56 Laboratory Diagnosis of Fungal and Algal Infections

Infectious Diseases of the Dog and Cat, 3rd Edition

CHAPTER 56 Laboratory Diagnosis of Fungal and Algal Infections

Spencer S. Jang

Richard L. Walker

Specific diagnosis of fungal and algal infections in animals requires laboratory procedures that include direct

microscopic examination and culture, frequently supported by serologic tests. Many such direct examinations and

primary cultures and some serologic tests are now well within the scope of in-office diagnostic procedures for

veterinary practice. The development of improved methods for mycologic diagnosis is directed at rapid procedures
using prepackaged identification kits and reagents, serologic kits, automated systems, and molecular techniques.

a high proportion of such cases, confirmation by a specialty laboratory will still be necessary to establish a definitive

diagnosis. Refer to

Appendix 5

, Laboratory Testing for Infectious Diseases of Dogs and Cats.

SPECIMENS FOR LABORATORY DIAGNOSIS

A satisfactory sample should be representative of the focus of infection and of adequate size to permit direct

examination and culture. Except in systemic infections suggesting fungemia and requiring blood culture, samples

should be obtained from the site of infection as indicated by lesions, signs, or symptoms. Because systemic

mycoses are usually acquired via the respiratory tract, lung tissue or airway exudates are preferred samples. In

certain disseminated mycotic infections, urine may also be an appropriate specimen to culture.

SAMPLE COLLECTION

When collecting skin scrapings for dermatophyte culture, clean the lesion, particularly the periphery, with 70%

alcohol. Avoid using iodine, which is harmful to dermatophytes. Surface antisepsis, when feasible, aids in

minimizing the collection of environmental bacterial and fungal contaminants and thereby ensures a meaningful

result. Before venipuncture, the skin must be disinfected by swabbing with 70% alcohol followed by 2% iodine.

Scrapings for dermatophyte culture are best obtained with a scalpel blade or the edge of a glass microscope slide

from the marginal, most active portion of the ringworm lesion. Use of a Wood's lamp may identify hairs infected

with certain dermatophyte species. The hair roots are then plucked with forceps for culture. Nails are collected by

clipping. The surface of heavily keratinized structures is scraped away for access to deeper portions. Claw surfaces

are disinfected with alcohol. The surface of skin pustules, nodules, vesicles, and so forth is disinfected, and

aspiration is done with sterile needle and syringe. A biopsy may be required if fungi fail to grow from the aspirate,

scraping, or swab. Normal tissue, along with portions from all zones of the lesion, should be taken. Opened skin

lesions are not disinfected or cleaned because such procedures may remove or kill the organisms of interest.

Swabs are of limited value in fungal isolation, and their use is discouraged. If no alternative collection method is

available, specimens received on swabs in a suitable transport medium (see

Chapter 33

) should be cultured

without delay. Swabs for direct smears, preferably, should not consist of cotton because recovery rates are poor,

and inexperienced observers may mistake cotton fibers for hyphae.

Blood for cultures are collected directly via syringe and needle into conventional and biphasic blood culture
bottles, automated blood systems, and lysis-centrifugation systems (see Isolation).

1,11

A fresh needle is used for

transfer of blood, 1 ml per 9 ml of culture medium, from syringe to the culture bottle. Blood samples taken from
indwelling intravenous (IV) catheters are not recommended.

1,11

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Urine is best taken by percutaneous cystocentesis, which ensures a sample uncontaminated by bacterial or fungal

flora in the lower genitourinary tract (see

Chapter 91

Stool specimen cultures for diagnosis of fungal infections of the gastrointestinal tract are generally misleading.

Biopsy specimens for histologic examination is best.

