Challenging frontiers: Canine sporotrichosis due to Sporothrix brasiliensis, first case in Chile

Abstract

We report the first case of Sporothrix brasiliensis in a dog at Santiago, Chile. From an initial biopsy obtained from the patient, a histopathological study was performed that oriented a fungal infection. The molecular diagnosis from the isolated colony confirmed the identity of the S. brasiliensis agent. Although the literature indicates itraconazole as the first treatment option, the patient was treated with terbinafine followed by itraconazole, without achieving a clinical cure. S. brasiliensis that spread rapidly in Latin America, hence highlight the importance of timely diagnosis that contribute to maintaining human and animal health.

2012 Elsevier Ltd. All rights reserved.

1. Introduction

Sporotrichosis is a mycosis caused by a group of fungi belonging to the genus Sporothrix [1], which is found in the environment, especially in soil, decaying wood, and plants. Although many Sporothrix spp. do not cause human or animal infections, this is not the case of the “pathogenic clade”, capable of infecting mammals and composed by S. brasiliensis, S. schenckii, S. globose and S. luriei [2]. S. brasiliensis can occur in humans, animals, and the environment, with a change between sapronotic, zoonotic and enzootic transmission. This fungus, unlike other species of the genus can be transmitted in its filamentous and yeast-like phase, a characteristic associated with its pathogenicity in mammalian hosts [3].

Over the past two decades, S. brasiliensis in cats and humans has emerged as a significant public health concern, primarily in Brazil, and subsequently in other regions in the Americas [4,5] and recently in Chile [6].

So far, canine sporotrichosis caused by S. brasiliensis had been detected only in Brazil, with the first report in 2013 with one case in Pelotas city and another in São Paulo city [7]. Presently, in the Metropolitan Region of Rio de Janeiro in Brazil, this species is the main etiologic agent associated with canine sporotrichosis [7].

There is recent evidence that the dog can be a facilitator of transmission to humans or other animals. The most common opportunity of dog infection by this fungus occurs during hunting or chasing activities, typically through thorn injuries, wood splinters, or through direct contact with sick cats [8]. Canine sporotrichosis is typically characterized by the presence of ulcers and/or nodules on the head, nose, ears, neck, thorax, or extremities, commonly appearing in a cutaneous or lymphocutaneous form [9]. On the other hand, disseminated forms and osteoarticular affectations are uncommon [10].

In Chile, the first and only report in animals appeared during 2022 in the Magallanes Region corresponding to two feral and one domestic cats [6]. More recently, S. brasiliensis was reported for the first time in a woman from the Valparaíso Region [11]. The objective of this report was to describe the first case of S. brasiliensis in a domestic dog in the Metropolitan Region, Chile.

2. Case presentation

The present case that we describe here occurred in the city of Santiago, Chile (33°27′24.98″S 70° 38′ 53.77″W). A 5-year-old, intact female, dalmatian was referred for a second opinion to a veterinary clinic on February 15th of 2023 with a one-year history of a gradually worsening skin condition with an ulcerative pattern involving the distal extremities and forehead. This patient had no history of having traveled to or from endemic areas where S. brasiliensis has been previously described, nor a history of being in contact with healthy or infected domestic cats. The owner reported that she had been treated with high doses of prednisone and antibiotics, but with poor improvement.

The dog presented at the physical examination with multi-focal scarring alopecia, erythema, various fistulas with several draining tracts and ulcerative skin lesions with pus (Fig. 1). The patient was mildly lethargic, with signs of moderate pain and body condition score 4/9. The lymph nodes were within normal parameters. A swab cytology was performed during the first appointment and showed neutrophilic inflammation, degenerate neutrophils, vacuolated macrophages, no microorganisms, or nucleated keratinocytes were found. In this opportunity, a bacterial culture and antibiogram of the section of the lesions was requested, along with a blood count and biochemical profile. While waiting for this result, the patient was administered a single dose of cefovecin at a dose of 8 mg/kg (Zoetis, Las Condes, Chile).

Fig. 1.

Ulcerated, moist and bloody patient lesions distributed on the forelimbs, hindlimbs and face.

