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446 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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Fungi are eukaryotic and can be either unicellular or multicellular organisms. Fungi have cell walls composed mainly
of chitins, glucan, and mannam with a membrane- bound cell
nucleus with chromosomes. e dominant sterol in the cytoplasmic membrane of fungi is ergosterol, compared with cholesterol in mammalian cells. ese organisms are heterotropic
(eg, require exogenous energy sources) and can reproduce by
either asexual (involving mitosis) or sexual (involving meiosis) cell division. Fungi may exist in a morphologic form that
results from sexual reproduction (teleomorph, or perfect state)
and a form that results from asexual reproduction (anamorph,
TABLE 19-1.
or imperfect state), in which each of the forms has its own
name (eg, the sexual form of Scedosporium apiospermum com-
plex is Pseudallescheria boydii). e use of separate anamorph
and teleomorph species names ended in January 2013, and
all legitimate names for the species can be used. It will likely
take a signicant period (eg, a decade or more) before these
nomenclatural changes of fungal organisms achieve stability
and acceptance.
Fungi have traditionally been categorized into mold, yeast, or
dimorphic fungi based on morphologic and structural features
(Table19-1).
14-18
a
YEASTS AND YEAST-LIKE ORGANISMS DIMORPHIC FUNGI
Candida
Cryptococcus
Rhodotorula
c
Saccharomyces cerevisiae
Trichosporon
Blastoschizomyces capitatus
Histoplasma capsulatum
Blastomyces dermatitidis
Paracoccidioides brasiliensis
MOLDS
Mucormycetes
d
Rhizopus
Mucor
Rhizomucor
Lichtheimia corymbifera
Apophysomyces elegans
Saksenaea vasiformis
Cunninghamella bertholletiae
Basidiobolus
Conidiobolus coronatus
Dematiaceous Fungi Dermatophytes Hyaline Hyphomycetes
Fonsecaea pedrosoi
Fonsecaea compacta
Rhinocladiella
Microsporum
Trichophyton
Epidermophyton occosum
Phialophora verrucosa
Pleurostomophora richardsiae
Phaeoacremonium parasiticum
Phialemonium
Cladophialophora
Scedosporium
Lomenospora prolicans
Verruconis gallopava
Exophiala jeanselmei complex
Exophiala dermatitidis
Hortaea werneckii
Stachybotrys chartarum
Curvularia
Bipolaris
Exserohilum
Alternaria
Molds (or moulds) are long, cylindrical, and
b
Sporothrix schenckii
e
Talaromyces marneffei
Coccidioides
b
Hormographiella aspergillata
Aspergillus
Penicillium
Paecilomyces
Purpureocillium lilacinum
Scopulariopsis
Emmonsia
Acremonium
Fusarium
Lecythophora
a
b
Coccidioides immitis
yeast- like colonies or cells at 35°C to 37°
c
CandidaC lusitaniaeClavispora lusitaniaeC krusei
Pichia kudriavzeviiC kefyrKlu veromyces marxianus); C guilliermondiiMeyerozyma guilliermondiiC lipolyticaYarrowia lipolytica
Candida
d
genera RhizopusMucorRhizomucorLichtheimiaAbsidia
Basidiobolus and Conidiobolus
e
Sporothrix schenckii°C to 30°C but is yeast like at 35°C to 37°
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 447
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threadlike (lamentous) fungi that form multicellular mycelium
or thallus, an intertwined mass of branching hyphae (tube- like
extensions or lament- like cells), with septa (having cross walls;
being septate) or pauciseptate. An asexual spore (conidium) is
produced on conidiophores, a specialized hyphal structure that
serves as a stalk, and macroconidia and microconidia may be
present. ermally, monomorphic molds can be divided into
four groups: (1) Mucormycetes, (2) dematiaceous fungi, (3) dermatophytes, and (4) hyaline hyphomycetes. Mucormycetes have
broad hyphae that are almost nonseptate, with asexual spores
(sporangiospores) formed by cleavage in a saclike structure (sporangium). e most common Mucormycetes observed in the
clinical laboratory are from the order Mucorales, which are associated with severe fungal infections referred to as mucormycosis.