Fluids and contents of abscesses are collected through aspiration by needle and syringe. Large volumes, adequate

for centrifugation, are best. Any granules should be included and characterized. Bone marrow is similarly sampled

by needle and syringe or core needle biopsy. At least 3 ml of cerebral spinal fluid (CSF) is desirable via lumbar or

cisternal puncture. For lung sampling, a transtracheal or bronchial wash or bronchial brushing is done (see

Chapter

Necrotic material or curettings and other surgically collected material should be handled aseptically pending

examination and culture. Corneal lesions are sampled by scraping several times with a sterile Kimura spatula or

nylon brush (see

Fig. 93-9

). Slide preparation and culture are done at the site and time of collection.

TRANSPORTATION AND PRESERVATION

For referral to a diagnostic laboratory, tissue and fluid samples should be shipped by the most expeditious route in

secure, sturdy, leak-proof containers. Accompanying information on the type of sample being submitted and any

clinical information and other circumstances will assist the laboratory clinician in selecting methods of processing

the sample, including appropriate media, incubation conditions, and safety precautions.

Specimens that cannot be promptly processed can be held in a suitable bacterial transport medium (see

Chapter

). They are refrigerated at 4° C but not frozen for periods of up to 12 to 15 hours. Refrigeration may delay

proliferation of slow-growing fungi for 1 to 2 days. Aspergillus and zygomycetes are sensitive to refrigeration. If a

specimen suspected of harboring a zygomycete cannot be promptly cultured, overnight storage at room

temperature in a bacteriologic transport medium is permissible. Some fungi have been recovered from specimens

up to 2 weeks in transit, but this type of delay is not recommended.

Urine specimens may be kept under refrigeration for up to 12 hours before culture. Most bacteria and yeasts will

multiply in urine kept at room temperature. The Urine C & S Transport Kit (Becton Dickinson, Franklin Lakes,

N.J.) delays growth of bacteria and Candida albicans for up to 48 hours at room temperature. Vaginal swabs in

transport medium or aspirates may be held under refrigeration before processing.

Blood culture bottles or lysis-centrifugation tubes (see Isolation), if subject to delay in processing, can be held at

room temperature for up to 16 hours. CSF and fluids from serous cavities and joints should be processed as soon

as possible. The presence of proteins and carbohydrates in CSF contributes to its qualities as a maintenance

medium. CSF should be held at room temperature if not cultured immediately.

Nasal curettings and excised polyps may be divided between sterile containers for culturing and jars of 10%

buffered formalin for histologic preparation aimed at diagnosing rhinosporidiosis (

Fig. 56-1

). Impression or scrape

smears of nasal tissue should be made before fixation of specimens (see

Fig. 83-3

). Tissue and bone marrow may

be moistened with a small amount of sterile saline if transport is delayed.

Skin scrapings, nails, and hairs can be collected in a clean envelope or a sterile culture dish for mailing. Skin

scrapings also can be held in place between glass slides taped at both ends. Such samples should not be kept in

tightly sealed containers because resulting accumulation of moisture can lead to overgrowth by saprophytes.

Storage is best at room temperature; refrigeration can be harmful for some dermatophytes.

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Fig 56-1 Rhinosporidium. Section of nasal polyp, dog. Thick-walled proliferating

spherules (H and E stain, ×500). (Courtesy Spencer Jang, University of

California, Davis, Calif.)

PROCESSING OF SPECIMENS

The complete processing of specimens involves direct microscopic examination, isolation, identification, and

serology. In the following sections, some of the procedures are considered, with particular reference to their

feasibility as in-office tests. No special equipment, beyond that required for basic clinical bacteriology, is needed

for fungal diagnosis. For incubation purposes, an undisturbed area where room temperature (approximately 25° C)

remains fairly constant is adequate. A hand lens (8 to 10×) or a dissecting microscope is helpful in the early

recognition of fungal colonies.

Direct Examination

The search for diagnostically significant fungal structures may involve preparation of stained or unstained wet

mounts, fixed stained smears, and histologic sections. Some of these techniques are simple and rapid and may

provide the clinician with a presumptive or even definitive diagnosis and a time-saving guide to therapy (

Table

56-1

Wet Mounts

Specimen material may be suspended on a slide in saline, water, or, preferably, 10% potassium hydroxide

(KOH), which clears the preparation of tissue admixtures, leaving fungal elements intact. Examination should

56.4

56.5

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begin under low power (100×) and with subdued light, with the condenser racked down to achieve maximal

contrast. When structures suggestive of fungal elements are seen, higher magnification (400×) is needed for

confirmation.

