Molecular identification of Phialophora oxyspora as the cause of mycetoma in a horse

Mandi J. Lopez Laboratory for Equine and Comparative Orthopedic Research, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803

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 DVM, PhD, DACVS
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Sandra O. Robinson Laboratory for Equine and Comparative Orthopedic Research, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803

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A. James Cooley Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, MS 39762

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Michael A. Prichard Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706

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Michael R. McGinnis Medical Mycology Research Center, Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555.

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DOI:
https://doi.org/10.2460/javma.230.1.84
Volume/Issue:
Volume 230: Issue 1
Online Publication Date:
01 Jan 2007
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Abstract

Case Description—An 18-year-old mare was evaluated for an oral mass that developed after extraction of a broken incisor.

Clinical Findings—An ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass involved the gingiva lateral and dorsal to the right first to third maxillary incisors. Osteolysis of the roots of the first and second right maxillary incisors and periosteal proliferation of the adjacent premaxilla margins were apparent on radiographs. Histologic examination of the mass revealed multiple coalescing and ramifying foci of abscess formation, each containing a well-defined, discrete, black mass (2 to 7 mm in diameter). Myriad fungal hyphae enmeshed in a black, granular, cementlike material were within each of the black structures. Mycetoma was the histologic diagnosis. The causative agent could not be identified via culture because of lack of distinguishing characteristics. Fungal DNA was isolated from frozen fungal cultures and paraffin sections. The D1/D2 domains of the large subunit P gene rDNA were amplified and sequenced. The sequences of the D1/D2 domains of both isolates were 96% homologous with those of Phialophora oxyspora.

Treatment and Outcome—The mass was surgically excised, the local area curetted, and the wound allowed to heal by second intention. Postoperative treatment consisted of administration of phenylbutazone and IV administration of sodium iodide followed by oral administration of potassium iodide. There was no evidence of recurrence 1 year later.

Clinical Relevance—Mycetomata should be a differential diagnosis for equine gingival masses. Identification of the fungal agent can be critical for selection of optimal treatments. Molecular methods may permit definitive identification when standard phenotypic-based identification criteria are inconclusive.

Abstract

Case Description—An 18-year-old mare was evaluated for an oral mass that developed after extraction of a broken incisor.

Clinical Findings—An ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass involved the gingiva lateral and dorsal to the right first to third maxillary incisors. Osteolysis of the roots of the first and second right maxillary incisors and periosteal proliferation of the adjacent premaxilla margins were apparent on radiographs. Histologic examination of the mass revealed multiple coalescing and ramifying foci of abscess formation, each containing a well-defined, discrete, black mass (2 to 7 mm in diameter). Myriad fungal hyphae enmeshed in a black, granular, cementlike material were within each of the black structures. Mycetoma was the histologic diagnosis. The causative agent could not be identified via culture because of lack of distinguishing characteristics. Fungal DNA was isolated from frozen fungal cultures and paraffin sections. The D1/D2 domains of the large subunit P gene rDNA were amplified and sequenced. The sequences of the D1/D2 domains of both isolates were 96% homologous with those of Phialophora oxyspora.

Treatment and Outcome—The mass was surgically excised, the local area curetted, and the wound allowed to heal by second intention. Postoperative treatment consisted of administration of phenylbutazone and IV administration of sodium iodide followed by oral administration of potassium iodide. There was no evidence of recurrence 1 year later.

Clinical Relevance—Mycetomata should be a differential diagnosis for equine gingival masses. Identification of the fungal agent can be critical for selection of optimal treatments. Molecular methods may permit definitive identification when standard phenotypic-based identification criteria are inconclusive.

An 18-year-old Quarter Horse mare was evaluated at the University of Wisconsin Veterinary Medical Teaching Hospital because of a mass over the right maxillary incisors. The right third incisor had been extracted approximately 18 months earlier after it was fractured by a kick from another horse. The owners reported no problems with the mare's health until they detected a growth in the area of the previous injury just prior to evaluation.