Two genera from the order Entomophthorales, Basidiobolus and
Conidiobolus, are less commonly observed Mucormycetes but
are responsible for subcutaneous infections in otherwise healthy
individuals. Dematiaceous fungi produce dark- colored colonies
of olive, brown, gray, or black due to melanin pigment in the cell
walls. Some of the common infections associated with dematiaceous fungi include chromoblastomycosis, phaeohyphomycosis, and mycetoma. Dematiaceous fungi also cause tinea nigra
and black piedra. Dermatophytes are most oen associated with
supercial fungal infections (tinea or ringworm) of the skin,
hair, and nails. ese lamentous fungi colonize the outermost
layer of the skin and digest keratin as a source of nutrients. e
three genera (Microsporum, Trichophyton, and Epidermophyton)
are dierentiated by their conidium formation (macroconidia
or microconidia). Hyaline hyphomycetes are colorless, septate
hyphae molds that produce conidia that may be either colorless or pigmented. Coccidioides immitis and Coccidioides posa-
dasii are known pathogens from this group whereas most other
organisms cause opportunistic infections in immunocompromised patients.
Yeasts (and yeast- like organisms) appear as round or oval
cells that are unicellular and generally reproduce at their surface by budding (blastoconidia). Some produce pseudohyphae
(an elongated chain of cells, like a chain of sausages, resembling hyphae; however, borders between cells are delineated by
marked constrictions), whereas others have true hyphae (tend
to be straighter and without constrictions at the septa), which
may be septate or without septa (aseptate). Ascospores, a sexual
spore in a saclike structure (ascus), are produced by only some
yeast. Yeasts are the most frequently encountered fungi in the
clinical microbiology laboratory and are considered opportunistic pathogens. Candida spp. and Cryptococcus spp. are among
the most common yeasts causing fungal infections. Yeasts are
not considered a formal taxonomic group but a growth form
of unrelated fungi (members of the phyla Basidiomycota and
Ascomycota).
ermally, dimorphic fungi have two distinct morphologic
forms in which their growth forms can change from a multicellular mold (in their natural environment or when cultured
at 25°C to 30°C) to budding, unicellular yeasts (during tissue
invasion or when cultured at 35°C to 37°C). Medically important dimorphic fungi include Histoplasma capsulatum, Blasto-
myces dermatitidis, and Paracoccidioides brasiliensis. Each of
these fungi is considered pathogenic and must be handled with
caution in the clinical laboratory.
The Identification of Fungi
e following section provides a summary of the common methods currently used in diagnostic testing of medically important
15-18
fungi.
morphologic characteristics, such as the color of the colonies,
the size and shape of cells, the presence of a capsule, and the
production of hyphae, pseudohyphae, or chlamydospores. Culture remains the “gold standard” in most clinical microbiology
laboratories and is the only method that allows subsequent susceptibility testing. NAAT, molecular characteristics, and/or proteomics (MALDI-TOF MS) are rapidly gaining a larger routine
role in fungal identication, particularly when morphologybased identication is atypical, confusing, or not helpful (eg,
organisms that fail to sporulate) and in cases in which precise identication is required (eg, epidemiologic studies).
Because no one test is perfect, it is oen necessary to perform
several diagnostic tests (both morphologic and genotypic meth
odologies) to maximize the accuracy of fungal identication.
Laboratory diagnosis of fungal infections includes direct microscopic examination, morphologic identication, isolation in culture, and use of non–culture- based methods, such as antigen
and/or antibody detection, 1,3-β- - glucan detection, molecular and nonmolecular diagnostic testing, and MALDI-TOF
MS.
cal specimens need to be selected, collected, and transported
to the laboratory for immediate processing.16 Because dierent
fungi are capable of causing infection at a number of anatomic
sites, specimens from the site of infection and peripheral blood
specimens should be considered and submitted for culture and
microscopic examination. Communication with the laboratory
regarding the clinical infection and suspected fungi is important
and may be useful for determining how best to process specimens safely and eciently, including pretreatment and staining
procedures, selection and incubation of media, and choice of
additional diagnostic testing. Early identication of the infecting fungal pathogen may have direct diagnostic, epidemiologic,
prognostic, and therapeutic implications.
opaque colonies) from molds (large, lamentous colonies that
vary in texture, color, and topography). Drawings, color plates,
and brief descriptions found in standard textbooks can serve as
guides and assist in the preliminary identication of fungi seen
on direct microscopic examination of clinical specimens.
