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Home , Conidiobolomycosis, Conidiobolus coronatus, Fluconazole, Flucytosine, Potassium iodide, Pseudallescheria boydii

CE Article #2

Fungal Infections of the Upper RespiratoryTract

Allison J. Stewart, BVSc(Hons), MS, DACVIM Elizabeth G. Welles, DVM, PhD, DACVP Tricia Salazar, MS, DVM Auburn University

ABSTRACT: Fungal infection of the upper respiratory tract presents diagnostic challenges and therapeutic dilemmas. Diagnosis of upper respiratory tract is based on clinical signs, identification of mass lesions during endoscopic examination, and collection of tissue samples by biopsy or by mucosal scraping. Cytologic or histologic examination of lesions may identify characteristic morphologic features of fungal organisms. The etiologic agent can be definitively identified by microbiologic culture, immunohistochemistry, polymerase chain reaction, or serology.

o retrospective or prospective studies new with good efficacy and reduced have compared various surgical toxicities have become available within the past N(debulking, laser, cryotherapy) and decade. Pharmacokinetic studies on newer medical (topical, intralesional , systemic) treat - drugs are being conducted in horses, and as ment options for fungal in horses. more drugs become available in generic form, Isolated case reports, small case series ,1,2 and a the cost of relatively more efficacious medical limited number of review articles 3 describe therapy will be dramatically reduced. This arti - empirical therapies. A multimodal approach is cle reviews the reported etiologies of , as well as often successful, but the relative benefits of the the diagnostic techniques and the medical and individual treatment components are unknown. surgical therapies for , equine respiratory tract Treatment options depend on the site and fungal granulomas. extent of the infection, accessibility to surgical intervention, the etiologic agent, the known FUNGI susceptibilities, evidence-based study The fungal kingdom comprises , , results from human medicine , and the financial fungal rusts, and mushrooms. Fungi are eukary - resources of the owner. otic organisms with a definitive cell wall made Although results of several small comparative up of chitins, glucans, and mannans. Within the studies on treating guttural pouch have cell wall, the plasma membrane contains ergos - been reported , to date, no terol, a cell membrane sterol that is frequently comparative studies have been targeted by antifungals. regulates the conducted to determine the permeability of the cell membrane and activity •Take CE tests ideal therapeutic plan to treat of membrane -bound enzymes. There are more • See full-text articles mucosal than 70,000 species of fungi, but only 50 CompendiumEquine.com or cryptococcal infections in species have been identified as causes of disease horses. In human medicine, in people or animals. Pathogenic fungi can be

CompENdium EQuiNE 208 may 2008 Fungal infections of the upper Respiratory Tract CE 209

divided into three groups: multinucleate septate fila - For nasal and nasopharyngeal lesions, specimens for mentous fungi, nonseptate filamentous fungi , and yeasts. cytology, histopathology , and culture can be obtained by Dimorphic fungi can change between forms , depending use of an endoscopically guided biopsy instrument. How - on environmental conditions. In soil and decaying mat - ever, these samples tend to be small, superficial , and often ter, the mycelial form usually is present and is composed nondiagnostic. In cultures, mucosal contaminants may of a collection of hyphae. The mycelia produce infective overgrow the organism of interest. Larger biopsy samples spores that can inoculate vertebrate tissue. from the nasal passages or the nasopharynx can often be obtained by use of a uterine biopsy instrument, which can CLINICAL SIGNS AND GROSS LESIONS be passed nasally with visual guidance from a flexible Mycotic granulomas of the upper respiratory tract have endoscope. Excisional biopsy or surgical debulking may be been found in the nasal passages, paranasal sinuses, performed through a sinus flap or via laryngotomy. nasopharynx, guttural pouch, and trachea of infected horses. The most common clinical signs of upper respira - Cytology tory fungal infection include unilateral or bilateral Fungal hyphae may be identified in airway fluid or in serosanguineous or mucopurulent nasal discharge as well impression smears from masses obtained by biopsy.

Fungal diseases of the upper respiratory tract include conidiobolomycosis, , , , pseudallescheriosis , and . as inspiratory or expiratory noise. Other clinical signs Some fungi have characteristic morphologic features that include coughing, facial deformation, and dyspnea can allow an early presumptive identification (Table 1). caused by partial blockage of nasal passages by granulo - matous masses. Fungal plaques in the guttural pouch are Histopathology often located over the arterial blood supply. Horses with Hyphae of certain fungi may be poorly visualized guttural pouch mycosis often have episodic serosan - using routine hematoxylin –eosin staining. Therefore , guineous nasal discharge. This may progress to poten - special stains (e.g., periodic acid-Schiff, Gridley’s fun - tially fatal epistaxis if there is erosion into an artery. The gus , Grocott-Gomori methenamine- ) can duration of clinical signs can vary from days to many be useful in staining histopathologic specimens. In months. Differentials for fungal granulomas of the respi - patients with chronic fungal infections, there is often ratory tract include ethmoidal hematoma, squamous cell evidence of extensive fibrosis on histopathology. carcinoma, amyloidosis, or exuberant granulation tissue. Microbiologic Culture DIAGNOSIS Some fungi have fastidious growth requirements and Diagnostic Samples may take up to several weeks to grow on culture media or Lesions in the nasal passages, turbinates, nasopharynx, may be overgrown by contaminant bacteria. Tissue sam - guttural pouch, trachea, and bronchioles can usually be ples submitted for microbiologic culture should be placed observed directly during endoscopic examination. in a prepared culture media and transported at room Masses in the paranasal sinuses may be observed using temperature. Specific culture media such as Sabouraud’s radiography. Computed tomography or magnetic reso - dextrose agar (Remel, Lenexa, KS), inhibitory agar, nance imaging provides detailed imaging of the equine or Mycobiotic (Remel), which contains cycloheximide skull and can determine the extent of lesions and degree and chloramphenicol, are useful. of bony invasion. A sterile rigid arthroscope or flexible endoscope can be passed through an 8- to 20- mm Molecular Techniques trephine that is drilled into the conchofrontal or maxil - Serologic tests that use immunodiffusion, 4 radioim - lary sinus to directly view some lesions within the munoassays, latex agglutination, complement fixation , or paranasal sinuses. ELISAs 5 are available to detect circulating antigens or may 2008 CompENdium EQuiNE 210 CE Fungal infections of the upper Respiratory Tract

