Diagnosis and Treatment of Nasal Aspergellosis In a 3 year old Dog
 
Katja Lang Senior Seminar Paper Cornell University College of Veterinary Medicine Clinical Advisor: Dr. Meredith Miller Basic Science Advisor: Dr. Dorothy Debbie February 16th, 2011

Keywords: Canine, Sinonasal aspergillosis, nasal discharge
 

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ABSTRACT:
A 3 year old, male intact, mixed breed dog presented to the Cornell Internal Medicine Service for evaluation of nasal discharge and periods of epistaxis. On presentation, he was severely cachectic. Initial diagnostics revealed mild hyperglobulinemia, mild hypoalbuminemia, and mild neutrophilia, consistent with inflammation. A B12/folate/TLI panel did not reveal any abnormalities. Computed tomography (CT) and rhinoscopy were recommended for a more definitive diagnosis. The CT showed a destructive sinorrhinopathy without mass effect. Visualization of fungal plaques within the nasal cavity was present on rhinoscopy. During the same anesthetic episode, the patient was treated with an intranasal and intrasinus infusion of clotrimazole. The histopathology confirmed the diagnosis of sinonasal aspergillosis.
CASE HISTORY:
A 3 year-old male intact mixed breed dog presented to the Cornell Internal Medicine Service on 8/18/10 for evaluation of nasal discharge and epistaxis. The owner first noticed that he began sneezing more frequently about 5 months prior. He was a dog that liked to sniff the ground on walks so the owner assumed there was a intranasal foreign body. In the middle of July 2010, the owner noticed mucoid nasal discharge that started in the left nostril but became bilateral. Additionally, the nasal discharge became progressively more copious. In August, he had two episodes of epistaxis where he bled a large amount but it stopped after a few minutes. Otherwise, the owner only noted a few drops a blood per day from the nose.
A week before presentation to the CUHA, he began to have diarrhea with spots of frank blood present. In the past 2 months, he lost about 20 pounds and his appetite was decreased. Lastly, his owners report that in the last few weeks he has been acting more lethargic. His referring veterinarian treated him with unsuccessful courses of Simplicef, ampicillin then doxycycline.
PHYSICAL EXAM FINDINGS:
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On presentation, our patient was quiet, alert and responsive. We were unable to obtain vital parameters because he was fractious. His body condition score was 2/9 (body weight = 21.5kg) and he appeared diffusely muscle wasted. There was a large amount of bilateral mucoid discharge. Depigmentation and crusting of the nasal planum and surrounding skin was present. The retrobulbar space was palpated and no masses were felt. There was no evidence of visual or palpable asymmetry or bony defects. Auscultation revealed clear lung sounds bilaterally and no murmurs or arrhythmias were heard. The abdomen felt soft and non-painful; there was no obvious organomegaly or masses. A thorough physical exam was not possible given his demeanor.
PROBLEM LIST & DIFFERENTIAL DIAGNOSES:
The problem list for our patient included: 1. Epistaxis 2. Bilateral mucoid nasal discharge 3. Depigmentation and crusting of the nasal planum 4. Hematochezia 5. Weight loss
Differentials for epistaxis include: coagulopathies (thrombocytopenia, thrombopathia, coagulation factor defects, acquired coagulopathies), increased capillary fragility (hyperviscosity syndrome, hypertension, hyperlipidemia, thromboembolic disease), neoplasia (nasal adenocarcinoma, lymphoma, benign polyps), bacterial infection, fungal rhinitis (aspergillus, histoplasma, cryptococcus), dental disease with oronasal fistula, nasal parasites (cuterebra, pneumoyssus, eucoleus), idiopathic eosinophilic or lymphoplasmacytic rhinits, and arteriovenous malformation. The differentials for nasal discharge include infectious rhinitis (viral, fungal, parasitic), inflammatory (allergic, lymphoplasmacytic rhinitis, nasopharyngeal polyp, acquired nasopharyngeal stenosis), neoplasia (adenocarcinoma, SCC, fibrosarcoma, osteosarcoma, chondrosarcoma, lymphoma, TVT), foreign body, congenital (cleft palate, ciliary dyskinesia), dental
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disease (oronasal fistula, tooth root abscess), and lastly trauma. Based on our normal coagulation panel and normal cell counts, a coagulopathy was less likely. Our top differentials based on clinical signs and history was sinonasal aspergillosis, neoplasia and lymphoplasmacytic rhinitis.