Microscopic examination of the clinical specimen can delineate morphologic features (Table 19-2) and oen provides
preliminary identication of many fungi (eg, Aspergillus spp.,
Mucormycetes, dematiaceous molds). Microscopic morphology can oen provide denitive identication of a mold whereas
the addition of biochemical tests, serology, nucleic acid- based
molecular testing, and/or MALDI-TOF MS are usually needed
for identifying the genus and species of most yeast and yeastlike fungi. Direct microscopic examination of properly stained
clinical specimens and tissue sections is usually the most rapid
(within a few minutes or hours) and cost- eective method for
Fungal identication has traditionally been based on
18-22
10,14,18-27
As with all types of infections, appropriate biologi-
e rst step is usually identifying yeast- like fungi (pasty,
14-17
-
448 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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ADDITIONAL TESTS
FOR IDENTIFICATION
by Candida albicans
Candida dubliniensis
Candida stellatoidea
Candida
detection of
Candida mannan
antigen and
antimannan
the antigen
and antibody
test should
together to
Carbohydrate
assimilation
diagnosis
+
+
Differentiation from
C gattii
melanin stains in tissue
bromthymol blue) agar
IN CULTURE SEROLOGIC TESTS
MORPHOLOGIC FEATURES
MICROSCOPIC
MORPHOLOGIC
FEATURES OF
β-
Clusters of
Variable
colonies usually
CLINICAL SPECIMENS MACROSCOPIC MICROSCOPIC ANTIGEN ANTIBODY
Round to oval budding
Assay Reagent)
indicated for
and terminal
diagnosis of
invasive fungal
infection and
should be used in
may have smooth
or slight constrictions
other diagnostic
elongated blastoconidia)
along the sides of either
Candida glabrata
in diameter) than other
forms
antigen
cells may have
to tan in color
budding yeasts of
based buds
may or may not be
POTENTIAL
CLINICAL
SPECIMENS
FUNGAL
TABLE 19-2.
ORGANISM
Candida
secretions or
systemic sites
Cryptococcus
neoformans
sites
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 449
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continued)
based
in ∼50% of cases on
has resulted in antigen
colony characteristics
and allergic
Aspergillus
enlarged vesicles
identify Aspergillus
Culture-
useful tool in the
diagnosis of invasive
are hyaline and
hematologic
malignancy
and stem cell
attractive as alternative
Aspergillus
β-
Assay Reagent)
indicated for
attractive as alternative
diagnosis of
invasive fungal
infection and
should be used in
other diagnostic
Aspergillus
fumigates
Aspergillus avus
dichotomously
45°
sites
Aspergillus
Aspergillus niger
surface mycelium
450 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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ADDITIONAL TESTS
FOR IDENTIFICATION
Culture results need to
Colony characteristics
are used to identify
are not routinely used
Various methods
for serologic
Various methods
for initial antigen
detection in
Demonstration of thermal
and Histoplasma
meningitis)
disseminated and
IN CULTURE SEROLOGIC TESTS
MORPHOLOGIC FEATURES
MICROSCOPIC
MORPHOLOGIC
FEATURES OF
POTENTIAL
CLINICAL
Differentiation of
various genera based
CLINICAL SPECIMENS MACROSCOPIC MICROSCOPIC ANTIGEN ANTIBODY
SPECIMENS
black in color
gray-
sites
on genus and
Colonies are
may be single or
dematiaceous
color
sites
tuberculate
macroconidia
°
°
often clustered
or intracellular
microconidia
°
budding yeasts
monocytes)
sites
°C
FUNGAL
TABLE 19-2.