The KOH digestion method is employed universally in preparation of cutaneous samples suspected of harboring

dermatophytes (see

Chapter 58

). The hair or skin scraping to be examined is placed into a drop of 10% KOH on

a clean slide (

Fig. 56-2, A

). Material may require soaking in KOH for a period before further processing. The

crusty material is teased apart with forceps or dissecting needles and covered with a coverslip. Gently press the

coverslip down to expel any bubbles. This preparation is passed over an open flame several times, but care must

be taken not to boil the mixture. This slide is examined immediately for the presence of arthroconidia or fungal

chains embedded in the material (see

Fig. 56-2, B and C

). If no organisms are initially observed, the slide is

reexamined in 30 minutes. A mixture of 20% KOH and 36% dimethyl sulfoxide or 25% KOH or sodium

hydroxide (NaOH) with 5% glycerol increases penetration and clarity of specimens. Nails may require up to 2

hours for better clearing.

India ink (Pelikan) or nigrosin (1% aqueous), when mixed on a slide with fluids or exudates containing

Cryptococcus neoformans, provides a dark background that outlines the large capsules surrounding the yeast

cells (see

Fig. 61-6,B

). Placing a drop of test material and a drop of India ink separately on a slide and then

adding a coverslip will allow for a proper mixture gradient to form. Less than 50% of culture-positive human
CSF samples are proved to be infected by this method.

Less generally available but useful methods of unstained wet mount study include phase microscopy, in which

the visibility of fungal structures against a background of tissue debris is improved, and fluorescent microscopy,

in which a specimen is prepared by mixing with an equal volume of 10% to 20% KOH and 0.5% calcofluor

white (Difco, Detroit, Mich.) on a slide. The specimen is examined on a fluorescent microscope equipped with

a 365-nm exciter filter and a barrier filter that will transmit light at 410 nm. The fungal wall will fluoresce

brilliantly. This procedure would be available at commercial diagnostic laboratories. See

Appendix 5

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Table 56-1 Direct Examination of Fungi in Clinical Specimens

STAIN OR REAGENT

USAGE

TEXT FIGURES/DISADVANTAGES

Gram

Stains bacteria, yeasts, and other fungi

Cryptococcus neoformans (

Fig. 56-3

);

Malassezia pachydermatis (

Fig. 56-4

);

Sporothrix schenckii (

Fig. 56-5

); some

fungi stain variably or not at all

Potassium hydroxide

Clearing tissue and cellular debris from

variety of specimens to provide greater

visibility of fungal elements

Dermatophytes (

Figure 56-2

); artifacts

develop after preparation time;

experience required

India ink

Observation for presence or absence of

capsules of fungal cells against a dark

background

C. neoformans (

Fig. 61-6

); problems of

artifacts can occur in the stain, poor

sensitivity

Calcofluor white

A fluorescent brightener binding to

polysaccharide such as cellulose and chitin

Detects variety of fungal elements;

requires a fluorescent microscope

Wright

Stain for cytologic examination of

peripheral blood, bone marrow, body

fluids, and organ impressions

Histoplasma capsulatum (

Fig. 60-8

);

Aspergillus (

Fig. 64-5

); Candida (

Fig.