Initial evaluation revealed an ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass arising from the gingiva lateral and dorsal to the right first to third maxillary incisors (Figure 1). The horse resisted manipulation of the mass. Results of the rest of the physical and oral examinations were unremarkable. Maxillary radiography revealed substantial osteolysis of the roots of the first and second right maxillary incisors (Figure 2). Associated osteolysis in adjacent bone and active aggressive periosteal proliferation of the adjacent dorsal and lateral margins of the right premaxilla were evident. A wedge biopsy of the mass was performed.

Figure 1—
Figure 1—

Photograph of a gingival mycetoma in a horse. The left first incisor is indicated with an arrow.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

Figure 2—
Figure 2—

Radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. On the right side, the third incisor is missing and there is osteolysis of the roots of the first and second incisors. A soft tissue mass, osteolysis in adjacent bone, and periosteal proliferation of the adjacent lateral margins of the right premaxilla are evident.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

Histologic examination of biopsy tissue revealed multiple irregular, well-defined, discrete, black masses (2 to 7 mm in diameter) separated by intense inflammation and fibrosis (Figure 3). Within the black granules was a myriad of fungal hyphae surrounded by black, finely granular, cementlike material. Hyphal elements varied from 4 to 5 μm in diameter and were septate and irregularly branched. Hyphae radiated toward the periphery of the granules and were often bounded peripherally by neutrophils, followed by mixed accumulations of epithelioid macrophages, plasma cells, lymphocytes, and fewer neutrophils enmeshed in a fibrous connective tissue stroma. An occasional finely serrated eosinophilic band along the margins of the black granules was suggestive of Splendore-Hoeppli material. Several lytic bony trabeculae were along 1 margin. The histologic diagnosis was pyogranulomatous stomatitis with multiple eumycotic pigmented fungal granules typical of a black-grain mycetoma.

Figure 3—
Figure 3—

Photomicrograph of wedge-biopsy section from the mycetoma in the horse in Figure 1. Notice a large, irregular, welldefined, discrete, black structure (extending from the lower left corner of the photomicrograph to the upper right corner) that was contained within a focus of abscess formation. Within the structure are myriad pigmented fungal hyphae (white arrowhead) enmeshed in a black, granular, cementlike material. Along the periphery of the structure are accumulations of eosinophilic substance typical of Splendore-Hoeppli material (S). Neutrophils (N) are in close proximity to the material, followed by epithelioid macrophages and large numbers of plasma cells and lymphocytes (P), all enmeshed in a fibrous connective tissue stroma. H&E stain; bar = 25 μm.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

Surgical excision of the mass was recommended, and treatment was instituted following owner approval. The horse was sedated with xylazine (1.0 mg/kg [0.45 mg/lb], IV) and glycerol guaiacolate (40.0 mg/kg [18.14 mg/lb], IV). General anesthesia was induced with ketamine (2.0 mg/kg [0.91 mg/lb], IV) and maintained with halothane in oxygen in a closed-circle system. Following aseptic preparation and appropriate draping of the mass and surrounding tissue, a full-thickness circumferential incision was made in the mucosa approximately 2.5 cm from the base of the mass. The mass was then excised with a combination of sharp and blunt dissection. Subsequently, the first and second right maxillary incisors were extracted, and the surrounding maxillary bone was curetted thoroughly. Following copious lavage with physiologic saline (0.9% NaCl) solution, the wound was left open to heal by second intention. Tissue was submitted for histologic examination and bacterial and fungal cultures.

Postoperative maxillary radiography confirmed removal of the rostral aspect of the maxilla and right incisors (Figure 4). Persistent heterogeneous opacities within the maxilla that extended caudally and bony proliferation of the dorsal maxillary surface were consistent with fungal osteomyelitis of the remaining rostral portion of the maxilla.

Figure 4—
Figure 4—

Postoperative radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. Incisors on the right side have been removed. Notice heterogeneous opacities within the maxilla that extend caudally and are consistent with persistent fungal osteomyelitis (arrows).

