diagnostic testing for fungal diseases

Diagnostic testing for fungal diseases

Robin F. Lane, DVMAssociate Veterinarian, Animal Hospital of High Park, Toronto, Ontario, Canada

Fungi are eukaryotic organisms (with a nucleus) similar to animal cellsalong with algae, higher plants, and protozoa. Unlike animal cells, fungihave rigid cell walls, and unlike plant cells, fungi lack chlorophyll. Morethan 200,000 species of fungi have been characterized. Most fungi performan essential function of returning nutrients back to the soil from plants anddigesting various biological materials like cornified epidermis (hair, horns,and nails) found in the environment. There are a large number of fungalspecies that are plant pathogens, with only approximately 200 species iden-tified or recognized to be pathogenic to animals and humans [1]. TheKingdom Fungi has its own specific eukaryotic physiology that makes fungiunique in their pathogenesis and drug resistance.

Saprophytic fungi are ubiquitous and normally found in the environment(soil, water, and plants). Saprophyte fungi can cause disease. Although animmunocompetent animal’s immune system can clear saprophytes, animmunosuppressed animal allows a saprophyte to invade tissues. In thesecases, saprophytes are known as opportunistic fungi, with a possible fataloutcome [2].

Dimorphic fungi are highly zoonotic [2] capable of causing disease in theimmunocompetent host. These fungi are present as molds in soils, enteringby way of the respiratory tract and becoming invasive yeast at the highertemperature found in the animal or human host [2]. Most dimorphic fungiare restricted geographically and also are known as endemic fungi andsystemic deep mycoses [2]. Most people exposed to these fungi do notbecome ill, and of those who become ill, less than 10% develop extra-pulmonary dissemination or systemic deep mycoses [2].

Animal Hospital of High Park, 3194 Dundas Street, West Toronto, Ontario M6P 2A3

Canada.

Robin F. Lane, DVM, is a part-time paid consultant for Antech Diagnostics.

E-mail address: [email protected]

1094-9194/03/$ - see front matter � 2003, Elsevier Inc. All rights reserved.

doi:10.1016/S1094-9194(03)00010-0

Vet Clin Exot Anim 6 (2003) 301–314

In animals, the medically important dimorphic fungi are Blastomycesdermatitidis, Coccidioides immitis, and Histoplasma capsulatum [3,4]. All threeof these are also endemic fungi [2].

Despite much advancement in laboratory diagnostics, mycology is stillmainly a visual science. Routine fungal identification depends onmacroscopicand microscopic morphology under defined media and conditions, and onbiochemical testing [1]. Fungi have two basic morphological forms known asyeast and molds. Yeast are single-celled, rounded and propagate by way ofbudding. Molds are multinucleated and grow in long filamentous patternsknown as hyphae. Mycelia are a mass of hyphae.

Some fungi are found only as molds, as seen by the hyphae of Aspergillusspecies. Most fungi grow as yeast and mold in different parts of their lifecycle, and some fungi, like Candida albicans, have a third morphologicalform known as pseudohyphae [5].

Fungi may cause disease in any animal or person. Mycotic infectionsin animals are diagnosed infrequently compared with bacterial infections [6],which may be in a small part because of the difficulty of accurate fungaldisease diagnosis. A relatively large number of birds are diagnosed withmycotic infections [7–10]. This is possibly because of the increased use ofantibiotics in sick birds, allowing opportunity for saprophytes to flourish[10]. Small mammals housed indoors rarely develop mycotic infections otherthan dermatophyte (ringworm) infection [11,12]. In reptiles, most fungalinfections are caused by saprophytes, which may be fatal in the immuno-suppressed host [13,14].

In people, severe and fatal mycotic infections are increasing rapidlybecause of immunosuppressive viral infections, immunosuppressive thera-pies in organ transplant and cancer patients, and long-term antibiotic andcorticosteroid use [1,2,15]. Often veterinary medicine follows the progressin the human medical field, especially in the arena of diagnostics andmedications [16,17–20].

