C H A P T E R 4

Isolation and Identification of FungiJane E. Sykes and Shelley C. Rankin

wKEY POINTS• Fungioftengrowmoreslowlythanbacteriaandcanhavespe-

cificculturemediarequirements.Somefungiaredangeroustoculturebecausetheyproduceairbornesporesthatmayleadtolaboratory-acquiredinfections.

• The clinician should tell microbiology laboratory personnel ifa fungal infection is suspected. Providing a list of suspectedpathogensfacilitatessafeandsuccessfulcultureofpathogenicfungi.Theanimal’sclinicalabnormalities,travelhistory,historyof immunosuppressivedrugtherapy,andtheresultsofcytol-ogyorhistopathologyshouldbetakenintoaccount.

I N T R O D U C T I O N A N D D E F I N I T I O N SFungi are eukaryotic microorganisms that secrete enzymes inorder to digest organic material in the environment. The resul-tantnutrientsareabsorbedthroughaspecializedfungalcellwall(knownaschemotrophic nutrition).Abasicunderstandingoffun-gal terminology facilitates communications between clinicians,microbiologists, and pathologists. Fungal organisms have twobasicmorphologicforms:1. Molds—moldsexistasfilamentsknownashyphae,whichmay

bedividedintocellularcompartments(septate hyphae).Amassoffilamentsisknownasamycelium.Agranuleformedbyacom-pactedmatoffungalfilamentsisaeumycetoma(asopposedtoanactinomycotic mycetoma;seeChapter42).

2. Yeasts—these are single-celled organisms that reproduce bybudding.Insomecases,budsdonotdetachfromtheparentcell,butinsteadformachain.Elongatedchainsofbudsmayresemblehyphae.Thesearetermedpseudohyphae(Figure4-1).Many pathogenic fungi exist as a mold in the environment,

but in tissues convert to a yeast form. These fungi are calleddimorphic fungi.AnexampleofadimorphicfungusisBlastomyces dermatitidis.

Fungireproducebyproducingspores.Sporescanbeproducedfollowing asexual or sexual reproduction. Asexual reproduction(throughmitosis)generatessporesthatareidenticaltotheparentcell.Sexualreproductioninvolvesthefusionofhaploidnucleifromtwohyphalstructures,which is followedbymeiosis.Theasexualstageofa fungus isknownasan anamorph,whereas thesexualstageisknownasateleomorph.Manyfungihavetwodifferentspe-cies names to reflect these stages. For example, the teleomorphofCryptococcus neoformansisknownasFilobasidiella neoformans.Formally, the teleomorph name is supposed to take precedenceandcanbeusedtodescribebothstages.1However,asexualrepro-ductionpredominates inmost fungi, includingduringgrowth inculture.Thus,certainfungi,suchasC. neoformans,aremorewidelyknownbytheiranamorphname.Inaddition,teleomorphsofsomefungalorganismshavenotyetbeendescribed.Someorganisms

29

• Specimensshouldbecollectedandtransportedasforbacterialculture.Refrigerationinhibitsgrowthofsomefungi.

• Becauseofincreasingdrugresistanceamongpathogenicfungi,thedemandforantifungalsusceptibilitytestinghasincreased,and for some fungal pathogens, culture and susceptibilitytesting before beginning antifungal drug therapy may beadvantageous.

canexistinmorethanoneasexualform,andthereforehavemorethantwonames.Forexample,Pseudallescheria boydii(teleomorph)hastwoasexualforms,Graphium fruticolaandScedosporium apio-spermum.Currently,thereisamovebymycologiststoidentifyonlya single name for a fungal organism (‘one fungus = one name’).Throughoutthisbook,thenamemostcommonlyusedinthelit-eratureisusedtorefertodifferentfungalpathogens.

Specimen Collection and Transport

Isolation of fungal organisms from clinical specimens can be facilitated by the use of special media. For many fungal spe-cies, this may be hazardous to laboratory personnel. Submission of specimens for culture of dangerous organisms such as Blas-tomyces dermatitidis, Coccidioides immitis, and Histoplasma

FIGURE 4-1  Histopathology of the thyroid gland from a 13-year old female spayed poodle mix with disseminated Candida albicans infection. The dog had been treated with prednisone, cyclosporin, and azathioprine  for  refractory  immune-mediated hematologic disease. Note intralesional pseudohyphae. H&E stain, 400× magnification.