65-1,A

), Prototheca (

Fig. 69-6

); limited use

Gomori's methenamine silver

Detection of fungal elements in histologic

section

Candida albicans (

Fig. 65-5

); not readily

available to most clinical laboratories

Periodic acid-Schiff reaction

Detection of fungal elements in histologic

section

C. albicans (

Fig. 65-6

); not readily

available to most clinical laboratories

Hematoxylin and eosin

Routine histologic stain used for

detection of some fungal elements

Rhinosporidium (

Fig. 83-4

); Coccidioides

immitis (

Fig. 62-8

); Prototheca (

Fig. 69-1

);

usually requires large numbers of fungi to

be detected with this stain

Fixed Smears

Gram stain is commonly done on most routine clinical specimens and will detect most fungi. It is of limited use

in differentiating fungi because most specimens will stain gram positive or unpredictably, and it produces

distortion in cell morphology. Yeasts can often be detected because they retain the primary crystal violet stain

(

Fig. 56-3

). Fungal cell walls often appear as unstained halos. The usefulness of Gram stain is generally limited

to smears in which Candida, Malassezia (

Fig. 56-4

), Geotrichum, Trichosporon, or the yeast form of Sporothrix

spp. (

Fig. 56-5

) is suspected.

Romanowsky-type stains, such as Wright, Giemsa Leishman's, and Diff-Quik, will stain many fungi, especially

yeasts, and are the stains of choice for the tissue phase of Histoplasma capsulatum (see

Fig. 60-8

). As with

Gram stain, fungal cell walls remain unstained by these procedures.

A modified periodic acid-Schiff (PAS) reaction is applicable to direct smears and colors mycotic structures and

some other extraneous and tissue components selectively red. This application is beyond the scope of most

routine office laboratory work but should be obtainable as a service through any histology laboratory (see

Appendix 5

Fluorescent microscopy has had some limited application in mycologic diagnosis. Currently, no fluorescent

diagnostic reagents are available commercially, and no diagnostic services use this approach.

Molecular-based methods for the direct detection of fungal pathogens have advanced substantially in recent

years and are finding their way into use in the clinical laboratory. At present, use of these methods is

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CHAPTER 56 Laboratory Diagnosis of
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predominately restricted to large clinical laboratories or reference laboratories because of the associated

expense and technical complexity of the tests. Refer to

Appendix 5

for laboratories performing these tests.

The most common molecular methods for identification of specific fungal agents employ either nucleic acid

hybridization, which use genus- or species-specific nucleic acid probes or nucleic acid amplification methods
such as the polymerase chain reaction (PCR).

PCR uses DNA primers to amplify specific segments of fungal

genome from the sample. In some cases, PCR is coupled with the use of internal probes or restriction fragment

length polymorphism analysis to improve sensitivity and specificity. Alternatively, DNA sequencing can be

performed on fungal-specific, PCR products amplified directly from tissues or fluids using universal primers

derived from conserved regions in the fungal genome. Once the DNA sequence is obtained, a database can be

searched to determine what the sequence most closely matches. This method allows for identification of

fastidious or noncultivable fungi.

Histology

Stained sections from biopsy and necropsy specimens often provide critical diagnostic information about

mycotic infections. Routine hematoxylin and eosin stain permits detection of the tissue phase of dimorphic

fungi causing systemic mycoses (coccidioidomycosis, histoplasmosis, blastomycosis, cryptococcosis). With

filamentous fungi, it may demonstrate hyphae in tissues, often providing an indication as to their septate or

nonseptate nature and whether they are pigmented or nonpigmented, and thereby helping with their

classification. More specific for fungi is the preferred Gomori's methenamine silver stain, which stains fungal

structures brownish-black against a pale green background, or a PAS reaction, which makes mycotic elements

appear dark red against a contrasting background, depending on the counterstain. Many laboratories use

hematoxylin and eosin as a counterstain or hematoxylin alone, which permits better pathologic characterization

of the lesion than do other procedures. Mayer's mucicarmine can be used to demonstrate capsules on C.

neoformans..

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CHAPTER 56 Laboratory Diagnosis of
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Fig 56-2 A, Hairs from a crusty skin lesion, in a KOH preparation under 40×

magnification with (B) arthrocondia along or on the hair shaft or (C)

chains of arthroconidia embedded in crusty material. (Courtesy

Spencer Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Fig 56-3 C. neoformans. Nasal granuloma of a cat. Budding forms, top; hazy

zone around the six cells below represent capsules (Gram, ×5000).

