Phaeohyphomycosis caused by Trematosphaeria grisea in an immunocompromised English springer spaniel

Abstract

A 10-year-old, male, farm-based springer spaniel that presented with subacute progressive left-sided central vestibular signs was diagnosed with meningoencephalitis of unknown origin. The patient received prednisolone (2 mg/kg/day orally), ciclosporin (5 mg/kg orally twice a day) and a single infusion of cytarabine (200 mg/m²). Six weeks later, neurological signs had resolved, but multiple cutaneous nodules developed on the limbs, trunk and face. Lesions discharged a dark purulent material without tissue grains. A ‘filamentous fungus’ was isolated, but discarded without identification. Aspirates from nodules showed pigmented, septate fungal hyphae, and culture of aspirates yielded Trematosphaeria grisea. Itraconazole (5 mg/kg once a day) and gradual tapering of immunosuppressive drugs led to resolution of nodular lesions over 4 months without neurological relapse. A fungal granuloma developed transiently 8 months after starting itraconazole. Immunosuppressive drugs were stopped 15 months after introduction without neurological relapse. Itraconazole was withdrawn 8 weeks later, 5 months after last relapse of skin disease.

BACKGROUND

To our knowledge, this is the first report of canine phaeohyphomycosis caused by Trematosphaeria grisea (formerly Madurella grisea Persoonia). In common with previous cases, the immunosuppressive therapy required for meningoencephalitis of unknown origin (MUO) was considered to be an important risk factor for the fungal infection. The timeline of the development of skin lesions in relation to the introduction of immunosuppressive therapy suggests proliferation of previously traumatically implanted fungus, leading to overt infection, as a consequence of impaired cell-mediated immunity.

CASE PRESENTATION

A 10-year 3-month-old, male, neutered springer spaniel that lived on a farm presented to the Royal Veterinary College's Queen Mother Hospital for Animals with a 2-week history of subacute progressive left-sided hemiparesis, vestibular ataxia and obtundation, consistent with left-sided central vestibular system neuroanatomical localisation. After comprehensive investigations, a diagnosis of MUO was made based on magnetic resonance imaging, consistent with multiple intra-axial T2-weighted hyperintensities in the medulla oblongata, mononuclear pleocytosis on cerebrospinal fluid (CSF) analysis (total nucleated cell count: 37 cells/uL, reference interval: <5 cells/µL), and exclusion of an infectious aetiology (negative CSF PCR for Toxoplasma gondii and Neospora caninum; negative serology [ELISA] for Ehrlichia, Dirofilaria, Borrelia, Anaplasma; negative serology [IFA] for tick-borne encephalitis virus [IgG and IgM], and Neospora caninum [IgG]).^{1, 2} As the survival time in cases of MUO treated with prednisolone is improved by concurrent use of immunosuppressive drugs,¹ the patient received combination treatment with prednisolone (2 mg/kg/day orally [PO]), a single constant-rate infusion of cytarabine (200 mg/m²) and ciclosporin (5 mg/kg PO twice a day [BID]).

Six weeks later, neurological signs had resolved, but multiple and rapidly progressing cutaneous nodules and plaques, which first developed on the hindlimbs, soon also affected the trunk (Figure 1), face and all four limbs. Rapid progression to crateriform ulceration (Figure 2) was observed with discharge of a dark purulent material without tissue grains, with subsequent formation of a dark/black crust, despite 7 days of empirical therapy with clindamycin (14 mg/kg PO once a day [SID]). Culture of swabs of the discharge submitted to a commercial laboratory by the local veterinary surgeon showed a widely resistant (but fluoro-quinolone susceptible) Enterobacter cloacae and a ‘filamentous fungus’ that was discarded without identification. Immediately before referral, lesions progressed further despite a 4-day course of marbofloxacin (2.2 mg/kg PO SID).

FIGURE 1

Ulcerated nodule with black purulent discharge on the dorsal midline of the 10-year-old, male, farm-based springer spaniel with phaeohyphomycosis caused by Trematosphaeria (Madurella) grisea. Hair has been clipped.

FIGURE 2

A crop of ulcerated papules and nodules on the flank of the 10-year-old, male, farm-based springer spaniel with phaeohyphomycosis caused by Trematosphaeria (Madurella) grisea. Hair has been clipped.

INVESTIGATIONS

Fine-needle aspirates from nodular lesions were transferred to glass slides, stained with modified Wright Giemsa stain (‘Diff-Quik’), and examined using a light microscope. Pigmented, septate fungal hyphae that focally formed chains of broad, almost spherical cells were occasionally observed among abundant pyogranulomatous inflammation (Figure 3).

