Sinonasal aspergillosis in dogs: a review

Abstract

Sinonasal aspergillosis is an uncommon, yet debilitating and often frustrating condition to treat in dogs despite years of research evaluating pathogenesis, diagnosis and treatment. The disease is most commonly caused by non-invasive fungal infection, thought to be secondary to altered innate and/or adaptive immune responses. Attempts to confirm this have however failed. A variety of conflicting opinions regarding the diagnosis and treatment of sinonasal aspergillosis exist. Often the use of a particular treatment protocol is based upon personal or regional preference. Evaluation of the veterinary literature demonstrates that the evidence base in support of individual treatment recommendations is weak. A number of recent publications have helped to expand the current knowledge base and therefore our understanding of important practicalities for both diagnostic options and treatment protocols. The following review examines the current evidence for the pathogenesis of sinonasal aspergillosis in dogs, as well as the various diagnostic options. The available evidence for frequently utilised -therapeutic options and their likely outcomes is also explored.

Infection And Immunity

Fungal infections of the nasal cavity are relatively uncommon in dogs (Sharp and others 1991b, Tasker and others 1999, Peeters and Clercx 2007, Meler and others 2008). A variety of infections are reported in the veterinary literature with Aspergillus spp., particularly Aspergillus fumigatus, most common (Peeters and Clercx 2007). These ubiquitous soil saphrophytes play important roles in environmental recycling, sporulating abundantly to produce vast quantities of conidia which are distributed via air currents (Latge 1999). Conidia are small enough (2 to 3 µm) to reach pulmonary alveoli and exposure occurs with inhalation, but disease does not develop in every individual (Latge 1999).

The capacity of fungal elements to result in infection may depend upon both host immunocompetence and virulence factors associated with the fungal organism. Trapping and removal of inhaled fungal elements by the respiratory tract mucociliary defences usually prevent further access. Where this fails, additional innate immune system mechanisms are employed, including the alternative complement system, phagocytic cells (neutrophils, macrophages and dendritic cells), natural killer and γδ T-cells which work to destroy pathogens intracellularly or by secretion of compounds extracellularly (Romani 2004, Shoham and Levitz 2005). Reduced mucociliary clearance, decreased phagocytic cell numbers or impairment in their capacity to destroy organisms can cause infection.

Stimulation of phagocytic cell surface receptors, particularly toll-like receptor (TLR)-4, by Aspergillus spp. conidia results in macrophage production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-1α and IL-1β. Hyphae result in IL-10 production via TLR-2-mediated pathways (Shoham and Levitz 2005). These factors initiate pathways that may counteract host protective mechanisms. Importantly, by processing and presenting antigen and aiding inflammatory mediator production, the innate immune system instructs the adaptive immune system which provides long-term protection (Romani 2004, Shoham and Levitz 2005). Dendritic cells play an important linking role by capturing and processing fungal antigen, by expressing lymphocyte co-stimulatory molecules and by migrating to lymphoid organs to secrete cytokines and initiate adaptive immune responses.

The array of cytokines released determines protection or susceptibility to infection by resulting in differentiation of CD4+ T-lymphocytes to either a T-helper (Th)-1 or Th-2 cell response (Shoham and Levitz 2005). Interferon (IFN)-γ, IL-6, TNF-α and IL-12 result in a Th-1 response, while a Th-2 response is seen when IL-4 and IL-10 predominate (Shoham and Levitz 2005). Immunological studies demonstrate that Th-1 biased responses convey protection with only mild or asymptomatic infection occurring, whilst Th-2 biased responses result in severe or allergic disease. Normally these responses are balanced to aid elimination of infectious agents, whilst limiting autoimmune injury (Romani 2004, Shoham and Levitz 2005).

Organism Virulence Factors

Fungal metabolites and secretory products produced by individual Aspergillus spp. may convey survival advantages by suppressing localised host immune factors or allowing immune system evasion. Aspergillus fumigatus possesses various fungal toxins and metabolites that may reduce mucociliary function. Gliotoxin has been implicated, although fumagillin and helvolic acid, amongst others, have similar effects (Tomee and Kauffman 2000). Reduced mucociliary clearance provides an opportunity for fungal elements to reach epithelial surfaces, resulting in further damage and potentially invasion. Invasion may be enhanced by improved adherence of fungal elements to host tissues via extracellular matrix and serum proteins including laminin, fibronectin, collagen, fibrinogen and complement component C3 (Tomee and Kauffman 2000).

In addition, fungal metabolites impair phagocytic functions that would normally destroy conidial and hyphal forms (Tomee and Kauffman 2000). Gliotoxin reduces adherence and phagocytosis of fungal elements, while aflatoxins affect phagocytosis, intracellular killing and spontaneous superoxide production. Complement binding and activation of bound opsonins, which normally enhance phagocytosis, are affected too, reducing susceptibility to destruction (Tomee and Kauffman 2000).

