Neuical NYC0L0uY

Alvln 8ey llores, 8M1, M1(ASCl), MP

Nycology
SLudy of lungl
LLymology: Creek 1erm M?kLS meanlng mushroom
CLher Lerms relaLed
uMalls, molds, mushroom, brackeL fungl, mlldew
Impoitance of Fungi .
lood Source (nuLrluon)
lood roducuon (8read Maklng, 8eer, ClLrlc Acld)
Anublouc Source
Symblosls (Mycorrhlzae) - aldlng ln absorpuon of waLer and
mlnerals from Lhe soll
More Lhan 100,000 specles, only a few (200) are paLhogenlc.
F0NuI
1PALLCP?1LS (wlLh Lrue nucleus)
Lukaryouc Crganlsms, nucleaLed
PeLeroLrophlc Crganlsms
lanL-llke buL LACk S1LMS and 8CC1S and does noL possess
CPLC8CP?LL
KEY BIFFERENCES
!"#$#%&'$()*%) ,-./( 0#%&'$(#
1ype of Cell Lukaryouc rokaryouc
Cell Membrane LrgosLerols are resenL no sLerols excepL ln
Mycoplasma
Cell Wall Composed of complex
CPC such as CLuCAnS,
MAnnAnS, CPl1ln
epudoglycan (nAC and
nAM)
Spores Sexual and Asexual (for
8eproducuon)
Lndospores (noL for
8eproducuon), some
asexual spores
MeLabollsm PeLeroLrophlc/
ChemoheLeroLrophlc
PeLeroLrophlc,
phoLoauLoLrophlc,
chemoauLoLrophlc
Cxygen 8equlremenL Aeroblc (Molds)
laculLauve Anaerobe
(?easLs)
Aeroblc, laculLauve
Anaerobe, Anaerobe
pP 8equlremenL pP 3 (acldlc) neuLral pP
Anublouc SensluvlLy olyenes, lmldazoles,
Crlseofulvln
enlcllllns, 1eLracycllnes,
Amlnoglycosldes
Fungi
LACk Chlorophyll
Absorbs nuLrlenLs
May ossess lncluslons such as:
vacuoles (conLalnlng LlluS)
Clycogen
Mulu-veslcular 8odles : SplLzenkorper" - organlzlng cenLer for
growLh and morphogenesls
Some may be CapsulaLed
More 8eslsLanL Lo Csmouc ressure (can grow ln hlgh salL
concenLrauons)
lungl may llve as heLeroLrophs, saproLrophs or as paraslLes
May be a normal ora of mouLh and lnLesunal LracL

F0NuI EXISTS IN 2 F0RNS

MCLu ?LAS1
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N0LBS vs YEASTS. what's the
uiffeience.
YEAST vs N0LBS
12345 6789
unlcellular, 8ound, non lllamenLous,
Can roduce seudohyphae
Mulucellular, lllamenLous
Capable of formlng P?PAL
8eproduce by 8uddlng, or 8lnary
llsslon
8eproduce by lragmenLauon and
Spore formauon
Crows aL 33-37C

laculLauve Anaerobe
Crows aL 8oom 1emperaLure

Aeroblc
Appear as MolsL, Creamy or 8uuery
Colonles wlLh an alcohol llke odor

Can be ldenued by 8lochemlcal 1esLs
Appear as ury, Couony wlLh a velveLy
Surface

Can be lu by appearance of spores and
hypha
1ake noLe. Some lungl exhlblL ulMC8PlSM ln whlch lL has Lwo forms of growLh,
MosL aLhogenlc lungl are ulmorphlc
YEAST
Nolus
Composed of P?PA - boslc sttoctotol oolt
Nolus
Pypha
uemouoceoos - plqmeoteJ










