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

Abstract

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.

Introduction

The outcome of a fungal infection depends on a complex interplay between the fungal pathogen and the host's immune response. Non-invasive fungal infections are those in which fungal elements are restricted to surface colonisation of the skin, hair, mucosa or airways. By contrast, in invasive fungal infections (IFIs), fungal elements penetrate tissues and can spread locally or by haematogenous and/or lymphatic dissemination. For example, in dermatophytosis, the most common non-invasive mycosis of cats, Microsporum canis infects the stratum corneum of the epidermis, hair shafts and hair follicles. However, occasionally, M canis penetrates the dermis to cause an IFI. ¹

Part 1 of this two-part article series describes the general diagnostic approach to cats with suspected IFIs or oomycoses, hereafter termed invasive fungal-like infections (IFLIs). Attention then turns to reviewing clinical features and providing diagnostic tips for the most common IFIs and IFLIs. The fungal pathogen Candida is not reviewed since there have been fewer than 10 cases of invasive candidiasis reported;^2-6 by contrast, non-invasive Candida species infections of the lower urinary tract are moderately frequent. ⁷ Algal infections, such as protothecosis, and other IFLIs, such as microsporidiosis and rhinosporidio-sis, are outside the scope of this review.^8-17

Agents of disease

The fungal kingdom is vast and currently includes 19 different phyla. ¹⁹ The most common moulds and yeasts that cause IFIs in cats are listed in Table 1. Yeasts are unicellular and reproduce asexually by budding to form a daughter cell, whereas moulds are multi-cellular filaments (hyphae) that reproduce asexually through fragmentation of hyphae or production of asexual spores (conidia). Many yeasts and moulds also reproduce sexually by meiosis. Some yeasts have three vegetative states - yeast, pseudohyphae and hyphae -which can make cytological identification in tissues more difficult. Some fungi (thermally dimorphic ascomycetes, Table 1) exist as moulds in their environmental reservoirs and switch to yeast forms in the higher temperatures of infected hosts. ²⁰

Table 1.

Classification of common invasive fungal pathogens in cats

Phylum	Class	Genus
		Hyaline (non-pigmented) hyphomycete moulds	Dematiaceous (pigmented) hyphomycete moulds	Dimorphic fungi
Ascomycota	Eurotiomycetes	Aspergillus species Microsporum species	Cladophialophora species Exophiala species	Histoplasma species † Blastomyces species † Coccidioides species †
	Sordariomycetes		Scedosporium species Lomentospora species	Sporothrix species †
	Dothideomycetes		Curvularia species Bipolaris species Cladosporium species
Basidiomycota	Tremellomycetes			Cryptococcus species
Mucoromycota*	Mucoromycetes	Rhizopus species Mucor species
Basidiobolomycota*	Basidiobolomycetes	Basidiobolus species
Zoopagomycota*
Entomophthoromycota*	Entomophthoromycetes	Conidiobolus species

^*Previously known as Zygomycetes. ^†Thermally dimorphic

Oomycetes are the most common fungal-like organisms to infect cats.

Most feline IFIs and IFLIs are not zoonotic. Rather, they are caused by shared environmental pathogens, which lead to disease in humans and animals alike, usually through inhalation or traumatic cutaneous inoculation. Sporotrichosis is a notable, highly zoonotic exception. M canis is also zoonotic in cats with dermatophytic pseudomycetoma and concurrent dermatophytosis.

General diagnostic approach to a possible invasive fungal or fungal-like infection

History and signalment

Knowledge of which fungi are ubiquitous saprophytes, occurring anywhere regardless of geographic location, and which are endemic or hyperendemic in particular regions is helpful when considering the likelihood and ranking of an IFI/IFLI in the differential diagnosis list (Figure 1; see also later discussion of the 'Diagnostic approach for selected invasive fungal and fungal-like infections'). With global warming, the geographic range of many environmental fungi is increasing. ²¹ Travel history is also important, because some IFIs (eg, cryptococcosis and histoplasmosis) can be latent and subclinical but can reactivate months to years later if the cat is immunosuppressed. ²²

Figure 1.

The geographic range of many environmental fungi is increasing in association with global warming and will likely continue to expand.

✜ Coccidioides species (blue) can be found in North, Central and South America, especially in Central/Southern California, Arizona, Utah, Nevada and New Mexico, with recent spread into South-Central Washington

✜ Cryptococcus gattii species complex (yellow) is present in Western Europe, Australia, Southern Africa, the Pacific Northwest and Western California. Cryptococcus neoformans species complex (not shown) is prevalent globally

✜ Blastomyces dermatitidis (pale orange) is especially concentrated around the waterways of Mississippi River systems and adjacent states of the USA. It is also endemic in Canada in some parts of Ontario, Quebec and Manitoba. Its range in Africa, India and the East Coast of North America is estimated based on human cases (pale blue)

✜ Pythium insidiosum (green) is reported in Brazil, Central America, South East Asia and the East Coast of Australia

✜ Histoplasma capsulatum (pink) is hyperendemic in regions of South and North America

✜ Sporothrix brasiliensis (red) is confined to Brazil, Argentina, Panama, Paraguay and the Magallanes region of Chile

Patient signalment can raise the ranking of IFI/IFLI on the differential diagnosis list. For example, Persian cats and related breeds are predisposed to some IFIs, including aspergillosis and dermatophytic pseudo-mycetoma. ²³ Fungal infections can occur in cats of either sex or any age, but some predispositions exist for certain IFIs/IFLIs (see 'Diagnostic approach for selected invasive fungal and fungal-like infections'). Moreover, while IFIs are commonly diagnosed in apparently immunocompetent cats, immuno-suppression from concomitant disease or immunomodulatory drugs, such as ciclo-sporin, ²⁴ is a risk factor for IFIs and also for haematogenous dissemination of an IFI.

Complete blood count, serum biochemistry profile and retrovirus testing

For mould IFIs and IFLIs, an inflammatory leukogram and mild to severe hyperglobuli-naemia are common abnormalities. ²⁵ Hyper-globulinaemia is present in ~65% of cases of sporotrichosis. ²⁶

Mild to moderate non-regenerative anaemia (normocytic, normochromic) is common in cats with cryptococcosis, sporotrichosis and histoplasmosis, but anaemia may be severe in cats with histoplasmosis.^26-29 Leukocyte changes are variable among infections caused by thermally dimorphic fungi. Neutrophilic leukocytosis, sometimes with a left shift, is common in both histoplasmosis and sporo-trichosis,^26,28 while monocytosis is a common finding in cases of cryptococcosis ²⁷ and coccidioidomycosis. ³⁰ Infiltration of bone marrow in cats with disseminated histoplasmosis may result in neutropenia, thrombocytopenia or pancytopenia. ³¹

Peripheral eosinophilia is not common overall, but, if present, should raise a flag for possible IFIs/IFLIs of any type. Hypoalbuminaemia is common in disseminated sporotrichosis and histoplasmosis, ²⁶ especially where there is gut involvement. Hypercalcaemia may be present due to monocyte and macrophage activation within pyogranulomas; increased expression of 25-hydroxyvitamin D₃ 1a-hydroxylase then results in hypervitaminosis D. This has been reported in histoplasmosis, blastomycosis and dermatophytic pseudomycetoma.^28,32,33 Other serum biochemical changes in IFIs/IFLIs reflect specific organ involvement.