ORGANISM
Dematiaceous
molds
Histoplasma
capsulatum
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 451
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continued)
useful
Antigen screening
test should be
antibody testing
β-
Assay Reagent)
indicated for
diagnosis of invasive
fungal infection
and should be used
Demonstration of
are not useful
diagnosis of
blastomycosis
sensitivity and
in conjugation
Histoplasma-
available to detect
antigenuria and
antigenemia in
disseminated
to test antigen in
Various methods
reliable) for
initial antibody
and other body
Coccidioides
does not distinguish
assays also
available and
more often used
antibodies against
Coccidioides
galactomannan)
Coccidioides)
°C to 30°
smooth conidia
37°
contoured yeast-
a broad base
arthroconidia
disjunctor cells
to differentiate
other small yeasts
Candida
°C to 30°
colonies vary from
in diameter) and
Blastomyces
dermatitidis
as “double contoured”)
sites
nonconidiating
colony
35°C to 37°
on a broad base
Coccidioides
°C or 37°
colonies initially
sites
immitis/
posadasii
cottony aerial
becomes gray-
arthroconidia may be
seen in cavitary and
necrotic lesions
452 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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ADDITIONAL TESTS
FOR IDENTIFICATION
β-
Coccidioides
test) for detection from
Assay Reagent)
indicated for
diagnosis of
invasive fungal
infection but has
a limited role
Demonstration of
LA test is
commercially
used but could
disseminated
based diagnosis
had failed)
IN CULTURE SEROLOGIC TESTS
MORPHOLOGIC FEATURES
MICROSCOPIC
MORPHOLOGIC
FEATURES OF
CLINICAL SPECIMENS MACROSCOPIC MICROSCOPIC ANTIGEN ANTIBODY
°°
rising at right
°°
colonies are
diameter) yeast- like
at the end of the
borne in rosette-
orange- gray
at 35°°
fusiform budding
or velvety and
bodies)
35°C—37°
colonies are
and yeast- like
POTENTIAL
CLINICAL
SPECIMENS
FUNGAL
TABLE 19-2.
ORGANISM
Sporothrix
schenckii
sites
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 453
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attractive as alternative
methods but are mainly
Demonstration of
in- house systems
being available
attractive as alternative
methenamine silver
studies but not
Assay Reagent)
for diagnosis
indicated for
diagnosis of
invasive fungal
infection and
should be used in
other diagnostic
been successful and
commercially available
°°
smooth
metulae bearing
length) or elongated
°°
the round to oval
at 35°°
35°°
budding does not occur
sites
arthroconidial
yeast cells divide
elongate
-
sites
Talaromyces
Penicillium)
marneffei
Pneumocystis
jirovecii
SourceMedical Microbiology
454 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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a preliminary diagnosis of fungal infection. In addition, microscopic detection of fungi can assist the laboratory in the selection of media and interpretation of culture results.
e Gram stain that is typically used for bacterial processing may also allow the detection of most fungi, especially Can-
dida spp., because the size of the smallest fungi is similar in
size to large bacteria; the presence of budding cells can also be
observed. A wide range of stains is available (Table19-3) to
assist in the rapid detection of fungal elements.
10,14,17,18
A common approach to wet preparations of specimens or smeared
dried material is to use a 10% solution of potassium hydroxide
(KOH) with or without uorescent calcouor white stain. e
strong alkaline KOH solution digests tissue elements to allow
better visualization of the fungi, while the calcouor white stain
binds to chitin and polysaccharides in the fungal cell wall, allowing it to appear white under ultraviolet light. Specic staining
techniques are oen used to outline morphologic features that
are diagnostic and distinctive of the suspected fungal organism
(eg, India ink stain for detection of a polysaccharide capsule of
Cryptococcus neoformans). In suspected cases of histoplasmosis,
the Giemsa or Wright stain is useful for detecting intracellular yeast cells within macrophages from blood or bone marrow
specimens.
Histopathologic stains are extremely valuable for identifying fungal elements in tissues and host tissue reactions to
fungal infection.