In general, any immunosuppressed animal is susceptible to fungalinvasion and disease [21,22], and fungal infection should be considered incases that are nonresponsive to broad-spectrum antibiotics. Ferrets treatedwith long term corticosteroids for lymphoma or insulinoma may becomeimmunosuppressed and develop mycotic disease [23].

Diagnosis of a fungal or mycotic infection is not as simple as culturingand identifying the presence of fungi [24]. Often fungi can be identifiedreadily, yet clinical signs, and therefore disease, may not be present. Evenwhen clinical signs are present, isolation of a fungi alone cannot assure thepractitioner of its pathogenicity [25].

The use of serology and polymerase chain reaction (PCR) amplificationmust be viewed as auxiliary, not primary diagnostic aids [26]. As in mostother diagnostics, the focus is on the patient, and the laboratory data areused to guide the practitioner.

302 R.F. Lane / Vet Clin Exot Anim 6 (2003) 301–314

General survey testing

Many times, the practitioner investigates mycotic diseases after there is noresponse by the patient to broad-spectrum antibiotics. Nonspecific changesseen in avian patients include a persistent leukocytosis [27] with a heterophiliaor monocytosis, yet there may be no hematological changes [28]. Anemia hasbeen associated with chronic inflammatory diseases like aspergillosis [29].

There has been an increase in the use of protein electrophoresis asa survey test in psittacines. This is the most reliable way to assess the majorprotein groups in avian species [30–32]. A recent study found that proteinelectrophoresis changes were the most consistent change in psittacines withaspergillosis [33].

With advanced cases of mycotic pneumonia, there may be pulmonary orair sac granulomas [23,34,35]. Survey radiographs of nasal cavities, lungs,and sinuses are used in localizing cryptococcal granulomas in marsupials [36].

Magnetic resonance imaging (MRI) has limited availability to theveterinary practitioner, yet it is a powerful imaging tool that has been shownto evaluate the brain, the eye, and abdominal cavity in birds [37]. It canassist in confirming and localizing fungal and other granulomatous lesionsin avian chronic sinusitis [38]. CT and MRI have major limitations in theirexpense and inaccessibility to the veterinary practitioner. Similar to MRI,but superior for bony evaluation, CT scans may have a role in aiding withdiagnosis of mycotic infections of sinuses in the avian patient [9,39]. Inpeople, MRI and CT scans have been useful in diagnosing fungal infections,because the extension of pathology across tissue planes strongly suggestsa fungal etiology [2].

Endoscopy allows direct visualization and sampling of mouth, trachea,upper gastrointestinal (GI) tract, upper respiratory tract, air sacs, and avianabdominal cavity [40–42].

Fungal testing methods

Many fungi species may be visualized with simple equipment and supplies,yet the practitioner must accurately interpret laboratory data to ensure thedata confirm an infection in the patient, and not a contaminant [4,10].Occasionally, an animal may be carrying potentially pathogenic fungi such asdermatophytes without obvious disease, and it is not understood fully whenthese animals will be a source of a zoonotic disease to other animals [11].

Gram stain

The gram stain can provide general morphology and information onnumber of yeasts, shape, size, presence and type of budding, and capsulethickness. Samples may be collected from skin, oral cavity, cloacal and fecalsamples. Yeasts are seen as round to oval dark blue-to-black organisms (3 to

303R.F. Lane / Vet Clin Exot Anim 6 (2003) 301–314

6 lm) usually 5 to 10 times larger than other gram-positive cocci (0.5 to1 lm) [4]. Fungal hyphae and megabacteria also may be identified [43]. Ongram stains, megabacteria are seen as large (approximately 90 lm) grampositive rods [21,44].

The limitations of gram stains include mistakes in stain protocol [45],possible confusion mistaking large gram-positive cocci for yeast [43], andthat hyphae do not stain uniformly [2].

Cytology

An advantage of cytology is the use of simple staining methods with morecellular detail to assess the presence of inflammatory cells [4,46]. Samplescan be collected from fine needle aspirates, impression smears, tracheal andlung washes, and nasal and sinus flushes [13,47,48]. In the avian patient,glass slide impressions of the skin have been described [28].