Results of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) testing reflect regional prevalence, and, although these retroviruses are not significantly associated with IFIs/IFLIs, testing is recommended. A positive test result is not a contraindication for IFI/IFLI treatment; however, cats with advanced FIV or FeLV infection may require longer courses of treatment.

Fungal-specific tests on urine, serum or other body fluids

Serum antibody or fungal antigen tests, as listed in Table 2, are especially useful in the early stages of a diagnostic investigation since test samples can mostly be procured without the need for general anaesthesia. Their use should be guided by the patient's clinical signs at presentation, as well as the travel history and geographic location. Aspergillosis and crypto-coccosis occur worldwide. While sporadic cases of histoplasmosis and sporotrichosis may also be seen in any geographic region, these, as well as blastomycosis, coccidioidomycosis and pythiosis, usually occur in endemic/hyper-endemic regions (Figure 1). Tests to detect fungal antibodies or antigens in serum or urine are not currently available for dermatophytic pseudomycetoma or phaeohyphomycosis.

Table 2.

Availability of antibody and antigen tests for invasive fungal and fungal-like infections in cats

Aspergillosis	Sporotrichosis	Histoplasmosis	Blastomycosis	Coccidioidomycosis	Cryptococcosis	Pythiosis
SERUM ANTIBODY
✓	✓	✘	✘	✓	✘	✓
✜ ELISA: >90% Se and Sp,34,35 but limited/no commercial availability ✜ AGID: 43% Se, >90% Sp 34	✜ ELISA: >90%Se and Sp, but limited/no commercial availability 37	✜ AGID and CF: low Se and Sp; no rigorous studies s studies	✜ RIA and EIA: commercial availability for dogs only ✜ AGID: ~20% Se; not recommended	✜ AGID: two tests - one to detect antibodies (IgG and IgM), the other to quantitate IgG by serial dilution of positive sera. 41 High Se (43/44 cats 30 and 39/39 cats 41 in two studies); Sp unknown. A positive titre indicates previous or active infection. A sick cat with consistent clinical signs and positive serology likely has active infection 42		✜ ELISA: limited Se and Sp data available, but likely similar to dogs with pythiosis - that is, high Se and moderate Sp 46
FUNGAL ANTIGEN
✘	✘	✓	✓	✘	✘	✘
✜ Sample: serum ✜ Detects: serum GM ✜ EIA: 23% Se, 78% Sp; not recommended 36		✜ Sample: urine (best), serum, BAL fluid ✜ Detects: Histoplasma capsulatum antigen ✜ EIA: >90% Se and Sp (urine).38,39 in localised May be negative infections. 40 High Sp for fungal vs non-fungal disease (~97%), but low Sp for fungal genus due to cross-reactivity with other fungi, especially Blastomyces species	✜ Sample: urine(best), serum ✜ Detects: fungal cell wall antigen (GM) also present in other fungal species ✜ EIA: 87% Se (serum), 94% Se (urine). Sp is low because of cross-reactivity with other fungi, especially Histoplasmaspecies	✜ Sample: serum, urine ✜ Detects: Coccidioides species GM antigen. Not evaluated in cats but not recommended in dogs due to low Se (~20%) 43	✜ Sample: serum, CSF ✜ Detects: polysaccharide capsule antigen ✜ CALAS: >90% Se and Sp 44 ✜ POC LFA test: 80-92% Se, >90% Sp. 45 May be negative in localised infections. False- positive results more likely at low titres

AGID = agar gel immunodiffusion; BAL = bronchoalveolar lavage; CALAS = cryptococcal antigen latex agglutination system; CF = complement fixation; CSF = cerebrospinal fluid; EIA = enzyme immunoassay; ELISA = enzyme-linked immunosorbent assay; GM = galactomannan; POC LFA = point-of-care lateral flow antigen; RIA = radioimmunoassay; Se = sensitivity; Sp = specificity

Diagnostic imaging

Thoracic radiography is useful when an IFI/ IFLI is suspected because the respiratory tract is the most common portal of entry and may be diseased. A predilection for upper or lower respiratory tract involvement, or both, depends on the infecting pathogen (Table 3). Because of the superimposition of anatomical structures, skull radiography is not sensitive for the detection of upper respiratory tract involvement; if suspected, CT examination of the head is recommended. CT changes in sino-orbital aspergillosis (SOA) may include increased soft tissue attenuation of the nasal cavities and sinuses, as well as turbinate lysis. ⁵¹ The degree of nasal cavity involvement ranges from mild to severe. Orbital masses are usually unilateral and ven-tromedial, with heterogeneous and peripheral rim contrast enhancement. Changes in thesinonasal cavity, orbit and paranasal bones are not specific for aspergillosis and may be indistinguishable from neoplasia (eg, nasal carcinoma or lymphoma) or other IFIs/IFLIs such as cryptococcosis, ²³ sporotrichosis or pythiosis. ⁵² If neurological signs are present, MRI of the head or spinal cord (depending on lesion localisation) is the preferred imaging modality. In SOA, orbital masses are hyperintense and heterogeneous on T1- and T2-weighted images, and enhance with con-trast.^53-55 If present, central nervous system (CNS) lesions show increased heterogeneous contrast enhancement. In blastomycosis with CNS involvement, there are focal or multifocal intra-axial masses with dural contact; lesions are hypointense on T2-weighted images and diffusion-weighted imaging, and show severe perilesional oedema and strong homogeneous contrast enhancement. ⁵⁶ In disseminated phaeohyphomycosis with CNS involvement, there may be focal or multifocal fungal abscesses; MRI features of these lesions in humans - which are likely similar in cats -include irregular walls and intracavitary projections caused by fungal hyphae. ⁵⁷ Fungal granulomas in cases of cryptococcosis are hypointense on T1- and hyperintense on T2-weighted images, often with well-defined rim enhancement after contrast administration.^58,59

Table 3.