Complete blood count test results revealed mild anisocytosis, poikilocytosis, polychromasia, degenerate neutrophils, left deviation, normocytic normochromic and a regenerative anemia; while blood biochemistry analysis elevated alkaline phosphatase (500 UI/L). The culture and antibiogram showed Staphylococcus pseudointermedius sensitivity to enrofloxacin; clindamycin; erythromycin and vancomycin. The dog was prescribed prednisone 1 mg/Kg (Laboratorio Chile, Ñuñoa, Chile) and enrofloxacin 5 mg/Kg (Rostrum, Drag Pharma, Quilicura, Chile) for 15 days; after that period, no signs of improvement were observed. An incisional biopsy, of one of the ulcerative lesions located on the right forelimb, was performed for histopathological analysis.

The histopathological study revealed hyperkeratosis, acanthosis, and ulceration were observed at the epidermal level. Whereas, dermal and subcutaneous tissue showed the presence of intense pyogranulomatous inflammation, which consisted mainly of epithelioid histiocytes, multinucleated giant cells. Infiltration with neutrophils and lymphocytes were present in the surrounding tissue. Numerous fungal elements were detected within the cytoplasm of histiocytes or occasional multinucleated giant cells. Typically, they were characterized by round to oval yeast-like structures with a thick, refractile cell wall, often appearing as clusters. Fungal elements were positive to PAS staining, indicating the presence of polysaccharide-rich fungal cell walls. Involvement of some small blood vessels with signs of vasculitis or thrombosis was observed associated to necrotic areas (Fig. 2).

Fig. 2.

A. Skin biopsy shows hyperkeratosis, acanthosis, and ulceration at the epidermal level, with a thick necrotic crust (thin arrow). Whereas the dermal and subcutaneous tissue showed the presence of intense nodular pyogranulomatous inflammation with fistulization (thick arrow). H/E stain 40x. B. At higher magnification the subcutaneous pyogranulomatous inflammation that consisted mainly of epithelioid histiocytes and lymphocytes , with the presence of less neutrophils. Some fungal elements, positive to PAS staining, were detected within the cytoplasm of phagocytes. PAS Stain 1000x.

Subsequently, a new sampling procedure of the remaining lesions was recommended to be taken and sent to the Clinical Microbiology and Microbiome Laboratory, of the School of Veterinary Medicine of the Andrés Bello University. While waiting for the results of the fungal culture, the patient was started on topical clotrimazole 1% twice daily (Laboratorio Chile, Santiago, Chile); baths with miconazole 2% + chlorhexidine 2% (Regepipel, Drag Pharma, Quilicura Chile) twice a week and oral terbinafine 40 mg/kg/once daily (Laboratorio Chile, Santiago, Chile) [12], for five weeks and a recheck was done, where the owner did not see any improvements.

The polyphasic identification was carried out in a BSL-2 biosafety cabinet. Initially, a direct microscopic examination of the sample with Diff Quick staining was performed, where scarse blastoconidia were observed. Then, the tissue sample was minced and seeded on Sabouraud agar glucose (SGA) containing chloramphenicol (0,05 g/L) and cycloheximide (0,4 g/L) (Merk, Rahway, NJ, USA), and incubated at 25 °C and 37 °C for 10 days. The macro and micromorphological characteristics of the colony allowed the presumptive identification of Sporothrix spp. Meanwhile, the genotypic study of the isolated colony included DNA extraction performed with the Quick-DNA Fungal/Bacterial kit (Zimo Research, Irvine, CA, USA), following the manufacturer’s instructions. PCR amplification was performed using universal primers ITS5, ITS4 and calmodulin. The sequences obtained were edited, assembled, and analyzed by comparing the nucleotide similarity using the BLAST tool of the National Centre for Biotechnology Information (NCBI). The sequences of isolates presenting 100% genetic identity to S. brasiliensis (access code OR852420, PP083686).

The minimum inhibitory concentration (MIC) was determined by broth microdilution, according to document M38-A2 from the Clinical and Laboratory Standards Institute (CLSI) (CLSI standard M38) [13]. The drugs tested were amphotericin B (AMB), fluconazole (FLC), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), voriconazole (VRC) and terbinafine (TRB) (Sigma-Aldrich, St. Louis, MO, USA). Briefly, inoculum of each strain of 1-5 × 10⁴ conidia/mL were prepared in sterile saline solution, including strains. MICs were determined by visual inspection after 48–72 h of incubation at 35 °C (CLSI standard M38) [13]. The azoles demonstrated good activity against the tested strains, showing MIC range of 0.25–1 μg/mL., with exception fluconazole was showed MIC >64 μg/mL. AMB showed MIC values between 0.5 and 1 μg/mL, however TRB show MIC >16 μg/mL.