Localised inhibition of host cell growth and subsequently enhanced fungal growth may also be facilitated by toxins. Again gliotoxin is largely implicated, reducing T-cell proliferation and activation of cytolytic T-lymphocytes, blood monocytes, fibroblasts and other cells (Tomee and Kauffman 2000). Toxins such as ribotoxins inhibit protein synthesis and are highly toxic to eukaryotic cells (Tomee and Kauffman 2000).

Mycotic Rhinosinusitis In Humans – Parallels With Canine Disease

In humans, fungal infections are grouped into those affecting immunocompromised or immunocompetent patients (Hamilos and Lund 2004). Immunocompetent patients are typically diagnosed with non-invasive infection such as allergic rhinosinusitis/bronchopulmonary disease or chronic erosive rhinosinusitis (Uri and others 2003, Barnes and Marr 2006). Granuloma formation has been reported in chronically obstructed paranasal sinuses (Latge 1999, Barnes and Marr 2006, Day 2009). Invasive fungal sinusitis, pulmonary infection and disseminated systemic fungal disease are more likely in immunocompromised patients (Latge 1999, Barnes and Marr 2006, Day 2009).

Canine Mycotic Rhinosinusitis

In previous studies of dogs with chronic nasal disease, mycotic rhinosinusitis occurred with a frequency of 7 to 34% (Sharp and others 1991b). Aspergillus fumigatus is most frequently isolated, although various other species including A. Niger, A. nidulans and A. flavus have been reported. Penicillium spp. and other fungal species are rare causes (Sharman and others 2010).

Marked nasal turbinate destruction is invariably identified, with extension of disease into periorbital soft tissue and destruction of the cribriform plate in severe cases. Destruction is attributed to host inflammatory responses and dermonecrolytic fungal toxins, rather than direct action of the fungal organisms involved as histopathologic studies suggest that infection is non-invasive in dogs (Peeters and others 2005). This differs from sinonasal aspergillosis (SNA) in cats where progression to periorbital disease is frequently reported and is more challenging to treat (Barrs and others 2012).

The pathogenesis of non-invasive fungal infections in dogs is poorly understood. Defects of innate and adaptive immune mechanisms are not typically observed in SNA (Romani 2004, Shoham and Levitz 2005, Peeters and others 2006). Concurrent disease or systemic immunodeficiency is not typically recognised; however, facial trauma, nasal foreign bodies and dental disease are occasionally implicated (Sharp and others 1991b, Peeters and Clercx 2007, Day 2009).

Limited evaluation of adaptive immunity has occurred in dogs (Peeters and others 2006, 2007). Cytokine profiling identifies significant up-regulation of mRNA for cytokines predominantly associated with a Th-1 response compared with normal controls or dogs with lymphoplasmacytic rhinitis (Peeters and others 2006, 2007, Vanherberghen and others 2012). Up-regulation of IL-8, IL-18 and TNF-α in SNA may promote phagocytic killing and recruitment of immune cells (Peeters and others 2006). Local immune responses may therefore be adequate to prevent invasion and dissemination, but insufficient to prevent inflammation. Up-regulation of IL-10 and TGF-β may relate to failure to clear infection, despite significant inflammation (Peeters and others 2007). Additional evaluation is required, before adjunctive immunotherapeutics can be recommended.

Signalment And Clinical Course

Mesocephalic and dolicocephalic dogs are most commonly diagnosed with SNA. Specific breed predispositions are not observed and brachycephalic breeds can be affected (Sharp and others 1991b, Zonderland and others 2002). Affected dogs are generally young to middle-aged with very young or very old dogs occasionally reported. A male predisposition is not consistently supported (Sharp and others 1991b, Zonderland and others 2002, Johnson and others 2006).

Nasal signs may be present for weeks to months or even years with chronic mucopurulent to purulent nasal discharge, nasal pain and nasal planum ulceration/depigmentation most commonly reported (Fig 1). Sneezing, epistaxis, decreased appetite and signs of depression may be observed. Epistaxis may be so severe that life-threatening anaemia develops requiring blood transfusion. Additionally in severe disease facial deformity, epiphora and seizures may be identified.

Figure 1. Mucopurulent nasal discharge and nasal depigmentation in a dog receiving treatment for sinonasal aspergillosis with enilconazole via indwelling catheters in the frontal sinuses and nasal cavity. Image courtesy of Associate Professor Peter Irwin, Murdoch University, Western Australia

Diagnostics

Although clinical findings and course of disease may increase suspicion for SNA, a combination of diagnostics encompassing diagnostic imaging [computed tomography (CT) or radiography], rhinoscopy/sinuscopy, histopathology, cytology, fungal culture and serology are recommended for definitive diagnosis. Other common causes of chronic nasal disease including neoplasia, nasal foreign bodies, rhinitis secondary to dental disease and idiopathic lymphoplasmacytic rhinitis should also be considered and excluded.