Typical radiographic findings in invasive fungal and fungal-like infections in cats

Phaeohyphomycosis	Aspergillosis	Sporotrichosis	Histoplasmosis
✜ Skull: usually normal ✜ Thorax: may be abnormal if there is haematogenous dissemination. Changes may include pulmonary mass(es) 47	✜ Skull: may be abnormal ✜ Thorax: usually normal in SOA, abnormal in invasive pulmonary aspergillosis and may be abnormal in DIA. Changes are not well characterised but may be similar to other causes of fungal pneumonia (eg, histoplasmosis, blastomycosis)	✜ Skull: may be abnormal ✜ Thorax: generally normal. May be abnormal (pulmonary infiltrates) in disseminated disease; changes are not well described ✜ Appendicular: lesions on the limbs are not usually associated with underlying bone or joint involvement	✜ Skull: usually normal ✜ Thorax: abnormal in up to 50% of cases - diffuse, linear, nodular or miliary interstitial infiltrates.31,50 Mixed patterns (bronchointerstitial/alveolar) and pleural effusions can also occur ✜ Appendicular: synovial effusions, pathological fractures, multifocal lytic metaphyseal lesions and proliferative periosteal reactions commonly affect more than one long bone/joint; carpus and tarsus frequently involved28,29
Blastomycosis	Coccidioidomycosis	Cryptococcosis	Oomycosis
✜ Skull: usually normal ✜ Thorax: usually abnormal, even in the absence of respiratory signs. Poorly defined soft tissue pulmonary masses, nodules or alveolar consolidation are most common. Also miliary interstitial infiltrates, cranial mediastinal mass and/or hilar masses 48	✜ Skull: usually normal ✜ Thorax: often abnormal, even in the absence of respiratory signs. Diffuse mixed pattern (alveolar/ bronchial/interstitial) consolidation is common; other findings may include hilar lymphadenopathy, pleural effusion, lobar consolidation and/or mass/nodule 30 ✜ Appendicular: lytic and proliferative lesions with periosteal new bone formation (fungal osteomyelitis) 30	✜ Skull: may be abnormal ✜ Thorax: usually normal in cats with Cryptococcus neoformans species complex infection. Abnormal in up to 40% with Cryptococcus gattii species complex infection. Diffuse, regional, nodular or occasionally miliary interstitial infiltrates, alveolar infiltrates, mediastinal mass, hilar masses and/or pleural effusion 49 ✜ Appendicular: lesions on the limbs are not usually associated with underlying bone or joint involvement	✜ Skull: may be abnormal ✜ Thorax: usually normal ✜ Appendicular: usually normal

DIA = disseminated invasive aspergillosis; SOA = sino-orbital aspergillosis

Advanced imaging is not reliable for definitive diagnosis of fungal granulomas since, for both CT and MRI, features overlap with those of neoplasia.^23,51,58,60

DIA = disseminated invasive aspergillosis; SOA = sino-orbital aspergillosis

Cytology

Cytology of fine-needle aspirates from masses, cytocentrifugation (cytospin) preparations of urine, bronchoalveolar (BAL) fluid, cerebrospinal fluid (CSF), brushings from conjunc-tivae or impression smears of ulcerated lesions can provide useful information. Fungal elements, if present, can usually be identified using Romanowsky-type stains, such as Wright, Giemsa and Diff-Quik. Depending on the pathogen, fungal elements may exhibit positive or negative staining (Figure 2). Additional tissue or fluid samples obtained aseptically for cytology can also be submitted for fungal culture to confirm pathogen identity and antifungal susceptibility.

Figure 2.

Diff-Quik stained smears showing fungal elements. (a) Positive-staining fungal hyphae in frontal sinus tissue of a cat with invasive fungal rhinosinusitis due to Aspergillus felis infection. (b) Intracellular negative-staining fungal elements in a fine-needle aspirate preparation from a retrobulbar mass of a cat with a phaeohyphomycosis caused by Phoma species

In general, cytology or culture of nasal swabs or washes cannot be used for definitive diagnosis since the nasal cavity can be contaminated with or colonised by fungal spores, which can readily be cultured, giving rise to a false-positive diagnosis of fungal infection. However, if large numbers of fungal elements (hyphae, yeasts) are visualised in the presence of inflammatory cells, there should be a high index of suspicion for fungal infection and a confirmatory test should be performed (eg, fungal antigen test or biopsy).

Surgical biopsies: procurement and transport to the laboratory

Surgical biopsies of involved tissues are often the best samples for definitive diagnosis, especially where less invasive (eg, antigen) tests are not available or where cytology is non-diagnostic. Definitive diagnosis is dependent on taking a representative biopsy of tissue. In many IFIs/IFLIs, fungal elements are only found in the necrotic centre of pathological lesions and are surrounded by a pyogranulomatous inflammatory reaction, which may contain a large proportion of eosinophils. Biopsies that sample only the periphery of a granuloma may yield negative fungal cultures, leading to a misdiagnosis on histology of eosinophilic granuloma complex or non-fungal sterile inflammation. The authors have observed this in cases of SOA and dermatophytic pseudomycetoma.

When performing surgical tissue biopsy from a cat with a possible IFI/IFLI, a good general rule of thumb is either to take one large biopsy and divide it into three portions, or to take three smaller individual representative biopsies. One sample (or portion) is placed in formalin, one is placed in sterile saline in a sterile container for culture and the remaining one is placed in a sterile container and frozen for later molecular diagnostics if fungal culture is negative (Figure 3). Hyphae can be damaged in tissue samples that are homogenised in the laboratory using tissue grinding or beads, as is common practice for bacterial culture, resulting in negative mould cultures. ⁶¹ The recommended technique for preparing tissues in the laboratory for fungal culture is to dice them into small (2 mm³) pieces using a sterile scalpel blade. Yeasts are less likely to be damaged by homogenisation than moulds.

Figure 3.

Representative tissue biopsies from a lesion (A) are placed in formalin (Bi) for histopathology, in sterile saline in a sterile container (Ci) for culture or in a sterile container (D) for frozen storage at -20°C. After embedding formalin-fixed samples in a paraffin block, a microtome (Bii) is used to cut thin sections for histological examination (Biii[a]). If fungal elements are identified on histology and fungal culture is negative, 6-10 urn thick formalin-fixed sections are cut from the paraffin block using the microtome and a sterile blade, and then placed in a sterile tube (Biii[b]) for DNA extraction and molecular identification. Tissue samples should be diced into small pieces (2 mm³) and cultured on fungal agar (Cii[a]). Molecular identification is most reliable using DNA extracted from fungal culture material (Ciii[a]). DNA can also be extracted from residual fresh tissue (Cii[b]) or from stored frozen tissue (D) for molecular identification

Samples for culture should be submitted to the laboratory at room temperature if they can be delivered within 2 h; otherwise, they should be refrigerated at 4°C until collection. ⁶²

Some fungal elements are hard to visualise on routine haematoxylin and eosin staining of histological preparations. Whenever fungal infection is suspected by the clinician, additional 'fungal' stains should be requested. This includes all cases in which histological reports of biopsied tissues describe a pyogranulomatous or granuloma-tous inflammatory response but where no fungal elements are detected. The two most common special stains used to detect fungal and fungal-like elements are periodic acid-Schiff and Grocott methenamine silver (Figures 4-7).

Figure 4.