30 SECTION 1 LaboratoryDiagnosisofCanineand

capsulatum is discouraged if the diagnosis can be obtained using other methods such as cytology, histopathology, and serologic testing. These organisms exist as yeasts in tissues (at 37°C) that are minimally hazardous to clinicians and laboratory person-nel, but grow as hyphae at lower temperatures and on culture medium, which generate spores that can be inhaled and cause disease.

Specimens that are commonly submitted for fungal culture include lower respiratory tract wash specimens, cerebrospinal fluid, urine, other body fluids, skin, eye, hair, or biopsy speci-mens from the nasal cavity, bone, or skin. Guidelines for speci-men collection are the same as for bacterial culture (refer to Chapter 3). The clinician should inform the laboratory what fungal species are suspected, because different fungi have differ-ent media preferences and growth conditions. As large a speci-men as possible should be collected. Care should be taken to avoid collection of contaminated specimens. Blood cultures may be useful for dogs and cats with disseminated fungal disease when lesions cannot be easily accessed. Detection of fungemia can sometimes be achieved using blood culture systems for bac-terial isolation. Lysis-centrifugation methods (Isolator tubes, Wampole Laboratories) may be more sensitive for detection of dimorphic and filamentous fungi in blood. Mycobacterial blood culture media (such as MB/BacT, BioMérieux) may also offer increased sensitivity for detection of fungemia.2

For dermatophyte culture, 10 to 12 hairs should be plucked from the edge of the lesion using sterile forceps and submit-ted without refrigeration in a clean dry paper envelope or ster-ile glass or plastic tube. Paper envelopes are preferred to tubes because tubes retain moisture that can promote overgrowth of contaminants. Scrapings of crusts at the edges of the lesion can also be submitted. Claw clippings can be collected after cleaning them with 70% alcohol to remove contaminating bacteria and fungi. Crumbling material from under the claw may be the best specimen, or the entire claw can be submitted.

If transport requires longer than 2 hours, appropriate trans-port media should be used or specimens should be refrigerated. When rapid transport to the laboratory is possible, storage and transport at room temperature is preferred. Fungal culture of specimens that are more than 24 hours old is not recommended because of increased likelihood of false-positive and false-nega-tive results. The laboratory should be informed if the patient is receiving antifungal drug treatment, which may interfere with the ability to culture fungus from the specimen.

Specimens should be labeled with the patient’s name, patient number, and source of specimen, as well as the date and time of collection, and labeled, packaged, and transported accord-ing to regional and global regulations for shipping of infectious substances (see Chapter 1).3 Careful packaging and transport is particularly important for specimens suspected to contain fungal pathogens. If dangerous organisms might be present, the laboratory should be warned in advance and the specimens marked appropriately.

Diagnostic Methods

Microscopic Examination of Direct SmearsExamination of a direct smear before culture allows rapid par-tial or complete identification of many fungi by the laboratory (Table 4-1, Figure 4-2). This can help the clinician make initial treatment decisions and guides media selection by the labora-tory. Using Gram stain, yeasts generally stain gram positive

FelineInfectiousDiseases

and molds tend to be gram negative.4 A potassium hydroxide (KOH) preparation may be performed to examine a specimen for dermatophyte hyphae and arthroconidia. The KOH clears keratinaceous and cellular debris, but fungal elements are left intact. If tissue granules are present, the laboratory will crush them, examine them for hyphae, and the granules can also be cultured. Fungal morphology may be altered in specimens from dogs and cats receiving antifungal drug treatment (Figure 4-4).

Special stains used in the laboratory for fungal identification include India ink, which is used to identify cryptococcal yeasts in cerebrospinal fluid (see Figure 62-8), and calcofluor white, which binds polysaccharides in the fungal cell wall and fluo-resces when examined using fluorescence microscopy (Figure 4-5, A). Other stains, such as lactophenol cotton blue (see Figure 4-5, B), are also used to identify fungi after they have been grown in culture.

In some situations, fungi are observed in tissues with histo-pathology but a culture of the causative organism is not avail-able, either because it has not been performed or because the organism has failed to grow. The morphology and staining characteristics of the organism on histopathology may also be useful for preliminary identification or allow full identification of the pathogen. Special stains used include Mayer’s mucicar-mine stain, which stains the cryptococcal polysaccharide cap-sule red; Gomori’s methenamine silver stain, which stains fungal organisms black; and periodic acid–Schiff, which stains fungal elements dark pink (Figure 4-6). If the identity of the fungus remains in doubt, an additional specimen should be collected for culture if possible, and the microbiology laboratory should be made aware of the morphology of the organism and the dif-ferential diagnoses. Complete identification of the organism using culture is useful for proper treatment and prognostication.