Diagnostic Imaging

Radiographic features of SNA are well described and detectable in most cases (Sullivan and others 1986). Accurate head positioning is imperative for diagnosis. Radiographs should include dorsoventral and lateral views of the skull, intraoral views of the nasal cavities and maxilla as well as a rostrocaudal view of the frontal sinuses. Intra-oral, dorsoventral and rostrocaudal views of the frontal sinuses allow for evaluation of nasal cavity symmetry and frontal sinus involvement (Figs 2 and 3) (Sullivan and others 1986).

Figure 2. Open mouth, ventrodorsal projection of the left and right nasal cavities showing a patchy increase in opacity within the mid to caudal left and right nasal cavities. An increased lucency is also present within the mid right nasal cavity indicating turbinate lysis

Figure 3. Rostrocaudal projection of the frontal sinuses showing an increased opacity within the left frontal sinus

Turbinate destruction within the nasal cavity is evident as wide-spread punctuate lucencies or a generally increased radiolucency. Mixed-density patterns or an overall increase in opacity may be seen with accumulation of fungal plaques, debris or discharge. Rostrocaudal views of the sinuses are essential to avoid misdiagnosis. Soft tissue density, hyperostosis and punctuate lucencies may be seen within the frontal bones on these views (Sullivan and others 1986).

CT improves sensitivity (88 to 92%) compared with radiographs (72 to 84%) (Saunders and van Bree 2003). Cavitary destruction of the turbinates (Fig 4), mucosal thickening and thickened and reactive maxillary, vomer and frontal bones may be evident using CT, particularly with subtle or unilateral changes (Fig 5) (Saunders and others 2002, Saunders and van Bree 2003). CT is more sensitive for cribriform plate involvement (Saunders and others 2002, Saunders and van Bree 2003).

Figure 4. Destruction of the nasal turbinates identified on computed tomography (CT) of the nasal cavity. Image courtesy of Murdoch University Veterinary Hospital, Western Australia

Figure 5. Marked hyperostosis of the frontal bone associated with severe involvement of the right frontal sinus, as identified using computed tomography (CT). Image courtesy of Murdoch University Veterinary Hospital, Western Australia

Magnetic resonance imaging (MRI) in canine SNA is considered more sensitive than CT for soft tissue change, whilst the opposite is true for evaluation of hyperostosis and lysis within the bones surrounding the nasal cavity. Despite this there is no demonstrable difference between CT and MRI for diagnosing nasal cavity mycoses (Saunders and others 2004).

Rhinoscopy And Sinuscopy

Direct visualisation with rhinoscopy may demonstrate turbinate destruction, mucoid nasal discharge and fungal plaques (Fig 6). It is important to remember that at least one study showed that 8 of 46 dogs (17%) had disease confined to the frontal sinuses that was only identified with sinuscopy (Johnson and others 2006). Sinus trephination may therefore be advantageous where nasal cavity disease is minimal to confirm fungal disease and additionally provides access for debridement of fungal plaques before treatment. In some cases significant nasal turbinate destruction allows direct sinus access by endoscopy via the nasal cavity without trephination.

Figure 6. Severe nasal turbinate destruction and fungal plaques within the nasal cavity of a dog affected by sinonasal aspergillosis. Image courtesy of Associate Professor Vanessa Barrs, University of Sydney Veterinary Teaching Hospital, New South Wales, Australia

Cytology And Histopathology Of Nasal Specimens

Cytology of nasal discharge is considered to have poor diagnostic accuracy in mycotic nasal disease. Fungal organisms found on nasal cytology could reflect normal nasal cavity colonisation or be present secondary to any chronic nasal cavity disease that reduces mucociliary clearance. Comparison of direct smears of nasal discharge, blind endonasal swabs, mucosal brushings of suspected lesions under endoscopic guidance and squash preparations from nasal biopsies collected under endoscopic guidance show the greatest accuracy for samples collected under direct visual guidance. Fungal elements were detected in 14 of 15 (93·3%) of cases using mucosal brushings and 15 of 15 (100%) using squash preparations of nasal biopsies. Detection rates were poorest with samples prepared from nasal discharge which identified fungal elements in two cases only (13·3%) (De Lorenzi and others 2006).

Histopathological examination is generally accepted as being more accurate (Fig 7), although in one histological study of 15 dogs with SNA, fungal elements were demonstrated in only 6 cases (40%). Interestingly, hyphae were not identified within or below the mucosal surface in any samples suggesting that infection is non-invasive (Peeters and others 2005). More recently, fungal hyphae were detected in 18 of 22 (82%) nasal biopsy samples collected under direct endoscopic guidance (Pomrantz and Johnson 2010). Sampling areas adjacent to plaques rather than plaques themselves, or erroneous identification of mucus or secondary mucosal irregularities could contribute to lower-than-expected detection rates.