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

Gross examination of the surgically resected maxillary mass revealed individual black granules ranging from 2 to 7 mm in diameter, although closely associated granules in clusters of 4 to 15 contiguous granules were common (Figure 5). Histologic features were similar to those of the original biopsy specimen. The irregular black granules consisted of fungal hyphae and intervening, finely granular, black material, although some granules had a few intermingled degenerated inflammatory cells. Hyphae were endogenously pigmented, often more conspicuously along the periphery. Large granules had a lamellar appearance and formed discontinuous concentric rings. The orientation of hyphae in a radial pattern along the periphery of the granules was consistently observed.

Figure 5—
Figure 5—

Photograph of a gingival mycetoma following excision in the horse in Figure 1. Notice numerous 2- to 7-mm, discrete, irregular, black, granular foci on the cut surface of the mass (arrows). Bar = 1 cm.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

α-Hemolytic Streptococcus sp and Actinobacillus sp were identified via aerobic bacterial culture of samples from the mass. Fungal culture performed in the Department of Pathology at the University of Texas Medical Branch, Galveston, Tex, resulted in a hyphal mass lacking differential structures after 3 months, which prevented standard phenotypic identification. The fungus was wooly, turned brown after initially being white, and produced a dark brown exudate (Figure 6). The presence of fungal structures in viable tissue confirmed the etiology of the infection. No evidence was found histologically to indicate that the isolated bacteria were pathogenic. All retained tissue samples that were not submitted for culture were embedded in paraffin following formalin fixation, and all fungal culture samples were stored at −80°C. Identification of DNA of the fungus from frozen fungal cultures and contained within paraffin sections was initiated.

Figure 6—
Figure 6—

Photograph of the fungal organism cultured from the mycetoma in the horse in Figure 1. Bar = 1 cm.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

The DNA from approximately 1 g of cultured fungal material and from 4 paraffin-embedded sections (10 μm thickness) of the surgically resected mycetoma was extracted and purified by use of a commercially available genomic DNA isolation kita and published methods.1,2 The D1/D2 domains of the LSUP rDNA3 were amplified with proprietary primers D1 and D2.b Polymerase chain reaction was carried out in 50-μL reactions containing 10 μL of template DNA, 2.5 μL (2 pmol) of each primer, and 25 μL of universal mastermix.c The PCR mixtures with either equine DNA or without template were used as negative controls. Polymerase chain reaction assay was performed with a PCR thermal cyclerd under the following conditions: 95°C for 7 minutes, followed by 35 cycles consisting of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds with a final extension at 72°C for 10 minutes. The PCR products were analyzed via electrophoresis on a 2% agarose gel. Specific 616–base pair fragments were seen for both fungal isolates, whereas no amplicons were apparent in the negative control lanes (Figure 7). The amplified products were purifiedf and subjected to bidirectional sequencing with an automated sequencerg and terminator chemical analysis as recommended by the manufacturer.h The D1 and D2 primers were used to sequence each DNA sample. Sequences were assembled and examined by use of sequencing software.i On the basis of homology searches in the GenBank database, the fungal culture and paraffin section DNA sequences were both determined to be 96% homologous with the LSUP rDNA of Phialophora oxyspora (accession No. AB100630).3

Figure 7—
Figure 7—

Agarose gel electrophoretogram of PCR products of the fungal pathogen recovered from culture (lane 2) and paraffin sections (lane 3) of the mycetoma in the horse in Figure 1. A 100–base pair ladder is in lane 1 (values on the left side of the figure [600, 700] indicate base pairs).e Lanes 4 and 5 contained negative controls of equine DNA and no template, respectively.

Citation: Journal of the American Veterinary Medical Association 230, 1; 10.2460/javma.230.1.84

The horse received phenylbutazone (2.0 mg/kg, IV) after recovery and for 2 days after surgery (1.0 mg/kg, PO, q 12 h). Additional postoperative treatment consisted of sodium iodide (1.0 mg/kg, IV) once weekly for 4 weeks and potassium iodide (0.5 mg/kg [0.23 mg/lb], PO, q 24 h) for 14 days. The horse was discharged 10 days after initial evaluation with instructions to monitor the horse for signs of iodide toxicosis, including dry, flaky skin; anorexia; lethargy; or epiphora. Intravenous administration of sodium iodide was performed by the referring veterinarian, and the owners completed oral administration of potassium iodide. One year after evaluation, the owners reported that the mass had not recurred and the surgical site appeared normal.