Dry smears can be stained with new methylene blue and Romanovsky’stype of stains (Wright’s) [4,10]. Slides of wet material can be stained withlactophenol cotton blue [10] and India ink [2]. On cytology, the finding ofseptate hyphae and conidia can support a diagnosis of Penicillium cyclopium[25], while budding yeast and hyphae can be seen with candidiasis [8]. Theinvasive yeast forms of dimorphic fungi have characteristic features, makinga presumptive diagnosis possible from cytology [2]. The finding of inflam-matory cells is seen with an active disease state in many, but not all,fungal infections [4].

Wood’s ultraviolet lampThis procedure can be useful; however fewer than 50% of M. canis

fluoresce, and none of the Trichophyton species fluoresce [12].

Potassium hydroxideA 10% to 20% solution of potassium hydroxide (KOH) dissolves most of

the organic matter and allows direct visualization of dermatophyte fungalelements in hair and skin scrapings [2,3]. This test has variable results [12];one reason may be that the fungal elements are difficult to visualize withouta contrasting dye like lactophenol cotton blue or blue-black ink [2].

Dermatophyte mediaCommercially available media allow for growth of and distinction

between other fungal contaminants and the dermatophytes Microsporumand Trichophyton species by using a color indicator in the media [1,4,49].These media are considered a nonspecies-specific test [49].

Serology

There are a limited number of antibody and antigen tests available toassist in the diagnosis of specific mycotic infections.


Antigen testsAntigen tests are commercially available for aspergillosis and cryptoco-

ccus [15,36,50]. Because antigen tests reveal only that the antigen of thetarget organism has been found, this test should not be used alone todiagnose a fungal infection. The advantage of antigen tests is they area nonspecies-specific test that can be used on any species. One considerationis whether the antigen selected by the test is present in different host speciesbecause of the possible different host and fungal interactions. Combinedwith other supporting laboratory data, the presence of specific fungalantigens can enhance the practitioners’ accurate diagnosis of a specificfungal disease. Decreasing titers may be useful for monitoring response totreatment [36], yet in cases without supporting evidence of clinical signs andlesions, leukocytosis, radiographic changes, or cytological and histopatho-logical evidence of fungi, a positive antigen test is not diagnostic for a fungalinfection.

Antibody testsAntibody tests are an indication of exposure and host response, not

necessarily of disease. Antibody tests are commercially available for avianaspergillosis [31,51,52], blastomycosis [4,50], and coccidioidomycosis [4].These tests depend upon the host’s functional immune system to producedetectable levels of antibodies for a long enough time frame for detection. IgGantibody tests detect antibodies that may persist for months to years, whereastests for IgM detect antibody detect levels that may last only for days toweeks.With antibody tests, a negative result is not proof that the host has notbeen infected, since a poor immune response, declining antibody levels, andtesting before seroconversion will all give a false negative antibody test resultsin an infected host [26]. Validation and sensitivity of many of the com-mercially available antibody tests have not been completed, yet ingeneral if the test is based on a direct interaction between the organism’santigen and the host’s antibody, then the antibody test is useful.Methodologyproblems with antibody testing occur when secondary antibodies are neededfor detection of the host antibody. Such issues are seen with the indirectfluorescent antibody (IFA) and antibody ELISA tests [26].

Polymerase chain reaction tests

The amplification process of PCR theoretically allows for one nucleicacid sequence to produce close to one million identical nucleic sequences,thus producing macroscopically detectable levels of DNA. These DNA-based tests have the highest sensitivity and specificity of all the commonassays used to diagnose infectious diseases, yet these tests detect only thepresence of target nucleic acid, not viable organisms. Recently there havebeen many labs offering a wide range of infectious disease testing usingPCR-based technology. Validation of PCR-based assays is of major


significance, and not to be underestimated. Depending on the sequence ofDNA amplified during the PCR reaction and whether there is backgroundcontamination, a positive test result may be useless. When PCR-basedtesting is interpreted incorrectly, the technology can be detrimental [26,52].

Histopathology

The most definitive diagnosis of a mycotic infection is with histopathol-ogy demonstrating the invasion of tissues by fungal elements [4,10,25,53].