Fungal CultureBefore culture, clinical specimens must be prepared for inocula-tion. For example, body fluids are usually first centrifuged by the laboratory to concentrate fungal organisms. The sediment is then resuspended in a smaller volume of supernatant and inocu-lated onto selective media. If biopsy specimens are submitted, the tissues are finely sliced, ground, or minced, which releases the organisms before they are inoculated onto the media.

Many types of media are available for isolation of fungi, and no single medium is optimal for all organisms. Many fungi grow on blood agar plates used for primary isolation of bacteria. Special media used for initial (primary) isolation of fungal organisms from tissues and body fluids are shown in Table 4-2. Other media are available for differentiation of fungal species (see Fungal Identification, later). Media that contain antibacterial agents (usually chloramphenicol or gen-tamicin) are used to culture specimens that might contain bac-teria. Cycloheximide is added to dermatophyte test medium to inhibit the growth of contaminant saprophytic mold spe-cies. This allows selective isolation of dermatophytes such as Microsporum canis.

Plates for fungal culture are sealed with an air-permeable tape in order to prevent exposure of laboratory personnel to potentially dangerous spores and to minimize plate contami-nation with airborne fungal organisms. Plates that grow fila-mentous fungi are opened only in a biological safety cabinet. Laboratories prefer to use media in tubes or bottles to reduce occupational exposure to aerosolized fungal elements. Fungal cultures are incubated at room temperature (22°C to 25°C)

31CHAPTER 4 IsolationandIdentificationofFungi

TABLE4-1Microscopic Descriptions of Some Common Fungal Pathogens of Dogs and Cats

OrganismYeast or Hyphae Diameter (µm) Description

Geographic Distribution

Blastomyces dermatitidis

Yeast 8-15 Round with thick, often refractile cell wall and broad-based budding (see Figure 60-8).

Southeastern and south central United States, upper Midwest region

Histoplasma capsulatum

Yeast 2-5 Oval to round, often found in clusters within macrophages (see Figure 61-7).

Ohio and Mississippi river valleys, especially in southern United States. Also Central America

Cryptococcus spp. Yeast 2-15 Round to oval, narrow-based bud-ding, sometimes forming chains of organisms. Large capsule (see Figures 4-4 and 62-8). Poorly encapsulated variants are rarely encountered.

Worldwide, common in Australia and western North America

Coccidioides spp. Spherule 10-200 Spherules vary in size and may contain endospores (“pomegranate-like”). Using cytology, they appear as empty and collapsed, deeply basophilic structures (see Figure 63-8).

Central California, Arizona, Nevada, Central and South America

Sporothrix schenckii

Yeast 2-6 Oval, round, or cigar-shaped, occasion-ally budding (see Figure 64-4). May be found intracellularly and extra-cellularly. Difficult to find in dogs but may be abundant in cats.

Temperate and tropi-cal zones worldwide. Most common in South and Central America

Malassezia spp. Yeasts and pseudohyphae

3-8 (yeast), 5-10 (pseudohyphae)

Round to oval; when budding, have the appearance of “footprints” (see Figure 59-2).

Worldwide

Candida spp. Yeast and pseudohyphae

3-4 (yeast), 5-10 (pseudohyphae)

Usually yeasts with a single bud or pseudohyphae with multiple constrictions (see Figure 67-3).

Worldwide

Dermatophytes Hyphae 3-15 Septate hyaline hyphae, arthroconidia may be visible along hair shafts (see Figure 58-8)

Worldwide

Aspergillus spp. Hyphae 4-6 Septate hyaline hyphae with uniform diameter that branch at 45° angles (see Figure 4-2).

Worldwide

Scedosporium spp., Paecilomyces spp.

Hyphae 3-12 Septate hyaline hyphae that tend to branch at wider angles than Aspergillus spp. (45° to 90°) (see Figure 4-6).

Worldwide

Basidiobolus and Co-nidiobolus (zygomy-cosis)

Hyphae 10-30 Wide, non- or poorly septate irregular hyaline hyphae, branching at right angles, may be twisted or folded. Poorly stained using hematoxylin and eosin stain (see Figure 4-3).