Table 1. Characteristic Morphologic Features of, and Availability of SerologicTests for, Fungal Organisms Reported to Cause Fungal Granulomas in the Equine Upper RespiratoryTract Cost and Agent Cytologic Morphology SerologicTest Laboratory

Cryptococcus Round, thin-walled, -like (5–10 µm) with Capsular antigen ELISA (antigen) $16 at UGA neoformans a large heteropolysaccharide capsule (1–30 µm) that 7 $18 at NMDA does not take up common cytologic stains. The capsule Latex agglutination (antigen) $20 at CSU is best stained with mucicarmine stain. The organisms show narrow-based budding and lack endospores. $30 at UT

Conidiobolus Broad, thin-walled, highly septate, irregularly Immunodiffusion is highly sensitive Serologic coronatus branched hyphae (5–13 µm) 31 ; often surrounded by and specific. A decreasing titer testing is acidophilic-staining, glycoprotein antigen –antibody correlates with disease resolution in not widely complexes known as Splendore-Hoeppli material . horses. 8,9 available.

Pseudallescheria Hyaline, nonpigmented, septate, randomly branched —— boydii hyphae (2–5 µm) with regular hyphal contours. The asexual form has nonbranching conidiophores with terminal conidia. The sexual form in culture has a cleistothecium (large, round body) and . 28

Coccidioides Relatively large, round spherules (20–80 µm; up to Agar gel immunodiffusion (antibody) $8 at NMDA 47,52, a immitis 200 µm) with a double-contoured cell wall. The for IgM and IgG $12 at CSU mature spherules are called sporangiospores (2–5 µm). CF (antibody) 47,51, b: may have some $12 at NMDA a false-positive results $15 at UT

Aspergillus Broad (2–4 µm), septate hyphae with parallel sides Galactomannan enzyme Platelia Aspergillus spp and acute, right-angled branching. immunoassay (sandwich antigen enzyme immunoassay) antigen test; 80.7% immunoassay by sensitive and 89.2% specific; some MDL reactivity to Penicillium , Alternaria and Paecilomyces spp Agar gel immunodiffusion $15 at UT (antibody): A. fumigatus only

Fungal panel — Agar gel immunodiffusion $28 at MDL (Aspergillus , Blastomyces , , and Histoplasma spp) $40 at CSU $55 at CU

aHiggins JC, Leith GS, Voss ED, et al. Seroprevalence of antibodies against in healthy horses. JAVMA 2005;226:1888-1892. bSmith CE, Saito MT, Simons SA. Pattern of 39,500 serologic tests in coccidioidomycosis. JAVMA 1956;160:546-552. CF = Complement fixation. CSU = Colorado State University College of Veterinary Medicine and Biomedical Sciences. Phone 970-297-1281; fax 970-297-0320; www.dlab.colostate.edu. CU = Cornell University Animal Health Diagnostic Center. Phone 607-253-3900; fax 607-253-3943; diagcenter.vet.cornell.edu. MDL = MiraVista Diagnostics Laboratory, Indianapolis, IN. Phone 317-856-2681; fax 317-856-3685; www.miravistalabs.com. NMDA = New Mexico Department of Agriculture Veterinary Diagnostic Services. Phone 505-841-2576; www.nmda.nmsu.edu/animal-and- plant-protection/veterinary-diagnostic-services. UGA = The University of Georgia College of Veterinary Medicine. Phone 706-542-5568; fax 706-542-5233; www.vet.uga.edu/dlab/index.php. UT = University of Tennessee College of Veterinary Medicine. Phone 856-974-5639; fax 865-946-1788; www.vet.utk.edu/diagnostic/ bacteriology.

