Abstract
This report describes a 6-year-old neutered male feline immunodeficiency-positive cat with repeated abdominal and thoracic effusions. The cat was diagnosed with and treated for lymphosarcoma but remission was short-lived and, on re-evaluation, a fungal peritoneal exudate was noted. Cytology of the organisms is described and the culture elucidated Cladosporium carrionii, an important cause of chromoblastomycosis. Treatment with itraconazole was unsuccessful in this case.
Case Report
Chromoblastomycosis (syn chromomycosis, Fonseca’s/Pedroso’s disease) is caused by a variety of wind-dispersed, saphrophytic moulds, including Cladosporium carrionii, Fonsecaea pedrosoi, Fonsecaea compacta and Phialophora verrucosa. A tropical disease affecting mainly humans walking barefoot or performing agricultural work, the initial injury that leads to infection often goes unnoticed. Some consider chromoblastomycosis to be an emerging ‘cosmopolitan’ disease, 1 but most cases in the literature are rural in their demography. This organism is most commonly seen in Australia, Venezuela and South Africa. 2 Infection by the dematiaceous fungus occurs by direct inoculation of conidia or hyphal fragments into the subcutaneous tissues of the person. 3 The organisms typically cause toenail infections or dermal nodules, which can progress along the lymphatics and can (rarely) spread to the lungs causing pneumonia. Lesions in humans can take 30 years to fully develop into verrucous (ie, wart-like) lesions. 1 When inhaled, these moulds may also be significant allergens. 4 Cladosporium infection has also been described in a patients with acquired immune deficiency syndrome (AIDS) and in a patient under immune suppression for a renal transplant. Members of this genus are renowned for their neurotropism.5–7
Chromoblastomycosis caused by C carrionii has never been reported in cats. Two previous publications reported fatal systemic involvement by Cladosporium bantiana and two cases of cerebral infection,8,9 while other articles describe various cutaneous, systemic and neurological manifestations of other species.7,10–14 Bouljihad et al 11 cite some of these other, older cases. We describe the first case of C carrionii in a feline immunodeficiency virus (FIV)-positive cat with strongly suspected concurrent lymphosarcoma and septicaemic fungaemia.
A 6-year-old neutered male cat weighing 4.45 kg with a history of dyspnoea, coughing, episodes of gagging and reduced activity was seen by the referring veterinarian. According to the owner, feline infectious peritonitis (FIP) had been diagnosed in the household previously. On initial evaluation the patient had mild mucous membrane pallor, a mild tachycardia (160 beats per min) and ascites, with concomitant loss in body condition. An indirect fundoscopic ocular examination and all other clinical parameters were normal. Blood smear, complete blood count and chemistry profile were performed (Table 1). The blood smear showed a mild anisocytosis, eosinophilia and atypical lymphocytosis.
Haematology and chemistry results
RBC = red blood cells; Hgb = haemoglobin; MCV = mean cell volume; MCH = mean cell haemoglobin; MCHC = mean cell haemoglobin concentration; RDW = red cell distribution width; Retic = reticulocytes; WBC = white blood cells; Diff = differential; MPV = mean platelet volume; PDW = platelet distribution width; ALP = alkaline phosphatase; ALT = alanine aminotransferase; BUN = blood urea nitrogen
Abdominal ultrasound examination was performed (Toshiba EccOcee, 6–8 MHz and 3.5–5.0 MHz probes) and it was noted that all intra-abdominal lymph nodes were enlarged and the kidneys had mild cortical hyperechogenicity with a medullary stripe sign. There was considerable ascites. Pinkish milky fluid (500 ml) was removed. Cytology revealed a large number of lymphocytes with many neutrophils and red blood cells, with scanty mast cells. The exudate had a total protein of 54 g/l and albumin of 23 g/l (0.8:1.0 albumin/globulin [A/G] ratio). No specific gravity was reported. The cytologist could not conclusively exclude FIP, but downplayed the likelihood of it. Lymphosarcoma was deemed more likely and a feline leukaemia virus/FIV tabletop test was done (Virbac SpeedDuo; Virbac), with a positive result for FIV. Subsequent to this, all other cats in this multi-cat household were tested and found to be FIV negative.
On day 2 the patient had stopped eating and the fluid had re-accumulated. Ultrasound-guided splenic and hepatic aspirates were negative for lymphoblasts but the liver aspirate had prominent neutrophil infiltrates. A regional abdominal lymph node that was aspirated had hyperplastic changes and clusters of polygonal cells with round nuclei and distinct cell borders. The clinical pathologist was concerned that the whorled appearance of some clusters might indicate a neoplastic, metastatic glandular or mesenchymal origin, but no histopathology was carried out. The patient was admitted for assisted feeding by syringe (Hill’s Prescription Diet a/d; Hill’s Pet Nutrition) and responded well. The patient was started on 5 mg prednisolone (Centaur Prednisolone; Bayer) q12h PO on day 4 of admission.