24,25
Histology laboratories commonly use
stains such as hematoxylin and eosin for these general purposes. Periodic acid-Schi and Gridley fungus stains can also
assist in visualization of fungal elements, especially if debris
is present in the tissue background. Special stains (eg, Gomori
methenamine silver, mucicarmine, and Fontana-Masson)
are useful for enhancing the detection of specic fungal elements (Table19-3) for a histopathological diagnosis of fungal
infections.
15,17,18
TABLE 19-3.
STAIN
(ABBREVIATION) DETECTION CHARACTERISTICS/COMMENTS
Alcian blue
Cryptococcus neoformans
Pneumocystis jirovecii
Cryptococcus
neoformansCryptococcus gattii
also be useful for Aspergillus fumigates
Aspergillus avusTrichosporon
Histoplasma
capsulatumPneumocystis
jiroveciiTalaromyces
marneffei
Pneumocystis jirovecii
cases of meningitis
observation of fungi
disseminated disease
CryptococcusNocardia)
are not stained
to infecting fungus
Aspergillus
differentiate from background
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 455
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TABLE 19-3.
STAIN
(ABBREVIATION) DETECTION CHARACTERISTICS/COMMENTS
AspergillusCandida albicans
Pneumocystis jirovecii
Cryptococcus neoformans
Nocardia
Blastomyces dermatitidis
Cryptococcus neoformans
material) and Cryptococcus gattii
Blastomyces dermatitidis and
Rhinosporidium seeberi
Commercial antibodies used in the
<50% of meningitis cases
Actinomyces and other actinomycetes are negative
Blastomyces dermatitidisNocardia
Toluidine blue Pneumocystis jirovecii
Source
Culture remains the gold standard for isolation and identication of fungi suspected of causing infection. Petri plates are
preferred over screw- cap tubes because of the larger surface area
and dilution of inhibitory substances in the specimens. However,
for laboratory safety reasons, most thermally dimorphic fungi
(eg, Histoplasma, Blastomyces, Paracoccidioides, T marneei) and
Coccidioides spp. are pathogenic and should be grown on slants
(ie, avoid the use of Petri plates and slide culture). A variety of
media are available for the isolation and cultivation of yeasts and
molds (Table19-4).
15-17
Sabouraud dextrose, brain heart infusion, and inhibitory mold agars are enriched media commonly
recommended to permit the growth and isolation of yeasts and
molds. Several media, with (selective) and without (nonselective) inhibitory agents, should be used because no one medium
is adequate for all the dierent types of specimens or organisms. Antibiotics such as chloramphenicol or gentamicin are
included as inhibitory substances of most bacterial contaminants, whereas cycloheximide is used to inhibit saprobes and
prevent the overgrowth of contaminating molds. Nonselective
media (without inhibitory agents) should be used with specimens from sterile sites, and when suspected, fungi are likely to
Pneumocystis jirovecii reddish blue or
be inhibited by cycloheximide (eg, Aspergillus fumigatus, C neoformans/gattii, Lomentospora prolicans, T [Penicillium] marneffei, some Candida spp., most Mucormycetes) or by antibiotics
(eg, Nocardia or other lamentous bacteria). Direct microbiological examination (outlined previously) of clinical specimens
can assist in the selection of media based on specimen type and
suspected pathogen. In addition, the choice of media will be
inuenced by the patient population, local endemic pathogens,
cost, availability, and laboratory preferences.
Proper temperature and adequate time for incubation are necessary to optimize the recovery of medically important fungi from
clinical specimens. Inoculated media should be incubated aerobically at 30°C. If an incubator at that temperature is not available,
then 25°C (room temperature) can be considered. Other temperatures (eg, 35°C to 37°C for thermally dimorphic organisms)
should be reserved for selected fungi that prefer a higher temperature. In general, yeasts are detected within 5days or less, dermatophytes within 1 week, and dematiaceous and dimorphic fungi
between 2 and 4 weeks. Cultures should be regularly reviewed
(eg, every day the rst week, every 2to 3days the second week,
twice during the third week, once weekly thereaer) to account
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