Discussion

Mycetoma is a chronic, proliferative lesion of the subcutaneous tissue and contiguous bones that contains granules and is caused by either actinomycetes or filamentous fungi.4 Mycetomata caused by actinomycetes are called actinomycotic mycetomata, and those caused by filamentous fungi are referred to as eumycotic mycetoma or eumycetoma. Lesions typically begin at a site of minor trauma and continue to spread locally over the ensuing months and years. The clinical manifestations of mycetomata vary, but the lesions are often localized and contain granulomas and abscesses that suppurate and drain exudate containing granules or grains through fistulous tracts. Size, color, shape, and texture of the granules vary with the causative agent.5

There are a number of pathogenic dematiaceous fungi, those with melanin or melaninlike pigment in the wall of their hyphae or spores or both.3 Many are wellknown agents of mycetoma.4,6-9 Antifungal treatment can now often be used to resolve fungal infections because of recent pharmacologic advances. However, appropriate identification of the causative agent is key to selection of an appropriate treatment.10 Identification of pathogenic dematiaceous fungi is typically accomplished via morphologic and physiologic analyses. These procedures are timeconsuming, require technical expertise, and are ineffective for identification of species with poor conidia production and a wide diversity in anamorphic life cycles.3 Methods to facilitate prompt and accurate fungal identification can directly influence treatment and prognosis, and the demand for such methods is rapidly increasing.

Fungi and most eukaryotes contain 80S ribosomes consisting of the large (60S) and small (40S) subunits. Each subunit rRNA contains a number of associated proteins; the large subunit contains the 25S to 28S (LSUP), 5.8S, and 5S rRNA molecules, and the small subunit contains the 18S rRNA molecule. Two other regions also exist in each subunit, the internal transcribed spacer region and the intergenic spacer region. Genes coding for rRNA subunits occur as multiple tandem repeats in nuclear DNA and contain strongly conserved and variable regions in their sequences.3,11 The LSUP contains 3 divergent domains (D1, D2, and D3) that are among the most variable regions within the entire gene.3 Ribosomal DNA has been widely used for identification of microorganisms, and the LSUP D1/D2 regions and internal transcribed spacer region sequences have been used to identify pathogenic fungi, including Phialophora spp.3,12

Phialophora spp are ubiquitous, dematiaceous, filamentous fungi that inhabit soil, wood, and decaying vegetation. Phialophora spp are among the common agents of phaeohyphomycosis and chromoblastomycosis in humans and other animals.13,14 Pheohyphomycosis is characterized by the fungus in host tissue with any combination of pigmented (melanin) yeastlike cells, pseudohyphaelike elements, and hyphae.3 Chromoblastomycosis is a chronic infection of cutaneous and subcutaneous tissue with verrucose lesions and round, brown, thick-walled, muriform cells (sclerotic bodies).3 Phialophora spp have also been identified as the cause of mycotic keratitis,15 bursitis,16 endophthalmitis,17 and prosthetic valve endocarditis.14 Mycetoma of the human foot caused by Phialophora spp has been reported on several occasions,9,18,19 but these fungi are not considered a common agent of mycetoma.7 Infection with Phialophora spp generally occurs on exposed tissue through traumatic inoculation with contaminated vegetative matter.20 The site of infection in the horse of this report was likely inoculated at the time of the original injury from a kick from another horse. To our knowledge, this is the first reported case of Phialophora-associated mycetoma in a horse.

Treatment of mycetoma is often complex and prolonged.9,18,19 Although mycetomata are generally encapsulated, which aids in isolation and removal of the lesion, surgical excision alone has a recurrence rate as high as 80% in humans.9 Complete removal of infected tissue is complicated by the locally invasive nature of the lesion. The most successful approach is reported to be surgical excision followed by prolonged systemic antifungal administration (with or without local administration).9,18,19 Successful treatment of an equine cutaneous mycetoma caused by Pseudallescheria boydii by use of surgical excision has been reported.5 In general, confirmed cases of mycetoma are rare in horses. In the case described here, conclusive identification of the fungal pathogen may have been important for further treatment, especially considering the maxillary osteomyelitis that remained following surgical excision. It is possible that the osteomyelitis progressed, although this was not reported by the owners, and follow-up radiographs were not available.