Figure 7. Histologic section of a nasal cavity biopsy showing an Aspergillus spp. conidial head and many small conidia. Many septate hyphae are also seen (arrow). H&E, scale bar = 10·08 µm. Image courtesy of Murdoch University Veterinary Hospital, Western Australia

Other histopathological findings include mucosal ulceration and inflammation with the inflammatory infiltrate usually comprising a mix of neutrophils and mononuclear cells, although predominantly lymphoplasmacytic infiltrates may be seen (Peeters and others 2005, 2008). Histopathological findings are consistent with human chronic, erosive, non-invasive mycotic rhinosinusitis (Peeters and others 2005, Day 2009).

Fungal Culture

Definitive diagnosis by fungal culture of biopsy samples has previously been considered to lack sensitivity and specificity because of potential for false-positive results in dogs with other primary nasal cavity disease (Harvey and others 1981, Sharp and others 1991b). Endoscopic guidance for sample collection improved sensitivity (75 to 96%) in recent studies (Billen and others 2009a, Pomrantz and Johnson 2010).

Laboratory methodology is very likely to contribute to disparate results between studies, although direct comparisons are often difficult as culture media and incubation temperatures vary and these can influence fungal growth. Recently, fungal culture yields from dogs with mycotic rhinosinusitis were found to be influenced by sample type (perendoscopic mucosal biopsies were superior to blinded swabs), and were greatly enhanced at 37^οC compared with incubation at room temperature (Billen and others 2009a). Time until positive culture was also reduced to a mean of 4·0±1·7 days at 37^οC compared to 9·2±3 days at room temperature (Billen and others 2009a). Blind endonasal swabs lacked sensitivity regardless of incubation temperature with only 1 of 16 (6·25%) and 3 of 16 (18·75%) positive cultures obtained at room temperature and 37^οC, respectively (Billen and others 2009a). Of interest is that no samples from normal dogs or those with non-fungal nasal disease produced fungal growth in two recent studies (Pomrantz and others 2007, Billen and others 2009a). This suggests that the predictive value of positive cultures is high and likely more specific to fungal infection than previously thought.

Dna Quantification

There is limited benefit in quantification of fungal DNA in nasal tissue. Using an A. fumigatus specific assay, results overlap between affected dogs, controls and those with lymphocytic plasmacytic rhinitis (LPR) or nasal neoplasia (Peeters and others 2008). Detection of fungal DNA in control samples likely represents filtration or nasal cavity colonisation.

Detection of fungal DNA in whole blood is also of little benefit. This is due to poor sensitivity (21%), as there is little communication with the vascular system, and poor specificity (45%) from a high frequency of false-positive results (Peeters and others 2008).

Serology

Agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and counter immunoelectrophoresis have all been used to assess SNA in dogs, although the latter is not widely available (Pomrantz and others 2007, Billen and others 2009b). Serology is widely regarded as unreliable because of variable sensitivities and specificities (Garcia and others 2001, Dial 2007, Pomrantz and others 2007, Billen and others 2009b). Two studies of AGID showed potential benefit (sensitivity of 67 to 76·5%, specificity of 98 to 100%), although SNA can obviously not be excluded with negative results (Pomrantz and others 2007, Billen and others 2009b). No significant difference is found between sensitivity (88·2%) or specificity (96·8%) for AGID versus ELISA (Billen and others 2009b). ELISA is potentially superior although quantification of the immune response has no proven benefit for canine disease and serial monitoring -throughout -therapy using AGID also does not appear a useful indicator of disease status (Pomrantz and Johnson 2010).

Antibody detection is not reliable for diagnosis in people with invasive aspergillosis (Young and Bennett 1971, Garcia and others 2001). Diagnosis relies instead upon detection of fungal antigens and measurement of galactomannan (GM), an Aspergillus spp. cell wall component (Aquino and others 2007, Woods and others 2007). Limited evaluation of GM in canine (and feline) SNA fails to consistently detect elevated levels, making it less useful for this presentation (Garcia and others 2001, Billen and others 2009b). False-positive results are frequently documented in young people, and those receiving certain antibiotic therapies, reducing specificity. This phenomenon has also been reported in cats (Whitney and others 2011).

Treatment

Despite similarities between human chronic, erosive rhinosinusitis and canine SNA, treatment differs significantly. In human patients endoscopic surgery to removal fungal plaques is curative without topical therapy or ongoing medical management with antifungal agents (Uri and others 2003). By contrast, treatment of dogs remains challenging despite available therapeutic options. In some cases a degree of uncertainty may remain despite a comprehensive diagnostic approach and in these circumstances the decision to treat may be based upon the degree of suspicion.

