History
A 2-year-old 19.5-kg (43.5-lb) spayed female German Shepherd Dog was referred to a veterinary teaching hospital for evaluation of a neck mass and possible hip dysplasia. The dog had been bred in Oklahoma and raised in Kansas and Michigan.
Clinical and Clinicopathologic Findings
Physical examination revealed fleas, mild hyperthermia (39.4°C [103°F]), slight lameness of the right hind limb with signs of pain on extension of the hip joint, and multiple enlarged lymph nodes or masses in the left and right superficial cervical, popliteal, and inguinal areas. There was marked wear of canine, premolar, and molar teeth with extensive exposure of the pulp cavities. Thoracic radiography revealed multiple soft tissue opacities, most consistent with an enlarged sternal lymph node and mild enlargement of the tracheobronchial lymph nodes. Pelvic radiography revealed enlargement of the medial iliac lymph node and punctate lysis of the caudal aspect of the right acetabulum with an associated irregular periosteal reaction, suggestive of osteomyelitis. Fine-needle aspirate specimens of the external masses were collected and submitted for evaluation. Results of a CBC were all within reference intervals. A serum biochemical profile revealed mild to moderate hyperproteinemia characterized by moderate hyperglobulinemia
On cytologic evaluation, all fine-needle aspirate specimens, including the aspirate specimen from the mass in the location of the left superficial cervical lymph node, were highly cellular and had similar characteristics (Figure 1). Cellularity was mainly attributable to an abundance of inflammatory cells, namely a mixture of degenerated neutrophils and macrophages (including multinucleated giant cells and epithelioid macrophages) along with fewer eosinophils, plasma cells, and small lymphocytes. Large mats of elongated structures that measured 15 to 25 μm in length and 5 to 8 μm in width and had parallel, 1- to 2-μm-thick, unstained walls with occasional pinching were observed extracellularly as well as in macrophages. Additionally, many small, round to oval (maximal dimension, 3 to 10 μm) organisms often with narrow-based budding were observed. The cytoplasm of these structures appeared moderately basophilic to amphophilic.
Photomicrographs of a fine-needle aspirate specimen from a prescapular mass of a 2-year-old German Shepherd Dog that was referred for evaluation of a neck mass and possible hip dysplasia. Microorganisms are present extracellularly in large hyphal mats (A) and appear to have been phagocytized by multinucleated giant cells. Hyphae measure 15 to 25 μm in length and 5 to 8 μm in width and have parallel, 1- to 2-μm-thick, unstained walls with occasional pinching. Many small, round to oval (maximal dimension, 3 to 10 μm) organisms, some with narrow-based budding, are also present. These organisms have moderately basophilic to amphophilic internal structures. In addition, a moderate number of small lymphocytes and few plasma cells are visible (B), suggesting a lymphoid origin for the prescapular mass. Modified Wright stain; in panels A and B, bar = 50 μm and 25 μm, respectively.
Citation: Journal of the American Veterinary Medical Association 257, 8; 10.2460/javma.257.8.813
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Additional Cytologic, Histologic, and Microbiological Findings
Cytologic findings were consistent with those of lymph node aspirate specimens, with evidence of marked mixed pyogranulomatous and eosinophilic inflammation associated with extracellular and intracellular fungal organisms. No neoplastic cells were identified. Overall, the findings were suggestive of hyphomycosis, possibly disseminated given the multiple locations affected. No pigments were identified; however, on the basis of the morphology of the hyphae and the presence of yeast-like swellings, phaeohyphomycosis was suspected. Culture and antimicrobial susceptibility testing for fungal organisms were performed.
Previously, the dog had been anesthetized and biopsy specimens of the mass in the area of the left superficial cervical lymph node were collected for histologic evaluation and microbial cultures. Histologic examination of sections of the left prescapular mass identified a lymph node that was nearly effaced by coalescing granulomas comprised of epithelioid macrophages and multinucleated giant cells. Eosinophils were frequently observed. The multinucleated giant cells contained variably sized, round to tubular or pleomorphic structures; such structures were also observed extracellularly. Septation, acute-angle branching, and budding of the structures were uncommon (Figure 2). These findings were indicative of pyogranulomatous lymphadenitis with intralesional and intracellular fungi.
Photomicrographs of a section of the prescapular mass. A—Notice the abundance of macrophages and multinucleated cells associated with fungal hyphae (asterisk). Moderate numbers of small lymphocytes and plasma cells are present, indicating an almost entirely effaced lymph node. H&E stain; bar = 100 μm. B—Rare eosinophils are also evident. The fungal elements are located extracellularly as well as intracellularly. Hyphae have parallel walls, rare septation, and acute-angle branching. Occasional yeast-like elements are visible (asterisk). H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 257, 8; 10.2460/javma.257.8.813
Fungal culture of a specimen of the left superficial cervical lymph node was performed. On the basis of colony morphology and morphology of the organisms, the fungal growth was identified as Bipolaris sp. Antimicrobial susceptibility testing revealed that the fungus was resistant to fluconazole and itraconazole and susceptible to posaconazole and voriconazole. The internal transcriber spacer region of the ribosomal RNA gene was amplified with a PCR assay, sequenced, and compared with a National Center for Biotechnology Information database (accession No. MH538935). Alignment results were 99.82% identical to Bipolaris sp, Curvularia sp, Cochliobolus sp, Setosphaeria rostrate, and Alternaria sp.