PYTHIOSIS. Histological sections of a skin biopsy sample from a 6-month-old female domestic shorthair cat with a granulating lesion on the right hindlimb. Pythium insidiosum infection was diagnosed within the dermis and subcutis. (a) On haematoxylin and eosin staining, ghost-like outlines of irregular hyphae (arrows) can be seen embedded in the magenta pink necrotic material. On periodic acid-Schiff (b) and Grocott methenamine silver (c) staining, irregular hyphae with non-parallel walls and tapering ends are identified. Bar = 10 um

Figure 5.

PHAEOHYPHOMYCOSIS. Histological sections of a skin biopsy sample from an adult domestic shorthair cat with a large ulcerated nodular skin mass. (a) Haematoxylin and eosin-stained section; within the chronic severe pyogranulomatous dermatitis and panniculitis, hyphal elements (3-4 um in width) are easily identified due to their melanised (brown) cell walls. Septate hyphae stain positively with periodic acid-Schiff (b) and Grocott methenamine silver (c). Bar = 10 um

Figure 6.

CRYPTOCOCCOSIS. Histological sections from a mass infiltrating the left external ear canal of a 9-year-old female neutered domestic shorthair cat with Cryptococcus neoformans infection. (a) Haematoxylin and eosin-stained section; sheets of refractile, round-to-oval yeasts, occasionally exhibiting narrow-necked budding (arrow), are surrounded by large unstained capsules. Yeast walls stain magenta with periodic acid-Schiff (b) and black with Grocott methenamine silver (c). Bar = 10 um

Figure 7.

DERMATOPHYTIC PSEUDOMYCETOMA. Histological sections from a resected nodule from a 10-month-old female neutered Exotic Shorthair cat. The patient had multiple subdermal nodules on the head and abdomen with pyogranulomatous dermatitis and panniculitis due to Microsporum canis infection. (a) Haematoxylin and eosin-stained section; mats of fungal hyphae with occasional bulbous ends are surrounded by neutrophils and a multinucleated giant cell (asterisk). Fungal hyphae stain magenta pink with periodic acid-Schiff (b) and brown/black with Grocott methenamine silver (c). Bar = 10 um

Adequate tissue should be sampled for submission for histology and for fungal culture. In practice, fungal cultures are often not requested at the time of biopsy, either because an IFI/IFLI is not suspected or because of client financial constraints.

Fungal culture

Optimal conditions for culture

The most reliable way of definitively identifying the cause of an IFI is by fungal culture (Table 4). However, it is underutilised in veterinary medicine. Morphological features of fungal colonies and microscopic examination of cultured fungal elements usually enable identification to genus level. Identification to species level may require one or more PCR assays or matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), using fungal culture material.

Table 4.

Optimal media and incubation temperatures for fungal isolation, and targets for PCR of DNA extracted from fungal culture for molecular identification ⁶³

Genus or group	Media *	Optimal growth conditions	Molecular targets for PCR assays
Aspergillus species	MEA, SDA, CzA	28-37°C	ITS1, ITS2 or ITS1-5.8S-ITS2, p-tubulin, calmodulin, actin
Basidiobolus species Conidiobolus species	SDA, IMA, PDA, CMA	30°C and 37°C; faster growth at 30°C. Difficult to grow, especially after prolonged transportation. Killed by refrigeration	ITS1, ITS2 or ITS1-5.8S-ITS2, 18S rRNA, Cyt b
Cryptococcus species	SDA, BSA, CGB	37°C (budding yeast)	ITS1, ITS2 or ITS1-5.8S-ITS2, D1/D2
Histoplasma species	BHIA	25-30°C (mould) 37°C (budding yeast)	ITS1, ITS2 or ITS1-5.8S-ITS2
Mucor species	PDA, CzA, water agar with 0.1% yeast extract	30°C or 37°C (depending on species). Difficult to culture; grinding specimens may damage fragile hyphae	ITS1, ITS2 or ITS1-5.8S-ITS2, Cyt b
Dematiaceous fungi (Phaeohyphomycetes)	SDA, MEA, IMA, PDA	25-37°C	ITS1, ITS2 or ITS1-5.8S-ITS2, actin, GPDH
Microsporum canis (dermatophytic pseudomycetoma)	PDA	25-35°C	ITS1, ITS2 or ITS1-5.8S-ITS2, D1/D2
Pythium species Lagenidium species	SDA, PDA, CMA, blood agar	34-37°C	ITS1, ITS2 or ITS1-5.8S-ITS2, COXII
Sporothrix species	BHIA	25°C and 37°C	ITS1, ITS2 or ITS1-5.8S-ITS2, D1/D2, P-tubulin, chitin synthase, TEF

^*Non-selective general purpose media are shown in bold

BHIA = brain-heart infusion agar; BSA = bird seed agar; CGB = canavanine glycine bromothymol blue agar; CMA = corn meal agar; Cyt b = cytochrome b gene; CzA = Czapek Dox agar; GPDH = glycerol-3-phosphate dehydrogenase; IMA = inhibitory mould agar; ITS = internal transcribed spacer; MEA = malt extract agar, PDA = potato dextrose agar, SDA = Sabouraud's dextrose agar, TEF = translation elongation factor

The success of fungal culture depends to a large extent on the expertise of the laboratory processing the sample, as well as the condition in which the biopsy is received, and the duration and conditions of transport. Reasons for negative fungal cultures include use of culture media and/or incubation temperatures that are not conducive for growth, as well as overgrowth by commensal or contaminating bacteria or fungi. The use of at least two different incubation temperatures and different types of media has been shown to increase the sensitivity of isolation of fungi from clinical specimens. ⁶⁴

It is well worth contacting the diagnostic laboratory to ascertain whether it:

• ✜ Routinely incubates fungal cultures at two different temperatures: most commonly 25° or 28°C, and 37°C; ²⁵

• ✜ Uses culture media supplemented with antimicrobials (usually gentamicin or chlor-amphenicol) when bacterial contamination of the sample is likely;

• ✜ Has the capacity to perform molecular testing and/or MALDI-TOF MS;

• ✜ Submits unusual isolates to a mycology reference laboratory for definitive species identification. Since many of the organisms that cause IFIs/IFLIs in cats are environmental saprophytes capable of infecting humans, mycology reference laboratories are well equipped to identify them.

Most thermally dimorphic species of fungi (eg, Sporothrix species, Histoplasma species) pose an inhalational infection risk to laboratory staff if grown as moulds, and culture of these species is not recommended routinely. If culture is required, laboratory staff should be warned in advance if fungal infection is suspected, as advanced biosafety level (BSL3) conditions to prevent inhalation of spores may be required.

