JOURNAL OF CLINICAL MICROBIOLOGY, June 2008, p. 21412143 Vol.
46, No. 60095-1137/08/$08.000 doi:10.1128/JCM.00205-08Copyright
2008, American Society for Microbiology. All Rights Reserved.
Pseudallescheria fusoidea, a New Cause of Osteomyelitis
Mark D. Lindsley,1* Josep Guarro,2 Raed N. Khairy,3 JoAnna
Williams,4Naureen Iqbal,1 and Preeti Pancholi4*
Mycotic Diseases Branch, Division of Foodborne, Bacterial, and
Mycotic Diseases, Centers for Disease Control andPrevention,
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 ofPathology,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
Pseudallescheriafusoidea, which resulted from traumatic
implantation after an injury sustained 3 years earlier.
CASE REPORT
An otherwise healthy 29-year-old male was admitted to
thehospital with progressive pain, intermittent swelling,
tender-ness, and erythema of the right foot. Three years earlier,
thepatient injured his foot while playing baseball, resulting in
alaceration of the skin which was subsequently left
untreated.Physical examination exhibited a diffusely swollen right
footwith tenderness and erythema; no sinus tract or drainage
wasobserved. The patient denied any fevers, chills, numbness, oruse
of medications. He also had no history of travel outside theUnited
States. Magnetic resonance imaging (MRI) of the rightfoot revealed
chronic osteomyelitis extending into the meta-tarsal shafts of the
first through the fifth digit. The patient wasinitially placed on
vancomycin therapy until a subsequent bonebiopsy demonstrated
fibrovascular tissue with chronic inflam-mation, including many
plasma cells, and fungal hyphal ele-ments (Fig. 1).
Culture of the bone tissue on potato dextrose agar
producedgrowth of a white, woolly colony that turned slightly gray
withage. Microscopic morphology revealed a hyaline mold with afew
pyriform conidia resembling Scedosporium sp. The isolatewas sent to
the Centers for Disease Control and Prevention(CDC) fungus
reference unit for confirmation. The mold wassubcultured onto
pablum agar, which induced abundant pyri-form conidia produced on
short, annellidic conidiophores (Fig.2). Optimal growth of the
isolate was observed at 30C, withslower growth at 25C and 37C,
restricted growth at 40C, andno growth at 45C. DNA was extracted
from the mold, andPCR amplification of the internal transcribed
spacer (ITS)region of the ribosomal DNA (rDNA) was performed using
20M each of the primers ITS5 (5-GGAAGTAAAAGTCGTAACAAGG-3) and ITS4
(5-TCCTCCGCTTATTGATATGC-3) (22), 0.2 mM of each deoxynucleotide
triphosphate, and1.25 U of Taq polymerase diluted in standard PCR
buffer
(Roche Diagnostics Corp., Indianapolis, IN). Two microlitersof
the DNA extract was added to the PCR mixture, and thetarget DNA was
amplified using an Applied Biosystems 9700thermocycler for 40
cycles of 94C, 51C, and 72C for 1 mineach. The sequence of the
amplified product was obtainedusing a BigDye Terminator version 3.1
cycle sequencing kit(Applied Biosystems, Foster City, CA) and
evaluated using a3730 DNA analyzer (Applied Biosystems). The
resulting se-quence was compared to those deposited in GenBank,
usingthe BLASTn program. The 569-bp sequence of the ITS
regionexhibited a 97 to 98% match to that of Pseudallescheria
boydii,Pseudallescheria angusta, and Pseudallescheria fusoidea. At
theRovira i Virgili University mycological laboratory in
Spain,further sequence analysis of the TUB region of the
-tubulingene, using the primers TUB-F/TUB-R (5, 7), identified
themold as Pseudallescheria fusoidea. The 544-bp sequence
dis-played a 99% similarity to the sequence of the type strain of
P.fusoidea (CBS 106.53). The isolate was placed into the CDCculture
collection (CDC no. B7315).
Subsequently, antifungal testing of the patient isolate
wasperformed at the CDC antifungal drugs unit, using the
brothmicrodilution assay, following the Clinical and
LaboratoryStandards Institute (CLSI) M38-A reference method (4).
Theculture and incubation conditions recommended by this docu-ment
for antifungal susceptibility testing of P. boydii were fol-lowed,
with the following exceptions: (i) the isolate was grownon pablum
agar, as this agar induced sporulation to a greaterdegree 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 4weeks of growth at 30C instead of
the recommended 7 days at35C. As recommended by CLSI document
M38-A, the MICwas determined after incubation at 35C for 72 h and
definedas the lowest drug concentration that caused complete
(100%)inhibition of growth. Antifungal susceptibility testing with
am-photericin B was determined using the Etest (17).
Susceptibil-ity to the echinocandins was interpreted by using the
minimumeffective concentration (MEC), which was defined as the
low-est concentration of a drug that produced a morphologicalchange
from filamentous growth to microcolonies. The P.fusoidea isolate
displayed elevated MICs against amphotericinB (32 g/ml),
fluconazole (256 g/ml), itraconazole (16 g/
* 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:
[email protected]. Mailing address for Preeti Pancholi: 1492 E.
Broad Street,Columbus, OH 43205. Phone: (614) 257-3488. Fax: (614)
257-2405.E-mail: [email protected].
Published ahead of print on 23 April 2008.
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ml), and ketoconazole (16 g/ml). MICs of 1 g/ml were ob-served
with both voriconazole and posaconazole. Of the threeechinocandins
tested, anidulafungin displayed the lowest MICof 0.015 g/ml, and
caspofungin and micafungin displayedhigher MICs of 8 g/ml and 4
g/ml, respectively.