Initially, tramadol and fluconazole were administered to the dog at the time of discharge from the hospital, pending results of fungal culture and antimicrobial susceptibility testing. Three weeks later, the peripheral lymph nodes had decreased in size, but there was marked muscle atrophy of the right pelvic limb and the lameness persisted; CBC and serum biochemical results were unchanged. Two months after the diagnosis had been made, the dog developed multiple joint swellings and cutaneous masses. The lymph nodes remained moderately enlarged. A neurologic examination of the dog revealed appropriate mentation and decreased menace responses bilaterally that were related to complete blindness of the right eye and partial blindness of the left eye. Additionally, nystagmus was present, and the dog circled. These findings were consistent with central (brain-associated) disease. The owner declined further diagnostic investigations. When the antimicrobial susceptibility results indicated resistance of the fungus to fluconazole and itraconazole, the dog's treatment regimen was changed to voriconazole with terbinafine. Clinical signs progressed including loss of proprioception of the left pelvic limb and complete blindness in both eyes. The dog died at home 6 months following diagnosis. Necropsy was declined by the owner.
Morphologic Diagnosis and Case Summary
Morphologic diagnosis: left prescapular pyogranulomatous lymphadenitis with intralesional and intracellular fungi consistent with Bipolaris sp.
Case summary: presumptive disseminated Bipolaris infection in a dog.
Comments
Diagnosis of phaeohyphomycosis can be challenging. Because phaeohyphomycosis is caused by a heterogeneous group of dematiaceous fungi that are considered environmental saprotrophs, isolation of these organisms in culture is often regarded as contamination. As such, the observation of fungal elements in cytologic or histologic specimens collected under sterile conditions underpins the importance of the culture findings.1 Identification of an infective fungal species on the basis of cytologic or histologic examination findings is extremely difficult to impossible; nonetheless, hyphal morphology may help distinguish between phaeohyphomycoses and hyalohyphomycoses. The hyphae of fungi associated with phaeohyphomycoses have thick walls, are variable in diameter, and have yeast-like swellings.2 The hyphae of fungi associated with hyalohyphomycosis have thin, regular walls, and yeast-like structures are absent.3 For identification, culture followed by macroscopic and microscopic examination of the organismal growth is still a reference method. Fungal culture also allows for antimicrobial susceptibility testing of isolated organisms. Newer identification techniques based on PCR amplification of the internal transcriber spacer regions followed by sequencing have been developed and may supersede morphology-based methods in the near future. However, currently, the accuracy of those techniques for mold identification is limited by the numbers of sequences available in DNA repositories,3,4 and results may not aid in antifungal selection, as they did in the case described in the present report.
Most pigmented molds are plant pathogens or saprotrophs and considered opportunistic pathogens of mammals. In animals as well as in humans, phaeohyphomycoses are considered an emerging disease; however, the cause for the increased number of reports is uncertain and could be attributable to an increase in administration of chemotherapy and immunomodulatory drugs in relation to immune-mediated diseases or organ transplantation,1,4 improvement of the detection techniques, or increased awareness of these infections within the medical community.5 Systemic Bipolaris infections have been sparsely reported in the veterinary literature6–8; of those 3 affected dogs, 2 were receiving immunomodulants (corticosteroids for one dog8 and corticosteroids and cyclosporine for the other7) at the time of hospital admission. The third affected dog had no history of previous treatment.6 In all those cases, the infection was systemic as evidenced by the presence of fungal elements in various organs at necropsy. The dog of the present report had not received previous immunomodulatory treatment, and it was presumed that the dog was immunocompetent. For this dog, the origin of the infection was unknown. At the time of the referral examination, there was no cutaneous lesion to suggest a previous penetrating wound that could have resulted in inoculation with the fungus. The marked dental changes with pulp cavity exposure suggested that the disseminated infection could have had an oral cavity origin. The dog's sinuses were not examined endoscopically or with diagnostic imaging to completely rule out an upper respiratory tract origin of the infection; however, at the referral evaluation, the dog did not have respiratory signs. For most reported infections, the origin of the infection remains unknown. With regard to cerebral phaeohyphomycosis, the route of exposure is still not understood, and spread of fungal organisms from the sinuses, orbits, or the ears as well as by a hematogenous route (evidenced by positive blood culture results) has been suspected in people.
When the infection is limited to 1 body location and the lesion is excisable, surgery can be curative. Unfortunately, lesions are often disseminated, and prognosis for affected animals is guarded to poor. Systemic antifungal treatment may be effective, yet the response of animals with phaeohyphomycosis to antifungal drugs is unpredictable. Some authors recommend aggressive surgical resection of infected tissues with wide margins and treatment with itraconazole for a minimum of 3 months.2 A more prolonged course of treatment with itraconazole should be considered when lesions are not resectable and when clinically there is an initial response of the patient to this drug. Other antifungal drugs, such as voriconazole, posaconazole, and amphotericin B lipid complex, are recommended when lesions recur or the disease is disseminated.1,2,4 It has been suggested that animals that develop phaeohyphomycosis while receiving immunosuppressive therapy have a better treatment response if the immunosuppressive treatment can be tapered and discontinued quickly, compared with the treatment response of animals that are receiving no immunosuppressive therapy.2 Development of treatment plans for affected animals is optimized when results of fungal culture and antimicrobial susceptibility testing are available.
Acknowledgments
Funding for Pierre Deshuillers was made possible with support from the Morris Animal Foundation (grant #D15FE-902).
The authors thank Dr. Reyes-Gomez for assistance with processing the images used in this article.
References
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6. Giri DK, Sims WP, Sura R, et al. Cerebral and renal phaeohyphomycosis in a dog infected with Bipolaris species. Vet Pathol 2011;48:754–757.
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8. Waurzyniak BJ, Hoover JP, Clinkenbeard KD, et al. Dual systemic mycosis caused by Bipolaris spicifera and Torulopsis glabrata in a dog. Vet Pathol 1992;29:566–569.