The most widely used antifungal agents in canine SNA treatment are the azole group comprising the benzimidazoles (thiabendazole), imidazoles (ketoconazole, clotrimazole, enilconazole, miconazole) and triazoles (fluconazole, itraconazole, posaconazole, voriconazole). These agents impede ergosterol biosynthesis, an integral component of fungal membranes, via the p450 enzyme system by blocking 14α-sterol demethylase resulting in lanosterol accumulation within fungal membranes (Hector 2005). Topical azoles such as clotrimazole and miconazole also have a direct lytic effect (Hector 2005). Although selective for fungi, individual azoles vary in their interactions with mammalian cytochrome p450 which can cause hepatotoxicity and a range of drug interactions of varying importance. Cutaneous vasculitis secondary to itraconazole administration has also been reported (Legendre and others 1996). Degree of protein binding, oral bioavailability and solubility also vary between azoles and frequently dictate the way they are used. Azoles such as clotrimazole and enilconazole have poor oral bioavailability and are administered topically (Hector 2005).

Topical application of clotrimazole formulated with a propylene-glycol base is associated with severe, life-threatening side-effects in dogs including nasopharyngeal swelling severe enough to necessitate temporary tracheostomy placement (Caulkett and others 1997, Peeters and Clercx 2007). These effects are not observed when a polyethylene glycol base is used (Mathews and Sharp 2006, Peeters and Clercx 2007). A small case series has documented nasal neoplasia following topical treatment with clotrimazole in a polyethylene glycol base, although a direct causal relationship has not been confirmed (Greci and others 2009).

Other classes of antifungal agents include allylamines, which inhibit ergosterol synthesis at an earlier step in the pathway than azoles, and echinocandins, which interfere with β-1,3-glucan synthesis, both important components of fungal cell walls in some species. These antifungals have not been extensively evaluated in dogs, but may in the future prove useful adjunctive therapies (Hector 2005).

Oral Therapy

Poor clinical responses are reported when oral azole antifungal agents alone are prescribed. Cure rates of only approximately 50% were achieved in older studies (Harvey 1984, Sharp and Sullivan 1986, 1989). Newer triazoles (fluconazole, itraconazole) improve success (70%) (Sharp and others 1991a). Positive clinical responses with fluconazole are interesting given its efficacy against Aspergillus spp. and other filamentous fungi is questionable and ineffective in vitro (Hector 2005). Improved effect is seen in vitro against Aspergillus spp. when fluconazole is combined with the allylamine terbinafine; however, this has not been evaluated in canine SNA (Mosquera and others 2002). Voriconazole and posaconazole are currently prohibitively expensive and unproven in canine SNA.

Limited response to oral antifungal agents is not surprising given histopathological studies show no fungal invasion. Duration of therapy required for cure is often markedly prolonged, increasing costs.

Topical Therapy

Topical antifungal administration remains the most widely used method of treatment in dogs. Various different techniques have been assessed including surgically implanted administration catheters in the frontal sinuses, endoscopic placement of temporary frontal sinus catheters, a non-invasive nasal technique, and trephination of the frontal sinuses with instillation of depot therapy. Topical therapeutic techniques allow direct penetration and therefore direct action on fungal plaques; however, frequently even with these methods multiple treatments are required. When assessing published reports using an “evidence-based medicine” approach, the majority of studies regarding the treatment of SNA are case-series describing a single treatment type (n=3) or case-series where two treatment types are compared (n=7). The level of evidence, using the scheme devised by Tivers and others (2012) (Table 1), for each of the major studies reporting outcome in canine SNA is included in Table 2. Care should particularly be taken comparing reported outcomes for the described techniques in the available studies as the method used to determine therapeutic success often varies from clinical reassessment alone to a comprehensive diagnostic reassessment including advanced imaging and rhinoscopy.

Table 1. Levels of evidence

Grade	Description
Scheme devised by Tivers and others (2012) ^a A cohort study is a study that follows a group of patients over a period of time and investigates the effect of a treatment or risk factor ^b A case-control study is one that examines the effect of a risk factor on the outcome for a group of patients with a disease compared to that of a matched control group without the disease
1a	Systematic review of randomised controlled trials (RCTs)
1b	Individual RCT (with narrow confidence interval)
2a	Systematic review of cohort studies*
2b	Individual cohort study (including low-quality RCT)
3a	Systematic review of case-control studies†
3b	Individual case-control study
4a	Lower-quality prospective cohort/case-control study – concerns regarding definition of comparison groups and/or objective (preferably blinded) nature of assessment and/or consideration of confounding factors and/or adequacy of follow-up
4b	Retrospective cohort/case-control study
4c	Case series – describing outcome for one treatment method with no control group
4d	Case series – describing novel aspect of management and providing some information regarding outcome
4e	Lower-quality case series – concerns regarding study design and/or ability to interpret information
5	Expert opinion without explicit critical appraisal, or based on physiology, bench research or “first principles”

Table 2. First and overall treatment success rates in previously published studies of mycotic rhinosinusitis