The patient was placed on a 6-week course of voriconazolebut did
not return for a follow-up examination until a yearlater. At that
time, he reported that his symptoms had im-proved after finishing
the initial antifungal course but had nowreturned due to new onset
of pain in his right foot. An X rayof the patients foot revealed
radiographic stability with con-tinued osteopenia and loss of bony
tissue as a result of theinitial infection. A bone biopsy was
performed; however, theresults of microbiological cultures were
negative. Unfortu-nately, histopathologic examination was not
performed. All
laboratory values were within normal limits, except for
anelevated C-reactive protein (50 mg/liter) value. Because
thepatient had a history of not being seen for extended periods
oftime and the possibility that he had an undetected infection,
hewas placed on broad-spectrum antibiotics for 6 weeks. Thepatient
was seen in the clinic 4 months after antibiotic therapywas
discontinued and continues to show clinical stability, withno
further progression of disease.
Osteomyelitis, an infection of bone and bone marrow,
occurseither secondarily by hematogenous spread or through
implan-tation due to physical trauma or surgery. Whereas
osteomyeli-tis is most often the result of bacterial infection,
fungal osteo-myelitis occurs less frequently, usually resulting in
a chronic,more indolent course of infection. Fungal osteomyelitis
follow-ing a traumatic injury is typically due to contamination of
thewound with soil containing fungi such as Fusarium spp. (12,18),
Scedosporium prolificans (19, 20), or Pseudallescheria boy-dii (6,
11, 13, 14). The clinical and radiological presentation offungal
osteomyelitis differs little from that of osteomyelitiscaused by
bacteria. If the infection is treated empirically, thediagnosis of
fungal osteomyelitis may be delayed and may noteven be considered
until standard antibacterial treatment fails.
This report describes for the first time a case of
fungalosteomyelitis caused by the mold Pseudallescheria
fusoidea,demonstrating the chronic and insidious nature of this
disease.Molds in the genus Pseudallescheria are commonly found in
thesoil in temperate regions of the world (10). The most
commonspecies isolated are Pseudallescheria boydii and
Scedosporiumapiospermum, traditionally considered the anamorphic
(asex-ual) state of P. boydii, but recent molecular studies have
dem-onstrated that they are two different species (8).
FIG. 1. Gomori methenamine silver-stained section of tissue
fromthe patients initial bone biopsy demonstrating invasion of
fungal ele-ments 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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A recent taxonomic investigation has demonstrated that P.boydii
is a complex of closely related but distinct species in-cluding P.
angusta, P. ellipsoidea, and P. fusoidea (7). Pheno-typic species
identification is based on the morphology of theirascospores (7,
21), observed during the teleomorphic state ofgrowth. However, when
they are in the anamorphic state,which is the state most often
observed in the clinical labora-tory, these species are generally
indistinguishable. Conse-quently, a mold phenotypically identified
as P. boydii, isolatedfrom patients with osteomyelitis (6, 11, 13,
14) or other clinicalforms of disease (2, 3, 16), may have been one
of these otherspecies, including P. fusoidea. To accurately
determine thespecies identification of these molds, genotypic
analysis is re-quired.
The rDNA gene sequence has been used most commonly forthe
molecular identification of fungal species. However, asin the
current case, the discriminatory power of the rDNAgene sequence was
not great enough to distinguish betweenspecies of this genus. The
use of an additional gene sequence,the -tubulin gene in this case,
was necessary to determine thefinal identification (7, 9). The TUB
region of the -tubulingene has been shown to be the most
informative in the phylo-genic analysis of P. boydii and its
relatives, as demonstrated ina previous multilocus sequence study
(7).
The number of fungal species known to cause significantdisease
is continually increasing (15). With more antifungaldrugs now
available for the treatment of fungal infections (1),determining
the specific identification of these fungi is impor-tant. Diagnosis
by histopathology alone may not be sufficient,since some of these
fungi may have similar morphologicalcharacteristics but vary
greatly in their drug sensitivities. Forexample, species of the
genus Aspergillus are typically suscep-tible to antifungal drugs
such as itraconazole and amphotericinB (15), while Pseudallescheria
boydii, which can be difficult todifferentiate from Aspergillus
histopathologically, is resistant tothese drugs (9). The P.
fusoidea isolate described in this reportdisplayed high MICs to
many of the commonly used antifungalagents including amphotericin
B, fluconazole, and itracon-azole. Antifungal drugs that
demonstrated the lowest MICsagainst P. fusoidea were the more
recently developed extended-spectrum azoles, voriconazole and
posaconazole, and theechinocandin anidulafungin. The MIC pattern
observedwith this isolate was consistent with that of others
reportedpreviously (9).
Currently, very little is known about the epidemiology of
P.fusoidea. Up to now, only three other strains of this species
areknown, but all have an environmental origin, i.e., the
typestrain was recovered from goat dung in India, and the othertwo
originated from forest soil of Cuba and Zaire. This is thefirst
isolate of P. fusoidea that has been recovered from aclinical
source and causes significant morbidity. Genotypicidentification of
more isolates from both clinical and environ-mental sources will be
necessary to further our understandingof the epidemiology and
public health significance of this or-ganism.Nucleotide sequence
accession numbers. The ITS region
and the TUB region sequences were submitted to GenBank(accession
numbers EU370965 and EU370966, respectively).
The findings and conclusions in this article are those of the
authorsand do not necessarily represent the views of the CDC.
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