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antibodies against several fungal organisms (Table 1) . reference method for molds (M38-P), but clinical appli - These tests have been used to assist in the diagnosis of cability is in its infancy. 12 It is likely that in vitro suscep - equine fungal infections caused by Cryptococcus spp ,6,7 tibility testing for yeasts and molds will evolve to allow coronatus, 3,8,9 and Aspergillus spp. 10 Because more selective use of antifungals, but to date, clinicians of cross -reactivity between some related fungal species, should not rely solely on results of these methods to care must be taken in interpreting test results. Many guide therapy. 12 healthy horses have antibodies against Aspergillus spp as a result of environmental exposure . The development of a commercial ELISA with less reactivity is promising. 5,11 Amphotericin B is a polyene antibiotic that combines Serologic testing is especially useful if biopsy samples with ergosterol in the fungal cell membrane and causes are difficult to obtain. an increase in cell permeability. Intravenous ampho - Immunohistochemistry, fluorescent in situ hybridiza - tericin B is one of the most efficacious antifungal thera - tion, and DNA probes can also be used to positively pies but can cause nephrotoxicity and phlebitis. Other diagnose fungal organisms in histopathologic sections. possible adverse effects include , , cardiac , hepatic and renal dysfunction, and hyper - ANTIFUNGAL THERAPEUTICS sensitivity reactions. 13 Reported doses range from 0.3 to During the past decade, there has been significant 0.9 mg/kg (diluted in saline ) IV administered over 1 progress in the development of new antifungals in human hour for approximately 30 days. Topical amphotericin B medicine and in the subsequent empirical use and deter - can also be used .14,15 mination of pharmacokinetic profiles in horses. Although systemic therapy is expensive, it has resulted in an increased incidence of successful treatment of fungal dis - derivatives in the class (e.g., eases in horses. As various antifungals become available in , , , enilconazole, keto - generic form, the cost of antifungal therapy for horses will conazole, , , , become more affordable. New antifungals differ in their ) kill fungi by inhibition of ergosterol mode of activity, toxicity, and propensity to interact with biosynthesis in the fungal cell membrane. other drugs. Therefore, antifungal therapy must be tai - is absorbed poorly in the nonacidified form 16 but can be lored to the etiologic agent, site and extensiveness of acidified for better absorption (30 mg/kg via nasogastric infection, adjunctive therapies, and financial resources. tube q12h mixed with 0.2 normal hydrochloric acid ). 17 Itraconazole (Sporanox; Janssen-Ortho, Toronto In Vitro Sensitivity Testing Ontario, Canada) solution is absorbed well orally For many years , clinically relevant antifungal suscepti - ( : 60%). A dose of 5 mg/kg PO q24h bility testing was nonexistent. Consequently, antifungal maintains concentrations above minimum inhibitory therapy in human medicine was largely empirical and concentration for susceptible yeasts and Aspergillus spp, based on evidence-based medicine rather than guided with no detectable adverse effects. 18 Itraconazole (Spo - by susceptibility data. Selection of antifungal therapy for ranox) suspension is very expensive , and because of the horses was based on recommendations from human lit - large volume required, administration by nasogastric erature, although it was limited by high expense and tube is recommended. The use of compounded itracona - lack of pharmacokinetic information specific to horses. zole is not recommended. Itraconazole is very unstable Currently, the National Committee for Clinical Labora - (requires a low pH) and, owing to its lipophilic nature, is tory Standards (NCCLS) has a published, approved ref - difficult to formulate in aqueous solution. erence method for susceptibility testing for yeasts Oral fluconazole at a of 14 mg/kg , fol - (M27-A). 12 This method is useful for interpreting sus - lowed by 5 mg/kg q24h, yields concentrations in ceptibility data for azole antifungals and in plasma, cerebrospinal fluid, synovial fluid, aqueous human medicine, but interpretation of results for humor, and urine above the minimum inhibitory con - amphotericin B has been problematic. 12 To date, no centration reported for several equine fungal patho- interpretive breakpoints exist for any antifungal agent gens. 19 However, fluconazole reportedly has minimal against or for azoles against any activity against filamentous fungi ( Aspergillus and Fusar - of the molds. The NCCLS has published a proposed ium spp). Low -dose oral fluconazole (1 mg/kg PO may 2008 CompENdium EQuiNE 212 CE Fungal infections of the upper Respiratory Tract

Figure 1. Surgical debulking of a mass via a conchofrontal sinus bone flap in a standing horse.

Access to the frontal sinus through a Significant hemorrhage resulting from Local subcutaneous infiltration with lidocaine. conchofrontal sinus bone flap. surgery to the conchofrontal sinus.

q24h) for at least 10 to 15 days has been anecdotally occur. toxicity is characterized by excessive successful in treating fungal keratitis. Compounded flu - lacrimation, a nonproductive cough, increased respira - conazole formulations are very stable. tory secretions, and dermatitis. 9 The recommended dose Voriconazole, a new broad -spectrum antifungal, of 20% is 20 to 40 mg/kg/day IV for 7 to is preferred for initial treatment of invasive aspergillosis, 10 days. 9,15,29 Orally administered iodide is available in , and cryptococcosis in human patients who two forms: cannot tolerate or have infections that are refractory to Inorganic iodide (10 to 40 mg/kg/day) is 20,21 • other therapeutic agents. A recently published pharma - available only in a chemical grade and is unstable in cokinetic study of voriconazole in horses showed excellent the presence of light, heat, and excessive humidity. 15,29 oral bioavailability and a long half-life. 22 The authors pro - posed a dose of 4 mg/kg PO q24h to achieve plasma con - • Organic ethylenediamine dihydroiodide (EDDI) is centrations greater than 1 µg/mL but recommended that commercially available (0.86 to 1.72 mg/kg of EDDI because of potential accumulation , multiple-dosing studies is equivalent to 20 to 40 mg/kg/day of the 4.57% 29 should be conducted before clinical use in horses .22 dextrose powdered form). Voriconazole is presently very expensive but is likely to Because of the risk for developmental congenital achieve therapeutic concentrations in the brain, retina , and in foals, administration of lungs. 20 Topical miconazole and enilconazole have success - should be avoided in pregnant mares . fully been used to treat guttural pouch mycosis. 23–26 Miconazole is suitable only for topical administration Surgical Therapy because it is toxic when given intravenously. Surgical debulking, endoscopic laser surgery, and cryotherapy can be used as primary treatments or as pre - Systemic Iodide Therapy medical therapy in treating fungal granulomas. Surgical Iodides have very little, if any, in vitro antibiotic effect, debulking can be performed through a conchofrontal or but they seem to have a beneficial effect on the granulo - nasomaxillary sinus flap or through a laryngotomy inci - matous inflammatory process. 27 Although the exact sion. Surgery via a sinus flap can be performed with the mode of action is unknown, in several successfully use of either sedation and local anesthesia in standing treated cases , iodides were used as primary or adjunctive patients (Figure 1) or general anesthesia in patients in therapy for fungal granulomas. 9,28 However, overall , the lateral recumbency (Figure 2). As with all sinonasal sur - efficacy of iodides is considered limited at best. The geries, there is a significant risk for hemorrhage, and the treatment is inexpensive, but toxicity and resistance can need for blood transfusion is not uncommon . If marked

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Figure 2. Surgical debulking via a conchofrontal sinus Figure 3. Endoscopic image of mycotic granulomas flap in a horse under general anesthesia and in lateral caused by C. coronatus in the nasal passages of a horse. recumbency.

