Eumycetoma and disseminated phaeohyphomycosis in a Sumatran tiger

Abstract

A 16-y-old female Sumatran tiger (Panthera tigris sumatrae) was evaluated for hyporexia. Examination revealed chronic kidney disease and a large subcutaneous axillary mass with draining tracts that contained numerous small black grains. Histologic examination revealed the presence of intralesional fungal hyphae. Persistent hyporexia and pyogranulomatous disease, as well as progressive cachexia and azotemia occurred despite treatment, and euthanasia was performed. Disseminated phaeohyphomycosis was diagnosed on postmortem examination, additionally affecting various lymph nodes, the nasal cavity, mesenteric adipose tissue, abdominal aorta, pericardium, and kidney. Fungal culture from a deep-tissue sample isolated a pure growth of Curvularia sp., a dematiaceous opportunistic fungus able to cause eumycetomas and/or phaeohyphomycosis. Phaeohyphomycosis is a rare but emerging condition, not previously reported as disseminated disease in an exotic carnivore, to our knowledge. Aggressive systemic antifungal treatment was unsuccessful, likely complicated by diagnostic challenges and concurrent renal disease. The presence of a swelling with abundant grains exiting draining tracts should direct clinicians to the diagnosis of a mycetoma, warranting early and aggressive treatment.

A 16-y-old, female Sumatran tiger (Panthera tigris sumatrae) was evaluated for hyporexia and weight loss. The patient had a history of oral eosinophilic granulomas and chronic bilateral idiopathic keratopathy, requiring occasional treatment with glucocorticoids and placement of episcleral cyclosporine implants (North Carolina State University, Raleigh, NC, USA; 12-mg implant in each eye). On anesthetized examination, the tiger appeared moderately dehydrated and was slightly underconditioned. In the left axillary region and along the cranioventral thorax were three ~5-cm round, irregular, fluctuant-to-firm subcutaneous pocketing masses that coalesced and were accompanied by pitting edema, erythema, and draining tracts. The tracts exuded a viscous, hemorrhagic-to-opaque fluid that contained numerous firm dark grains (Fig. 1). Chronic bilateral corneal changes and a focal proliferative lesion on the soft palate were noted, consistent with the history.

Figures 1–4.

Eumycetoma and phaeohyphomycosis in a Sumatran tiger. Figure 1. Numerous firm black grains exiting a draining tract, accompanied by a viscous, hemorrhagic-to-opaque fluid. Figure 2. Cut section of the mass on postmortem examination, highlighting the abundant tan, opaque fluid containing numerous <1-mm black grains. Figure 3. Fungal hyphae are variably pigmented, frequently have 8–15-µm, terminal bulbous dilations (presumed chlamydospores; arrows) and are surrounded by pyogranulomatous inflammation. H&E. Figure 4. Special staining highlights the irregularly septate and occasionally branched hyphae. Gomori methenamine silver stain. Bar = 75 µm.

Blood and urine were obtained for routine testing, and values were compared to species-specific reference intervals (RIs) when possible. ¹⁸ Important abnormalities included elevated symmetric dimethylarginine (SDMA) compared to previous values (1.45 µmol/L), high-normal creatinine (312 µmol/L; RI: 53–371 µmol/L), hypercalcemia (total calcium: 2.95 mmol/L, RI: 2.1–2.9 mmol/L; ionized calcium: 0.77 mmol/L), and hyperglobulinemia (53 g/L; RI: 23–52 g/L). Urine was inappropriately dilute (USG 1.014), consistent with chronic kidney disease. Draining fluid from the mass was obtained for culture and cytology, which showed predominantly neutrophilic, mixed-cell inflammation. Histology performed on punch biopsies of the swelling showed pyogranulomatous cellulitis with intralesional fungal hyphae that were variably pigmented, septate, with parallel walls and acute angle branching. Approximately one month later, fungal culture was reported as 1+ growth of a few Phaeoacremonium parasiticum.

Following examination, supplemental fluids, anti-nausea, antacid, antibiotic, and anti-inflammatory steroid treatments were administered empirically. The patient was prescribed mirtazapine (Alkaloida Chemical; 0.33 mg/kg PO q48h × 2 doses), maropitant (Cerenia, Fareva; 1 mg/kg PO q24h × 11 doses), and prednisolone (Lloyd; 0.5 mg/kg PO q24h).

One week later, the tiger was immobilized for a recheck examination and initiation of antifungal therapy. Its weight between these exams had reduced by 16%. The subcutaneous coalescing mass was unchanged. Recheck bloodwork showed worsening azotemia (creatinine 583 µmol/L; RI: 53–371 µmol/L), SDMA (2.05 µmol/L), hypercalcemia (3.05 mmol/L; RI: 2.1–2.9 mmol/L), hyperglobulinemia (54 g/L; RI: 23–52 g/L), and moderate eosinophilia (1.7 × 10⁹/L; RI: 0–0.6 × 10⁹/L). A fresh sample of draining fluid from the mass was submitted for next-generation sequencing targeting the V1-V2 regions of 16S rRNA and internal transcribed spacer 1 (ITS1; Microgen Diagnostics), as well as superficial fresh-frozen tissue from the mass for fungal ITS2 rDNA PCR (Zoological Medicine and Wildlife Disease Laboratory, University of Florida, Gainesville, FL, USA), and both were negative for fungal species. ²⁵ Hair shafts and fluid from a draining tract were negative for growth of dermatophytes on dermatophyte test medium. The tiger was administered general supportive therapies in addition to reconstituted amphotericin B lipid complex (Abelcet, Exelead; 1 mg/kg IV). Itraconazole (Pet Health Pharmacy; 5 mg/kg PO q24h × 14 doses) was prescribed.

