Invasive Fungal Infections and Oomycoses in Cats: 1. Diagnostic approach.

CLINICAL RELEVANCE: In contrast to superficial fungal infections, such as dermatophytosis, invasive fungal infections (IFIs) are characterised by penetration of tissues by fungal elements. Disease can spread locally within a region or can disseminate haematogenously or via the lymphatics. The environment is the most common reservoir of infection. Since fungal spores are airborne, indoor cats are also susceptible to IFIs. Some environmental fungi are ubiquitous and present globally, while others are endemic or hyperendemic within specific geographic regions. Zoonotic pathogens include Microsporum canis, Sporothrix schenckii and Sporothrix brasiliensis. AIM: In the first of a two-part article series, the approach to the investigation of feline IFIs and oomycoses is reviewed. As well as tips for diagnosis, and information on the ecological niche and distribution of fungal pathogens, the review covers clinical presentation of the most common IFIs, including cryptococcosis, histoplasmosis, blastomycosis, coccidioidomycosis, sporotrichosis, phaeohyphomycosis, aspergillosis and dermatophytic pseudomycetoma, as well as the oomycoses pythiosis, lagenidiosis and paralagenidiosis. In Part 2, the spectrum of activity, mechanisms of action, pharmacokinetic and pharmacodynamic properties and adverse effects of antifungal drugs are reviewed, and the treatment and prognosis for specific IFIs and oomycoses are discussed. EVIDENCE BASE: The review draws on published evidence and the authors' combined expertise in feline medicine, mycology, dermatology, clinical pathology and anatomical pathology.

Complete clinical response to combined antifungal therapy in two cats with invasive fungal rhinosinusitis caused by cryptic Aspergillus species in section Fumigati.

Cryptic species in Aspergillus section Fumigati are increasingly reported to cause invasive aspergillosis in humans and animals. These infections are often refractory to treatment because of intrinsic antifungal resistance. We report two cases of invasive fungal rhinosinusitis in domestic cats caused by A. udagawae and A. felis. Clinical signs resolved after combined therapy including posaconazole, caspofungin and terbinafine. Both cases remained asymptomatic more than 2 years from initial presentation.

Disseminated invasive aspergillosis caused by Aspergillus felis in a cat.

A 2-year-old male desexed Ragdoll cat with a 1-year history of sneezing and nasal discharge presented with a large subcutaneous cervical mass, identified as the right medial retropharyngeal lymph node on computed tomography (CT). A right orbital mass, destructive sino-nasal cavity disease and multiple pulmonary nodules were also identified. Aspergillus felis was cultured from the lymph node. After treatment with posaconazole and liposomal amphotericin B the lymph node enlargement and orbital mass resolved but left frontal sinus involvement and pulmonary lesions persisted despite additional caspofungin therapy. The cat was euthanized 14 months after diagnosis with dysphagia and chronic progressive exophthalmos. A meningeal granuloma with intravascular fungal hyphae was identified at post-mortem and A felis was cultured from the left frontal sinus and a right retrobulbar fungal granuloma. This case demonstrates that disseminated disease is a possible sequel to invasive fungal rhinosinusitis caused by A felis in cats.

Fungal Rhinosinusitis and Disseminated Invasive Aspergillosis in Cats.

Fungal rhinosinusitis, including sinonasal aspergillosis (SNA) and sino-orbital aspergillosis (SOA), is the most common type of aspergillosis encountered in cats. Other focal forms of aspergillosis including disseminated invasive aspergillosis occur less frequently. SOA is an invasive mycosis that is increasingly recognized and is most commonly caused by Aspergillus felis, a close relative of Aspergillus fumigatus. SNA can be invasive or noninvasive and is most commonly caused by A fumigatus and Aspergillus niger. Molecular methods are required to correctly identify the fungi that cause SNA and SOA. SNA has a favorable prognosis with treatment, whereas the prognosis for SOA remains poor.

cyp51A Mutations, Extrolite Profiles, and Antifungal Susceptibility in Clinical and Environmental Isolates of the Aspergillus viridinutans Species Complex.