hemorrhage is anticipated, a blood transfusion should be passages, the trachea , or the soft palate can be observed prepared for preoperatively. If marked hemorrhage endoscopically (Figure 3). The histologic appearance of occurs , temporary bilateral carotid artery occlusion can conidiobolomycosis is similar to those of and be used (up to 16 minutes) to reduce intraoperative . Basidiobolus spp and P. insidiosum hemorrhage. 30 Direct pressure provided by gauze pack - tend to infect subcutaneous tissue of the limbs and ing is often required for 48 to 72 hours after surgery. trunk, whereas C. coronatus generally infects the mucous Surgery is often combined with local injection of membranes of the upper respiratory tract. Although amphotericin B or adjunctive medical therapy. Treat - nasal pythiosis has occasionally been reported, P. insidio - ment has been unsuccessful in many horses that were sum is not a true fungus but rather is classified among treated for many months by medical and surgical means, the Oomycetes and is discussed elsewhere. 32 Hyphae of thereby necessitating placement of a permanent tra - C. coronatus are thin-walled, highly septate, and irregu - cheostomy as a salvage procedure to prolong life for larly branched (5 to 13 µ m). The lesions typically have breeding or for pasture soundness. large numbers of eosinophils and fewer macrophages, neutrophils, plasma cells , and lymphocytes that surround ETIOLOGIC AGENTS hyphae (Figure 4). The definitive diagnosis is based on Disease Complex microbiologic culture, immunodiffusion, or polymerase Organisms within the phycomycosis (subcutaneous chain reaction .3 Detection of serum antibodies by mass due to fungus or bacteria) disease complex 31 immunodiffusion is considered highly sensitive and spe - include C. coronatus, Basidiobolus spp , and Pythium cific 9 and can be used to monitor response to treatment. insidiosum , which are in the class Zygomycetes and A nested polymerase chain reaction pythiosis assay has order . Fungi within this order are also been used to identify C. coronatus .33 opportunistic. They infect immunocompetent animals Conidiobolomycosis lesions can be treated with surgi - and tend to form nondisseminating subcutaneous cal excision, cryotherapy , or long-term administration of masses. In contrast, other members of Zygomycetes iodides or antifungals. 3 Pharyngeal masses can be (Rhizopus, Mucor , and Absidia spp ) often disseminate to approached via laryngotomy and visualized with a flexi - cause systemic disease. ble endoscope. Surgical debulking followed by intrale - sional injection of amphotericin B (20 mg) via an 8 -inch Conidiobolomycosis spinal needle through the surgical incision, followed by C. coronatus is a saprophytic fungus that causes granu - 10 days of a topical mixture of amphotericin B (20 mg), lomatous lesions of the upper respiratory tract in horses. potassium penicillin (100,000 IU) , and dimethyl sulfox - Single to multiple granulomatous lesions in the nasal ide (30 ml) , was successfully used to treat pharyngeal may 2008 CompENdium EQuiNE 214 CE Fungal infections of the upper Respiratory Tract

Figure 4. Conidiobolomycosis lesions characteristically contain large numbers of eosinophils and fewer numbers of Figure 5. Cytologic smear of a biopsy sample of a nasal macrophages, neutrophils, plasma cells, and lymphocytes. mass from a horse. C. neoformans show their characteristic C. coronatus has broad, thin-walled hyphae ( white arrows ). The hyphae wide, nonstaining capsule ( large arrow ) and narrow-based budding are surrounded by acidophilic-stained glycoprotein antigen–antibody (small arrow ). (modified Wright’s stain; bar = 40 μm) complexes ( black arrows ), which are known as Splendore-Hoeppli material . (Hematoxylin–eosin stain; bar = 40 μm; courtesy of Jennifer Taintor and Joe Newton, Auburn university) could be considered when parenteral treatment is not economically feasible. 3 A vaccine using C. coronatus anti - infection with C. coronatus in one horse .14 There are gen from broth cultures was unsuccessful in treating several other reports of successful treatment of nasopha - seven horses with conidiobolomycosis. 3 Similar ryngeal infection with C. coronatus using topical or immunotherapy has been very successful in treating intralesional amphotericin B .14,15,34,35 It is important to horses with pythiosis. 37 remember that long-term therapy and reevaluation are essential because recurrence is possible. 15 Cryptococcosis A mare with C. coronatus granulomatous tracheitis was Cryptococcosis is commonly caused by C. neoformans successfully treated with iodide therapy (44 mg/kg IV of (var neoformans and var gattii )— a ubiquitous, sapro - 20% sodium iodide for 7 days ; then 1.3 mg/kg PO of phytic, round, basidiomycetous yeast-like fungus (5 to EDDI q12h for 4 months, then q24h for 1 year, then 10 µ m in diameter) with a large heteropolysaccharide once per week). Excessive lacrimation was an occasional capsule (1 to 30 µ m in diameter) that does not take up adverse effect of the iodide but resolved if therapy was common cytologic stains (Figure 5). The capsule, which discontinued for 1 day. 9 forms a clear halo when stained with India ink, is C. coronatus cultured from a nasal mass in one horse immunosuppressive and antiphagocytic. Capsular anti - was found to be resistant in vitro to ketoconazole and gens that are secreted into the host’s body fluids bind itraconazole. 36 Treatment with itraconazole (3 mg/kg opsonizing antibodies before they reach the organism. 1 36 PO q24h for 4 ⁄2 months) was ineffective. Oral flu - Equine cryptococcosis occurs at a relatively high fre - conazole was successful in treating two pregnant mares quency in Western , 6 and there is an epidemio - with nasal conidiobolomycosis. 2 One mare was adminis - logic relationship between C. neoformans var gattii and tered 2 mg/kg PO q12h for 8 weeks ; the other mare was the Australian river red gum tree ( Eucalyptus camaldu - treated at currently recommended dosages (a loading lensis ) as well as between C. neoformans var neoformans dose of 14 mg/kg followed by 5 mg/kg q24h PO) 19 for 6 and avian (particularly pigeon) excreta. 6 Cytologic or weeks. The lesions in the second horse were more exten - histopathologic identification is very reliable for diagno - sive but resolved more rapidly than those in the horse sis because of the characteristic morphology of the treated with the lower dose. 2 Intralesional injection of yeast. Differentiation of various cryptococcal species is azole antifungals, such as miconazole or fluconazole, to possible based on culture characteristics, serologic iden - treat conidiobolomycosis has not been reported but tification, DNA sequencing, or immunohistochemical