Over the next 12 d, the patient was immobilized 3 times for reassessment and intravenous antifungal therapy. Because of progressive azotemia, nonresponsive granulomatous disease, and poor body condition with persistent hyporexia, euthanasia was performed one month after initial diagnosis. The tiger was immobilized and then euthanized with pentobarbital sodium and phenytoin sodium (Euthasol, Virbac; 25 mL IV).

On postmortem examination, there was generalized anemic pallor of mucosal surfaces. In the left axilla was a large fluctuant mass expanding the axillary subcutis, measuring 30 × 18 × 6.5 cm. Overlying this mass were numerous variably sized crusted to slightly ulcerated areas of skin (presumed draining tracts). On cut section, the mass was relatively well-demarcated, firm-to-fluctuant, and composed of numerous interconnected cavitations. These cavitations were lined by tan-to-pink-to-black, firm tissue and filled with abundant thin, tan, opaque fluid that contained numerous <1-mm, firm, gritty, black grains (Fig. 2). The regional lymph nodes associated with the mass, as well as distant lymph nodes in the abdominal cavity, were mildly to moderately enlarged and diffusely black on cut section. Firm, black, 0.3–1.2-cm nodules were disseminated throughout the thoracic and abdominal cavities. Specifically, nodules were found expanding the rostral nasal mucosa, adventitia of the abdominal aorta, pericardium, mesenteric adipose tissue, and left renal cortex. The kidneys were mildly asymmetric, and there was evidence of fibrosis throughout.

Microscopic examination of the pigmented nodules within the subcutaneous mass and other sites of dissemination (nasal mucosa, lymph nodes, renal capsule) identified dense aggregates of variably pigmented (colorless-to-yellow-to-brown) hyphal structures that were irregularly septate, exhibited branching, and had frequent terminal, 8–15-µm dilations that resembled chlamydospores (Figs. 3, 4). These fungal hyphae were surrounded predominantly by epithelioid macrophages and neutrophils, admixed with fewer lymphocytes, plasma cells, and rare eosinophils. The final diagnosis was severe locally extensive pyogranulomatous cellulitis with myriad intralesional pigmented fungal hyphae (phaeohyphomycosis) and widespread multisystemic dissemination.

Fungal culture of a fresh deep-tissue sample from the subcutaneous mass grew a pure culture of Curvularia sp. Fungal ITS2 rDNA PCR (Zoological Medicine and Wildlife Disease Laboratory) performed on formalin-fixed, paraffin-embedded (FFPE) tissue from the main lesion was positive. Successfully amplified products of the expected base pairs were excised and purified (QIAquick gel extraction kit; Qiagen) following the manufacturer’s instructions. The purified gel pieces, including the primer, were submitted to Genewiz for Sanger sequencing. The final product was edited, analyzed, and compared with sequences previously entered in GenBank. Based on the sequence, a Trichosporonaceae fungus (95% identical to Cutaneotrichosporon daszewskae and 94.8% identical to Cutaneotrichosporon terricola) was confirmed. Other findings included bilateral severe chronic lymphoplasmacytic and eosinophilic interstitial nephritis with fibrosis and glomerulosclerosis, an oral eosinophilic granuloma, which is well reported in tigers, ²⁰ and keratitis with corneal edema and uveitis in both eyes.

Phaeohyphomycosis is the term, separate from mycetoma, used to describe infection caused by dematiaceous fungi. ²² These pigmented fungi are ubiquitous and commonly saprophytic. ⁷ They are generally recognized as rare but emerging opportunistic pathogens, particularly in endotherms. ¹⁷ The condition has been documented in a wide range of species, including ectotherms, birds, ruminants, horses, dogs, and cats.^{4,5,7,11,15,17,19,22,24} Disease may be associated with immunocompromised status. Some dematiaceous fungi are also capable of forming a mycetoma, an infection of skin and subcutaneous tissue by fungi (eumycetoma) or filamentous bacteria (actinomycetoma) that is uniquely accompanied by swelling, draining tracts, and grains, which in eumycetomas contain abundant fungal hyphae.^8,9,13,16 These grains are a mix of fungal and host matrix components, including fungal polysaccharide cytoskeleton products. ¹³ Depending on the causative agent, grains can range from pale to black. ⁸ A useful diagnostic aid is to crush the grains after placement on a slide in 10% potassium hydroxide, allowing one to better visualize infectious organisms.^2,13 Samples collected for testing should contain numerous grains, given that these represent concentrated aggregates of the causative agent. It is best to aspirate grains from an unopened area or, less ideally, rinse extruded grains in 70% alcohol prior to fungal culture to decrease potential bacterial contamination.^2,8