(Courtesy Spencer Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Fig 56-4 Malassezia pachydermatis. Ear exudate, dog. Broad-based budding

yeast cells (arrows) have “shoe print” appearance (Gram, ×5000).

(Courtesy Spencer Jang, University of California, Davis, Calif.)

Fig 56-5 Sporothrix schenckii. Cutaneous exudate of a cat. Note budding yeasts,

oval, rod, and cigar-shaped forms (Gram, ×2000). (Courtesy Spencer

Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Isolation

Inoculation of a suitably prepared specimen on any appropriate medium is required. Preparation of specimens may

include centrifugation of fluid samples, grinding of biopsy and other tissues, surface sterilization of necropsy

specimens by searing, repeated washing of granules from mycetomas with saline, or filtration of CSF and blood.

Scrapings, swabs, and blood may be inoculated directly without further preparation.

Because many pathogenic fungi, when propagated on agar media, constitute airborne health hazards, laboratories

often prefer tubes and bottles to Petri plates for isolation purposes. If plates are used, they should be secured with

oxygen-permeable tape. All examinations of cultures producing aerial mycelium should be carried out in biologic

safety cabinets. Most fungi grow on media used routinely in microbiologic diagnosis (

Table 56-2

). These media

should be used when the sample is obtained from an uncontaminated site, such as the central nervous system,

internal organ, or joints. These sites have no resident flora and are not exposed to the external environment.

Samples originating from cutaneous sources or mucous membranes that harbor such flora are cultured on selective

media that may contain broad-spectrum antibacterial and antimycotic agents (see

Table 56-2

) for the suppression

of bacteria and nonpathogenic fungi, respectively. A low pH (no higher than 6.0) of the medium may further limit

bacterial overgrowth. Yeasts can be selectively recovered from specimens heavily contaminated with bacteria by

propagation on a medium of pH 3.5 to 4.0. Fungal cultures are optimally incubated at 25° C to 30° C. Incubation

at 37° C allows bacterial overgrowth and causes failure of some fungal pathogens to grow. An atmosphere of 40%

to 50% humidity is favorable for most fungi.

The Isolator system (Wampole Laboratories, Cranbury, N.J.) has improved the number and rate of fungal
isolations from blood.

This system involves lysis and centrifugation of 10 ml of blood. The supernatant is

removed from the upper stopper, and the concentrate is removed from the bottom stopper and plated. The isolation
rates of H. capsulatum, C. immitis, and C. neoformans have improved with the Isolator.

Vented broth bottles

used for bacterial blood cultures should not be expected to detect fungi other than certain yeasts. Biphasic bottles

contain 50 ml of brain-heart infusion broth and brain-heart infusion agar. A biphasic bottle is inoculated with 10

ml of blood, vented, and incubated in an upright position. After daily examination, it is tilted so that the broth

floods the agar surface. Automated blood culture systems have been used and are an efficient means for

laboratories dealing with large volumes of blood cultures.

Identification

Microscopic morphology of fungal reproductive structures is the most helpful criterion for identification. Other

criteria are macroscopic colonial features under different conditions of incubation, nutritional and metabolic

properties, antigenic characteristics, and pathogenicity for experimental animals.

Agar cultures are examined daily during the first 2 weeks of incubation and twice weekly thereafter. Allow 4 to 6

weeks for slow-growing fungi. Some common zygomycetes (Mucor, Rhizopus spp.) grow rapidly and abundantly,

filling a tube or Petri dish within 2 or 3 days. Fruiting bodies may be visible as black specks in the colorless

mycelium (

Fig. 56-6

). Aerial mycelium is grossly less prominent but more intensely pigmented by the presence of

fruiting structures with Aspergillus (

Fig. 56-7

) and Penicillium spp. (

Fig. 56-8

). Some fungi produce soluble

pigment that diffuses through the medium (e.g., Microsporum canis). In others, pigment is confined to parts of the

organism and may be best observed either on the surface or on the reverse side of the colony. The colonial surface

varies according to mycelial growth patterns from smooth (“glabrous”) to powdery, velvety, and cottony. Yeasts,

which form no or little pseudomycelium, produce mucoid, creamy, pasty, or waxy colonies.