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staining. 38 Serologic testing with latex agglutination to identify cryptococcal capsular antigen has been useful in the diagnosis of equine cryptococcosis, with resolution of lesions being correlated with declining serum titers. 7 Cryptococcosis in horses is primarily associated with , rhinitis (Figure 6), , and abortion. Reports of successful medical treatment are rare. 7,39

Pseudallescheriosis boydii is a saprophytic ascomycete (the telomorph is apiospermum ). Infection with P. boydii most commonly involves the extremities ; in human patients , it is known as Madura foot . Hyphae of or Aspergillus spp within tissue cannot be differentiated unless they are cultured. P. boydii cultured Figure 6. Endoscopic image of a mycotic granuloma from the nasal cavity and the sinus of a horse with caused by C. neoformans in the nasal passages of a horse. chronic, malodorous nasal discharge was susceptible in vitro to miconazole, ketoconazole, , and clotrimazole (Figure 7). After debridement and flush - Rhinosporidiosis ing of the plaque, 5 g of 2% miconazole cream was rarely causes polypoid growths infused twice daily for 4 weeks through lavage tubing in the nasal, vaginal , and ocular mucous membranes of that had been passed into the nasal passage through a horses. 44–46 On gross examination, mottled small white hole drilled in the frontal bone and sinuses. Sodium foci may be interspersed throughout the granuloma. 46 iodide was administered systemically for 4 days fol - We are not aware of any reports of treatment of R. see - lowed by for 14 days. The nasal cavity beri in the literature. was free of infection 30 and 60 days after treatment had been initiated. 28 Coccidioidomycosis Nasal mycosis caused by P. boydii has been reported in Coccidioides immitis is a soil saprophyte that grows in two other horses, both of which were euthanized. 40,41 In semiarid areas with sandy, alkaline soils. 47 In the environ - one horse, an attempt was made to curette the nasal ment, C. immitis exists as a mycelium with thick-walled,

Clinical signs of fungal diseases include nasal discharge, respiratory noise, facial distortion, or airway obstruction with upper respiratory tract granulomas. Guttural pouch mycosis may cause intermittent serosanguineous nasal discharge or epistaxis. plaques, which was followed by topical treatment via a barrel -shaped arthroconidia. Inhaled arthroconidia can surgically placed drain that was flushed daily with dilute enlarge to form nonbudding spherules, which incite an povidone - for 10 days, followed by natamycin inflammatory reaction. Most infections are pulmonary or solution for 2 weeks. No improvement was noted , and systemic and have been reviewed elsewhere. 47 Localized, the horse was euthanized. 41 P. boydii has also been iso - recurring nasal granulomas have also been reported. 48–50 lated from the of two of 60 healthy donkeys and Nasal masses can be very slowly progressive. One horse uterine fluid from horses with chronic uterine infec - developed a coccidioidal ethmoid mass 6 years after tion. 42,43 Miconazole, itraconazole, ketaconazole , and moving from to a nonendemic area. 48 The mass voriconazole are active in vitro against P. boydii , which was surgically removed ; 5 years later , the clinical signs generally responds poorly to amphotericin B. Topical recurred and the horse was euthanized because of the miconazole is effective for superficial lesions. 41 extensiveness of the lesions .49 may 2008 CompENdium EQuiNE 216 CE Fungal infections of the upper Respiratory Tract

Figure 7. Nasal and nasopharyngeal masses due to P. boydii in a quarter horse gelding with chronic, malodorous nasal discharge. (Courtesy of R. Reid Hanson, Auburn university)

mucosanguineous nasal discharge and the most rostral P. boydii fungal plaque as observed by Adhesions caused by P. boydii in the mass within the external nares. endoscopic examination. nasopharynx as observed by endoscopic examination.

C. immitis is difficult to culture. However, serology is very useful in diagnosing infection, and decreasing titers are associated with clinical improvement. 47 Oral itra - conazole and fluconazole have been used to treat osseous and pulmonary coccidioidomycosis. 51,52 Nasopharyngeal lesions can be treated surgically or medically, and long- term reevaluation is recommended.

Aspergillosis Aspergillosis in horses has been reviewed. 11 Aspergillus spp have broad (2 to 4 µ m in diameter) , septate hyphae with parallel sides and acute right -angled branching. They have a propensity for vascular invasion. A definitive diagnosis can be made by culture or staining using immunohistochem - istry or immunofluorescence (Fusarium spp and P. boydii 53,54 appear similar on histologic examination ). Serologic Figure 8. Endoscopic image of guttural pouch mycosis. 5,11 diagnosis has occasionally been useful but is often unreli - (Courtesy of John Schumacher, Auburn university) able. Aspergillus spp are very common in the environment, especially in moldy feed and bedding. 5 Aspergillus spp are the most common cause of guttural pouch mycosis (see below) and can also cause plaques in the nasopharynx. 35 cessfully with natamycin solution via an indwelling In two horses, mycotic rhinitis was treated successfully facial catheter in the caudal maxillary sinus. 1 with itraconazole (3 mg/kg PO q24h for 120 days, which achieved concentrations that exceeded the mini - Guttural Pouch Mycosis mum inhibitory concentration for the organism) .36 There are several excellent, detailed reviews of gut - Additional successful treatments in two horses included tural pouch mycosis. 11,55,56 Mycotic infection of the gut - topical natamycin (25 mg in 100 ml of saline) flushed tural pouch generally occurs on the dorsal wall of the via an endoscope as well as powder insufflated medial compartment or on the lateral wall of the lateral up the affected nostril. Another horse was treated suc - compartment (Figure 8). Mycotic ulcerations are typi -