DIAGNOSTIC RESULTS:
The diagnostic plan included computed tomography (CT) followed by rhinoscopy under general anesthesia. Pre-anesthetic blood work included a complete blood count (CBC) and chemistry panel. We felt it was necessary to run a coagulation panel before taking biopsies of the nasal cavity considering the history of epistaxis. Additionally, a B12/folate/TLI panel was performed to rule out any underlying small intestinal and/or pancreatic diseases considering his substantial weight loss and hematochezia. The CBC showed mildly elevated neutrophilia (10.9 thou/uL; 5.7-14.2) consistent with inflammation?. The chemistry panel showed moderate hypoalbuminemia (2.5 g/dL; 3.14.1) and mild hyperglobulinemia (3.8 g/dL; 1.9-3.6) consistent with inflammation. The coagulation panel and B12/folate/TLI was within normal limits.
Thoracic radiographs were performed to rule out metastasis if the nasal process was neoplastic before going through with further diagnostics. The radiographs revealed no abnormalities. Computed tomography (CT) was performed and showed a destructive sinorhinopathy without mass affect. The left ventral-nasal concha were absent and air freely communicated with the left maxillary recess. Portions of the nasal septum were lost with communication between passages. Associated with the left side of the ethymoid labyrinth and left frontal sinus, the mucosal linings were thickened. The left front sinus was fluid filled and contained tiny gas bubbles. There was mild thickening of the rightventral-nasal concha.
A rhinoscopy was performed on the patient under general anesthesia in sternal recumbency. Visualized were several white, fuzzy fungal plaques in the left nasal passage. Three small pinch biopsies were taken from the nasal cavity and submitted for histopathology. Histopathology showed a suppurative rhinitis with intralesionsal fungal hyphae that was consistent with nasal aspergillosis. The examined slides consisted almost
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entirely of mats of basophilic to eosinopholic fungal hyphae that were 6-8 m in diameter, parallel-walled, containing septate with dichotomous 45 degree branching. There were sporadic degenerate neutrophils and bone fragments.
TREATMENT:
The frontal sinuses were entered using an Illinois bone marrow instrument and used to flush the nasal passages with 0.9% sodium chloride. A large amount of mucoid nasal discharge was evacuated. 60mL of clotrimazole solution was infused through each side of the frontal sinuses and allowed to bathe the nasal cavity and sinuses for an hour. Additionally, two catheters were placed into the nasal cavity (WORK ON THIS). Postoperatively, he received 80 mg carprofen subcutaneously to decrease pain and inflammation. He recovered well from anesthesia and received hydromorphone intravenously once at night. He was sent home to the care of his owners with tramadol, metronidazole and drontal plus.
DISCUSSION:
Aspergillus fumigatus, the most common species that affects canines, are a opportunistic, saprophytic fungus that are ubiquitous in the environment, though they are most commonly found in compost, stables, and barns.4 Aspergillus niger, Aspergillus nidulans, and Aspergillus flavus are occasionally involved. Aspergillus is known to cause both sinonasal and disseminated disease, but they are two different disease entities. Disseminated disease is reported most commonly in immunosuppressed, German Sheppard dogs. The clinical signs are reflective of the organ systems affect but most commonly involve the disc spaces, and kidney.4 This paper will only focus on sinonasal aspergillosis (SNA), which is considered to occur in immunocompetent dogs and is localized to the nasal cavity. Sinonasal aspergillosis usually occurs as a primary disease but may be secondary to other nasal pathology such as foreign body, neoplasia, etc.12
Canine sinonasal aspergillosis is a common problem in middle aged mesaticephalic and dolichocephalic breeds.1,2 It is reported that between 12-34% of dogs with chronic
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nasal disease are affected.2,3,8 It is the second most common cause of nasal discharge after neoplasia.1,11 The mean age of dogs affected by this disease ranges in studies from 3.34.4 years, although juveniles and geriatric patients can be affected.2,4 This is in contrast to dogs with nasal neoplasia that have a mean age of 9.5. One study found that 80% of dogs are 7 years and younger and 30% are 3 years and younger.5 There is a predisposition for males and the breeds most frequently affected are Rottweilers, German Shepards, Golden Retrievers and Labrador Retrievers.1,3,5, 12