Long-term iodide treatment was initiated following surgical excision of the mycetoma in the horse described here because of substantial cost constraints. It is possible that surgical excision was curative. As indicated previously, there are abundant reports documenting recurrence of mycetoma in humans following surgical excision and systemic administration of antifungal medications.6,9 Conversely, successful treatment of mycetoma is reported less frequently and has included combined systemic administration of flucytosine and itraconazole19 and itraconazole alone6 following mass excision. Given the paucity of information regarding treatment of this condition in horses, concerns about the established osteomyelitis, budgetary limitations, and reports of recurrence of mycetoma in humans, a low-cost, well-established systemic treatment was initiated.21

A molecular approach to conclusively identify the causative organism was undertaken when results of standard culture methods were inconclusive. Because of the lack of fresh or frozen tissue samples, DNA was isolated from frozen fungal cultures and paraffin-embedded sections. Advances in PCR, DNA sequencing, and the availability of species-specific DNA sequences make a molecular approach to fungus identification feasible. This method of identification is not necessarily warranted in cases in which standard methods are diagnostic; however, given the importance of fungal identification for case management and establishment of prognosis, DNA identification can be invaluable.

ABBREVIATION

LSUP

Large subunit P
a.

BDtract Genomic DNA isolation kit, Maxim Biotech Inc, San Francisco, Calif.

b.

Applied Biosystems, Foster City, Calif.

c.

AmpliTaq Gold PCR Master Mix, Applied Biosystems, Branchburg, NJ.

d.

PTC-200 thermal cycler, Bio-Rad Laboratories, Hercules, Calif.

e.

EZ Load 100–base pair molecular ruler, Bio-Rad Laboratories, Hercules, Calif.

f.

Montage PCR Centrifugal Filter Device, Millipore Corp, Billerica, Mass.

g.

ABI3700 automated sequencer, Applied Biosystems, Branchburg, NJ.

h.

Big Dye Terminator, Applied Biosystems, Branchburg, NJ.

i.

Sequencer, Gene Codes, Ann Arbor, Mich.

References

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Contributor Notes

The authors thank Drs. John Salmen, Jeffery A. Meixner, and Amy Grooters for technical assistance, and Applied Biosystems, Foster City, Calif, for providing the D1 and D2 primers.

Address correspondence to Dr. Lopez.
Cover Journal of the American Veterinary Medical Association
Journal of the American Veterinary Medical Association
Publication Date:
01 Jan 2007
  • Figure 1—
    View in gallery
    Figure 1—

    Photograph of a gingival mycetoma in a horse. The left first incisor is indicated with an arrow.

  • Figure 2—
    View in gallery
    Figure 2—

    Radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. On the right side, the third incisor is missing and there is osteolysis of the roots of the first and second incisors. A soft tissue mass, osteolysis in adjacent bone, and periosteal proliferation of the adjacent lateral margins of the right premaxilla are evident.

  • Figure 3—
    View in gallery
    Figure 3—

    Photomicrograph of wedge-biopsy section from the mycetoma in the horse in Figure 1. Notice a large, irregular, welldefined, discrete, black structure (extending from the lower left corner of the photomicrograph to the upper right corner) that was contained within a focus of abscess formation. Within the structure are myriad pigmented fungal hyphae (white arrowhead) enmeshed in a black, granular, cementlike material. Along the periphery of the structure are accumulations of eosinophilic substance typical of Splendore-Hoeppli material (S). Neutrophils (N) are in close proximity to the material, followed by epithelioid macrophages and large numbers of plasma cells and lymphocytes (P), all enmeshed in a fibrous connective tissue stroma. H&E stain; bar = 25 μm.

  • Figure 4—
    View in gallery
    Figure 4—

    Postoperative radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. Incisors on the right side have been removed. Notice heterogeneous opacities within the maxilla that extend caudally and are consistent with persistent fungal osteomyelitis (arrows).

