Disseminated histoplasmosis in a cat rescued in Fortaleza, Brazil, and successfully treated with itraconazole – First case report identified molecularly

An unneutered female cat of unknown age presented bloody lesions on the edematous face, and respiratory signs. Cytology and culture from the skin sample collected with fine-needle aspiration showed yeasts inside activated macrophages, and fungal growth characteristic of Histoplasma spp., which was molecularly confirmed that was Histoplasma capsulatum var. capsulatum. The cat was successfully treated with oral itraconazole (10 mg/kg/daily) for 120 days. This is the first case report of feline histoplasmosis confirmed molecularly in Brazil.

Feline histoplasmosis is considered endemic in United States [1,11], and there are a few reports in cats around the world, as in Italy [12], Germany [2], Thailand [13], Japan [14], Costa Rica [8], among other countries, but little is known about the cases in Brazil. According to Table 1, most of these reported cases occur in the state of Ceará [3][4][5], although no other cases have been found in the other Northeastern States. Additionally, histoplasmosis was reported in cats living in Southeastern Brazil, as in the states of Rio de Janeiro [6,7] and Minas Gerais [9], and in Southern Brazil, as in the Rio Grande do Sul [10,15], in which the fungal identifications were performed by phenotypic analysis by culture [3][4][5][6]10,15] or only cytology [7,9]. However, the feline cases of histoplasmosis in Brazil are underestimated, since the notification is not mandatory and, besides that, the diagnosis is often delayed or it is misdiagnosed due to the lack of specialized laboratories in mycological diagnosis in our reality.
Treatment for histoplasmosis is based on the use of antifungal drugs, such as itraconazole, fluconazole, and amphotericin B, until lesions gradually regress [1,11]. The majority of cases in Brazil were resolved following antifungal therapy with itraconazole alone [3][4][5][6]15], or in combination with amphotericin B [7] or even with ketoconazole [9]. However, cases without resolution may evolve to either death or euthanasia [5,10], which further complicates the control of this disease.
This study aimed to report a case of disseminated histoplasmosis in a cat rescued in Fortaleza, Ceará (Northeast Brazil).

