Introduction

Cryptococcosis is the most common systemic fungal disease in cats worldwide.^123–4 Infections with Cryptococcus species may also occur in several other mammalian species, including dogs and humans, and even in birds and reptiles.^1,5 Feline cryptococcosis is caused by basidiomycetous encapsulated yeasts of the genus Cryptococcus (Tremellomycetes: Tremellaceae) belonging to the Cryptococcus neoformans–Cryptococcus gattii complex.^2,4,67–8 Based on molecular characterisation methods, this complex includes eight genotypes (VNI, VNII, VNIII, VNIV, VGI, VGII, VGIII, VGIV) with varying geographical distribution, virulence and antimicrobial susceptibility.^2,4 The environmental reservoir of C neoformans is usually related to bird faeces, particularly pigeon droppings. However, this yeast has also been found in decaying trees, wood and plant debris, waterways and soil, all usually contaminated with bird excrement.^5,9,10

The primary route of infection in cats is the nasal cavity, although, more rarely, transmission can also occur via cutaneous inoculation of fungal forms.^1,2,6,11 The incubation period varies from months to years, with the source of infection often remaining unknown.^1,2,6,11,12 The most frequent clinical manifestation of feline cryptococcosis is associated with the nasal form, but the disease can occur in several other distinct clinical forms, with involvement of the central nervous system (CNS), ocular, cutaneous, lymph nodes, and even pulmonary, abdominal and periarticular connective tissues.^3,4 Ocular lesions are a common manifestation of systemic cryptococcosis (observed in about one-third of clinical cases), primarily manifesting as multifocal chorioretinitis.^1,13

A definitive diagnosis of cryptococcosis can be established using cytological examination, serology for the detection of antibodies (cryptococcal antigen latex agglutination test), fungal culture, histopathology and PCR.^1,2,4,6 PCR allows identification of the implicated species and genotype.⁶ The treatment of feline cryptococcosis usually combines surgical excision of localised granulomas and administration of antifungal azole drugs, such as fluconazole, itraconazole and ketoconazole. However, cats with CNS infection and/or systemic disease often need treatment with amphotericin B plus flucytosine.^2,6,14 Therapy should be maintained until at least 2–4 months after the resolution of clinical signs.⁶ The prognosis for feline cryptococosis is good to excellent when the disease is diagnosed in the early stages.^1,5,12 Nevertheless, CNS involvement negatively affects prognosis.²

Here, we describe, to the best of our knowledge, the first published clinical case of cryptococcosis in domestic animals in Portugal, which simultaneously represents, to the best of our knowledge, the first clinical case of blepharitis due to C neoformans without concurrent naso-ocular signs in a cat.

Case description

A 2-year-old male domestic shorthair stray cat was presented to the Hospital Veterinário de Trás-os-Montes (Vila Real, Portugal) with an open wound of the upper and lower lids of the right eye (Figure 1), and a history of facial pruritus.

Figure 1

Mucopurulent discharge from an open wound in the right upper and lower lids

On physical examination, fever and bilateral parasitic otitis due to Otodectes cynotis were detected. In addition, ophthalmological examination revealed mucopurulent discharge from the upper and lower lids. Slit-lamp biomicroscopy showed a normal anterior segment. Intraocular pressure, measured by applanation tonometry (Tono-Pen Vet; Medtronic Solan), was within the normal reference interval (10 mmHg in OD and 11 mmHg in OS; reference interval 20–25 mmHg). No funduscopic alterations were detected in either eye by direct and indirect ophthalmoscopic examination. Based on these findings, blepharitis was diagnosed. The differential diagnoses included bacterial, parasitic, viral, fungal and allergic blepharitis, or an eyelid neoplasia. Complete blood cell count revealed a leukocytosis with neutrophilia with left shift, and the biochemical profile tests were within normal range. A serum sample was positive for feline leukemia virus (FeLV) antigen and antibodies to feline immunodeficiency virus (FIV), using a commercial enzyme-linked immunosorbent assay test (Urinovet; Bionote) for both viruses.