The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles. The cyp51A gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. cyp51A sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type A. fumigatus M172A/V and D255G, previously associated with A. fumigatus azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant A. fumigatus Some novel mutations were identified only among isolates with high azole MICs. However, cyp51A homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For A. viridinutans complex isolates, cyp51A mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.

Long term survival of a dog with disseminated Aspergillus deflectus infection without definitive treatment.

Canine disseminated fungal infection by Aspergillus species carries a guarded to grave prognosis as they often rapidly progress and are refractory to treatment with many euthanased soon after diagnosis. This case report describes a 2.5 year old female spayed German Shepherd Dog diagnosed with disseminated Aspergillus deflectus infection for which definitive treatment was declined by the owners. With only palliative management the dog survived three years and two months before succumbing to chronic kidney disease.

Canine rhinitis caused by an uncommonly-diagnosed fungus, Scedosporium apiospermum.

A Golden Retriever cross was presented with a four week history of violent sneezing and licking at the nasal planum. Nasal mycosis was diagnosed and Aspergillus sp. presumed the causative agent, until culture, PCR and DNA sequencing showed that Scedosporium apiospermum, an uncommonly diagnosed, yet emerging, fungal pathogen, was the agent responsible. Debridement of the fungal plaques and systemic itraconazole therapy resulted in complete resolution of clinical disease. We discuss the current literature on S. apiospermum, review its clinical significance and question the validity of its 'complex' taxonomy.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

Background: The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. Methods: A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Results: Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Conclusions: Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Identification of pathogenic Aspergillus isolates from captive birds in Australia.

Aspergillosis is a major cause of severe respiratory disease in birds. The prevalence of cryptic section Fumigati and other non-Aspergillus fumigatus species as causative agents is unknown. Species identity was determined in 30 isolates from affected birds from zoos, pet birds and poultry by PCR of the ITS1-5.8S-ITS2 and partial ß-tubulin genes. The most prevalent isolate was A. fumigatus sens. str. in 87% (26) cases. Other Aspergillus species were identified in 13% (4) cases, including A. restrictus (1), A. flavus sens. str. (2), and A. nidulans-clade (1). This is the first report of A. restrictus causing avian disease.

One-health pathogens in the Aspergillus viridinutans complex.

Cryptic species in Aspergillus section Fumigati are increasingly recognised as pathogens in humans and animals. The A. viridinutans complex (AVC) has recently expanded to comprise 10 species, of which six are known to be pathogenic, including A. udagawae, A. felis, A. pseudofelis, A. parafelis, A. pseudoviridinutans, and A. wyomingensis. They cause locally invasive and disseminated invasive disease syndromes, including chronic pulmonary aspergillosis and invasive aspergillosis in humans, invasive fungal rhinosinusitis in cats, and disseminated invasive aspergillosis in dogs. In contrast to A. fumigatus, AVC species are characterized by higher minimum inhibitory concentrations (MICs) of antifungal drugs and the infections they cause are typically more chronic and more refractory to therapy. This review, of relevance for one-health practitioners, explores the history of the AVC as well as current phylogenetic relationships, secondary metabolite production, environmental distribution, clinical syndromes, and antifungal susceptibility patterns.

Discovery of Aspergillus frankstonensis sp. nov. during environmental sampling for animal and human fungal pathogens.