Treatment type	Number of dogs	First treatment success (%)	Overall treatment success (%)	Level of evidence^*
^a As outlined by Tivers and others (2012) ^b Includes five dogs from Sharp and Sullivan (1986) ^c Twelve dogs (50%) were considered cured based upon re-examination which included radiologic examination and rhinoscopy. A further eight dogs (33%) were possibly cured based upon the absence of clinical signs (n=7) or the absence of disease at post-mortem examination following death from unrelated disease (n=1). Two dogs failed treatment and did not receive further therapy. ^d A total of 23 dogs were included in the study. For Group A dogs (n=13) 10 had a successful first treatment (76·9%) and 12 dogs were successful following multiple treatments. As Group B dogs routinely received a second treatment regardless of whether disease was determined to be present upon reassessment, first treatment outcome in this group could not be assessed. Twenty dogs (86·9%) were reported as having successful outcomes when all treatments were considered ^e All 12 of the dogs in the topical enilconazole + depot bifonazole group were cured after a second treatment. Three of the five dogs that required additional therapy following the first treatment also received enilconazole plus bifonazole for the second treatment. Two of the five dogs received topical bifonazole depot therapy alone
Indwelling catheters – enilconazole
• Sharp and Sullivan (1986)
º Enilconazole (10 mg/kg) + ketoconazole (10 mg/kg)	5	100	NA	4d
• Sharp and others (1993)
º Enilconazole (10 mg/kg) + ketoconazole (10 mg/kg)	7^†	85·7	100	4e
º Enilconazole (10 mg/kg) only	24	50 (83)^‡	NA
Temporary trephination – 1% clotrimazole
• Mathews and others (1998)	37	62	83·7	4b
• Friend and others (2002)	23	76·9^§	86·9	4e
• Sharman and others (2010)	24	54·1	NA	4b
Non-invasive catheter soaks – 1% clotrimazole
• Mathews and others (1998)	23	69·5	91·3	4b
• Sharman and others (2010)	45	40	NA	4b
Non-invasive catheter soaks – 1% enilconazole
• Zonderland and others (2002)	19	47·3	89·5	4a
• Saunders and others (2003)	36	55·5	94·4	4d
• Schuller and Clercx (2007)	15	46·6	93·3	4d
Endoscopically inserted catheters 2% enilconazole
• Zonderland and others (2002)	7	85·7	100	4a
• Schuller and Clercx (2007)	12	58·3	83·3	4d
Topical and depot clotrimazole therapy
• Sissener and others (2006)	14	86	86	4d
• Sharman and others (2010)	10	70	NA	4b
Depot bifonazole therapy – Billen and others (2009a,2009b)
• Including topical enilconazole soak	12	58·3	100^¶	4a
• Depot therapy alone	5	60	60

Indwelling catheters and enilconazole

Original topical therapeutic techniques involved instillation of enilconazole (10 mg/kg) twice daily for 7 to 14 days, via catheters surgically implanted into the nasal cavity (Fig 1). For the five dogs treated initially, therapy was 100% successful with resolution of nasal signs in all cases (Table 2) (Sharp and Sullivan 1986). Subsequent cures were reported in 20 of 24 (83%) of cases, 50% based on reassessment with radiography, rhinoscopy and mycological examination and 33% with clinical scoring alone (Sharp and others 1993).

Although successful, prolonged hospitalisation and morbidity led to declining popularity. Complications including catheter dislodgement, inappetence, pytalism and aspiration pneumonia were reported and dogs also become intolerant of daily administrations requiring sedation for each treatment. Disease extension into periorbital regions appears to limit efficacy (Sharp and others 1993). This may be due to inadequate penetration of antifungal agents to this region; however, as disease is considered non-invasive this may be more likely to reflect inadequate attenuation of the inflammatory response.

Temporary trephination and non-invasive infusion

Alternative techniques aimed to avoid prolonged hospitalisation and potential complications. Frontal sinus trephination for temporary catheterisation (Fig 8) was the first of these to be described (Davidson and others 1992). This was well -tolerated with an excellent outcome in 21 dogs (Davidson and others 1992). Further evaluation gave first and overall treatment success of 62% and 83·7%, respectively (Mathews and others 1998). No significant difference was found when a routine second treatment was administered compared with dogs receiving only one (Friend and others 2002).

Figure 8. A dog undergoing treatment via temporary trephination of the frontal sinuses

Non-invasive protocols eliminate trephination altogether. The simplest technique involves blind placement of catheters into the nasal cavity and rotation of the head through 360^ο to improve distribution to the sinuses. The procedure is of at least 1 hour duration, but may be longer where significant debridement of disease is required. Minor side-effects seen with trephination (emphysema, incision site infections) are avoided. Enilconazole and clotrimazole have both been evaluated at varying concentrations with outcomes varying from 46·6 to 69·5% for first treatment outcome and 89·5 to 94·4% for overall success (Table 2), albeit in small numbers of dogs for some studies (Bray and others 1998, Mathews and others 1998, Friend and others 2002, Zonderland and others 2002, Saunders and others 2003, Schuller and Clercx 2007). A non-invasive protocol using enilconazole as a 10% solution was unsuccessful in four dogs (Bray and others 1998).