Cytological examination of a Diff-Quik-stained appositional smear showed numerous polymorphic neutrophils and macrophages, together with spherical yeast cells compatible with Cryptococcus species (Figure 2). Direct examination of the pathological sample performed with Gram staining confirmed a few spherical and ovoid encapsulated yeasts. The sample was then inoculated into three Sabouraud dextrose (Liofilchem) and potato dextrose (Difco) agar plates.¹ Plates were sealed with parafilm and invertedly incubated in the dark at 37ºC. After 10 days, small colonies were noted to be growing on both media. Isolates were confirmed in fresh preparations and with Hiss stainings (Figure 3). Stained colonies showed polymorphic and encapsulated yeasts compatible with Cryptococcus species. India ink-stained smears of the isolates revealed negatively coloured capsules compatible with Cryptococcus species. DNA extracted from culture was amplified by PCR with specific primer URA5 followed by restriction fragment length polymorphism (RFLP) analysis, with the restriction enzymes Sau96I and HhaI in a double digest. The molecular type was determined by comparing the obtained URA5 RFLP pattern with the respective standard patterns for each molecular type, allowing the identification of C neoformans genotype VNI.¹⁵

Figure 2

Appositional smear showing numerous polymorphic neutrophils and spherical yeast cells with a prominent unstained capsule compatible with Cryptococcus species (Diff-Quik; scale bar = 20 µm)

Figure 3

Microscopical examination of fungal culture: cells of encapsulated yeasts compatible with Cryptococcus species (Hiss staining; scale bar = 20 µm)

Upon diagnosis of cryptococcosis, the cat was treated with itraconazole (5 mg/kg, Itrafungol; Esteve Veterinaria) combined with amoxicillin and clavulanic acid (12.5 mg/kg, Nisamox; Norbrook Laboratories), permethrin, nystatin, triamcinolone acetonide and neomycin sulfate ear ointment (Oridermyl; Vétoquinol) and imidacloprid/moxidectin spot-on application (Advocate; Bayer). Blepharitis and otitis were completely resolved 1 month after onset of treatment (Figure 4). Three months later the antifungal therapy was suspended. At this time the cat appeared clinically normal.

Figure 4

Blepharitis had completely resolved 1 month after treatment. Small scars can be noted in the upper and lower lids

Discussion

In Europe, cryptococcosis is usually rare or sporadic.^6,16 Retrospectives studies have shown that there is no breed or sex predisposition.⁶ Cats of any age can be affected.¹ Stray and rural cats seem to be more exposed to Cryptococcus, but indoor urban cats can also be infected through infected pigeon droppings.^1,6 The occurrence of the disease in cats is higher than in other domestic animals, probably owing to their grooming behaviour, which increases the probability of fungal delivery to the nasal cavity.^6,17

Localised cutaneous lesions, as in the present case, probably result from contamination of cat-scratch injuries with viable basidiomycetous encapsulated yeasts.^1,11 Here, the infection was only expressed in a gap in the integrity of normal cutaneous barriers.¹¹ As a result, the cat recovered faster with a shorter duration of therapy than others. The role of immunosuppression cannot be excluded in the pathogenesis of feline cryptococcosis.^1,6,7 Some studies suggest that cats co-infected with FeLV and FIV are more predisposed to cryptococcosis, but this conclusion is not shared by others.^7,12,18

Having been discovered about 100 years ago, cryptococcosis is a non-contagious systemic mycosis currently responsible for an estimated 625,000 human deaths due to cryptococcal meningitis in patients with human immunodficiency virus/AIDS each year worldwide.¹⁹ Cats, dogs and other infected animals do not represent a direct threat to public health as infection is acquired from contaminated environments, but they may act as sentinel species. The knowledge of local fungal habitats may be useful in planning preventative measures for both human and animal infections.⁶ In an environmental survey carried out in the municipality of Vila Real, C neoformans was found in the majority of sampled cages of domestic racing carrier pigeons (genotypes VNIV, VNI and VNIII) and in a few samples of decaying Eucalyptus leaves (genotype VNI).¹⁰

Conclusion

To the best of our knowledge, this is the first clinical case report of feline blepharitis due to C neoformans without concurrent naso-ocular signs. In addition, besides an anecdotal abstract about infection in a cat and reports of an infected free-living toad and a goat,^16,20,21 this is the first published clinical case of cryptococcosis in pets in Portugal.

The current findings, together with those from recent reports of the infection in domestic animals, should alert the veterinary community to this underdiagnosed disease. Additionally, in Portugal and other European countries, cryptococcosis should be included in the differential diagnosis of feline blepharitis.