By day 6 the ascites had recurred and the patient appeared clinically anaemic. Milky red fluid (800 ml) was drained from the thoracic cavity. An echocardiogram carried out by the referring general practitioner did not show evidence of hypertrophic cardiomyopathy and the patient was referred for suspected stage V lymphosarcoma.
On admission at the specialist centre (day 9), an additional fluid sample was withdrawn from the abdomen and sent to a laboratory for FIP PCR and a second opinion on the cytology. The sample was turbid-cloudy, with a specific gravity of 1.030 and a protein level of 5.4 g/100 ml. It was tested using a reverse transcriptase nested PCR that targets the NS gene of the feline coronavirus genome; the result was negative for coronaviral nucleic acid and cytologically positive for lymphosarcoma.15–18 In the interim, the patient was treated with a modified COP (cyclophosphamide, Oncovin and prednisolone) protocol consisting of 0.15 ml vincristine (Abic-Vincristine 2 mg/2 ml; Teva Pharmaceuticals) and 0.50 ml of injectable subcutaneous (SC) prednisolone (Prednisolone 1% Kela; Bayer) while awaiting the report. The latter treatment was repeated daily thereafter (days 11–14) as the patient appeared to have responded well to treatment (clinically and radiographically). Radiographs showed re-accumulation of thoracic fluid on day 15, and this was aspirated. The COP treatment was halted and oral prednisolone and 50 mg of cephalexin PO q12h (Ceporex 50 mg; Schering-Plough) were started. Two days after discharge the owner reported that the patient was doing well, and on day 20 the patient was administered one 50 mg cyclophosphamide tablet PO (Endoxan; Sanofi-Aventis) along with a repeat injection of vincristine. On day 27, a repeat haematology was performed (Table 1), and the patient administered 0.1 ml of SC mepyramine maleate (Mepyramine; Kyron Laboratories), 0.2 ml of intravenous (IV) metoclopramide (Clopamon; Aspen Pharmacare) and 2.1 ml of doxorubicin by slow IV infusion over 45 mins (Doxorubicin-Sol PCH 50 mg; Pharmachemie). Fluid (150 ml) fluid was drained by thoracocentesis after radiographs revealed an effusion, and it was decided to initiate a doxorubicin-based protocol.
By day 34 the ascites had recurred and, for the patient’s comfort, 300 ml was drained. A post-doxorubicin haematology examination was performed. On day 46 the abdominocentesis was repeated (650 ml) but the patient re-presented on day 48 for a new mass the owner had noted on the skin of the right side of the neck. The nodule was 1 cm in diameter, firm and dermal. It was aspirated along with fluid from the abdomen and sent for culture. In-house cytology revealed what was thought to be a fungal organism on Diff-Quik staining (Figures 1–3) and thus chemotherapy was suspended and the patient started immediately on itraconazole 10 mg/kg q12h (Sporanox Oral Solution; Janssen Pharmaceutica).
Branching hyphae of Cladosporium carrionii and pyogranulomatous reaction in a dermal nodule aspirate. Diff-Quik stain (Kyron Laboratories) (magnification x 1000)
Branching hyphae seen on ascitic fluid preparation. Diff-Quik stain (Kyron Laboratories) (magnification x 1000)
Branching hyphae of Cladosporium carrionii and pyogranulomatous reaction in ascitic fluid. Diff-Quik stain (Kyron Laboratories) (magnification x 1000)
Various tissue samples and a peritoneal fluid sample were submitted to a veterinary pathology laboratory (Vetdiagnostix) for fungal culture and identification of the organisms seen in the Diff-Quik smears. Based on the morphology of the organisms observed cytologically, Histoplasma and Lagenidium were initially the strongest differentials. Samples were plated onto Sabouraud’s dextrose agar (Oxoid Ltd) and incubated aerobically at 30°C. Cladosporium carrionii (now Cladophialophora carrionii) is a relatively slow-growing organism and usually matures within 18 days. The samples were submitted on 4 November 2008 and a definitive identification, based on the phenotypic characteristics of the culture, was available on 12 December 2008. Antigen tests were not available. Macroscopically, the colonies had a dark surface and were flat with a slightly raised centre. They were covered with velvety, dull grey, grey–green or purplish-brown mycelia. The reverse of the culture was black. Microscopically, the hyphae were septate and darkly pigmented with lateral and terminal conidiophores in various sizes. These conidiophores produced long, branching chains of brown, smooth-walled, oval-to-pointed conidia that varied in size from 2.5 to 4.5–7.0 µm. These conidia were easily dispersed with handling and had relatively pale scars of attachment (hila) when compared with other Cladosporium species. 2 This satisfied the criteria for identification of C carrionii (Table 2). 2 Disc diffusion sensitivity testing was carried out according to the Kirby-Bauer method, 19 and the organism was found to be sensitive to ketoconazole, miconazole and clotrimazole. It was resistant to amphotericin B, nystatin and flucytosine.