Dogs with this disease are considered to be systemically immunocompetent, but there is speculation that mucosal immune dysfunction predisposes certain dogs to infection.4 This fungus spreads via aerosolization of its 2-3 micrometer condia.2 Their concentration in air is high at about 1-100 conidia per m3.13 It is estimated that most hosts inhale hundreds of conidia daily, but they are cleared mainly by cell-mediated immunity.2 This organism must adhere to the mucosa and resist phagocytosis. Adhesion occurs by hydrophobic proteins that cover conidia, the "rodlet layer", which bind to albumin and collagen.13 Gliotoxin, is an important virulence factor responsible for inhibition macrophage phagocytosis, T-cell activation/proliferation and can induce macrophage apoptosis.2,13 In vitro studies have shown that A. fumigatus can limit lymphocyte transformation and impair peripheral blood lymphocyte proliferation.1 There are other toxic metabolites produced by the fungus including, aflatoxins and ribotoxins , which interfere with the mucociliary apparatus, neutrophilic phagocytosis and opsonization.1 Aspergillus fumigatus causes damage to the host by releasing an endotoxin that is both hemolytic and dermonecrotic.2 Another substantial amount of damage is caused by the host immune response itself.1 Dogs with SNA have upregulated mRNA Il-8 and monocyte chemoattractant protein (MCP), which explains why the main inflammatory infiltrate are lymphocytes, plasma cells and neutrophils. The main interleukin responsible for suppressing the immune response is Il-10, which is thought to both limit the extent of local damage and/or inhibit the host ability to clear the infection. The cause of increase of Il-10 is unknown. 1
The three hallmark clinical signs are profuse mucohemorrhagic nasal discharge with periods of epistaxis, ulceration, crusting and/or depigmentation of the nasal planum and discomfort of the facial region. The depigmentation almost exclusively occurs in
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fungal disease due to the toxins release.5 Nasal depigmentation is reported in several immune-mediate dermatologic disorders but these usually do not result in nasal discharge. The nasal discharge in both neoplasia and sinonasal aspergillosis usually start unilateral and progress to bilateral, whereas dental disease and foreign bodies idiopathic rhinitis remain unilateral. In contrast, allergic, infectious, or immune-mediated rhinitis tend to show clinical signs bilaterally.11 Another easy, inexpensive test to perform to differentiate is to put a glass slide or cotton tip in front the nares to look for airflow. Loss of airflow is suggestive of a mass lesion, whereas an increase in airflow is more suggestive of nasal aspergillosis.4 Less common clinical signs that can occur when this disease becomes especially severe are lethargy, inappetance, ocular involvement (epiphora, exophaltamus, keratitis), and neurologic signs (seizures, dullness).
The diagnosis of aspergillosis requires several tests include imaging (radiographs, CT or MRI), rhinoscopy, histopathology, culture and serology. The gold standard involves direct visualization of fungal plaques in the nasal cavity or the frontal sinus via rhinoscopy or sinuscopy or the observation of fungal elements on cytology or histopathology.1 Some studies find it necessary to have three positive results to confirm the diagnosis as some positive results can be misleading. It is always best to take biopsy samples after imaging to avoid hemorrhage and other artifacts.12
Imaging is a critical part establishing a diagnosis and treatment plan for SNA. With the advent of more sophisticated imaging like MRI and CT, radiographs are not commonly used. On radiographs, you cannot appreciate the nasal septum, and caudal recesses. Additionally, the cribiform plate, naso-orbital wall and vomer bone are only visualized when they are compromised.8 MRI and CT are more sensitive and far superior at evaluating the extent of disease. They also eliminate superimposition of bony structures and nasal turbinates. Radiographs have a sensitivity of 72%, whereas CT is 88%.1 There were no research articles that clearly an advantage for using MRI over CT. Many radiologists prefer CT because it is better at assessing bony lysis. However, CT may be difficult to interpret if fluid accumulations are excessive.2 If neoplasia is higher on a differential list based on signalment or clinical signs, MRI may be preferred. One study found that MRI was superior for diagnosing neoplasia and may be useful for differentiating thickened mucosa versus fungal colonies.1,8 Evaluating the cribiform
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plate, periorbital region, and frontal sinus is critical to deciding what treatment options are best for the patient. CT is the optimal imaging modality to elevate the frontal sinus because it can discriminate air and bone well.