Case
An unspayed female cat of unknown adult age was rescued from a rural property where she was in contact with other cats and dogs. The new owner noticed ulcerative lesions on the animal's face two months prior, which worsened to the point that the cat was rescued and immediately taken to the veterinary clinic for evaluation on day zero. Physical examination revealed edematous and hemorrhagic lesions on the face, involving the right supra/infraorbital and nasal regions. A lesion was observed on the left region of the face, median to the eye (Fig. 1). The cat weighed 2.5 kg and showed apathy, mild diarrhea, mucopurulent nasal secretion, sneezes, moderate dehydration, enlarged submandibular lymph nodes, and subtle pulmonary crackles in lung auscultation. The ocular and oral mucous membranes were healthy pink and moist, and the body temperature was 38.2 • C (considered normal), without any indication of pain during abdominal palpation.
Blood samples were collected for complete blood count and serum biochemistry profile (alanine aminotransferase -ALT, and creatinine). After previously cleaning the area, skin lesions samples were collected by FNA and scrapings and swabs for cytology and microbiological cultures. Cytology using Romanowsky staining was performed with samples from ulcerative, erythematous, and hemorrhagic lesions from various areas on the face, mainly in infraorbital and supraorbital regions.
Cytological findings showed hypercellularity of non-degenerate neutrophils. Moreover, activated macrophages showed intracytoplasmic structures featured as small-sized oval-to-round yeast with an extracellular surrounding halo and eccentric nucleus shaped as punctate or halfmoon (Fig. 2). Intracellular and extracellular coccoid bacteria were also noted, indicating a secondary infection. These results indicated a pyogranulomatous infiltrate with infection suggestive of Histoplasma spp. Based on these results, treatment was initiated on the same day (day zero) with oral itraconazole (ITL 25mg®, 10 mg/kg, two times a day) along with the topical application of ketoconazole 2% (Navitae Ltda.®) in the lesions after cleaning with 0.9% NaCl solution. Considering the secondary infection, the cat was admitted for same day hospital treatment and received three intramuscular applications of amoxicillin trihydrate (Agemoxi L.A.®, 20 mg/kg/daily), every two days, along with a subcutaneous application of ketoprofen (Ketoflex® 1%, 2 mg/kg/daily), for four days.
Thoracic radiographs and abdominal ultrasound were performed on day 3 of treatment. The abnormal findings included lymphadenomegaly, that was observed in the lymph nodes of the gastroduodenal (0.78 cm), kidney (0.99 cm) and hepatic (0.95 cm) regions, in which the echotexture was slightly reduced and rounded, and an increase of the size liver, suggesting hepatic inflammatory process. Therefore, the frequency of administration of itraconazole was reduced at the same dose (10 mg/ kg/daily), while the topical cream ketoconazole was stopped. A food supplement (Nutrisana Spirulina®, 2.5 mg/feed, daily) began on day 4 until the +100 days.
Considering that the gold standard for the diagnosis of histoplasmosis is the fungal culture associated with the reversion of the filamentous phase of the fungus to the yeast phase, samples of each clinical lesion collected by FNA was seeded on Saboraud, Saboraud plus chloramphenicol and Mycosel agar, and incubated at 25 • C in aerobiosis. On the 15th day of incubation, all culture media showed fungal growth of a whitish cottony texture on the obverse (Fig. 3a) and brownish color on the reverse. Microscopically, hyaline septate hyphae and rounded and spiculated macroconidia were observed at a magnification of 400 × , from 8 to 15 μm (Fig. 3b). For the filamentous phase reversion to yeast, colonies were seeded on BHI agar (Brain Heart Infusion) plus 5% sheep blood and incubated at 35 • C. The phase reversal showed a bright and creamy colony, microscopically characterized by small oval yeasts (1)(2)(3)(4)(5) μm in diameter), confirming the fungal dimorphism. Due to biological risk, handling of the pathogen was carefully performed in a laminar flow hood.  Fragments of the fungal colony obtained from the culture were submitted to DNA extraction following molecular diagnostic by the polymerase chain reaction (PCR) in order to confirm the pathogenic agent. The RYP1 gene (263 bp) was amplified using the following primers OAS1057 (5 ′ -ACC CTT GCA GCT TAC AAC CT-3 ′ ) and OAS1058 (5 ′ -TCC GTC CAT CGC TTA ATA CC-3 ′ ), besides specific amplicons (300 bp) to RYP1 genes, according to Nguyen and Sil [16]. The amplified products were analyzed by electrophoresis on 1% agar gel using Blue Green (Loading Dye) as a fluorescent dye and visualized in a UV-transilluminator. Negative and positive controls were used from a human histoplasmosis case. The fungal sample from the feline case was identified as Histoplasma capsulatum var. capsulatum. The sequence of this isolate was previously deposited online on Genbank (number MK893850.1) and it was identified by data bank analysis with NCBI BLAST, in which presented between 97.44% and 100% identity with 102 human clinical isolates and one feline isolate.
The antifungal treatment with itraconazole was performed for 120 days, and the patient showed improvement of clinical signs from the first 30 days of therapy. No issued of vomiting or diarrhea were noted during the treatment. The evolution of the clinical treatment showed the complete remission of the cutaneous lesions until the final period of treatment (Fig. 4). Although no new imaging exams were performed due to the financial condition of the owner, the cat became pregnant one year after this episode and gave birth to healthy kittens. Afterward, the cat was spayed and has been living a healthy life without any recurrences.