537
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Table 56-2 Isolation Media for Fungi

MEDIUM (COMMERCIAL SOURCE) SELECTIVE FEATURES

PRINCIPAL USE/LIMITATION IN

MYCOLOGIC DIAGNOSIS

Blood agar (Remel Labs, Lenexa,

Kan.)

Highly nutritious for most fungi

General purpose, converts some dimorphic

fungi to yeast form; noninhibitory,

nonselective, easily overgrown

Potato flake agar (Remel Labs,

Lenexa, Kan.)

Low pH, highly nutritious for most fungi

To induce sporulation of fungi

Inhibitory mold agar (Remel Labs,

Lenexa, Kan.)

Gentamicin and chloramphenicol for

bacterial suppression

General purpose; not for dermatophytes

Sabouraud's dextrose agar (Remel

Labs, Lenexa, Kan.; marketed as SAB

DUET with DTM in a

two-compartment plate by Bacti-lab,

Mountain View, Calif.)

Low pH, modest nutritional quality;

addition of chloramphenicol and

cycloheximide inhibits bacteria and some

fungi

General purpose; added antibiotics for

isolation from contaminated environment

such as recovery of dermatophytes;

cycloheximide inhibits

Cryptococcus;Aspergillus, Scedosporium

apiospermum, (Pseudallescheria boydii),

some Candida spp.; chloramphenicol

inhibits some yeast

DTM (Remel Labs, Lenexa, Kan.;

Bacti-lab, Mountain View, Calif.)

Gentamicin, tetracycline, and

cycloheximide are inhibitors; glucose and

phenol red are indicators

Isolation of dermatophytes, which turn

yellow medium to red in 48 hours; not for

sporulation; may produce atypical colonial

growth; natural pigmentation obscured;

nondermatophytes turn yellow medium to

red eventually

RSM (marketed as DERM DUET with

DTM in a two-compartment plate by

Bacti-lab, Mountain View, Calif.)

Cycloheximide and chloramphenicol are

inhibitors; glucose and bromothymol blue

are indicators

For dermatophytes, which turn blue

medium green early; prompt conidial and

pigment development permits

identification; color change of RSM not as

intense as of DTM with some

dermatophytes

DTM, Dermatophyte test medium; RSM, rapid sporulation medium.

Low-power (25 to 50×) microscopy helps in early detection of mycelial growth and such diagnostic features as

macroconidia and microconidia of dermatophytes (

Figs. 56-9

56-10

56-11

). Once colonial growth is established,

identification is based largely on microscopic examination for hyphal characteristics: septate versus nonseptate,

pigmented (dematiaceous) (

Fig. 56-12

) versus nonpigmented (hyaline), and conidia and their supporting

structures. This step obviously involves the opening of a culture vessel and should be done only by trained,

experienced personnel and under conditions in which exposure to people and animals and contamination of the

environment can be avoided.

At the in-office laboratory level, it is feasible to make teased preparations or transparent cellophane tape

lactophenol aniline blue mounts (LPAB, Remel Labs, Lenexa, Kan.) from mold cultures by using appropriate

precautions. Diagnostic features are usually better preserved in their natural interrelationships in cellophane tape

mounts (

Figs. 56-13

and

56-14

). In the absence of a laminar flow biosafety cabinet, such attempts should be

restricted to macroscopically positive dermatophyte cultures.

The slide culture procedure probably exceeds the capabilities of most veterinary practices and should be left to

clinical laboratories. This procedure permits study of undisturbed fungal structures by using LPAB (

Figs. 56-15

and

56-16

The germ tube test allows rapid differentiation of C. albicans (see

Chapter 65

) from most of the other, usually

nonpathogenic, Candida spp. Serum, 0.5 to 1 ml, is inoculated lightly with suspect growth and incubated at 35° C

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

for 2 to 3 hours. A drop of the suspension is then examined microscopically (100× and 400×) for the presence of

germ tubes sprouting from yeast cells (blastoconidia) of C. albicans (

Fig. 56-17

Fig 56-6 Mucor in culture. Sporangia form on sporangiophores; note nonseptate,

broad hyphae (Lactophenol analine blue, ×2000). (Courtesy Spencer

Jang, University of California, Davis, Calif.)