Invasive fungal infections (IFI) due to species in Aspergillus section Fumigati (ASF), including the Aspergillus viridinutans species complex (AVSC), are increasingly reported in humans and cats. The risk of exposure to these medically important fungi in Australia is unknown. Air and soil was sampled from the domiciles of pet cats diagnosed with these IFI and from a nature reserve in Frankston, Victoria, where Aspergillus viridinutans sensu stricto was discovered in 1954. Of 104 ASF species isolated, 61% were A. fumigatus sensu stricto, 9% were AVSC (A. felis-clade and A. frankstonensis sp. nov.) and 30% were other species (30%). Seven pathogenic ASF species known to cause disease in humans and animals (A. felis-clade, A. fischeri, A. thermomutatus, A. lentulus, A. laciniosus A. fumisynnematus, A. hiratsukae) comprised 25% of isolates overall. AVSC species were only isolated from Frankston soil where they were abundant, suggesting a particular ecological niche. Phylogenetic, morphological and metabolomic analyses of these isolates identified a new species, A. frankstonensis that is phylogenetically distinct from other AVSC species, heterothallic and produces a unique array of extrolites, including the UV spectrum characterized compounds DOLD, RAIMO and CALBO. Shared morphological and physiological characteristics with other AVSC species include slow sporulation, optimal growth at 37°C, no growth at 50°C, and viriditoxin production. Overall, the risk of environmental exposure to pathogenic species in ASF in Australia appears to be high, but there was no evidence of direct environmental exposure to AVSC species in areas where humans and cats cohabitate.

Sinonasal aspergillosis in a British Shorthair cat in the UK.

Case summary A 13-year-old, castrated male, British Shorthair cat presented for investigation of chronic, intermittent, bilateral epistaxis and stertor. CT revealed severe asymmetric bilateral intranasal involvement with extensive turbinate lysis, increased soft tissue attenuation and lysis of the sphenopalatine bone and cribriform plate. On retroflexed pharyngoscopy, a plaque-like mass occluded the choanae. Rostral rhinoscopic examination revealed extensive loss of nasal turbinates, necrotic tissue and mucosal fungal plaques in the left nasal cavity. The right nasal cavity was less severely affected. The nasal cavities were debrided extensively of plaques and necrotic tissue. Aspergillus fumigatus was isolated on fungal culture, and species identity was confirmed using comparative sequence analysis of the partial ß-tubulin gene. On histopathology of nasal biopsies, there was ulcerative lymphoplasmacytic and neutrophilic rhinitis, and fungal hyphae were identified on nasal mucosa, consistent with a non-invasive mycosis. The cat was treated with oral itraconazole after endoscopic debridement, but signs relapsed 4.5 months from diagnosis. Residual left nasal fungal plaques were again debrided endoscopically and oral posaconazole was administered for 6 months. Fourteen months from diagnosis, the cat remains clinically well with mild intermittent left nasal discharge secondary to atrophic rhinitis. Relevance and novel information This is the first case of rhinoscopically confirmed sinonasal aspergillosis to be diagnosed in a cat in the UK. Endoscopic confirmation of resolution of infection is useful in cases where mild nasal discharge persists after treatment.

Azole resistance in canine and feline isolates of Aspergillus fumigatus.

Azole resistance is an emerging cause of treatment failure in humans with aspergillosis. The aim of this study was to determine if azole resistance is emerging in Aspergillus fumigatus isolates from canine and feline sino-nasal aspergillosis cases. Susceptibilities of isolates collected between 1988 and 2014 from 46 dogs and 4 cats to itraconazole, posaconazole, voriconazole, fluconazole and ketoconazole were assessed using Sensititre YeastOne microdilution trays; and to enilconazole and clotrimazole, following the CLSI M38-A2 standard. For the majority of isolates MICs were high for ketoconazole, low for enilconazole and clotrimazole, and less than established epidemiological cut-off values for itraconazole, posaconazole and voriconazole. One canine isolate from 1992 had multiazole resistance and on Cyp51A gene sequencing a mutation associated with azole resistance (F46Y) was detected. There is no evidence of emerging azole resistance among A. fumigatus isolates from dogs and cats and topical azole therapy should be effective against most isolates.

Nasofacial infection in a cat due to a novel bacterium in Neisseriaceae.