Biopsies are used in the antemortem diagnosis of fungal diseases froma variety of sites. Modern endoscopy allows access and biopsy samplingfrom a number of internal organs [40], including reptilian lung biopsies [54].

There are many stains used in histopathological examination of affectedtissues to specifically identify fungal elements. These stains can be added toexisting histopathological sections without the need to resubmit or resec-tion tissues. Periodic acid-Schiff (PAS), Bauer’s and Gridley, Grocott’sstain, Gomori methenamine silver stain are among available fungal stains[7,10,53,55].

Culture

Culture is the only diagnostic test that detects viable organisms [26].Many fungi have two different names because different names were givento the sexual and asexual forms of the same fungi. In culture, often onlythe asexual form is obtained and identified [1]. Yeasts are identified bymicroscopic morphology and biochemical tests [1]. Molds generally areidentified by macroscopic colony characteristics and microscopic details [2].

Samples from various sites, including skin, oral, fecal, sinus and lungwashes, and internal organ sampling can be cultured for fungi [13,24].

Media selection for fungal culturing is not as specific as bacterial culture,and most fungi will grow on routine bacteriological media. Fungal culturingstarts with a general media plate like Sabouraud’s agar; then selective mediaare used to remove the background or ubiquitous fungi. There are a largenumber of media, with and without various additives and antibiotics,available for fungal culture [1,24]. For example, selective media are neces-sary for dermatophyte isolation because of the large amount of nonpatho-genic background fungi present on the skin.

The practitioner should inform the laboratory when a specific fungus issuspected, especially among the zoonotic dimorphic fungi, as this will helpthe laboratory select the appropriate culture media. The optimal incubationtemperature of fungi is between 25�C and 30�C. For reptilian fungalcultures, an incubation temperature of 20�C to 24�C has been suggested[14,21,24]. Candida and Aspergillus species grow quickly, and colonies can beseen in 1 to 5 days [2]. Many other fungi species have slow growth taking5 to 6 weeks to appear [2].


A positive fungal identification along with the source of the sample mayaid the practitioner in determining if the organism is capable of causingdisease. Accurate diagnosis requires the patient to have clinical signsconsistent with the disease. For example, culturing Aspergillus species maybe of no significance in a bird with no clinical signs [10,55]. Persistent orrecurrent lesions with repeated isolation of the same organism from thelesion increases the likelihood of the pathogenic nature of the fungi [25].

Zoonotic issues with the dimorphic fungi should not be underestimated.With the exception of the dermatophyte media, the private practitionershould avoid in-hospital fungal culture. With these dimorphic fungi, onlythe yeast grows at the usual incubator temperatures, and these structureswhile invasive, are not contagious. Still, when the plates are discarded, thecooler temperatures allow the mold form to develop, and these molds arehighly contagious and zoonotic.

Specific fungal diseases

Candidiasis

There are many Candida species, which gain entry to the body by wayof nonrespiratory routes [2]. Candida species can be normal inhabitants ofthe GI tract, often seeing one organism per high power field (40 timesmagnification) [8,49,70].

Cytology and gram stains reveal Candida species as small (3 lm to 6 lm)oval budding yeast with a narrow base [10,47,56]. C. albicans infections mayhave hyphae and pseudohyphae in addition to the yeast form [1,2,47].

In bird crop infections, Candida species can be a primary pathogen or actas an opportunistic invader [10,24]. In birds, the presence of hyphae is rare,and is a sign of invasive fungal disease [10,47]. Patients with lesions orclinical signs suggestive of candidiasis, with increased number of yeast oncytology or gram stains with inflammatory cells, often are given a tentativediagnosis of candidiasis and started on treatment [10,29,47].

Candidiasis rarely is documented in animals other than birds; howeverCandida species cause extensive disease in immunosuppressed people,ranging from simple mucosal colonization to fatal multiple organ invasion[2]. The author has cultured C. albicans from dermatological lesions ofa guinea pig’s upper lip frenulum, and in reptiles, Candida species have beencultured from hepatic and pulmonary lesions [57].