Identification of Cladosporium (syn Cladophialophora) carrionii
The patient was seen again on day 57 for a repeat abdomino- and thoracocentesis, and assisted feeding over the next 2 days with other nutritional products (Royal Canin Recovery Cans [Royal Canin South Africa] and Eukanuba High Calorie cans [Cube Route South Africa]). It was seen over the subsequent weeks (days 63, 68, 69, 70, 72, 74, 76 and 77) for repeated thoracic and abdominal drainage. The referring veterinarians also administered 10 mg (then 5 mg after 5 days) furosemide q12h PO (Lasix 20 mg tablets; Sanofi-Aventis) and half a 0.0625 mg digoxin tablet q12h PO (Lanoxin Paediatric tablets; Aspen Pharmacare) based on an echocardiogram obtained under diazepam and ketamine sedation. The referring veterinarians did not give a reason for the inclusion of the digoxin. The patient began vomiting on day 74 and the digoxin was stopped again for 2 days before restarting. Thoracic radiographs on day 77 revealed marked effusion, and itraconazole, furosemide and digoxin were continued. Serum levels of digoxin were not measured, nor were liver enzymes.
By day 84 the patient had marked dyspnoea and was admitted by the referring veterinarian for drainage under fentanyl + diazepam + propofol thoracocentesis but experienced cardiopulmonary arrest within minutes of anaesthesia and could not be resuscitated. Post mortem was not permitted by the owner.
The genus Cladophialophora/Cladosporium comprises eight species, namely C arxii, C bantiana, C boppi, C devriesi, C emmonsii, C minourae and C modesta, 20 in addition to the species under discussion, which is the most common pathogen of the group and in cases of chromoblastomycosis in general. In the past and in this case, identification relied on morphological and physiological characteristics and antigen detection after culture. However, because of the growth characteristics of these organisms, diagnosis can be delayed by up to 3 weeks. However, a Japanese group has described a PCR technique for identification of a species- and genus-specific primer of this organism, which could aid in the diagnosis of future cases. 20
Cladosporium species produce lipids responsible in some manner for the granulomatous pathology associated with infection. 4 In addition, the organisms produce urease, gelatinase and lipase, and the organism F pedrosoi also produces a phospholipase. These products may be important for the organism to invade and survive in the host extracellular matrix, to evade the immune reactions and for acquisition of nutrients. 3 Both the lipid and protein products may contribute to inflammation.
Immunity to the organisms causing chromoblastomycosis is difficult to describe. Esterre et al 1 tried to describe the immunodominant antigenic determinants in F pedrosoi and C carrionii. They described some patients who still had antibodies (principally IgG1, but also IgM and IgA) up to 1 year after clinical cure with an effective agent (terbinafine) compared with unaffected patients with a variety of other endemic diseases, and non-responders. They showed that C carrionii-infected patients were worse responders, as measured by antibody decrease. Immunity to these organisms seems to be T helper cell 2-mediated. 1
Treatments described as effective for chromoblastomycosis include 5-fluorocytosine, 21 ketoconazole, 5 itraconazole5,22,23 and terbinafine.1,24 Antonello et al 25 state that 5-fluorocytosine should be combined with an azole as its action of inhibiting the synthesis of fungal nucleic acids is unlike that of other substances, which typically act on the fungal membrane, and could act as a potentiating agent. Chromoblastomycosis is considered a challenging but potentially curable disease. 25
Conclusions
In the case of this cat, the infection was most likely facilitated, if not aggravated once established, by the concurrent lymphosarcoma, FIV and chemotherapy. FIV infection is considered to be common in South Africa (in one survey, 22.2% of domestic cats were positive). 26 Nonetheless, the patient remained relatively stable on itraconazole for a few weeks. It is debatable whether the mycosis or the neoplasia (or both) was the cause of death. Itraconazole has resulted in hepatotoxicity at higher dosages, although this was not noted in this patient as blood tests were never performed to monitor transaminases. Owing to improved absorption over the encapsulated form, when dosed on an empty stomach the itraconazole syrup administered can be given at a dosage of 25–50% of the standard 20 mg/kg/day advocated. 24 Similarly, the penultimate therapy with digoxin and furosemide likely did little to aid or aggravate the clinical situation, but was probably erroneous, being based on an echocardiogram under sedation, which is known to affect echocardiographic parameters. 27 Digoxin is a P-glycoprotein substrate, and concurrent administration of P-glycoprotein inhibitors such as itraconazole should be done cautiously. Lastly, the risk of contagiosity to the other cats in the same household by C carrionii is probably negligible unless they are also found to be FIV positive.
Footnotes
Acknowledgements
We acknowledge Drs Steve Wimberley and James Webster, Westville Veterinary Hospital, for the referral and input into the final article, and Dr Denis York, Molecular Diagnostic Services, for the PCR information.
Conflict of interest
The authors do not have any potential conflicts of interest to declare.
Funding
The authors received no grant from any funding agency in the public, commercial or not-for-profit sectors for the preparation of this case report.