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Fig 56-7 Aspergillus deflectus. Columnar conidial head resembling a briar pipe and

biseriate arrangement of phialides (Lactophenol analine blue, ×500).

(Courtesy Spencer Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-8 Penicillium. Fruiting heads give brushlike appearance (Lactophenol

analine blue, ×500). (Courtesy Spencer Jang, University of California,

Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-9 M. canis in culture. Rough and thick-walled multicellular spindle-shaped

macroconidia. Note curved, pointed ends (Lactophenol analine blue,

×2000). (Courtesy Richard Walker, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-10 Microsporum gypseum in culture. Numerous multicellular, fairly

thin-walled macroconidia with rounded ends (Lactophenol analine blue,

×2000). (Courtesy Spencer Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-11 Trichophyton mentagrophytes in culture. Spherical microconidia and

one thin-walled, multicellular, cigar-shaped macroconidium (arrow)

(Lactophenol analine blue, ×2000). (Courtesy Spencer Jang, University

of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-12 Cladophialophora bantiana in culture. Oval conidia occurring in chains.

Note dematiaceous (dark-pigmented) appearance of some conidia and

mycelium (arrows) (Lactophenol analine blue, ×2000). (Courtesy

Spencer Jang, University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
Fungal and Algal Infections

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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-13 Paecilomyces in culture. Ovoid chains of conidia attached to a phialide

(arrow) that is usually tapered (not visible) (Lactophenol analine blue,

×2000). (Courtesy Spencer Jang, University of California, Davis, Calif.)

Fig 56-14 Pseudallescheria boydii (= asexual stage of Scedosporium apiospermum).

Single elliptical conidia attached to tips of conidiophores arising along

the hyphae (Lactophenol analine blue, ×2000). (Courtesy Spencer Jang,

University of California, Davis, Calif.)

CHAPTER 56 Laboratory Diagnosis of
Fungal and Algal Infections

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Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-15 H. capsulatum in culture. Tuberculate macroconidia (Lactophenol

analine blue, ×2000). (Courtesy Spencer Jang, University of California,

Davis, Calif.)

Commonly used differential media include Czapek agar (Difco, Detroit, Mich.) for the differentiation of

Aspergillus spp.; cornmeal agar for the demonstration of chlamydospores in C. albicans and their absence in most
other Candida spp.; Trichophyton agars (Remel Labs, Lenexa, Kan.) for the identification of Trichophyton spp.

by their growth factor needs; Christensen's urea agar (Difco, Detroit, Mich) for production of urease by
Cryptococcus, Rhodotorula, Trichophyton,

and Trichosporon spp. and CHROMagar Candida (Hardy

Diagnostics, Santa Maria, Calif.) for culture and identification of C. albicans, C. krusei, C. tropicalis, and
Trichosporon spp.

For definitive identification of the dimorphic fungi Coccidioides immitis, H. capsulatum, and Blastomyces

dermatitidis antisera are commercially available (ImmunoMycologics, Norman, Okla.) for use in agar
immunodiffusion exoantigen test. The antigen is prepared from an extract of mycelial growth.

7,10

Fungal isolates can be identified to genus or species level, or both, by many molecular-based methods. Universal

primers to regions in the 28S rRNA gene have been used in PCR to amplify DNA that is then tested with specific
nucleic acid probes.

The use of a multiplex PCR approach for identification of a battery of commonly

encountered yeasts and molds has also been described.

Commercially available DNA probes such as the

Accuprobe system (Genprobe Inc., San Diego, Calif.) are available for identification of the important fungal

agents, such as C. immitis, B. dermatitidis, and H. capsulatum.