FIGURE 3

Mycelium of Trematosphaeria (Madurella) grisea comprising multiple branching septate hyphae surrounded by amorphous purple debris and degenerate neutrophils in a needle aspirate from the 10-year-old, male, farm-based springer spaniel with multiple ulcerated cutaneous nodules discharging black purulent material. Modified Wright's stain.

A fine-needle aspirate from a cutaneous nodule was inoculated onto Sabouraud's dextrose agar plates containing chloramphenicol (Thermo Scientific PO0161A) and incubated at 26°C, 32°C and 37°C. Multiple small colonies were evident within 48 hours of incubation at 32°C; these matured into large grey/green floccose colonies (black reverse pigmentation) with a raised umbonate centre (Figure 4). Growth at 37°C was very poor. Sub-cultures submitted to UK HSA Mycology Reference Laboratory were identified as T. grisea by phenotype and matrix-assisted laser desorption time-of-flight mass spectroscopy (MALDI-TOF MS). Molecular confirmation was obtained by comparing the sequences of ITS1 and D1/D2 subunit of the 26S rRNA gene using previously reported PCR primers and conditions,^{3, 4} with those available in GenBank. Our isolate showed greater than 99.5% sequence homology with the ITS1 (using primers ITS4/ITS5, Genbank accession number PP752064) and D1/D2 regions (using primers NL1/NL4, Genbank accession number PP756677) of the University of Tennessee Health Science Center's DI16-354 strain of T. grisea (GenBank accession numbers LN907497, LT796898). Susceptibility testing according to CLSI broth microdilution method M38-A2, as described previously,⁵ showed the following minimum inhibitory concentrations (MICs): amphotericin B: 1 mg/L; itraconazole (ITZ): 0.25 mg/L; terbinafine: <0.03 mg/L; these are low MICs suggestive of in vitro susceptibility, although there are no established breakpoints for this fungus.

FIGURE 4

Mature colonies (4 week's growth) of Trematosphaeria (Madurella) grisea on Sabouraud's dextrose agar, isolated from the 10-year-old, male, farm-based springer spaniel with phaeohyphomycosis.

DIFFERENTIAL DIAGNOSIS

Differential diagnoses considered for the multiple discharging cutaneous nodules were primarily of sterile or infectious (bacterial, mycobacterial, fungal) granulomata involving the dermis and/or panniculus. Recent isolation of a ‘filamentous fungus’ and current cytological observation of a pigmented, septate fungus confirmed a diagnosis of phaeohyphomycosis; absence of fungal grains in discharge excluded eumycotic mycetoma.

LEARNING POINTS/TAKE-HOME MESSAGES

• Cutaneous mycoses may develop following prior or contemporaneous inoculation of saprobic fungi when the host receives immunosuppressive therapy for concurrent diseases such as meningoencephalitis of unknown origin; Trematosphaeria (Madurella) grisea can be added to the list of pigmented fungi that may cause disease in dogs.
• Carefully examined fine-needle aspirates from nodular lesions may demonstrate the fungal pathogen and provide an inexpensive and less invasive method for sourcing material for fungal culture, when compared with conventional histopathology and minced tissue culture of infected tissue biopsies.
• Pigmented fungi that cause phaeohyphomycosis are often poorly responsive to medical therapy, and in cases where surgical excision of the lesion(s) is impractical, medical treatment lasting many months can be anticipated, with a goal of extending therapy for at least 2 months past clinical resolution.
• A ‘therapeutic conflict’ can be anticipated in phaeohyphomycosis cases requiring ongoing immunosuppressive therapy for concurrent disorders; while the prognosis for successful medical therapy is enhanced by discontinuation of ciclosporin and rapid tapering of glucocorticoids, withdrawal of immunosuppression risks loss of control/relapse of other serious internal diseases.
• Commercial diagnostic laboratories must be actively discouraged from simply discarding potentially pathogenic fungi that they lack the expertise to identify, at least until after they have consulted with the submitting veterinary surgeon. Where appropriate, isolates should be retained or forwarded to specialist laboratories for accurate identification, as the fungi associated with phaeohyphomycosis show significant species-specific differences in antifungal susceptibility.