Molecular diagnostics

Many veterinary diagnostic laboratories now offer molecular assays for identification of fungal pathogens. Molecular identification is most reliable when DNA for PCR and sequencing is extracted from fungal cultures of clinical specimens. ⁶⁵ Fungal DNA can also be extracted directly from formalin-fixed paraffin-embedded tissue (FFPET) or from fresh or frozen clinical specimens (Figures 3 and 8). ⁶⁶ Panfungal PCR primers are used to target part of the ribosomal RNA (rRNA) gene cluster present in multiple copies in yeasts, moulds and oomycetes (Table 4 and Figure 8). ⁶⁷

Figure 8.

(A) DNA extracted from fungal culture material, formalin-fixed paraffin-embedded tissues or fresh or frozen tissues can be subject to PCR and sequencing to determine fungal genus and species complex or species. (B) The methodology uses panfungal PCR primers to amplify targets within the multicopy ribosomal RNA gene cluster of fungi, which contains hypervariable regions. ITS = internal transcribed spacer; IGS = intergenic spacer

Sequencing the entire ITS1-5.8S-ITS2 region (~700 nucleotides) within the rRNA gene cluster amplified from fungal culture DNA extracts enables identification at least to genus and species complex level (Figure 8).^23,65 DNA extracted from FFPET may be degraded or fragmented and DNA quality is also influenced by the duration of formalin contact and number of fungal elements (hyphae, yeasts) present in the FFPET paraffin block, reducing the sensitivity of PCR assays (Figures 3 and 8). However, amplification of the shorter ITS1 (~290 nucleotides) or ITS2 regions (~330 nucleotides) is often possible.^25,67 In one study, where fungal elements were identified on histology of lesions from animal tissues, ITS2 PCR of FFPET DNA enabled identification to genus level in 65% of samples; in 96% of these, the identified fungus was consistent with the morphological identification on histological examination. ⁵⁰

Antifungal susceptibility testing

Broth microdilution methods are the most reliable means of determining in vitro anti-fungal susceptibility. There are two reference methods - one from the Clinical and Laboratory Standards Institute (CLSI) and one from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both use a 96-well format, broth media (RPMI 1640) and a minimum inhibitory concentration (MIC) endpoint (ie, complete inhibition of growth determined by visual inspection).

Commercial kits are also available, such as Sensititre YeastOne (TREK Diagnostic Systems), which utilises antifungals in serial two-fold dilutions on a 96-well, dried colori-metric broth microdilution panel. It has shown high interlaboratory reproducibility and moderate to high correlation with the reference methods, dependent on the fungal genus.^68,69

Although clinical MIC breakpoints, which correlate in vitro data (including susceptibility testing results and drug pharmacokinetics/ pharmacodynamics) with clinical trial outcomes, are still lacking in veterinary medicine, these are available for many fungal pathogens in humans and are broadly applicable. Also, epidemiological cut-off values (ECVs) of MICs are useful to detect decreased susceptibility or resistance in vitro. ECVs are upper limit values set by determining the distribution of MICs for wild-type isolates. The wild-type population is most commonly defined as those isolates around the modal MIC ± one two-fold dilution, which encompasses >95% of strains.

Diagnostic approach for selected invasive fungal and fungal-like infections

Cryptococcosis

• ✜ Aetiological agents Cryptococcus neoformans species complex (C neoformans, C deneoformans), C gattii species complex (C gattii, C deuterogat-tii, C tetragattii, C decagattii, C bacillisporus) and their hybrids.^70,71 Although not truly thermally dimorphic (ie, C neoformans exists as a yeast in pigeon guano), all Cryptococcus species switch to a filamentous form to produce basidiospores during sexual reproduction in the environment.

• ✜ Ecological niche C neoformans species complex is primarily isolated from pigeon guano and less frequently from organic matter in soil. ⁷² C gattii species complex has been found in wood, leaves, bark and plant debris of more than 50 tree species, including Eucalyptus species. ⁷³

• ✜ Geographic distribution The C neoformans species complex is distributed worldwide. ⁷² The distribution of the C gattii species complex is illustrated in Figure 1.

• ✜ Transmission Airborne infectious particles (basidiospores or desiccated yeast cells) are inhaled and deposited in the nasal cavity and/or sinuses. Less frequently, transmission is by ingestion of infectious propagules or inoculation through penetrating skin wounds. ⁷⁴

• ✜ Clinical forms of disease Nasal, ²⁷ CNS, ⁴² systemic ⁷⁵ and cutaneous^76,77 forms of disease are described, but there is considerable overlap between them. Nasal signs may be subtle or absent in CNS and other forms. Both Cryptococcus species complexes can cause all clinical forms of disease.

• ✜ Clinical presentation Nasal disease - Signs of chronic rhinitis (nasal discharge, sneezing, stertor, variable epistaxis) and mandibular lymphadenopathy are com-mon. ²⁷ Progressive osteolysis of the sinonasal cavity can lead to proliferative or ulcerative nasofacial skin and mucosal lesions, and facial deformity. Otitis media and vestibular signs may also occur. ⁷⁸ Extension of disease into the lower respiratory tract to cause pneumonia and pleural effusion occurs less commonly.^79,80

CNS disease - Extension of nasal disease through the cribriform plate or haemato-genously can cause ocular involvement (chorioretinitis, optic neuritis, uveitis), meningoencephalomyelitis and/or fungal CNS granulomas. Presentations include behavioural changes, altered mentation, inappetence, sudden-onset blindness, ataxia, spinal pain and seizures.^59,81 Increased intracranial pressure can result in severe acute obtundation.

Systemic and cutaneous disease - Systemic and cutaneous forms of cryptococcosis (Figure 9) signal advanced disease resulting from haema-togenous spread. ⁷⁵ Cutaneous lesions include non-pruritic, solitary or multiple nodules, abscesses and/or ulcers, with or without peripheral lymphadenopathy. ⁷⁷ Systemic presentations reflect organ involvement, ranging from skeletal (osteomyelitis, arthritis) to multi-organ disease (gastric and intestinal lesions, hepatitis, pancreatitis, renal abscessation, bladder wall infection, adrenal gland granulomas, generalised lymphadenomegaly).

Figure 9.

(a,b) A 6-year-old female neutered domestic shorthair cat in Hong Kong with large fleshy subcutaneous masses infiltrating the external ear canal caused by Cryptococcus neoformans. Images courtesy of Dr Edmund Cheung

Histoplasmosis

• ✜ Aetiological agents Histoplasma capsula-tum (H capsulatum var capsulatum, H capsula-tum var farciminosum, H capsulatum var duboisii).

• ✜ Ecological niche Soil, bat and bird guano harbour the organisms. ⁸³ Organic fertilisers and potted plant soil can also be sources of infection for indoor cats. ⁸⁴

• ✜ Geographic distribution H capsulatum is distributed worldwide, especially in tropical and subtropical regions. Hyperendemic areas include the Ohio/Mississippi River Valleys of South-central and Midwestern USA, Latin America (Figure 1), as well as some parts of Southeast Asia and India. ⁸⁵

• ✜ Transmission Airborne spores (micro-conidia) of the filamentous form are inhaled and deposited in the lungs, before transforming into yeasts.