Tropical and subtropical regions of the Americas, Africa, southeast Asia, and Australia

Phialophora, Exophiala, Cladophialophora, Bipolaris (phaeohy-phomycosis or dema-tiaceous molds)

Hyphae 1.5-6 Pigmented hyphae, budding cells with single septa and chains of swollen round cells (see Figure 68-5). May form granules.

Worldwide

Pythium insidiosum Hyphae 4-9 Sparsely septate, hyaline hyphae with 90° branches, detectable using hema-toxylin and eosin stain

Tropical, subtropical, and some temperate regions of the world.

32 SECTION 1 LaboratoryDiagnosisofCanineand

FIGURE 4-2  Direct  smear  of  an  aspirate  from  a  mediastinal  mass  of  a  6- year-old female spayed  labrador  retriever with disseminated Aspergillus deflectus  infection. Note hyphae with parallel walls that branch at 45° angles. Stain uptake by some hyphae is poor. Wright’s stain, 1000× oil magnification. (Image courtesy Dr. William  Vernau, University of California, Davis.)

A

B

FIGURE 4-4  A, Cerebrospinal fluid cytology (Cytospin preparation) from a dog with newly diagnosed cryptococcosis. Note the thick capsule, which appears as a halo around the organism, and narrow-based budding. Wright-Giemsa stain, 1000× oil magnification. B, Cytology of a  lymph node aspirate from a cat that had been treated for cryptococco-sis with antifungal drugs. The architecture of the organisms is distorted. Diff Quik stain,  1000 × oil magnification.

FelineInfectiousDiseases

FIGURE 4-3  Histopathology  showing  severe  pulmonary  zygomycosis  at  necropsy in an 11-year-old mixed-breed dog that had been diagnosed with diabetic ketoacidosis before euthanasia. H&E stain, 100× magnification.

A

B

FIGURE 4-5  Special stains used for identification of fungal isolates following direct smear preparation. A, Calcofluor white stain of a mass of Aspergillus fumigatus hyphae. 1000×  oil  magnification.  B,  Lactophenol  cotton  blue  preparation  of  the  mold  phase  of Histoplasma capsulatum. 1000 × oil magnification.

33CHAPTER 4 IsolationandIdentificationofFungi

A B

FIGURE 4-6  Histopathology of the kidney from a 2-year-old female spayed German shepherd with disseminated paecilomycosis. A, Periodic acid Schiff stain. The fungal hyphae stain bright pink. B, Gomori’s methenamine silver stain, which stains the fungal hyphae black. 400× magnification.

TABLE4-2Special Medium Types Used to Culture Fungi That Infect Dogs and Cats

Type of Medium Additives IndicationsSabouraud dextrose agar None Cultivation of all fungi.

Brain-heart infusion agar None Cultivation of all fungi.

Potato dextrose agar or potato flake agar

None Cultivation of all fungi. Especially useful for opportu-nistic molds. Induces sporulation.

Inhibitory mold agar Chloramphenicol, sometimes gentamicin Cultivation of all fungi when bacterial contamination is suspected or possible.

Dermatophyte test medium Chloramphenicol, gentamicin, cycloheximide

Dermatophyte isolation. Shows a color change when dermatophytes grow on the medium.

or 30°C, a lower temperature than that used for routine cul-ture of bacteria. In general, cultures should be examined daily for 2 weeks and twice weekly for an additional 2 to 4 weeks, because the growth of some organisms may be very slow. In general, rapidly growing fungi grow in 1 to 3 days, those with intermediate growth in 5 to 9 days, and slow growers take up to 4 weeks. Yeast colonies are smooth and resemble bacterial colonies, and molds grow in colonies that are dry, wrinkled, or heaped (Figure 4-7).

Fungal IdentificationAfter growth in culture, fungi are identified based on visual characteristics such as colony morphology and color. Light microscopy is useful to evaluate the microscopic morphology of yeasts and to determine the presence of septate or nonseptate hyphae and fruiting structures for molds (Table 4-3). Increas-ingly, DNA sequence information has been used to identify fungi (such as after PCR of D2 or ITS regions of the 23S rRNA gene). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry holds considerable promise for identification of fungi in the future (see Chapter 3).5 Molds that produce pigment are known as dematiaceous fungi (see Figure 4-7, B), and those that do not are known as hyaline

fungi. A variety of broth or solid media are also used for fun-gal identification. For example, Candida species can be differ-entiated using Candida bromcresol green (BCG) agar, because each species has a specific colonial morphology and color on this medium. Test strips are commercially available that allow multiple biochemical identification tests for yeasts to be per-formed simultaneously (e.g., API 20C AUX Yeast Identification Kit, bioMérieux) (see Figure 3-8), so identification can be made within 1 to 2 days.