Results of the hematological profile showed mild anemia and the serum biochemical exam was within normal standards. According to some authors, non-regenerative normocytic-normochromic anemia [5,7,10,12,15] may occur in these cases with a normal white cell count [1,5,9,10,13]. Other studies also described that elevated ALT activity can be observed [1,12,14], which did not occur in this case. Although a new blood exam was recommended, the owner did not authorize it. Thoracic radiographs and abdominal ultrasounds are recommended for evaluating the increase or decrease of the organs and, in our case, the cat had mild evidence of a hepatopathy and mesenteric lymph node enlargement. Additionally, histoplasmosis can be diagnosed using antigen detection on serum or urine [1], although this antigen detection assay was not performed in the reported feline.
The cases of histoplasmosis are regional in their distribution [1,5]. It is known that H. capsulatum is endemic throughout large areas of the temperate and subtropical regions of the world and tends to prefer areas with warm (mean ambient temperature of 22 • C to 29 • C) and humid conditions, which are generally between latitudes 45 • north and 30 • south [1]. The feline patient came from Fortaleza (Ceará, Northeastern Brazil), a geographical region where several feline cases have been reported [3][4][5]. This city is characterized by a rainy tropical climate, ranging from humid to sub-humid [17], providing climatic conditions in which should be taken into consideration during the patient's clinical investigation. The present case of feline histoplasmosis was confirmed after cytology, fungal culture, and molecular identification.
Similar cutaneous lesions in cats may be caused by fungal pathogens belonging to the clinical clades of Sporothrix spp. and Cryptococcus spp. complex [2], as well as by feline eosinophilic granuloma or squamous cell carcinoma [18]. Both Sporothrix spp. species and Histoplasma spp. may appear as small, round, or oval cells under direct microscopic examination, but only Sporothrix spp. show cigar-shaped yeast cells. Considering that the felines cases of sporotrichosis are often and this fungal genus is also dimorphic, like Histoplasma spp., a definitive diagnosis requires a fungal culture [18,19].
In the Cryptococcus spp. complex, yeast cells are round, commonly deeply basophilic, with a round, oval or elliptical nucleus and a characteristically wide mucopolysaccharide capsule that does not stain with the common rapid Romanowsky-type dyes. These thick non-staining capsules, occasionally with narrow-based budding, differ from the Histoplasma spp. yeast cells that have a thin poorly stained cell wall [19]. Additionally, the cryptococcosis cases in animals can be easily diagnosed by immunochromatography with latex antigen agglutination testing in serum specimens [20].
In our patient, the cytological findings revealed a pyogranulomatous inflammatory process with unaltered and degenerated neutrophils and activated macrophages. Inside these cells, oval-to-round structures suggestive of H. capsulatum were identified.
Direct examination, fungal culture, and phase fungal reversal were fundamental for the confirmation and diagnosis of histoplasmosis in our case. The diagnosis made by culture mediums (Sabouraud agar, BHI agar, Potato Dextrose agar, Lactrimel agar, and Malt agar) [19] is considered as the gold standard for histoplasmosis diagnosis [3][4][5][6]8,10,15], but the manipulation of the agent must be carefully performed in a safety cabinet due to the pathogenic potential [1]. However, one disadvantage lies in the difficulty of microscopically recognizing histoplasmosis by non-experienced mycologists, since other fungi may also present tuberculated macroconidia which appear as similar to Histoplasma spp., and may be mistaken for Chrysosporium spp. and Sepedonium spp [19]. At specific temperatures, only H. capsulatum assumes distinct forms. Under ambient temperature, a morphological macro colony of cottony appearance and micro morphologically, the mycelial form is constituted of hyaline hyphae, septate and branched, with smooth microconidia, which is the infecting form and tuberculated macroconidia. At 37 • C, it appears as a macroscopically bright and creamy colony and is microscopically characterized by small oval yeasts (about 1-5 μm in diameter) [1,19].
We reported a feline case of disseminated histoplasmosis in Fortaleza (Ceará, Northeastern Brazil), an endemic region where cases of this disease in cats is increasing. This study highlighted the importance of cytology as a fast, simple, and low-cost diagnostic auxiliary tool in veterinary routine, allowing for the rapid introduction of antifungal therapy, followed by the confirmation of the infection by H. capsulatum var. capsulatum through fungal culture and molecular identification. The molecular identification by PCR for amplification of the RYP1 gene allowed confirmation of the pathogen and, to our knowledge, this is the first case report of feline histoplasmosis confirmed molecularly in Brazil. The correct identification of the etiological agent is essential for therapeutic success, where the cat showed complete remission of the cutaneous lesions after 120 days of therapy with oral itraconazole, without any adverse effects. Additionally, the evolutionary follow-up of antifungal treatment has been demonstrated in this study.

Ethical form
The authors declare that the consent was obtained from the patient's owner.

Declaration of competing interest
There are none.