TREATMENT

Upon diagnosis of phaehyphomycosis, ITZ was introduced at 5 mg/kg SID. Marbofloxacin was withdrawn, the prednisolone dose was reduced to 1.4 mg/kg on alternate days, and ciclosporin maintained at original dose BID for 2 further weeks, then reduced to SID.

OUTCOME AND FOLLOW-UP

Skin lesions markedly improved within 3 weeks of starting ITZ, with reduced size and number of the now dried but crusted nodules (Figure 5), although fungal elements were still evident on needle aspiration. Five weeks later, the only two remaining nodular lesions had further reduced in size. ITZ and ciclosporin were continued SID at previous doses, while prednisolone was further reduced to 1 mg/kg every other day (EOD). Over the next 30 weeks, the prednisolone and ciclosporin doses were gradually reduced to 0.25 mg/kg EOD and 5 mg/kg EOD, respectively, while ITZ was continued at 5 mg/kg SID. There was no relapse of MUO, and skin lesions were absent, except for one cutaneous nodule with black discharge that erupted on the trunk 1 month before that visit (8 months after original diagnosis). Needle aspiration showed pyogranulomatous inflammation and pigmented fungi as before. Over the next 11 weeks, prednisolone was further tapered then withdrawn, and ciclosporin dosing frequency was reduced to every third day without incident. After a further 8 weeks, ciclosporin was also withdrawn, although ITZ therapy was extended for a final 8 weeks. No relapse of skin or neurological disease was observed over a subsequent 19-week follow-up period.

FIGURE 5

Lesions of phaeohyphomycosis caused by Trematosphaeria (Madurella) grisea in the 10-year-old male springer spaniel, illustrated in Figure 2, after 4 weeks of therapy with itraconazole (at 5 mg/kg once a day). While erythematous papules remain, the previous crateriform ulcers have healed.

DISCUSSION

T. grisea is a dematiaceous fungus of the order Pleosporales that is most often associated with black grain mycetoma or phaeohyphomycosis^{6, 7} in human patients in endemic areas (South America, Latin America and the Caribbean).⁸ By contrast, in Europe it is regarded as an environmental (water) saprobe and rarely implicated in human disease.⁹ We are unaware of any previous reports of isolation of this fungus from dogs in the United Kingdom or globally, although a wide array of other pigmented fungi have been implicated in canine phaeohyphomycosis.¹⁰

Identification of fungi of this type typically requires specialist expertise and approaches, in part due to the infrequent production of conidia or other characteristic structures when cultured,⁷ and it is not surprising that a commercial veterinary diagnostic laboratory was unable to identify their isolate in the first instance. However, as recent studies have reported significant species-specific differences in antifungal susceptibility in the causative agents of subcutaneous phaeohyphomycoses and eumycetoma, accurate identification is paramount in directing appropriate antifungal interventions¹¹; discarding the isolate without further consultation is not appropriate. While rRNA gene sequencing might be regarded as the molecular ‘gold standard’ method for fungal identification (which provided further confirmation of our isolate's identity), the cumbersome nature of this process makes it especially challenging in a commercial diagnostic environment; this has prompted evaluation of other more rapid techniques, such as MALDI-TOF MS, as initially used in this case,⁷ and an isothermal rolling circle amplification molecular method.⁹

The epidemiological features of this case parallel observations in a human patient whose T. grisea phaeohyphomycosis was activated at the site of a previous penetrating injury sustained years previously, within 2 months of receiving immunosuppressive drugs following cardiac transplantation.¹² We speculate that in view of the rapid lesion development, this farm-dwelling springer spaniel is more likely to have acquired the fungus by multiple prior traumatic cutaneous inoculations from the environment, rather than systemic dissemination following inhalation,¹³ which only developed into an overt cutaneous mycosis following introduction of the immunosuppressive therapy for MUO.¹¹ Multiagent immunosuppressive therapies, especially those incorporating ciclosporin, have been previously associated with multifocal cutaneous phaeohyphomycoses in dogs.^{14, 15} The 6-week interval from onset of immunosuppressive/ciclosporin treatment to development of fungal skin lesions in the present case fits well with median values of 31¹⁴ and 43 days¹⁵ reported in these two series of eight cases. These relatively short intervals suggest that activation of latent cutaneous fungal inocula may occur more frequently than is appreciated,¹¹ especially when one considers that ciclosporin is commonly administered for periods of months to years; cases that develop as a consequence of new environmental acquisition would be expected to develop at any stage during ciclosporin treatment and not within a few weeks after introduction.