CASE SUMMARY: A 2-year-old female spayed domestic shorthair cat was presented for a progressive subcutaneous nasofacial swelling. Histology of biopsy tissue revealed pyogranulomatous inflammation and large numbers of gram-negative capsulated bacterial coccobacilli within macrophages. The isolate was fastidious and grew after 6 days under microaerophilic conditions in a candle jar. The molecular identity of the isolate, from comparative sequence analysis of the 16s rRNA gene, is an as yet to be classified bacterial species within a novel genus of Neisseria. Infection resolved after 7 months of antimicrobial therapy with doxycycline and trimethoprim sulfamethoxazole. There has been no further recurrence of clinical signs in a 3 year follow-up period. RELEVANCE AND NOVEL INFORMATION: Cats are susceptible to nasofacial infections as a result of traumatic inoculation of environmental bacteria, fungi and protozoa. We report a novel pathogen in the Neisseriaceae family, identified by 16 sRNA comparative sequence analysis, as a cause of nasofacial infection in a cat, and its subsequent successful treatment with combination antimicrobial therapy.

Gastrointestinal granuloma due to Candida albicans in an immunocompetent cat.

A 3.5 year-old cat was admitted to the University of Melbourne Veterinary Teaching Hospital for chronic vomiting. Abdominal ultrasonography revealed a focal, circumferential thickening of the wall of the duodenum extending from the pylorus aborally for 3 cm, and an enlarged gastric lymph node. Cytology of fine-needle aspirates of the intestinal mass and lymph node revealed an eosinophilic inflammatory infiltrate and numerous extracellular septate acute angle branching fungal-type hyphae. Occasional hyphae had globose terminal ends, as well as round to oval blastospores and germ tubes. Candida albicans was cultured from a surgical biopsy of the duodenal mass. No underlying host immunodeficiencies were identified. Passage of an abrasive intestinal foreign body was suspected to have caused intestinal mucosal damage resulting in focal intestinal candidiasis. The cat was treated with a short course of oral itraconazole and all clinical signs resolved.

What causes canine sino-nasal aspergillosis? A molecular approach to species identification.

On the basis of phenotypic identification methods, Aspergillus fumigatus is reported as the most commonly identified aetiological agent of canine sino-nasal aspergillosis (SNA). However, definitive identification of Aspergillus spp. using phenotypic features alone is unreliable. The aim of this study was to determine the molecular identities of fungal species causing SNA in dogs. Genomic DNA was extracted from 91 fungal isolates from 90 dogs diagnosed with SNA in Australia, the USA and Belgium, and the ITS1-5.8S-ITS2 ribosomal DNA and partial ß-tubulin regions were sequenced. Eighty-eight of 91 (96.7%) isolates were identified as A. fumigatus and 3/91 (3.3%) belonged to Aspergillus section Nigri spp. (Aspergillus tubingensis: 2/91; Aspergillus uvarum: 1/91). These findings confirm that A. fumigatus is the most common aetiological agent of canine SNA. This is the first report to document a pathogenic role for A. tubingensis and A. uvarum in dogs.

Feline aspergillosis.

Feline aspergillosis includes sinonasal aspergillosis (SNA), sino-orbital aspergillosis (SOA), other focal invasive forms, and disseminated disease. SOA is an invasive mycosis that is being increasingly recognized, and is most commonly caused by a recently discovered pathogen Aspergillus felis. SNA can be invasive or noninvasive and is most commonly caused by Aspergillus fumigatus and Aspergillus niger. Molecular methods are required to correctly identify the fungi that cause SNA and SOA. SNA has a favorable prognosis with treatment, whereas the prognosis for SOA remains poor.

Type III hemifacial microsomia in a kitten.

The case described herein presented with craniofacial malformations resembling hemifacial microsomia (HFM), a congenital disorder described in humans. To our knowledge, this is the first reported case of HFM in a domestic cat.