The use of contrast is controversial in the literature. Some studies indicate contrast may help improve differentiation between mucosa or fluid and between necrotic and vascularized soft tissue. Other studies found post-contrast not helpful due to the fact that attenuation measurements had a high degree of error due to mixing of complex structures and varying densities in diseased nasal cavity.9
When evaluating an MRI or CT, it is usually possible categorize the patterns into destructive without mass effect versus non-destructive with mass effect sinorhinopathies. Establishing which category the images fits into allows one to refine the differential list. Non-destructive sinorrhinopathy without mass effect include sinonasal aspergillus, and idiopathic lymphoplasmacytic rhinitis (WORK ON THIS). Destructive rhinopathies with mass mass include neoplasia, cryptococcus?? SNA is considered destructive rhinopathy because the common features associated with on CT include moderate to severe turbinate destruction with variable amounts of soft tissue in nasal cavities, a rim of soft tissue along frontal bone, maxillary recess and nasal bones and hyperostosis of the maxillary, vomer and frontal bones.1,8 Other differentials that lead to nasal turbinate loss is neoplasia and idiopathic lymphoplasmacytic rhinitis (LPR). Neoplasia is more likely to show more significant bone loss and replacement with soft tissue density.1 One study looking at the CT findings in LPR, found that 70% of cases had turbinate loss and 42% had frontal sinus involvement.11 Although certain etiologies can have classic imaging features, a definitive diagnosis should be based on biopsy and histopathology to confirm a diagnosis.
Rhinoscopy can be useful for both diagnosis and treatment. Sampling without visualization may be unrewarding; the sensitivity is high if you biopsy directly from the fungal plaque.4 It is important to visualize the nasopharynx via the ventral nasal meatus prior to entering the dorsal nasal meatus to inspect the cribiform plate and the opening to the frontal sinus. Entering the dorsal nasal meatus first may cause hemorrhage and therefore impair visualization of the ventral nasal meatus.2 Entering the ventral nasal meatus first avoids this potential complication. The most typical findings with SNA are mucopurulent nasal discharge, destruction of nasal turbinates, and white-to-green fuzzy
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fungal plaques.1,2 The fuzzy appearance is caused by the conidiophores or fruiting bodies. Rhinoscopy is not helpful for evaluating bony structures, such as the nasal septum and cribiform plate, so advanced imaging should precede this. Additionally, fungal plaques may only be present in the frontal sinus. In one study evaluating distribution of fungal plaques, 17% of canines had plaques only located in the sinuses but not in the nasal cavity.4 This information would change where one placed the scope to take biopsies and where the antifungal medication needed to reach during treatment. It has been shown that debridement of the fungal plaques during rhinoscopy improves patient outcome as it allows the antifungal medications to penetrate deeper.1,4
Histopathology can be confirmative if the correct area is sampled.12 The specificity of this test is about 100% but sensitivity varies depending on sample site.1 Knowledge of the lesion location based on the imaging studies can be extremely helpful in deciding where to biopsy. It is also important to measure the distance from the nares to the medial canthus to avoid damage to the cribiform plate with the arthroscope.12 Aspergillus have characteristic features on histopathology, but a culture is needed for definitive diagnosis and to help differentiate from other fungi, like Penicillum species. The key features are a dense conglomeration of hyphae that branch dichotomously at 45 angle and identification of septate and conidia.6 The hyphae should have terminal bulbs with swellings.2 Some texts, require hyphal invasion into the mucosa for definitive diagnosis, whereas others believe Aspergillus species are not able to invade the mucosa.2,5
Culture and serology are controversial tests. The most commonly used serology tests are agar gel immunodiffusion (AGID), Elisa and Counterimmunoelectrophoresis, which all look for antibodies. Agar gel immunodiffusion (AGID) is commonly used but cannot quantify antibiodies, whereas the ELISA can. The sensitivity of AGID ranges in studies from 31% to 68%, which means over one-third of affected dogs would have a negative result.1,4 AGID has a high specificity (98%), indicating false positive are rare. On the contrary, one study found 15% of dogs with nasal neoplasia test positive on AGID.4, 5 In human medicine, the sandwich ELISA is used and is very sensitive and
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specific. Culture alone is misleading because Aspergillus can be part of normal flora.1 The sensitivity of culture varies from 40-77% and can yield false-positive results in 3040% of normal dogs and dogs with neoplasia. Serology has not been found to be useful for monitoring improvement post-treatment as positive titers may persist for more than 2 years for the ELISA and more than 5 years for AGID.