Interpretation of Fungal Culture Results

False-negative and false-positive results are very common with fungal culture, and results must be interpreted in light of the organism grown, the animal’s clinical condition, and the results of cytology and histopathology.

Specimens That Test NegativeNegative test results can occur as a result of inadequate speci-men size (such as when swabs or aspirates are submitted), when there are inadequate organism numbers at the site of specimen collection, when overgrowth with contaminating bacteria or saprophytic fungi occurs, or when there is loss of organism

34 SECTION 1 LaboratoryDiagnosisofCanineand

A

B

FIGURE 4-7  A, Growth of Cryptococcus gattii yeasts on potato flake agar. B, Clado-phialophora bantiana, a pigmented (dematiaceous) mold, isolated from canine brain tis-sue on potato flake agar.

FelineInfectiousDiseases

viability during specimen transportation. If the patient is receiv-ing antifungal drugs but has not completely cleared the infec-tion, culture may be negative.

False-negative test results also occur when the organism is not inoculated onto the correct media or sufficient time is not allowed for growth. It is critical that clinicians inform the lab-oratory if a fungal infection is suspected, and what organism might be involved. Poor laboratory quality control procedures may also lead to false-negative test results. Use of an accredited laboratory can help minimize such problems.

Specimens That Test PositiveThe detection of fungal organisms within a specimen does not always imply that the organism is causing the animal’s clinical signs. Contamination by saprophytic fungi, especially Aspergil-lus, is the most common cause of false-positive fungal cultures. Organisms are more likely to be clinically significant when cyto-logic examination of a stained smear also demonstrates the pres-ence of fungal organisms.

Antifungal Susceptibility Testing

Increasing resistance to antifungal agents has been docu-mented for a variety of fungal pathogens. This has led to an increased demand for antifungal susceptibility testing by clinicians. Fortunately, the reproducibility of antifungal sus-ceptibility testing has improved greatly in recent years, and breakpoints (see Chapter 3) have been established for treat-ment of many fungal infections in human patients. Although breakpoints are still lacking for veterinary patients, antifun-gal susceptibility testing allows comparison of the activity of two or more antifungal drugs, and serial testing of isolates from a single patient allows the development of resistance to be monitored over time. Methods of susceptibility testing that have been adapted for yeast and mold infections include broth microdilution, the Etest, and disk diffusion (see Chapter 3).

TABLE4-3Classification of Fungi That Are Known to Infect Dogs and Cats

Phylum Class Example CharacteristicsZygomycota Zygomycetes Absidia, Rhizopus, Conidiobolus,

BasidiobolusAseptate hyphae. In asexual reproduction, spores

are abundantly produced in a closed sac called a sporangium. Sexual reproduction results in a single, large thick-walled zygospore.

Basidiomycota Basidiomycetes Cryptococcus, Malassezia Sexual reproduction leads to haploid production of basidiospores on a basidium.

Ascomycota Archiascomycetes Pneumocystis Sexual reproduction leads to haploid production of ascospores in a sac-like structure (ascus). Organisms are more commonly identified based on characteristics of asexual reproduction.

Hemiascomycetes Candida spp.Euascomycetes Histoplasma, Blastomyces,

Microsporum, TrichophytonAspergillus spp.Fusarium spp.Pseudallescheria spp. (teleomorph

of Scedosporium spp.)

C

Common antifungal agents tested include amphotericin B, itraconazole, fluconazole, voriconazole, posaconazole, caspo-fungin, griseofulvin, and flucytosine (Figure 4-8). Limitations of antifungal susceptibility testing include the slow growth of fungal pathogens when compared to bacteria, and the fact that fungi may grow as yeasts or molds depending on media and incubation conditions. Partial inhibition of growth over

35HAPTER 4 IsolationandIdentificationofFungi

a wide range of antifungal drug dilutions may also occur (a phenomenon known as trailing). Criteria have been set for interpretation of MICs in the presence of trailing for differ-ent antifungal drugs.6 MALDI-TOF mass spectrometry can be used to determine antifungal drug susceptibility among fungal pathogens and in future may overcome some of the difficulties that currently exist.7