  • Figure 5—
    View in gallery
    Figure 5—

    Photograph of a gingival mycetoma following excision in the horse in Figure 1. Notice numerous 2- to 7-mm, discrete, irregular, black, granular foci on the cut surface of the mass (arrows). Bar = 1 cm.

  • Figure 6—
    View in gallery
    Figure 6—

    Photograph of the fungal organism cultured from the mycetoma in the horse in Figure 1. Bar = 1 cm.

  • Figure 7—
    View in gallery
    Figure 7—

    Agarose gel electrophoretogram of PCR products of the fungal pathogen recovered from culture (lane 2) and paraffin sections (lane 3) of the mycetoma in the horse in Figure 1. A 100–base pair ladder is in lane 1 (values on the left side of the figure [600, 700] indicate base pairs).e Lanes 4 and 5 contained negative controls of equine DNA and no template, respectively.

Figure 1—

Photograph of a gingival mycetoma in a horse. The left first incisor is indicated with an arrow.
Figure 2—

Radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. On the right side, the third incisor is missing and there is osteolysis of the roots of the first and second incisors. A soft tissue mass, osteolysis in adjacent bone, and periosteal proliferation of the adjacent lateral margins of the right premaxilla are evident.
Figure 3—

Photomicrograph of wedge-biopsy section from the mycetoma in the horse in Figure 1. Notice a large, irregular, welldefined, discrete, black structure (extending from the lower left corner of the photomicrograph to the upper right corner) that was contained within a focus of abscess formation. Within the structure are myriad pigmented fungal hyphae (white arrowhead) enmeshed in a black, granular, cementlike material. Along the periphery of the structure are accumulations of eosinophilic substance typical of Splendore-Hoeppli material (S). Neutrophils (N) are in close proximity to the material, followed by epithelioid macrophages and large numbers of plasma cells and lymphocytes (P), all enmeshed in a fibrous connective tissue stroma. H&E stain; bar = 25 μm.
Figure 4—

Postoperative radiographic image of the rostral aspect of the maxilla of the horse in Figure 1. Incisors on the right side have been removed. Notice heterogeneous opacities within the maxilla that extend caudally and are consistent with persistent fungal osteomyelitis (arrows).
Figure 5—

Photograph of a gingival mycetoma following excision in the horse in Figure 1. Notice numerous 2- to 7-mm, discrete, irregular, black, granular foci on the cut surface of the mass (arrows). Bar = 1 cm.
Figure 6—

Photograph of the fungal organism cultured from the mycetoma in the horse in Figure 1. Bar = 1 cm.
Figure 7—

Agarose gel electrophoretogram of PCR products of the fungal pathogen recovered from culture (lane 2) and paraffin sections (lane 3) of the mycetoma in the horse in Figure 1. A 100–base pair ladder is in lane 1 (values on the left side of the figure [600, 700] indicate base pairs).e Lanes 4 and 5 contained negative controls of equine DNA and no template, respectively.
  • 1

    Keim P, Price LB, Klevytska AM, et al. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 2000;182:2928–2936.

    • Search Google Scholar
    • Export Citation
  • 2

    Truman R, Fontes AB, De Miranda AB, et al. Genotypic variation and stability of four variable-number tandem repeats and their suitability for discriminating strains of Mycobacterium leprae. J Clin Microbiol 2004;42:2558–2565.

    • Search Google Scholar
    • Export Citation
  • 3

    Abliz P, Fukushima K, Takizawa K, et al. Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis. FEMS Immunol Med Microbiol 2004;40:41–49.

    • Search Google Scholar
    • Export Citation
  • 4

    McGinnis MR, Fader RC. Mycetoma: a contemporary concept. Infect Dis Clin North Am 1988;2:939–954.

  • 5

    McEntee M. Eumycotic mycetoma: review and report of a cutaneous lesion caused by Pseudallescheria boydii in a horse. J Am Vet Med Assoc 1987;191:1459–1461.

    • Search Google Scholar
    • Export Citation
  • 6

    Ahmed AO, Desplaces N, Leonard P, et al. Molecular detection and identification of agents of eumycetoma: detailed report of two cases. J Clin Microbiol 2003;41:5813–5816.

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