Treatment success for sinonasal aspergillosis has changed drastically in the last few decades. In the 1980's, the treatment consisted of systemic antifungals. Oral antifungal medications are expensive, poorly soluble/absorptive and have a narrow margin of safety. It is reported in the literature that oral thiabendazole and ketoconazole have a 40-50% efficacy and fluconazole and itraconazole are 70% effective.1 The common side effects associated with these antifungal medications are hepatoxicity, anorexia, vomiting, and cutaneous vasculitis. About 5-10% of dogs treated with itraconazole experience hepatotoxicity. A new generation triazole, voriconazole, holds promise for refractory cases, but has not been used extensively.5 In the 1990's, topical anti-fungal drugs began to be used and were more efficacious and carried less side effects. Surgically implanted catheters were placed into the nasal cavity and frontal sinus and flushed twice daily for seven to fourteen days. This procedure had a fairly high success rate of 80% but carried several complications with the indwelling catheter. The most commonly reported complications were subcutaneous emphysema, premature removal of catheter, inappetance and ptyalism.2,7 This method is not longer in vogue after the advent of a onetime treatment. A one-hour infusion with topical antifungal medications has a greater success rate and decreased hospitalization time. In refractory cases, surgical procedures are still in practice. These involve temporary rhinostomy with topical povidone iodine dressing or rhinotomy and surgical debridement. 2
The two most commonly used topical antifungal medications are eniconazole and clotrimazole. Ergosterol is a crucial part of the fungal cytoplasmic membrane and these medications block ergosterol biosynthesis, leading to increased membrane permeability.7 Each of these medications have properties that make them distinctive, but the overall success rate is comparable. It is unclear whether switching these drugs for the second treatment or vice versa is beneficial.2 To increase success rate endoscopic guided catheter placement into the frontal sinus has been used.9 Retreatment is suggested if the clinical
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signs have not resolved in two weeks. This is based on the mean and median resolution of discharge in patients following SNA treatment.7 The paper evaluating eniloconazole treatment felt that rhinoscopic evaluation was the best way to determine a success since clinical signs improved in all patients.2,3 Topical infusions are contraindicated in dogs that have lytic lesions in the cribiform due to leakage into the nervous tissue, but there are reports of patients treated with eniconazole without any complications.3 Owners should be warned of the risk, and offered systemic therapy as an alternative.1
Clotrimazole is unique in that at high enough concentrations, it can directly damage the fungal membrane.1 Topical infusion results in a complete cure after a single treatment in 65% and 87% after two or more treatment. These statistics were based on follow-up conversation with the referring veterinarian and owners.7 Another study that used followup rhinoscopy found a 48% success rate after one treatment and overall success rate of 67%.6 They concluded that clinical signs and serologic tests were not predictive of response to treatment. A smaller study involving 14 dogs examined the use of 1% clotrimazole cream following a 5-minute flush with 1% clotrimazole. This study had a success rate of 86% and may be useful for decreasing anesthetic time.1 The most commonly reported post-clotrimazole treatment complications are nasal discharge and sneezing.6 Additionally, clotrimazole can cause pharyngeal edema, irritation and upper airway obstruction due to its vehicle, propylene glycol and isopropanol.1,6 Propylene glycol has also been reported to cause vehicle-induced menigitis. There are human marketed 1% clotrimazole solutions (Taro and Teva Pharmaceuticals) that use polyethylene glycol as their vehicle, instead of the veterinary brands (Vetoquinol USA Inc) that use propylene glycol. The Internal Medicine service at Cornell University has found that using the human brand has eliminated this complication.14 Lastly, some postulate that dogs experience prolonged recovery due to systemic absorption and hepatic enzyme inhibition. 5
Eniconazole is reported to be less toxic. It is also has active antifungal properties in its vapor phase up to 1 cm which allows further distribution throughout the nasal cavity.1 The success rate is reported to be about 57% after one treatment and 92-94% after more than one treatment.3 There were no reports of relapses in any of the dogs during the 8 month follow-up period. All patients were treated with a ten-day course of antibiotics
