported that his symptoms had improved after finishing the initial antifungal course but had now
returned due to new onset of pain in his right foot. An X ray
of the patients foot revealed radiographic stability with continued osteopenia and loss of bony tissue as a result of the
initial infection. A bone biopsy was performed; however, the
results of microbiological cultures were negative. Unfortunately, histopathologic examination was not performed. All

Osteomyelitis, an infection of bone and bone marrow, occurs

either secondarily by hematogenous spread or through implantation due to physical trauma or surgery. Whereas osteomyelitis is most often the result of bacterial infection, fungal osteomyelitis occurs less frequently, usually resulting in a chronic,
more indolent course of infection. Fungal osteomyelitis following a traumatic injury is typically due to contamination of the
wound with soil containing fungi such as Fusarium spp. (12,
18), Scedosporium prolificans (19, 20), or Pseudallescheria boydii (6, 11, 13, 14). The clinical and radiological presentation of
fungal osteomyelitis differs little from that of osteomyelitis
caused by bacteria. If the infection is treated empirically, the
diagnosis of fungal osteomyelitis may be delayed and may not
even be considered until standard antibacterial treatment fails.
This report describes for the first time a case of fungal
osteomyelitis caused by the mold Pseudallescheria fusoidea,
demonstrating the chronic and insidious nature of this disease.
Molds in the genus Pseudallescheria are commonly found in the
soil in temperate regions of the world (10). The most common
species isolated are Pseudallescheria boydii and Scedosporium
apiospermum, traditionally considered the anamorphic (asexual) state of P. boydii, but recent molecular studies have demonstrated that they are two different species (8).

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The findings and conclusions in this article are those of the authors
and do not necessarily represent the views of the CDC.

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A recent taxonomic investigation has demonstrated that P.

boydii is a complex of closely related but distinct species including P. angusta, P. ellipsoidea, and P. fusoidea (7). Phenotypic species identification is based on the morphology of their
ascospores (7, 21), observed during the teleomorphic state of
growth. However, when they are in the anamorphic state,
which is the state most often observed in the clinical laboratory, these species are generally indistinguishable. Consequently, a mold phenotypically identified as P. boydii, isolated
from patients with osteomyelitis (6, 11, 13, 14) or other clinical
forms of disease (2, 3, 16), may have been one of these other
species, including P. fusoidea. To accurately determine the
species identification of these molds, genotypic analysis is required.
The rDNA gene sequence has been used most commonly for
the molecular identification of fungal species. However, as
in the current case, the discriminatory power of the rDNA
gene sequence was not great enough to distinguish between
species of this genus. The use of an additional gene sequence,
the -tubulin gene in this case, was necessary to determine the
final identification (7, 9). The TUB region of the -tubulin
gene has been shown to be the most informative in the phylogenic analysis of P. boydii and its relatives, as demonstrated in
a previous multilocus sequence study (7).
The number of fungal species known to cause significant
disease is continually increasing (15). With more antifungal
drugs now available for the treatment of fungal infections (1),
determining the specific identification of these fungi is important. Diagnosis by histopathology alone may not be sufficient,
since some of these fungi may have similar morphological
characteristics but vary greatly in their drug sensitivities. For
example, species of the genus Aspergillus are typically susceptible to antifungal drugs such as itraconazole and amphotericin
B (15), while Pseudallescheria boydii, which can be difficult to
differentiate from Aspergillus histopathologically, is resistant to
these drugs (9). The P. fusoidea isolate described in this report
displayed high MICs to many of the commonly used antifungal
agents including amphotericin B, fluconazole, and itraconazole. Antifungal drugs that demonstrated the lowest MICs
against P. fusoidea were the more recently developed extendedspectrum azoles, voriconazole and posaconazole, and the
echinocandin anidulafungin. The MIC pattern observed
with this isolate was consistent with that of others reported
previously (9).
Currently, very little is known about the epidemiology of P.
fusoidea. Up to now, only three other strains of this species are
known, but all have an environmental origin, i.e., the type
strain was recovered from goat dung in India, and the other
two originated from forest soil of Cuba and Zaire. This is the
first isolate of P. fusoidea that has been recovered from a
clinical source and causes significant morbidity. Genotypic
identification of more isolates from both clinical and environmental sources will be necessary to further our understanding
of the epidemiology and public health significance of this organism.
Nucleotide sequence accession numbers. The ITS region
and the TUB region sequences were submitted to GenBank
(accession numbers EU370965 and EU370966, respectively).

CASE REPORTS