Culture without lesions simply confirms the presence of viable yeast, andwhen possible, a biopsy with histopathology of affected tissue is re-commended [10,29,47].

Aspergillosis

Aspergillus species are opportunistic fungi found in soils as a mold. Thereare over six hundred Aspergillus species, with less than 12 species known to


cause infection in people [2]. Aspergillus fumigatus is the most commonisolated species with human and animal infections, with A. niger, and A.flavus isolated less frequently [2,10,55].

On gross exam, visible green masses are associated with A. fumigatusand brown masses with A. niger [4] . On cytology, Aspergillus species arecomposed of tubular filaments with regular septa and irregular branchingat a 45� angle [4]. If present, the fruiting body has elongated spores givingthe characteristic ‘‘holy water sprinkler’’ from which came the nameAspergillosis [1,55].

Infection in animals is usually caused by overwhelming numbers ofinhaled spores in an immunosuppressed patient [10,35,55]. Infection isrelatively common in birds [10,28,35,55], less common in reptiles[6,13,14,34,57,58], and rare to absent in small mammals.

Clinical signs and lesions and supporting diagnostic test results should helpto confirm active aspergillosis. To tentatively diagnose a case of aspergillosis,there should be at least two to three of the following signs: a persistentleukocytosis, abnormal plasma electrophoresis (EPH), radiographic gra-nulomatous changes, clinical signs, and elevated Aspergillus serological titers[24,31,32,52,55]. Endoscopic examination with cytology and culture of avianair sacs often is needed to finalize a diagnosis of aspergillosis [10,35,52].

Mycotoxins

Various species of fungi, including Aspergillosis and Fusarium species,which grow as molds on grain crops and food are capable of making a varietyof mycotoxins [59–61]. In birds, mycotoxins are not reported commonly andare difficult to confirm. Usual sources of mycotoxins are from nuts, seeds,and other foods that have become moist, allowing mold proliferation andmycotoxin production [59]. A limited number of mycotoxins can be identifiedfrom foods and stomach contents [4,59–61]. Dry samples should be sub-mitted in paper or cloth bags to avoid mold growth.

Megabacteria

Once thought as a bacterium, this organism recently has been identifiedby PCR-based technology as a fungus and given the proposed name AvianGastric Yeast [62]. This fungus has been reported in free-ranging andcaptive psittacines, passerines, ratites and gallinaceous birds [44].

Although PCR-based technology definitively identified this organism asfungal, the antemortem diagnosis of megabacteria is based on wet mountsand gram stains of fresh droppings [63]. Gram stains reveal a large(approximately 90 lm) gram-positive rod [24,44]. In the budgie, the presenceof organisms indicates infection, but because of intermittent shedding, thereis no correlation between numbers of organisms on the fecal wet mount anddegree of infection [63].


Dermatophytosis

Common dermatophytes (ringworm) include the fungi Microsporumcanis, M. gypsem, and Trichophyton mentagrophytes. Dermatophyte specieshave zoonotic potential [10,11,13,57,64]. Dermatophytes occur as anunusual parasite of the skin of mammals, birds and reptiles [10,12,14,25,54,55,57].

Microsporum species fluorescence with a Wood’s ultraviolet lamp hasbeen demonstrated in reptiles [22], yet fluorescence is not a consistentfinding [12,22], and T. mentagrophytes does not fluoresce [12].

Culture is reported to be more sensitive than KOH preparations[3]. The results from KOH preparations have been variable [12]. Com-mercially available dermatophyte media using a color indicator will identifyMicrosporum species and Trichophyton species [1,4]. These media arenonspecies-specific and have grown dermatophytes in several mammals [64].

Fungassay (Blue Ridge Biologicals, Inc., Hickory, NC), DermatophyteTest Media (Pittman Moore, Mundelein, IL),and Derm Duet (Bacti Lab,Mountain View, CA) dermatophyte media are available for the hospitaltesting. These may be useful in reptiles and birds. Fungal species other thanMicrosporum species and T. mentagrophytes require culturing at a commerciallaboratory for isolation and identification.

Skin biopsies and histopathology have demonstrated invading fungalelements in ferrets [12], birds [25], and reptiles [54], and these are diagnosticin any species.