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because the authors postulated most patients had concurrent bacterial rhinitis. This same study found that infusing 2% directly into the frontal sinus was more successful than 1% eniconazole into the nasal cavity. Rhinoscopic debridement in this same paper decreased the number of treatments.3 The authors attempted to quantify the rhinoscopy and clinical signs to see if it was related to the number of treatment needed till complete cure. The scores were not statistically different for patients that required two or more treatments. Side effects were most commonly reported in the first 24 hours and included profuse nasal discharge and sneezing.3 Rare side effects are scarring and occlusion of the nares or frontal sinus entrance and severe bleeding.1,3
Several studies looked at the relationship between CT findings and treatment outcomes. One study found that it was not possible to accurately predict the outcome of the treatment using topical eniconazole based on the severity of the CT lesions using topical eniconazole.9 In comparison, one study found it was possible to inform an owner that their pet would most likely only need one treatment if the score < 8. The maximum score was 24 and was based on the amount of abnormal soft tissue and degree of turbinate destruction.7
The long-term prognosis for SNA is good after successful treatment. Some dogs may experience episodic or permanent mild nasal discharge due to the turbinate loss. The excessive turbinate loss likely predisposes dogs to antibiotic-responsive bacterial rhinitis and about 25% of dogs experience this.4 Another paper felt this was much less common and represented only 1/60 dogs in that article7. One study looked at the long-term outcome of dogs treated with eniconazole infusion. This study found that 50% had mild episodic to permanent nasal signs and 50% were asymptomatic. The reinfection or relapse rate in this study was 3/27 dogs or 11% which occurred at 2, 23 and 36 months post treatment.
In rare instances, sinonasal tumors have been reported within 13 to 30 months. No causal relationship between the tumors and the medications, inflammatory reaction or fungus has been definitive. The human literature has linked chronic sinonasal inflammation to nasal cancers.10 Interestingly, clotrimazole and its vehicle, polyethylene glycol, are reported to be anti-carcinogenic and have low toxicity, so it is likely that a toxin produced by the fungus or the immune system are responsible.10
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References:
1Peeters D, Clercx C. Update on Canine Sinonasal Aspergillosis. Vet Clin Small Anim 2007; 37: 901-916.
2Greene, Craig. Infectious Diseases of the Dog and Cat. City; Saunders, 2006.
3Zonderland J, Stork C, Saunders J, et al. Intranasal infusion of enilconazole for treatment of sinonasal aspergillosis in dogs. J Am Vet Med Assoc 2002; 221: 1421-1425.
4Nelson W, Cook A. When to consider aspergillosis in dogs. Veterinary Medicine 2010.
5Benitah N. Canine Nasal Aspergillosis. Clin Tech Small Anim Pract 2006; 21: 82-88.
6Pomrantz J, Johnson L. Repeated rhinoscopic and serologic assessment of the effectiveness of intranasally administered clotrimazole for the treatment of nasal aspergillosis. J Am Vet Med Assoc 2010; 236: 757-762.
7Mathews K, Davidson A, Koblik P, et al. Comparison of topical administration of clotrimazole through surgically placed versus nonsurgically placed catheters for treatment of nasal aspergillosis. J Am Vet Med Assoc 2004; 225: 1703-1712.
8Saunders J, Clercx C, Snaps F, et al. Radiographic, magnetic resonance imaging, computed tomographic, and rhinoscopic features of nasal aspergillosis in dogs. J Am Vet Med Assoc 1998; 213: 501-506.
9Saunders J, Duchateau L, Storck C, Bree H. Use of computed tomography to predict the outcome of a noninvasive intranasal infusion in dogs with nasal aspergillosis. Can Vet J 2003; 44: 305-311.
10Greci V, Stefanello D, Giancamillo M, Mortellaro C. Sinonasal tumor in 3 dogs after successful topical treatment for frontal sinus aspergillosis. Can Vet J 2009; 50: 11911194.
11Windsor R, Johnson L, Herrgesell, E, De Cock H. Idiopathic lymphoplasmacytic rhinitis in dogs: 37 cases (1997-2002). J Am Vet Med Assoc 2007; 224: 1952-1957.
12Ettinger S, Felman E. Textbook of Veterinary Internal Medicine. St. Louis; Elsevier, 2010.
13Karkowska-Kuleta J, Rapala-Kozik M, Kozik A. Fungi pathogenic to humans: molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Acta Biochimica Polonica 2009; 56: 211-224.
14Barr, S, Rishniw, M. Questions contents of clotrimazole solution: J Am Vet Med Assoc 2010; 236: 163-1644.
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