Although we found no other reports of disseminated phaeohyphomycosis or mycetoma in a non-domestic felid, there is a report of cerebral phaeohyphomycosis in a female lion. ¹⁴ There is also a report of eumycetoma in a domestic Persian cat, secondary to a Microsporum canis infection. ⁹ M. canis is classically known for causing dermatophytosis. An atypical form of dermatophytosis is associated with pseudomycetomas, which lack the characteristic true grains noted above. ³ Persian cats appear more susceptible to forming pseudomycetomas than other species, which may be the result of a breed-related immunodeficiency. ¹⁰ Given that M. canis has caused disease in tigers, dermatophyte testing was performed in our case, and was negative. ²¹ Although dermatophyte culture results are obtained quickly, other fungal cultures generally are quite laborious and fungi are slow to grow. In our case, the initial fungal culture was not obtained until after the patient was euthanized, ~1 mo later. It is recommended that cultures for mycetomatous agents be held for ≥4 wk. ⁸

The fungal agents grown via cultures in our case, P. parasiticum and Curvularia sp., are both pigmented fungal species that can produce black grains. ⁸ They are considered facultative pathogens of soil or plants and are known to infrequently cause infections in humans and animals.^1,12 It is speculated that these organisms are usually introduced from penetrating trauma or inhalation and are more commonly seen in immunocompromised patients. ¹ However, an invasive case of phaeohyphomycosis, caused by Curvularia sp. in a canine patient, was found within hair follicles and supports that infection can occur without a penetrating injury. ⁷ The only fungal organism identified via PCR, Cutaneotrichosporon sp., is not readily found in the literature. It is a basidiomycetous yeast, ²³ not reported to cause eumycetoma or phaeohyphomycosis, nor is it a pigmented fungus. The sample used for this PCR was from FFPE tissue, and this likely affected the usefulness of this test. Histologically, the morphology of the fungus in our case more closely resembles Curvularia sp. than Cutaneotrichosporon sp. Based on this observation and the fact that the organism grew as a pure culture from a fresh deep-tissue sample, Curvularia sp. is implicated as the causative agent in our case. P. parasiticum was cultured from a superficial draining tract prior to initiation of treatment, and is likely a contaminant or coinfection that responded to antifungal therapy.

Infections resulting in phaeohyphomycosis or eumycetomas are largely considered opportunistic. The development in the host is often slow, and persistence after inoculation depends on the host’s immune response and the microorganism’s ability to evade the host’s defenses.^1,13 Furthermore, time from initial exposure to observation of draining tracts from a mycetoma can take up to years in some cases, making it challenging to determine when and where exposure occurred. ⁸ At the time of diagnosis, this tiger was housed alone in an enclosure with access to soil, mulch, and various plants. A definitive point of entry was not determined, and no other tigers housed in the same area have developed similar disease. As noted, disease can be associated with immunocompromise. Numerous comorbidities in the tiger’s history were likely associated with some type of aberrant immune response, rather than immunosuppression, namely the chronic idiopathic ocular lesions and eosinophilic granuloma. The tiger was also treated with numerous courses of oral steroids; however, the doses were considered anti-inflammatory rather than immunosuppressive and the last course was completed one month before the onset of clinical signs. Immunosuppression from the scleral cyclosporine implants was considered unlikely, but still investigated. Serum cyclosporine level at the time of euthanasia (Clinical Pharmacology Laboratory, Auburn University, AL, USA) was <25 ng/mL (below the limit of detection). It is unknown if environmental stress, concurrent renal disease, or other undetermined factors contributed to significant immunosuppression in our case.

Treatment options for severe fungal infections often include a prolonged course of antifungal therapy. The treatment of choice for small mycetomas in human medicine is surgical excision or, with larger lesions of fungal origin, often several months of oral antifungal therapy followed by evaluation for surgical debulking. However, long-term treatment success is challenging.^6,8 In our case, surgical removal of the axillary mass was unrealistic given lesion size, location, and debilitation of the patient. Had surgery been attempted, a cure was unlikely based on disease severity at the time of diagnosis and dissemination found on postmortem examination. Response to oral antifungal therapy could not be evaluated given the patient’s hyporexia and poor medication compliance. Additionally, given the challenges we noted with fungal culture, tailored selection of an antifungal agent based on susceptibility results was not possible. Regular intravenous antifungal therapy alone was insufficient in our case. In people, amphotericin B is not considered a curative agent, ⁸ but when combined with itraconazole it was successful in treating a man with phaeohyphomycosis secondary to Curvularia tuberculata. ²⁶ Clinicians should take the presence of grains from a soft tissue swelling with draining tracts seriously, given that this presentation is pathognomonic for a mycetoma, and rapidly determine if the grains contain fungal hyphae. To give the patient the best opportunity for recovery, immediate and aggressive treatment is warranted.