CHAPTER 56 Laboratory Diagnosis of
Fungal and Algal Infections

Page 20 of 23

Infectious Diseases of the Dog and Cat, 3rd Edition

Fig 56-16 Blastomyces dermatitidis in culture. One-celled conidia on short

conidiophores, “lollypop” appearance (Lactophenol analine blue,

×2000). (Courtesy Spencer Jang, University of California, Davis, Calif.)

Fig 56-17 Germ tube test. Formation of germ tube (arrow) characteristic of C.

albicans. Blastoconidia on left (wet mount, ×2000). (Courtesy Richard

Walker, University of California, Davis, Calif.)

541

CHAPTER 56 Laboratory Diagnosis of
Fungal and Algal Infections

Page 21 of 23

Infectious Diseases of the Dog and Cat, 3rd Edition

DNA sequencing of specific regions of the fungal genome can also be used for identification purposes. The

ribosomal RNA genes are most commonly used because they contain conserved areas present in most fungi and

therefore serve as sites for amplification of DNA from a variety of different fungi with a single pair of primers.

The rRNA genes also contain variable regions that are used in sequence analysis for making the identification. At

present, the most commonly used region is the D2 variable region of large subunit rRNA gene. Commercial

sequencing kits are available (MicroSeq D2 rDNA Fungal Identification System, Applied Biosystems) that use

this region, but the method is complex and generally relegated to use by reference laboratories that have access to

DNA sequencing facilities. A few commercial laboratories provide nucleic acid sequencing services for

identification of fungal isolates (Accugenix, Newark, Del. and MIDI Labs, Newark, Del.). Sequence analysis of

the internal transcribed spacer (ITS) regions (ITS-5.8S rRNA-ITS2) has also been used to identify a large number
of fungal species.

Oomycetes in the genus Pythium and Lagenidium are not true fungi but produce fungal-like

granulomatous diseases (see

Chapter 67

). PCR has been used to determine the specific cause of these infections.

Real-time PCR has been used to test peripheral blood for the diagnosis of invasive aspergillosis in people.

panfungal PCR has been used to detect Candida and Aspergillus in the blood of people with suspected
disseminated infections.

Molecular-based test results should be combined with clinical findings and morphologic features of the agent in

tissues or in culture, or both, before making a final diagnosis.

Among miniaturized prepackaged identification kits for yeast and algae isolates belonging to the genus Prototheca

spp. isolates, the API 20C (bioMerieux Vitex, Inc., Hazlewood, Mo.), the Uni-yeast-tek system (Remel Labs,
Lenexa, Kan.), and RapID yeast plus panel (Remel Labs, Lenexa, Kan.) are used.

All rely on tests that are

scored to yield a single profile code, which is listed in a code book and translates into a species name.

Identification can be completed within 4 hours to several days and more conveniently and generally more rapidly

than by conventional methods. Rapid multiple biochemical test systems lack the database for the more unusual
yeasts. Automated systems such as Vitex Yeast biochemical card (bioMerieux Vitex, Inc., Hazlewood, Mo.)

and

Microbial Identification System (MIS), (Microbial ID [MIDI], Inc., Newark, Del.) require continued expansion

and updating of the veterinary database for improved quality.

Serologic Testing

Reagents designed to detect antigens and antibodies in body fluids are becoming commercially available in

increasing numbers. An antigen detection kit, the C. neoformans latex agglutination test (Meridian Diagnostics,

Cincinnati, Ohio), is one of several kits available that has been adapted for use in office laboratories (see

Chapter

Table 61-3

, and

Appendix 6

). Testing for antigens for Histoplasma capsulatum is available on a commercial

basis (see

Chapter 60

and

Appendix 6

The accuracy of this procedure for use in dogs and cats is not known. An

enzyme-linked immunosorbent assay has been developed for the serologic diagnosis of pythiosis (see

Chapter 67

and

Appendix 6

For a discussion of serum antibody testing of particular fungal infections, see the respective

chapters.