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Figure 10. Photomicrograph of a histologic section showing Aspergillus spp in a fatal case of guttural pouch mycosis. The hyphae of Aspergillus spp are septate and show dichotomous branching. (Hematoxylin–eosin stain; bar = 30 μm; courtesy of Arno Wünschmann, university of minnesota)

of laryngeal hemiplegia, dysphagia, facial paralysis , or Figure 9. Fatal guttural pouch mycosis in a quarter horse mare that had a 2-week history of intermittent, Horner’s syndrome. unilateral epistaxis. The mare had been referred for arterial Guttural pouch mycosis is most often caused by coil embolization. An acute, profuse hemorrhagic episode occurred, and the mare died in 7 minutes. cally located over the internal carotid artery and less commonly located over the external carotid artery or maxillary artery. Plaques can vary in size and can expand to cover the entire roof of the pouch. Infection may erode through the median septum into the neighboring pouch, causing bilateral infection. Horses generally have intermittent mucopurulent to serosanguineous nasal discharge or epistaxis. 56 Erosion of the internal carotid artery or , less commonly , the external carotid artery or maxillary artery can result in fatal hemorrhage (Figure 9). If guttural pouch mycosis associated with epistaxis is untreated, 50% of affected horses will die .55 Bouts of epistaxis are unpredictable, but several usually occur before a fatal bleeding episode. Endoscopy or placement of lavage catheters carries a significant risk for dislodging fungal plaques or previ - ously formed clots, which can result in further hemor - rhage. Secondary neurologic abnormalities may result if thrombi from the internal carotid artery dislodge and embolize to the brain, causing subsequent cerebral dys - function. Inflammation can affect cranial nerves that traverse the guttural pouch, leading to the development may 2008 CompENdium EQuiNE 218 CE Fungal infections of the upper Respiratory Tract

Aspergillus spp, particularly A. nidulans (Figure 10). is possible, and new etiologies will likely be reported in Infections with Mucor , Penicillium , Paecilomyces , the future. Determination of the specific fungal agent , Rhizopus , and Alternaria spp have also influences the choice of the antifungal in medical man - been reported . Surgical arterial occlusion by either bal - agement but may be irrelevant in surgical intervention. loon catheter technique or transarterial coil is the pre - Drugs available for systemic therapy are becoming more ferred treatment for guttural pouch mycosis; there is affordable (especially fluconazole), but it has not been little evidence for the necessity of adjunctive medical determined whether medical therapy with a sole agent is therapy 56,57 (e.g., oral or topical thiabendazole; topical more or less efficacious or cost effective than are combi - nystatin, natamycin, miconazole, or enilconazole ; oral or nations of surgical debulking, intralesional injection , systemic iodides ). and/or the use of systemic antifungals. If surgical options are not available, topical medica - tions can be applied via an indwelling catheter (a tem - REFERENCES porary Chamber’s catheter or long -term commercial 1. Greet TRC. Nasal aspergillosis in 3 horses. Vet Rec 1981;109:487-489. guttural pouch catheter) that is advanced through the 2. Taintor J, Crowe C, Hancock S, et al. Treatment of conidiobolomycosis with fluconazole in two pregnant mares. J Vet Intern Med 2004;18:363-364. guttural pouch opening. may also be 3. Taintor J, Schumacher J, Newton J. Conidiobolomycosis in horses. Compend sprayed directly on the lesion through the biopsy portal Contin Educ Pract Vet 2003;25:872-876. of a flexible endoscope. The dorsal location of the 4. Rezabek GB, Donahue JM, Giles RC, et al. in horses. J Comp lesions makes treatment difficult , and there is always the Pathol 1993;109:47-55. risk for fatal hemorrhage. 5. Guillot J, Sarfati J, de Barros M, et al. Comparative study of serological tests for the diagnosis of equine aspergillosis. Vet Rec 1999;145:348-349. Intranasal 2% miconazole has been used in the resolu - 6. Riley CB, Bolton JR, Mills JN, et al. Cryptococcosis in 7 horses. Aust Vet J 23 tion of four cases of guttural pouch mycosis. In two 1992;69:135-139. cases, 70 mg of injectable miconazole solution was 7. Begg LM, Hughes KJ, Kessell A, et al. Successful treatment of cryptococcal diluted in 10 mL of normal saline ; in two other cases , pneumonia in a pony mare. Aust Vet J 2004;82:686-692. 400 mg of warmed gynecologic miconazole cream was 8. Kaufman L, Mendoza L, Standard PG. Immunodiffusion test for serodiag - nosing subcutaneous . J Clin Microbiol 1990;28:1887-1890. infused into the guttural pouch once daily for 1 week, 9. Steiger RR, Williams MA. Granulomatous tracheitis caused by Conidiobolus then every other day for 2 weeks. Treatment using the coronatus in a horse. J Vet Intern Med 2000;14:311-314. gynecologic cream was continued twice per week for an 10. Moore BR, Reed SM, Kowalski JJ, et al. Aspergillosis granuloma in the additional 3 weeks. All horses recovered uneventfully .23 mediastinum of a nonimmunocompromised horse. Cornell Vet 1993;83:97- 104. Although enilconazole is not available in the United 11. Blomme E, Del Piero F, La Perle KMD, et al. Aspergillosis in horses: a States, it has been used topically as a 0.9% or 1.7% solu - review. Equine Vet Educ 1998;10:86-93. tion to successfully treat four cases of guttural pouch 12. Gubbins PO. Fungal infections . In: Fink MP, Abraham E, Vincent J-L , mycosis caused by Rhizopus , Aspergillus , Penicillium , Kochanek PM, eds. Textbook of Critical Care . 5th ed. Philadelphia: Elsevier Mucor , Chrysosporium, and Alternaria spp. 25,26 Saunders; 2005;1345-1357. 13. Chandna VK, Morris E, Gliatto JM, et al. Localized subcutaneous cryptococ - Systemic treatment of guttural pouch mycosis has cal granuloma in a horse. Equine Vet J 1993;25:166-168. also been successful using itraconazole and topical 14. French DD, Haynes PF, Miller RI. Surgical and medical management of 24 enilconazole. Itraconazole (5 mg/kg PO for 3 weeks) rhinophycomycosis (conidiobolomycosis) in a horse. JAVMA 1985;186:1105- was administered , and topical enilconazole (60 mL) 1107. was later sprayed directly onto the mycotic lesion using 15. Zamos DT, Schumacher J, Loy JK. Nasopharyngeal conidiobolomycosis in a horse. JAVMA 1996;208:100-101. endoscopic guidance for 2 weeks. 24 Medical therapy 16. Prades M, Brown MP, Gronwell R, et al. Body fluid and endometrial concen - may be a more economically feasible treatment option trations of ketaconazole in mares after intravenous injection or repeated gav - than surgical arterial occlusion and can be considered age. Equine Vet J 1989;21:211-214. when neurologic deficits are present in the absence of 17. Nappert G, VanDyck T, Papich M, et al. Successful treatment of a fever asso - epistaxis. ciated with consistent pulmonary isolation of Scopulariopsis sp in a mare. Equine Vet J 1996;28:421-424. 18. Clode AB, Davis JL, Salmon J, et al. Evaluation of concentration of voricona - CONCLUSION zole in aqueous humor after topical and oral administration in horses. Am J Conidiobolomycosis, aspergillosis, cryptococcosis, Vet Res 2006;67(2):296-301. coccidioidomycosis , and pseudallescheriosis are the most 19. Latimer FG, Colitz CMH, Campbell NB, et al. of flu - conazole following intravenous and oral administration and body fluid con - commonly reported causes of fungal granulomas in the centrations of fluconazole following repeated oral dosing in horses. Am J Vet equine respiratory tract. Infection with other organisms Res 2001;62:1606-1611.