An important aspect of the diagnosis of cutaneous mycoses caused by environmental fungi is proof of fungal invasion of host tissue, and not merely surface contamination of skin, ulcers or sinus tracts. Accordingly, histopathology and tissue culture of aseptically obtained skin biopsy specimens is often recommended¹⁰ and certainly preferred to culture of surface swab samples. Samples for fungal culture should be chopped into small pieces but not homogenised, as that will reduce the viability of fungal hyphae. Aspiration of lesions for cytology and culture offers an inexpensive and less invasive alternative that may confirm fungal tissue invasion,^{14, 16} as in this case, with benefits for both the patient and owner. Laboratory personnel and clinicians should have an awareness of the sample type when considering the pathogenic significance of isolation of an environmental fungus from skin lesions, although observation of fungal hyphae on direct microscopic examination greatly enhances the significance of the subsequent culture.

ITZ is commonly recommended in cases of phaeohyphomycosis, for periods ranging from 3–6 months after surgical resection of solitary lesions, to 9–12 months in cases managed medically.¹⁰ The relapse of a solitary lesion in these cases that developed after 8 months of ITZ treatment, albeit in the face of ongoing immunosuppressive drugs, emphasises the need for prolonged therapy. Some authors suggest an ITZ dose of 10 mg/kg, although in studies of dogs with blastomycosis, fewer side effects have been reported with the 5 mg/kg dose used in this case.¹⁷

A ‘therapeutic conflict’ can be anticipated in phaeohyphomycosis cases requiring ongoing immunosuppressive therapy for concurrent disorders; while the prognosis for successful medical therapy is enhanced by discontinuation of ciclosporin and rapid tapering of glucocorticoids,^{10, 18} withdrawal of immunosuppression risks loss of control/relapse of other serious internal diseases. By way of example, one dog in the series reported by Dowling and colleagues¹⁴ died from a presumptive relapse of immune-mediated haemolytic anaemia after ciclosporin was discontinued following a diagnosis of opportunistic invasive fungal infection. Immunosuppressive drugs should be carefully tapered to the lowest possible maintenance dose while antifungal therapy is administered.

AUTHOR CONTRIBUTIONS

Erin Brennan: Conducted the dermatological examinations; devised and monitored the treatments, and wrote the original draft of the manuscript. Theofanis Liatis: Conducted the neurological examinations; devised and monitored the treatments, and reviewed and edited the manuscript. Elizabeth Johnson: Identified the fungal pathogen; contributed to discussions on epidemiology, and reviewed and edited the manuscript. Sian-Marie Frosini: Performed the molecular confirmation of the fungus's identification, and reviewed and edited the manuscript. Ross Bond: Conducted the dermatological examinations; isolated the fungus; devised and monitored the treatments, and wrote the original draft of the manuscript.

ACKNOWLEDGEMENTS

We thank Professor Steven DeDecker for supervising the neurological evaluation of the patient, and Ruth King and Menaka Menikpurage for technical support in the laboratory.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest.

FUNDING INFORMATION

Self-funded.

ETHICS STATEMENT

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The investigations and treatments in this case were conducted under the auspices of the UK Veterinary Surgeons Act following receipt of informed consent from the dog's owners. Additionally, the owners provided informed consent specifically for the reporting of this case at conferences and/or by scientific publication.

IMAGE QUIZ

Figure 2.

A 10-year 3-month-old, male, neutered springer spaniel receiving immunosuppressive therapy for meningoencephalitis of unknown origin presents with multiple ulcerated cutaneous nodules with black purulent discharge without tissue grains (Figure 1 or 2).

MULTIPLE-CHOICE QUESTION

What is your most favoured diagnosis?

POSSIBLE ANSWERS TO MULTIPLE-CHOICE QUESTION

1. Mycetoma

2. Idiopathic sterile nodular panniculitis

3. Deep staphylococcal pyoderma

4. Phaeohyphomycosis

5. Nocardiosis

CORRECT ANSWER

4. Phaeohyphomycosis.

The history of lesion development during immunosuppressive therapy favours a cutaneous infection rather than idiopathic sterile nodular panniculitis (whose discharge is more often a yellow colour and where immunosuppressive therapy may be curative/preventive). In mycetoma and nocardiosis, tissue grains (‘sulphur granules’) can be expected, and in nocardiosis the discharge may resemble ‘tomato soup’, rather than the black discharge as in the current case. A black discharge may suggest infection with a pigmented fungus, as in this case. That said, the colour of exudates can only be used as a guide, and microbiological tests should be used to verify the nature of any pathogen.