Blastomycosis

Blastomyces dermatitidis, a rare endemic fungus, is restricted geograph-ically to the Midwest, and along United States–Canada border from theGreat Lakes to the east coast [2]. It rarely has been isolated from soil, withreports of outbreaks in people following exposure to decaying wood inmoist environments [2].

Cytology has characteristic broad-based budding and large sized yeaststructures (8 lm to 20 lm) [4].

Blastomycosis is an uncommon mycotic disease in animals. Affectedferrets have pulmonary changes on radiographs, with diagnosis on cytologyand culture of lung aspirates or tracheal washes [23,65].

Coccidiodiomycosis

Coccidioides immitis, an endemic fungus, is restricted geographically tothe arid, sandy, alkaline soils of the southwestern United States andendemic areas in South America [2]. Cytology reveals characteristic large-sized (20 lm to 80 lm) spherules and small internal endospores (2 lm to5 lm) [2,4].


Coccidiodiomycosis is a rare disease reported in ferrets [23,65] andreptiles [58] with exposure to soils of endemic areas. Widespread dis-semination to a variety of organs occurs frequently [2,23]. Osteomyelitisis common. Radiographic lesions with confirmation on cytology of aspiratesis diagnostic [23,65].

Histoplasmosis

Histoplasma capsulatum, an endemic fungus, is restricted to specificgeographic areas, especially areas fertilized by bat or bird droppings. H.capsulatum has been recovered from the soil along the Ohio, Missouri, andMississippi River valleys, and along United States–Canada border from theGreat Lakes to the east coast, including areas of northern Florida andeastern Texas [2,66].

Cytology reveals this small budding yeast (2 lm to 5 lm) in macrophagesfrom any body fluid [4]. The small size yeast may be missed on cytology, andthe morphology of H. capsulatum can be confused with C. (Torulopsis)glabrata [1,2].

There are limited reports of histoplasmosis in reptiles [58] and birds[10,66]. Antemortem diagnosis is challenging and based on cytology andhistopathology [10], with culture being less reliable than biopsy in dogs andcats [3]. In cats, bone marrow and buffy coat smears yield the mostconsistent diagnosis [4].

Cryptococcus

This fungi is found in the soil universally. There are two varieties of thefungi. The first variety is Cryptococcus neoformans var. neoformans, which isan opportunistic pathogen [2,49]. This is the most common cause of fungaldisease in people with immunosuppression [2]. The second variety, C.neoformans var. gatti appears to be a primary pathogen, which grows only inthe soils under Red Gum eucalyptus trees in Australia and other tropicalareas including southern California [5,64].

On cytology, the characteristics are thick-walled mucoid yeasts (5 lm to20 lm) with narrow based budding. These yeast often are seen with littleinflammation, and culture is seldom necessary [3].

There are many reports of cryptococcosis in marsupials in Australia[5]. Cryptococcosis is a rare disease in ferrets [23,65], birds [10,55], andreptiles [58]. Antemortem diagnosis in birds and reptiles is challenging[10,34,55].

The cryptococcal antigen latex agglutination test (CALAS) is a highlysensitive and specific antigen test that diagnoses cryptococcosis. This testcan be run on body fluid samples, including serum, plasma, and cere-brospinal fluid [15,36]. Both varieties of C. neoformans can be detected bythe CALAS test [15]. This antigen test may be useful in evaluating avianand reptile patients for cryptococcosis.


It is reported that in marsupials [36], cats, and dogs [3] that thecryptococcus serum titers usually correlate with the severity of the disease,and decreasing titers are indicative of treatment success and prognosis [3,36].

Pneumocystosis

Once considered protozoa, Pneumocystis carinii recently has beenclassified as a fungus [67] that commonly infects the lungs of people withimmunosuppression [2]. Cytology reveals a small, extracellular, nonbuddingyeast 5 lm to 8 lm in size [2]. In animals, pneumocystosis often is found inthe wild shrew in California [67] and has been reported in immunosup-pressed ferrets on long-term corticosteroids with diagnosis on cytology fromtracheal lavages [23].

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