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20. Jeu L, Piacenti FJ, Lyakhovetskiy AG, et al. Voriconazole. Clin Ther 46. Blackford J. Superficial and deep mycoses in horses. Vet Clin North Am Equine 2003;25:1321-1381. Pract 1984;6:47-58. 21. Fleming RV, Walsh TJ, Anaissie EJ. Emerging and less common fungal 47. Ziemer EL, Pappagianis D, Madigan JE, et al. Coccidioidomycosis in horses : . Infect Dis Clin North Am 2002;16:915-933. 15 cases (1975-1984). JAVMA 1992;201:910-916. 22. Davis JL, Salmon JH, Papich MG. Pharmacokinetics of voriconazole after 48. Reed SM, Boles CL, Dade AW, et al. Localized equine nasal coccid - oral and intravenous administration to horses. Am J Vet Res 2006;67:170-175. ioidomyces granuloma. J Equine Med Surg 1979;3:119-123. 23. Giraudet AJ. Medical treatment with miconazole in four cases of guttural pouch mycosis. J Vet Intern Med 2005;19:485. 49. Hodgin EC, Conaway H, Ortenburger AI. Recurrence of obstructive nasal coccidioidal granuloma in a horse. JAVMA 1984;184:339-340. 24. Davis EW, Legendre AM. Successful treatment of guttural pouch mycosis with itraconazole and topical enilconazole in a horse. J Vet Intern Med 50. Pappagianis D. Epidemiology of coccidioidomycosis . In: McGinnis MR, ed. 1994;8:304-305. Current Topics in Medical Mycology . New York: Springer-Verlag ; 1988:199- 25. Vannieuwstadt RA, Kalsbeek HC. Guttural pouch mycosis : local treatment 238. with an indwelling through-the-nose-catheter with enilconazole . Tijdschr 51. Foley JP, Legendre AM. Treatment of coccidioidomycosis osteomyelitis with Diergeneesk 1994;119:3-5. itraconazole in a horse: a brief report. J Vet Intern Med 1992;6:333. 26. Carmalt JL, Baptiste KE. Atypical guttural pouch mycosis in three horses. 52. Higgins JC, Leith GS, Pappagianis D, et al. Successful treatment of Coccid - Pferdeheilkunde 2004;20:542. ioides immitis pneumonia with fluconazole in two horses. J Vet Intern Med 27. Plumb D. Veterinary Drug Handbook . 4 th ed. Ames: Iowa State University 2005;19:482-483. Press; 2002. 53. Tunev SS, Ehrhart EJ, Jensen HE, et al. Necrotizing mycotic vasculitis with 28 . Davis PR, Meyer GA, Hanson RR, et al. infection of cerebral infarction caused by Aspergillus niger in a horse with acute typhlocoli - the nasal cavity of a horse. JAVMA 2000;217:707-709. tis. Vet Pathol 1999;36:347-351. 29 . Scott DW, Miller WH. Fungal skin diseases . In: Scott DW, ed. Equine Der - matology . Philadelphia: WB Saunders ; 2003:311-312. 54. Thirion-Delalande C, Guillot J, Jessen HE, et al. Disseminated acute con - comitant aspergillosis and in a pony. J Vet Med 2005;52:121- 30 . Embertson RM. Upper airway conditions in older horses, broodmares and 125. stallions. Vet Clin North Am Equine Pract 1991;7:149-164. 55. Hardy J, Leveille R. Diseases of the guttural pouches. Vet Clin North Am 31 . Miller RI, Campbell RSF. The comparative pathology of equine cutaneous Equine Pract 2003;19:123-158. phycomycosis. Vet Pathol 1984;21:325-332. 32 . Chaffin MK, Schumacher J, McMullan WC. Cutaneous pythiosis in the 56. Lepage OM, Perron MF, Cadore JL. The mystery of fungal infection in the horse. Vet Clin North Am Equine Pract 1995;11:91-103. guttural pouches. Vet J 2004;168:60-64. 33 . Grooters AM, Leise BS, Lopez MK, et al. Development and evaluation of an 57. Leveille R, Hardy J, Robertson JT, et al. Transarterial coil embolization of the enzyme-linked immunosorbent assay for the serodiagnosis of pythiosis in internal and external carotid and maxillary arteries for prevention of hemor - dogs. J Vet Intern Med 2002;16:142-146. rhage from guttural pouch mycosis in horses. Vet Surg 2000;29:389-397. 34 . McMullan WC, Joyce JR, Hanselka DV, et al. Amphotericin-B for treatment of localized subcutaneous phycomycosis in horses. JAVMA 1977;170:1293- 1298. 35 . Hanselka DV. Equine nasal phycomycosis. Vet Med Small Anim Clin ARTICLE #2 CE TEST 1977;72:251-253. This article qualifies for 2 contact hours of continuing CE 36 . Korenek NL, Legendre AM, Andrews FM. Treatment of mycotic rhinitis with itraconazole in three horses. J Vet Intern Med 1994;8:224-227. education credit from the Auburn University College of Veterinary Medicine. Subscribers may take individual 37 . Newton J, Schumacher J. Pythiosis . In: Robinson NM, ed. Equine Current Therapy . 4th ed. Philadelphia: WB Saunders; 1997:396-397. CE tests or sign up for our annual CE program . 38 . Lester SJ, Kowalewich NJ, Bartlett KH, et al. Clinicopathologic features of an Those who wish to apply this credit to fulfill state relicensure unusual outbreak of cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases requirements should consult their respective state (January to July 2003). JAVMA 2004;225:1716-1722. authorities regarding the applicability of this program. 39 . Boulton CH, Williamson L. Cryptococcal granuloma associated with jejunal CE subscribers can take CE tests online and get real-time intussusception in a horse. Equine Vet J 1984;16:548-551. scores at CompendiumEquine.com . 40. Johnson GR, Schiefer B, Pantekoek JFCA. Maduromycosis in a horse in Western Canada. Can Vet J 1975;16:341-344. 41. Brearley JC, McCandlish IAP, Sullivan M, et al. Nasal granuloma caused by 1. Which statement about fungal cell structure is Pseudallescheria boydii . Equine Vet J 1986;18:151-153. incorrect ? 42. El-Allawy T, Atria M, Amer A. Mycoflora of the pharyngeotonsillar portions a. Fungal cell walls are composed of chitins, glucans , and of clinically healthy donkeys in Assuit. Assuit Med Vet 1977;4:63-69. mannans . 43. Carter ME, Menna ME. Petriellidium boydii from the reproductive tracts in b. Fungal cell membranes contain ergosterol . mares. N Z Vet J 1975;23:13. c. Fungi are prokaryotes , as are bacteria . 44. Myers DD, Simon J, Case MT. Rhinosporidiosis in a horse. JAVMA 1964;145:345-347. d. Fungi growing in decaying matter are usually present in the mycelial form. 45. Lengfelder KD, Pospischil A. Rhinosporidiosis in the horse : histological and electron microscopic study of a case. Zentralbl Veterinarmed B 1980;27:326- e. dimorphic fungi can change between forms , depend - 339. ing on environmental conditions . may 2008 CompENdium EQuiNE 220 CE Fungal infections of the upper Respiratory Tract