1 2 3 4 5 6 7 8 9 10 11 12 A POS AND

0.015AND0.03

AND0.06

AND0.12

AND0.25

AND0.5

AND1

AND2

AND4

AND8

AB0.12

B MF0.008

MF0.015

MF0.03

MF0.06

MF0.12

MF0.25

MF0.5

MF1

MF2

MF4

MF8

AB0.25

C CAS0.008

CAS0.015

CAS0.03

CAS0.06

CAS0.12

CAS0.25

CAS0.5

CAS1

CAS2

CAS4

CAS8

AB0.5

D FC0.06

FC0.12

FC0.25

FC0.5

FC1

FC2

FC4

FC8

FC16

FC32

FC64

AB1

E PZ0.008

PZ0.015

PZ0.03

PZ0.06

PZ0.12

PZ0.25

PZ0.5

PZ1

PZ2

PZ4

PZ8

AB2

F VOR0.008

VOR0.015

VOR0.03

VOR0.06

VOR0.12

VOR0.25

VOR0.5

VOR1

VOR2

VOR4

VOR8

AB4

G IZ0.015

IZ0.03

IZ0.06

IZ0.12

IZ0.25

IZ0.5

IZ1

IZ2

IZ4

IZ8

IZ16

AB8

H FZ0.12

FZ0.25

FZ0.5

FZ1

FZ2

FZ4

FZ8

FZ16

FZ32

FZ64

FZ128

FZ256

POS, positive control; AND, anidulafungin; MF, micafungin; CAS, caspofungin; FC, 5-flucytosine; PZ, posaconazole; VOR, voriconazole; IZ, itraconazole; FZ, fluconazole; AB, amphotericin B.

A

BFIGURE 4-8  A, Minimum inhibitory concentration (MIC) assay for a Candida krusei isolate. Growth in the presence of the antifungal drug is characterized by formation of a pellet on the bottom of the well and a change in the color of the medium from purple to pink. B, Schematic of the same microtiter plate showing antifungal drug dilutions. Organisms are defined as susceptible, intermediate, or resistant based on MIC cutoffs (breakpoints or interpretive criteria) set by regulatory bodies such as the Clinical and Laboratory Standards Institute in the United States. The report to the clinician would read susceptible to anidulafungin (0.12 µg/mL), susceptible to micafungin (0.25 µg/mL), susceptible to caspofungin (1 µg/mL), intermediate susceptibility to flucytosine (8 µg/mL), susceptible to voriconazole (0.12 µg/mL), susceptible to itraconazole (0.25 µg/mL), and resistant to fluconazole (128 µg/mL). The MICs for posacon-azole and amphotericin B were reported as 0.25 µg/mL and ≤0.12 µg/mL, but the organism was not classified as susceptible or resistant because interpretive criteria for this organism-drug combination were not available.

36 SECTION 1 LaboratoryDiagnosisofCanineand

REFERENCES 1. Pitt JI, Samson RA. Nomenclatural considerations in naming spe-

cies of Aspergillus and its teleomorphs. Stud Mycol. 2007;59:67-70. 2. Taniguchi T, Ogawa Y, Kasai D, et al. Three cases of fungemia in

HIV-infected patients diagnosed through the use of mycobacterial blood culture bottles. Intern Med. 2010;49:2179-2183.

3. World Health Organization. A guide for shipping infectious substances. 2009. http://www.who.int/ihr/infectious_substances/en/index. Last accessed May 11, 2012.

4. Shea YR. General approaches for direct detection of fungi. In: Ver-salovic J, Carroll KC, Funke G, et al. eds. Manual of Clinical Micro-biology. 10th ed. Washington, DC: ASM Press; 2011:1776-1792.

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5. Iriart X, Lavergne RA, Fillaux J, et al. Routine identifica-tion of medical fungi by MALDI-TOF: performance of the new VITEK MS using a new time-effective strategy. J Clin Microbiol. 2012;50(6):2107-2110.

6. Johnson EM, Espinel-Ingroff AV, Pfaller MA. Susceptibility test methods: yeasts and filamentous fungi. In: Versalovic J, Carroll KC, Funke G, et al. eds. Manual of Clinical Microbiology. 10th ed. Washington, DC: ASM Press; 2011:2020-2037.

7. Marinach C, Alanio A, Palous M, et al. MALDI-TOF MS-based drug susceptibility testing of pathogens: the example of Candida albicans and fluconazole. Proteomics. 2009;9:4627-4631.