Although experimental models demonstrate improved distribution to the sinuses using a non-invasive technique, limited evaluation of distribution in clinical disease is reported (Richardson and Mathews 1995, Mathews and others 1996, Sharman and others 2012). In one study, six dogs were treated using a non-invasive technique (with 1% clotrimazole, mixed with ioxaglate to a 20% solution) and assessed with CT at completion of a 1 hour infusion (Mathews and others 1996). Good distribution throughout the nasal cavity and frontal sinuses was achieved, although the volume of antifungal agent within the sinuses varied (Mathews and others 1996). Frontal sinus disease was present in only a small number of the included cases, however in one was considered to have diminished the volume of infusate entering this region (Mathews and others 1996). Although this study demonstrated distribution to the frontal sinuses, subsequent studies reported high rates of single treatment failures. A requirement for further therapy was theorised to result from inadequate or impaired sinus distribution in dogs with significant sinus disease. Modification of the technique involved placement of additional catheters within the frontal sinuses, via flexible endoscopy, to improve distribution. Improved distribution has not been confirmed with further imaging studies. First treatment outcome does appear improved; however, this has only been in small numbers of dogs (Zonderland and others 2002, Schuller and Clercx 2007).

Despite increased invasiveness, techniques utilising trephination offer an opportunity to debride disease within the frontal sinuses and ensure patency of the nasal ostium, both likely key factors improving distribution and influencing outcome. No studies evaluate and compare distribution using a trephination technique with that achieved using a non-invasive soak in clinical cases. A recent study of dogs with SNA performed by the authors demonstrated that distribution to the frontal sinuses and nasal cavity via temporary trephination was achievable; however, retention of clotrimazole and enilconazole was poor, as assessed by CT 5 minutes after initial infusion was completed. This likely reflects continued loss of agent to the nasal cavity and leakage from the nares or nasopharynx (Sharman and others 2012). Distribution and retention were independent of severity assessed by CT, clinical or rhinoscopic scoring.

It would seem that whilst trephination improves access for debridement, and ensures delivery of antifungal agent to the sinuses, a demonstrated poor retention confirms the necessity for continuous infusion using this technique. Additionally the study by Sharman and others (2012) demonstrated retention of saline used to flush the nasal cavity which may have impaired filling and distribution of antifungal agent. Ensuring frontonasal ostia patency and the contribution of flushing to debridement likely outweigh any negative impact, but sufficient time for saline drainage, or suctioning of residual saline before infusion may be beneficial.

Short infusion and depot therapy

Both non-invasive and temporary trephination techniques require long durations of anaesthesia.

Combined trephination, short clotrimazole (1%) soak and application of clotrimazole (1%) cream to the frontal sinuses (10 to 20 g per sinus) were therefore developed to reduce procedural time (Sissener and others 2006). Application of more viscous cream was postulated to improve antifungal agent retention, thus improving contact time and treatment success. Clotrimazole cream also moved slowly from the sinuses to the nasal cavity, theoretically allowing improved contact for this region also. Procedure duration and hospitalisation were greatly reduced and good success achieved with 12 of 14 (86%) clinical cures (Sissener and others 2006). In a recent retrospective series, depot therapy was successful in 7 of 10 (70%) of dogs on first treatment alone, although this was not statistically significant compared to other treatment types (Sharman and others 2010). Depot therapy with bifonazole cream via per-endoscopic frontal sinus catheters has also been described and in combination with debridement and enilconazole infusion resulted in 7 of 12 (58·3%) clinical cures (Billen and others 2010).

Experimentally, retention of commercially available cream in vitro using glass funnels is poor compared to compounded 1% clotrimazole gels made from hydroxypropyl cellulose, poloxamer and carboxymethyl cellulose in various concentrations. Until recently, distribution of only the compounded gels had been evaluated in vivo, although not in clinical cases (Mathews and others 2009). A recent study has since demonstrated good distribution of a commercial 1% clotrimazole cream (Canestan cream; Bayer) within the sinuses, with retention of at least 96 hours duration (Hayes and Demetriou 2012). This was again within normal cadavers and therefore may not adequately reflect retention in disease as already discussed (Hayes and Demetriou 2012).

Improved retention and therefore contact may improve outcome; however, ideal contact times for successful therapy with any of the topical agents are not established.

Other topical therapies

Other topical protocols are described. Povidone-iodine (10%) painted onto turbinates or povidine-iodine impregnated cadexomer dressings (Iodoflex; Smith and Nephew) following rhinotomy and debridement both proved successful in small numbers of dogs (Pavletic and Clark 1991, Moore 2003). Combination treatment with surgical rhinotomy, enilconazole soak and adjunctive oral itraconazole has also been evaluated (Claeys and others 2006). Dogs initially treated had recurrence of disease associated with the bone flap and cerclage wires, but therapy in additional cases was successful where bone flaps were discarded (Claeys and others 2006).