2. Which statement about guttural pouch mycosis d. Successful medical treatment of cryptococcosis has is incorrect ? never been reported in horses . a. Horses usually present with episodic mucopurulent e. C. neoformans has a large heteropolysaccharide cap - to serosanguineous nasal discharge . sule that is immunosuppressive and antiphagocytic . b. Fatal epistaxis can occur if there is erosion into an artery . 6. An epidemiologic association among the Aus - c. The most common cause is Aspergillus spp. tralian river red gum tree, bird droppings , and d. Fungal plaques frequently involve the retropharyngeal ______has been described . lymph nodes on the ventral wall of the medial com - a. Alternaria spp partment of the guttural pouch . b. Aspergillus spp e. Cranial nerve dysfunction can lead to the develop - c. C. coronatus ment of laryngeal hemiplegia, dysphagia, facial paraly - d. C. neoformans sis, or Horner syndrome. e. P. boydii

7. Guttural pouch mycosis is most often caused by 3. Which statement about antifungals is correct ? infection with a. Amphotericin B is an azole antifungal . a. Aspergillus spp . b. intravenous amphotericin B can be nephrotoxic . b. C. neoformans. c. The use of compounded itraconazole is recom - c. Alternaria spp . mended to reduce costs of therapy . d. P. boydii . d. miconazole is an inexpensive, safe , and effective intra - e. C. coronatus . venous antifungal . e. Ketoconazole has good oral bioavailability . 8. The preferred surgical treatment of guttural pouch mycosis is 4. Which statement about infection with C. corona - a. arterial occlusion by balloon catheter or transarterial tus is incorrect ? coil . a. C. coronatus is a round, basidiomycetous yeast-like b. curettage viaViborg’s triangle . fungus with a large heteropolysaccharide capsule. c. stylohyoid ostectomy . b. Hyphae of C. coronatus are thin-walled, highly septate, d. jugular vein occlusion . and irregularly branched. e. trephination via the nasomaxillary sinus into the pouch as well as topical therapy . c. The lesions typically have large numbers of eosinophils and fewer macrophages, neutrophils, plasma cells, and lymphocytes surrounding the hyphae. 9. Aspergillus spp typically have d. infection generally results in granulomatous lesions in a. thin-walled, highly septate hyphae with irregular the nasal passages, trachea, or soft palate. branching . e. Successful resolution has been obtained with intrale - b. broad , septate hyphae with parallel sides and acute , sional amphotericin B, oral fluconazole , and oral itra - right -angled branching . conazole . c. narrow , nonseptate hyphae with tapering sides. d. round , yeast-like cells with a large heteropolysaccha - ride capsule . 5. Which statement about C. neoformans is incor - e. nonencapsulated cells with broad-based budding . rect ? a. Cytologic or histopathologic identification is very 10. Which azole antifungal is toxic if administered reliable for diagnosis because of characteristic mor - systemically? phology . a. itraconazole b. Resolution of cryptococcal lesions is correlated with b. fluconazole decreasing serum titers. c. ketaconazole c. Cryptococcosis in horses is primarily associated with d. miconazole pneumonia, rhinitis, meningitis , and abortion . e. voriconazole

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