• ✜ Clinical forms of disease Pulmonary or disseminated disease is seen, the latter most commonly involving infiltrates in the spleen, liver, bone marrow, eyes and gastrointestinal tract. ⁸⁶

• ✜ Clinical presentation Chronic and disseminated disease is common. Clinical signs are usually non-specific and frequently include lethargy, inappetence, weight loss and peripheral lymphadenomegaly, while fever is variably present.^31,86-88 Respiratory signs may include dyspnoea and/or tachypnoea (affecting ~50-60% of cases). ⁸⁹ Cough or nasal discharge is less common.^90,91 Ocular signs, including conjunctivitis, blepharitis (eyelid swelling), chorioretinitis, uveitis and/or optic neuritis, occur in up to 25% of cases.^40,92,93 Musculo-skeletal signs, including lameness and joint effusions, are present in up to 20% of cases.^28,31,89 Less common presenting signs include cutaneous nodules or ulcers (~15% of cases), diarrhoea (~10%), CNS signs (~5%) and/or jaundice. ⁸⁹ Clinical examination often identifies peripheral and visceral lymphadenopathy, along with cranial abdominal organomegaly.

Figure 10.

Diff-Quik stained smear from the left mandibular lymph node of a cat with cryptococcosis, showing sheets of round yeasts with a non-staining capsule and occasional narrow-necked budding (arrows). An inflammatory response with neutrophils and macrophages is also apparent. x 40 objective

Figure 11.

Diff-Quik stained smear from a mesenteric lymph node of a cat with disseminated histoplasmosis, showing numerous intracytoplasmic round yeast forms within macrophages. x 100 objective

Blastomycosis

• ✜ Aetiological agent Blastomyces dermatitidis.

• ✜ Ecological niche B dermatitidis is found in soil, especially near waterways in wooded areas in endemic regions. Infection can occur in indoor-only cats.97

• ✜ Geographic distribution The organism is endemic in the USA, Canada and Africa, especially around the Great Lakes and Ohio/ Mississippi River Valleys (USA), with recent evidence of expansion to New York State and Saskatchewan (Figure 1). ⁹⁸

• ✜ Transmission Airborne conidia (spores), 2-10 |jm in diameter, are inhaled and deposited in the lungs.

• ✜ Clinical forms of disease Disease may be pulmonary or disseminated.

• ✜ Clinical presentation Blasto mycosis is uncommon in cats, which are far less susceptible to infection than dogs. The disease is similar in clinical presentation to histoplasmosis, although dermatological involvement is more common and lymphadenomegaly less common.^48,97,99

Coccidioidomycosis

• ✜ Aetiological agents Coccidioides immitis and Coccidioides posadasii.

• ✜ Ecological niche Coccidioides species favour warm, arid to semi-arid regions in alkaline, sandy soil, growing especially after rains have abated but soil is still moist.

• ✜ Geographic distribution Distributed throughout the Americas, especially Arizona (~60% of all reported human cases), central/southern California (35% of cases), Texas, Utah, Nevada and New Mexico (Figure 1), these fungi are becoming more widespread in the USA, with, for example, recent spread to Washington. Isolated case reports of infection in China in people with no travel history to hyperendemic regions are increasing in frequency.^21,100

• ✜ Transmission Hot, dry conditions result in desiccation of hyphae, and their maturation and fragmentation to become infective arthro-conidia; these are dispersed in air and dust by wind, and inhaled. Arthroconidia may also be transported on fomites to cause infection.

• ✜ Clinical forms of disease Infections may be respiratory or disseminated, with frequent involvement of skin, eyes, bones/joints, CNS and/or viscera.

• ✜ Clinical presentation Der ma tological lesions (nodules, non-healing wounds, draining lesions or plaques), especially on the trunk and distal extremities, and/or respiratory signs (chronic cough, respiratory distress) are most common, often accompanied by nonspecific signs (fever, inappetence, anorexia, weight loss). Less commonly, lameness, ocular signs (chorioretinitis, uveitis, panophthal-mitis, retinal detachment) or CNS signs are _reported.^{30,41,42,101,102}

Figure 12.

A 5-year-old male neutered domestic longhair cat from Arizona, USA, presented with severe weight loss (50% body weight decline), anorexia, dehydration and progressive hindlimb paralysis. (a) A right lateral thoracic radiograph showed a cranial mediastinal mass (asterisk) and a lytic-proliferative osteomyelitic lesion of the T2 vertebral process (arrow). (b) Lytic and proliferative osteomyelitis of the body and vertebral process of L4, extending along the ventral edges to involve L3 and L5, was causing the paralysis due to compression of the spinal cord at L4. The cat was positive for coccidioidomycosis on serology, with an anti-Coccidioides IgG antibody titre of 1:64. Cytological samples were not available. In areas endemic for Coccidioides species, consistent clinical signs and positive antibody titres are an indication for antifungal therapy. This cat progressively improved after treatment with fluconazole, before being lost to follow-up 7 months after diagnosis

Sporotrichosis

• ✜ Aetiological agents Sporothrix schenckii species complex (S schenckii, S brasiliensis, S globosa, S luriei, S mexicana, S albicans).

• ✜ Ecological niche These fungi are found in decaying organic matter in soil or water, and dead plant matter (bark, wood, thorns, moss, etc).

• ✜ Geographic distribution S schenckii is distributed worldwide, especially in temperate, subtropical and tropical regions. S brasiliensis is confined to Brazil, Argentina, Chile, Panama and Paraguay (Figure 1).^103,104

• ✜ Transmission Routes of transmission include traumatic cutaneous inoculation of yeasts/conidia (via contaminated soil, plant foreign bodies [eg, splinters] or during fighting) and inhalation of conidia. Disease is mainly spread horizontally cat to cat through contaminated fight wounds (cat bites or scratches) in urban outbreaks in, for example, South America and Malaysia, where S brasiliensis and S schenckii, respectively, are hyperendemic.^26,105-107

• ✜ Clinical forms of disease Disease may occur as focal cutaneous, mucocutaneous and systemic forms.

• ✜ Clinical presentation Free-roaming entire male cats in urban areas are most commonly affected, presenting with multifocal, exudative, encrusted ulcers and ulcerated nodules or plaques, especially on the ear tips, nose, digits and tail base.^26,105 Mucosal involvement (nasal, conjunctival, oral and/or genital) occurs in over 30% of cases.^26,108 Nasal signs, especially sneezing, and peripheral lymphadenomegaly are common. Other ocular signs include serous discharge, blepharitis and/or uveitis. ¹⁰⁸ Mucosal and cutaneous lesions can extend to involve adjacent tissues, including muscle, cartilage and bone. Primary granulomatous conjunctivitis can also occur in the absence of other lesions, characterised by epiphora, con-junctival hyperaemia and conjunctival follicles/ nodules. ¹⁰⁹ Severe inflammation and thickening of the conjunctiva can produce a mass-like appearance. Haematogenous dissemination to multiple organs, especially the lungs and liver, has been described with S brasiliensis, which is more thermotolerant than S schenckii. ¹⁰⁵

Dermatophytic pseudomycetoma

• ✜ Aetiological agents Zoophilic dermato- phytes M canis and Trichophyton mentagro-phytes (rare).