While these protocols result in good success in small numbers the ability to extensively debride disease is very likely to have improved success rates, rather than the type or way in which the topical agent was applied. These methods are invasive and therefore less attractive as first-line therapies.

Reasons For Treatment Failure

Treatment failure is often attributed to disease severity, and criteria designed to assess severity, including clinical, rhinoscopic and radiographic scoring systems, have been evaluated. Initial evaluation of CT scoring to predict outcome, using a 1-hour clotrimazole (1%) infusion, found reasonable sensitivity (71 to 78%) and specificity (79 to 93%) (Mathews and others 1998, Saunders and others 2003). However, results were contradicted in a subsequent study. This second study, using a 1-hour enilconazole (1%) infusion, found that although a high sensitivity was achievable (100%), the specificity was low (30%). CT therefore appears unhelpful in predicting therapeutic success although it is possible that variation in the application of the same scoring system between different investigators, or the use of a different antifungal agent, may have contributed (Saunders and others 2003).

In a recent prospective study by the authors, focal disease on occasion had a poorer outcome in comparison with those dogs whose disease resulted in severe turbinate destruction, despite the latter having a higher severity score with currently reported schemes (unpublished data). This could reflect more difficult access with focal disease and therefore insufficient debridement. Certainly outcome appears improved with significant exposure of the nasal cavity using invasive treatments (Pavletic and Clark 1991, Moore 2003, Claeys and others 2006). Equally, severe turbinate destruction improves access and potentially drug distribution in the nasal cavity. However, in a recent prospective study, whilst debridement was considered complete on visual assessment via rhinoscopy, CT demonstrated significant residual disease indicating that the former is not always a reliable means of assessment (Sharman and others 2010).

Factors potentially influencing outcome in a multi-centre retrospective study found only younger age to be significantly associated with success (Sharman and others 2010). Subjectively severity was not associated with outcome, although dogs with unilateral disease were 2·7 times more likely to have a successful first treatment compared with bilateral disease. Application of previous severity scoring systems was unable to be performed because of the study's retrospective nature (Sharman and others 2012).

Antifungal resistance is likely insignificant. Although susceptibility testing is not routinely performed in veterinary medicine, high concentrations of antifungal agents are achieved locally with topical treatment protocols, making it unlikely to be important for canine SNA.

Inadequate distribution and retention are far more likely reasons for failure. Theoretically, improved retention of antifungal agents should improve treatment outcome by increasing contact time and penetration of fungal plaques. However, ideal retention times have not been developed. Whether retention of agents within the frontal sinus and nasal cavity for days or weeks is better than hours is unknown. Variations in frontal sinus and nasal cavity anatomy within and between breeds may also influence distribution and retention and therefore outcome (Burrow and others 2012).

An inability to predict first-treatment success rate despite comparable treatment protocols is frustrating, but likely multi-factorial. Severity of disease, minor modification of technique, particular antifungal agent chosen, experience of the treating clinician and the ability and extent to which debridement is performed are all potential factors.

Summary And Conclusions

SNA is an uncommon, debilitating and challenging condition in dogs. Infection resembles non-invasive, chronic rhinosinusitis in humans, with only small numbers of dogs exposed to inhaled fungal elements developing infection. Most likely this is explained by altered innate and adaptive immune responses, although mechanisms for failure of these responses are unproven. Evaluation of innate mechanisms suggests a response that is -sufficient to limit infection, but inadequate to eliminate the organism (Peeters and others 2006). Diagnosis itself can be challenging, relying upon a combination of clinical findings and diagnostics.

Topical antifungal administration is preferred; however, the evidence base is currently insufficient to support a recommendation for one individual treatment protocol over another. Multiple treatments are required in approximately 50% of patients, regardless of the protocol chosen (Pomrantz and Johnson 2010, Sharman and others 2010). Treatments allowing extensive debridement and/or direct administration of antifungal agents to the frontal sinuses may be more likely to be successful (Pavletic and Clark 1991, Mathews and others 1998, Friend and others 2002, Zonderland and others 2002, Moore 2003, Claeys and others 2006, Sissener and others 2006, Schuller and Clercx 2007). Predicting first-treatment outcome based on clinical findings or advanced imaging findings remains challenging (Mathews and others 1998, Saunders and others 2003). Scoring systems may guide therapy, but an assumption that severity alone is responsible for outcome appears misguided. Severity of destruction may not correlate with the amount of fungal infection, and scoring systems fail to assess subtle change that may significantly impact outcome.

Almost certainly distribution and adequate retention of antifungal agents in regions of residual disease are important. Additional factors such as treatment protocol, antifungal agent used and experience of the treating clinician are also likely to impact outcome.

Conflict of interest

None of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.