The patient was still non-responsive after five weeks with terbinafine, so it was started with itraconazole (10 mg/kg/sid) for two weeks. During the course of this new antifungal drug the owner reported that the dog had pain and difficulty walking so declined therapy because and decided to euthanize the patient of the poor-quality life.

3. Discussion

Here we report the first case of canine sporotrichosis by S. brasiliensis in Santiago, Chile. Similar cases in dogs have only been reported in Brazil [7]. In Chile, S. brasiliensis was described for the first time in domestic and feral cats, in the Magallanes Region (52°36′28″ S 71°30′28″ W) [6]. More recently, this species was also reported for the first time in a woman in the Valparaíso Region (32°45′0.00″ S 71°19′59.99″ W) [11] at 2200 km and 121 km in a straight line respectively from the current report, which allows us to assume that it is an isolated infection event.

In general, dogs have been found to have a lower prevalence of sporotrichosis than cats [14]. For example, Montenegro et al. [15] found that of the total samples analyzed from cats, 58% were positive for Sporothrix spp., while only 8% of the dogs sampled were positive for S. brasiliensis. In addition to the above, a study conducted by Schubach et al. [14] demonstrated that of 44 dogs positives for sporotrichosis, only 6 had yeast elements, which highlights the low fungal load and the low probability of transmission [16]. However, the evidence found does not allow us to affirm that all dogs have a low possibility of transmission, and the fact that this fungus can be transmitted by three different types of routes [6].

Regarding the patient’s signs, these were consistent with those reported in previous literature [7,9,18]. The dog in this report was diagnosed with a cutaneous presentation without lymph node involvement and an ulcerative pattern involving the distal extremities and forehead, which is consistent with Gómez-Gaviria et al. [17] findings, where the most frequent form of disease was cutaneous, with 54.5% of the cases of the dogs studied, and with Sepúlveda Boechat et al. [7] where of the total of 47 dogs, 19 reported cutaneous lesions and 19 mucosal and cutaneous lesions. While in Barreto Fernandes Figueiredo et al. [19] report the most frequent presentations were the mucosal form and the cutaneous localized/fixed form. In general, all the previously mentioned reports described ulcerations on the extremities and the forehead or other areas of the face. Also, the patient’s blood parameters showed similarities with previous reports, highlighting the pyogranulomatous inflammation [20] and the normocytic normo-chromic regenerative anemia [18].

Among the treatments available for canine sporotrichosis, itraconazole has been the most used to date, either alone or in association with other drugs [7]. Furthermore, according to Gremião, it is a first-line antifungal for the treatment of human and animal sporotrichosis [5].

Before receiving the results of the antifungal test, the patient began treatment with the antifungal agent terbinafine, but no clinical signs of recovery were detected, and later demonstrated not to have sufficient “in vitro” activity. There is evidence that terbinafine has previously been used for the treatment of canine sporotrichosis with successful results [13], however it is not the first-choice antifungal. Itraconazole was then prescribed for two weeks until clinically reevaluation. However, the owner opted for euthanasia at another veterinary center.

The authors highlight the importance of a prompt diagnosis, after which appropriate decisions can be made about the best individual treatment for each patient along with an adequate follow-up [5]. It is recommended that relevant studies should be carried out in case of suspicion of sporotrichosis to prevent the spread of the disease in case of confirmation. In addition, it is strongly advised to follow the recommendations for responsible ownership of pets and companion animals. As a result, the contact between domestic and wild animals is reduced and therefore the possibility of transmitting the fungus to wildlife. This highlights the importance of addressing the problem from a One Health perspective [7].

Considering the rapid expansion of S. brasiliensis in Brazil during the last decade [5], the authors emphasize the need to take measures not only to be able to detect and confirm the infection but also to propose strategies to prevent and control this fungus in Chile, a country that is just beginning to observe the first cases of sporotrichosis due to S. brasiliensis. To finally obtain successful results, it is necessary to follow the One Health approach as a key point for the development of appropriate and effective measures, with participation and contributions of all members of society.

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Fondos Científicos PURINA 2022.

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Handling Editor: Dr Adilia Warris