• ✜ Ecological niche Cats and rodents are reservoirs of M canis and T mentagrophytes, respectively.

• ✜ Geographic distribution These pathogens are distributed worldwide.

• ✜ Transmission Infectious hyphal fragments (arthroconidia) are transmitted by direct contact with an infected animal or indirectly from a contaminated environment. Derma to -phytosis develops when arthroconidia adhere to the epidermis and germinate in the stratum corneum. ¹¹² Cutaneous dermatophytic pseudo-mycetoma occurs most commonly in cats with pre-existing dermatophytosis, after rupture of an infected hair follicle and release of fungal hyphae into the dermis. Contamination of the abdomen with infected hairs/hyphae or arthroconidia during surgical procedures (eg, neutering) can give rise to abdominal dermatophytic pseudomycetoma. ¹¹³

• ✜ Clinical forms of disease Cutaneous disease is most common, but disease may also be extracutaneous.

• ✜ Clinical presentation Persian cats, especially spayed females over 3 years of age, are predisposed.^114-124 Genetic studies have revealed that the Persian breed has multiple single nucleotide polymorphisms (SNPs) in a gene encoding the anti microbial peptide calprotectin. These SNPs are thought to be associated with susceptibility to dermatophytosis. ¹²⁵ The cutaneous form of disease is characterised by one or more soft-to-firm nodules or plaques, which may be ulcerated (Figure 13) and sometimes contain variably light-to-dark tissue grains. Depending on chron-icity, lesions range from a few millimetres to over 10 cm in diameter, and can occur anywhere on the body.

Figure 13.

Dermatophytic pseudomycetoma in a 6-year-old British Shorthair cat in Hong Kong. The cat, which had a chronic history of dermatophytosis caused by Microsporum canis, developed ulcerated masses on the tail initially, and subsequently on the trunk. Image courtesy of Dr Edmund Cheung

Occasionally, regional lymph nodes draining primary lesions are involved. ¹ Extra-cutaneous disease may also include abdominal, nasal and oral locations.^{1,33,113,114,126} Large abdominal masses have been diagnosed months to years after ovariohysterectomy, including a uterine stump mass in one cat, ¹¹⁴ and disseminated abdominal dermatophytic pseudomycetoma in another. ¹¹³ Clinical signs in affected cats included constipation, tenes-mus and dysuria. Invasive fungal rhinitis with oral cavity involvement, caused by M canis, was reported in an adult cat. ¹²⁶

Aspergillosis

• ✜ Aetiological agents

Sino-orbital aspergillosis (SOA) - cryptic species (that look alike morphologically) in Aspergillus section Fumigati, especially A felis and A udagawae.

Disseminated invasive aspergillosis (DIA) and invasive pulmonary aspergillosis (IPA) - species in Aspergillus section Nigri and Asper-gillus section Fumigati.

• ✜ Ecological niche These organisms thrive in organic matter in soil.

• ✜ Geographic distribution They occur worldwide.

• ✜ Transmission Disease is tranmitted by inhalation of airborne fungal spores (conidia).

• ✜ Clinical forms of disease Invasive fungal rhinosinusitis, typically SOA, is most common; DIA and IPA are uncommon. Sinonasal aspergillosis, which is usually a non-invasive form of fungal rhinosinusitis, ²³ is outside the scope of this review.

• ✜ Clinical presentation Persian cats and related breeds, including the Himalayan, Ragdoll and British Shorthair, are predisposed to SOA. Diabetes mellitus, a risk factor for invasive forms of aspergillosis in humans, has been reported in some cats with SOA and IPA.^127,128 Similar to mucormycosis, cats with DIA are usually immunocompromised (eg, from feline infectious peritonitis [FIP], FeLV infection, pan-leukopenia, immunomodulating drugs). ²³

Cats with SOA are usually presented with periorbital swelling; inhaled conidia deposited in the sinonasal cavity germinate into hyphae, which invade the sinonasal mucosa causing granulomatous rhinitis, osteolysis of the orbital bone and spread of infection into the orbit. Occasionally, cats present when infection is still confined to the nasal cavity, but clinical signs at presentation are typically associated with progressive expansion of a ventromedial orbital fungal granuloma, and include unilateral exophthalmos, conjunctival hyperaemia and discharge, nictitating membrane prolapse, central corneal ulceration, an oral mass or ulcer in the ipsilateral pterygo-palatine fossa and mild mandibular lymph-adenomegaly (Figure 14). If nasal signs (eg, sneezing, discharge) are not detected at presentation, there may be a history of these over the previous 6 months. As disease progresses, exophthalmos may become bilateral and other signs may occur, including paranasal soft tissue swelling (Figure 14), which can cause severe facial distortion, and CNS signs, including seizures, nystagmus, circling, facial muscle fasciculation, hyperaesthesia and blindness. Haematogenous or lymphatic dissemination to medial retropharyngeal lymph nodes has been described in association with a ventral cervical mass in two cats.^129,130

Figure 14.

Sino-orbital aspergillosis caused by Aspergillus fells infection in a 2-year-old neutered male domestic shorthair cat in Sydney, Australia. (a) The cat has exophthalmos, third eyelid prolapse and conjunctival discharge in the right eye, as well as right nasal discharge and facial distortion. (b) These signs are associated with a right orbital mass that has invaded the oral cavity, causing a bulge in the right pterygopalatine fossa (arrow)

DIA in immunosuppressed cats has been described as presenting with non-specific signs, including lethargy, anorexia, weight loss and fever. Lung and gastrointestinal involvement is most common. ¹³⁰

Dematiaceous fungal infections (phaeohyphomycosis)

• ✜ Aetiological agents There are multiple genera of melanin-pigmented dematiaceous fungi, the most common being Alternaria, Cladophialophora, Microsphaeropsis, Lomento-spora and Exophiala species.^131-134

• ✜ Ecological niche These fungi can be found in soil, plants (as endophytes or pathogens) and even transiently on feline skin (Alternaria and Cladophialophora species). ¹³⁵

• ✜ Geographic distribution While being distributed worldwide, there are regional differences in feline infections; for example, Alternaria species are most common in the UK and Europe, ¹³² and Microsphaeropsis arundinis is most common in Australia.

• ✜ Transmission Disease is transmitted via traumatic cutaneous or ocular inoculation, or inhalation, with subsequent haematogenous dissemination.

• ✜ Clinical forms of disease Cutaneous disease is most common (Figure 15), but rhinosinusitis, and pulmonary and disseminated forms of disease also occur.

• ✜ Clinical presentation Male outdoor-roaming cats are at highest risk, but infection can occur in cats without outdoor access (eg, via inhalation). Skin lesions are single or multifocal nodules, plaques or draining tracts/non-healing wounds, especially on the pinnae, digits or nasal planum. Lesions may be poorly circumscribed and non-pigmented or dark/purplish in appearance due to melanised fungal cell walls. Multifocal lesions indicate haematogenous dissemination. Clado -phialo phora species are neurotropic and cause CNS signs often referable to cerebral or cere-bellar disease, such as seizures, circling, obtundation, ataxia and tremors. Focal lung lesions are also possible; a cat presenting to the University of Sydney with a cough was found to have a single pulmonary granuloma caused by Clado phialo phora bantiana, with no other organ involvement. ⁴⁷

Figure 15.

A 14-year-old female neutered domestic shorthair cat that presented with a subcutaneous mass beneath the right eye, which was identified as a pigmented mould infection. (b) On CT examination of the head, the mass was found to have invaded the right orbit

Mucormycosis and entomophthoromycosis (both previously known as zygomycosis)

• ✜ Aetiological agents Pathogenic moulds including Entomophthoromycetes (eg, Coni -diobolus species), Basidiobolomycetes (eg, Basi-diobolus species) and Mucoromycetes (eg, Mucor, Rhizomucor, Rhizopus and Cokeromyces species) (Table 1).

• ✜ Ecological niche Basidiobolus and Conidio-bolus species are soil saprophytes found in association with plant debris and are also gut commensals of amphibians, fish, reptiles and insectivorous bats.^136,137 Most other Entomoph-thoromycetes are obligate insect parasites. Mucoromycetes occur in diverse environments, including soil, water and air. Mucor species have also been detected as contaminants on healthy cats' skin, but are not commensals. ¹³⁸

• ✜ Geographic distribution Some of these moulds are ubiquitous and occur worldwide; others (eg, Basidiobolus and Conidiobolus species) occur in tropical and subtropical regions, including Africa, Asia, Australia and South America. ¹³⁶

• ✜ Transmission Disease is transmitted by inhalation, ingestion or inoculation of fungal spores through trauma or insect bites. ¹³⁹

• ✜ Clinical forms of disease Gastrointestinal, pulmonary, mucocutaneous and disseminated forms of disease are recognised.

• ✜ Clinical presentation Entomophthoro-mycetes generally cause localised cutaneous or nasopharyngeal infections in immunocom-petent hosts. ¹⁴⁰ Conidiobolomycosis and basidiobolomycosis have been reported in dogs, but not to date in cats. Mucoromycetes are typically associated with acute, rapidly progressive disease in immunocompromised hosts; for example, peritonitis caused by Cokeromyces recurvatus was reported in a cat with intestinal lymphoma-associated jejunal perforation. ¹⁴¹ Disseminated mucormycosis has been diagnosed in cats with concurrent FIP, panleukopenia or FeLV infection. ¹⁴² Mucor-mycosis has also been described in cats with no known comorbidities. These have included a 14-year-old domestic shorthair cat with a subcutaneous nasal mass; a 2-year-old Siamese with tracheobronchial infiltration, presenting with severe dyspnoea; and two Persian cats, both under 1 year of age, with severe nodular and ulcerative gastroduodenal infiltration, one presenting with weight loss and acute haema-temesis, and the other with weight loss, fever and abdominal distension.^143-146

Oomycoses (pythiosis, lagenidiosis and paralagenidiosis)

• ✜ Aetiological agents Oomycete protists in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including Pythium insidio-sum, Lagenidium species and Paralagenidium species. Their cell walls contain cellulose and beta-glucans but, unlike fungi, they do not contain chitin, nor do their cytoplasmic membranes contain ergosterol. ¹⁴⁸

• ✜ Ecological niche These organisms are associated with soil and standing bodies of fresh water.

• ✜ Geographic distribution Oomycetes causing feline IFLIs occur in tropical and subtropical regions, as well as some temperate regions such as California and Arizona. Feline pythiosis has been reported in the US Gulf States (in particular Louisi-ana),^149,150 Brazil^151-154 and Spain. ¹⁴⁵ The authors have also diagnosed cases in Hong Kong. Reports of pythiosis in dogs in Australia (Northern Territory, Queensland and New South Wales),^155-157 Thailand and Ma lay sia reflect the endemic presence of oomycetes.

• ✜ Transmission In surface water, oomy-cetes produce motile biflagellate asexual zoo spores with marked chemotactic attraction to decaying plants and to epithelial surfaces of vertebrate hosts. Infection is the result of encys-tation and invasion of damaged skin or gastrointestinal mucosa.

• ✜ Clinical forms of disease Cutaneous/ subcutaneous disease (Figure 16) is common, while gastrointestinal disease is uncommon. Overall, IFLIs are less common in cats than in dogs. Disease can occur at any age but cats under 12 months of age appear to be predisposed to cutaneous disease. ¹⁵⁰ No other risk factors have been identified.

• ✜ Clinical presentation

• Cutaneous/subcutaneous - Subcutaneous masses are seen, especially in the neck, trunk, inguinal and perianal areas, nasopharynx and / or sino-orbitally. Sometimes these lesions are ulcerated with multiple fistulous tracts. They also occur at the tail base or extremities. Perianal lesions may present as a subcutaneous mass encircling the anus and can extend intrapelvically.

• Gastrointestinal pythiosis - Small intestinal masses develop, with mesenteric lymph node involvement. ¹⁴⁴ , ¹⁴⁹ , ¹⁵² , ¹⁵⁴ Presenting signs include combinations of anorexia, vomiting, haema-temesis, diarrhoea and weight loss. Palpable abdominal mass(es) may be present and, owing to the insidious course of disease, may be very large (>10 cm diameter) at presentation.

Figure 16.

(a) An 8-month-old female domestic shorthair cat, which had been adopted as a stray in Hong Kong, developed firm, progressive swelling of the right hind leg. (b) A CT scan taken by the primary veterinarian showed diffuse subcutaneous tissue infiltrates with increased soft tissue attenuation. (c) The affected tissue was surgically debulked at the time that diagnostic biopsies were collected. Histopathology revealed granulomatous inflammatory tissue containing irregular fungal hyphae. These were identified by PCR as Pythium insidiosum. Images courtesy of Dr Edmund Cheung

Key Points

• ✜ Invasive fungal and fungal-like infections in cats most commonly present with cutaneous, subcutaneous and/or respiratory signs, but in disseminated disease can have a variety of different clinical presentations, depending on organ involvement.

• ✜ These infections should be on clinicians' diagnostic radars and can be definitively diagnosed using a combination of different techniques that are readily accessible in clinical practice.