Pyallne/Monlllaceous- no plgmenL


Nolus
M?CLLluM - aggregaLes or mass of P?PA
ConslsLs of: 1PALLuS (vegeLauve oruon), AL8lAL, and
8L8CuuC1lvL A81
! #$%&' () *$+&,-.
1. vegeLauve mycella
grow ln or on Lhe medlum
absorbs nuLrlenLs from Lhe medlum
2. Aerlal
grow above Lhe surface of Lhe agar
forms mosL of Lhe vlslble parL of Lhe colony
3. lerule or reproducuve mycella
from whlch Lhe reproducuve sLrucLures arlse
Nolus
CLher lorms of vLCL1A1lvL P?PAL
8ACCuL1 P?PAL - resemble Lennls racqueL
Sl8AL - cork screw llke (seen ln 1rlchophyLon)
nCuuLA8 - enlarged knoLs of closely LwlsLed hyphae
lAvlC CPAnuLLlL8 - anLler llke appearance (seen ln
1tlcbopbytoo scboeoleloll)
LC1lnA1L 8CulLS -looks llke LeeLh of a comb
8PlZCluS - rooL llke processes found ln kblzopos
Bimoiphic Fungi
Bimoiphic Fungi
Nannei of Repiouuction
lungl LxhlblL 8oLh Sexual and Asexual lorms of 8eproducuon
Asexual 8eproducuon glves rlse Lo an AnAMC8P or
lML8lLC1 S1A1L
Sexual 8eproducuon glves rlse Lo a 1LLLMC8P or
1LLCMC8P or L8lLC1 S1A1L
1here are lunCl LhaL do noL possess a SLxuAL S1A1L
lunCl lML8lLC1l (ueuLeromycoLa)
Lxamples: Candlda, 1orulopsls, LpldermophyLom
Nannei of Repiouuction
hases on Lhe lungal Llfe Cycle
A. Somauc hase - feedlng or Lrophlc phase
- Lhrough producuon of exLracellular enzymes used Lo dlgesL
nuLrlenLs ln Lhe subsLraLum and for nuLrlenL absorpuon
8. 8eproducuve hase
ASLxuAL (formauon of spores and conldla)
SLxuAL

Asexual Repiouuction
nC luSlCn of nuCLLl
Spore roducuon ls Lhrough dlerenuauon of spore bearlng
hypha
Asexual 8eproducuon lncludes
A. lragmenLauon
8. 8uddlng -formauon of blasLoconldla (ln yeasLs)
C. llSSlCn
u. Spore lormauon
5potooqlospotes
coolJlom
Asexual
Repiouuction
Sporanglospores (Asexual Spores)
- borne ln a sporangla (sac llke
sLrucLure whose enure conLenLs
are converLed Lhrough cleavage
lnLo one 1 or more spores)
Asexual Repiouuction
Conldlum - produced ln a manner LhaL does noL lnvolve
cleavage, produced slngly or ln long chalns or clusLers by
conldlophores
Aspergillus with typical sporangium
showing the conidiophore, vesicle,
phialides and conidia in chains
Conidiospores
Phialides
Vesicle
Conidiophore
Coniuiogenesis
CAn CCCu8 ln 1WC lC8MS
1. 8lasuc - proLoplasm of Lhe conldlogenous cell ls blown ouL
or blasLed Lo form a conldlum. 1hls can be seen ln many
yeasLs such as Candlda.
2. 1halllc - no developmenL of conldlum unul a sepLum ls
formed beLween Lhe conldlum and Lhe parenL cell. 1he
conldlum orlglnaLes from Lhe whole of Lhe parenL cell.
ex. uermaLophyLes and Coccldloldes
Blastic Foims
PCLC8LAS1lC - Lhe parenL cell wall layers are lnvolved ln
blasuc daughLer cell developmenL
A. 8lasLoconldla - slmplesL form and produced by
buddlng. seudohypha forms as ln cases of Candlda
8. oroconldla - formed by Lhe daughLer cell by pushlng
Lhrough a mlnuLe pore ln Lhe parenL cell
Ln1L8C8LAS1lC - Lhe ouLer cell wall does noL paruclpaLe ln
Lhe process
A. hlaloconldla - conldla emerges from a phlallde
as seen ln Asperglllus and hlalophora
8. Annelloconldla - as Lhe conldla are released, a dlsuncL
rlng of cellular maLerlal ls le leavlng behlnd a dlsuncL saw
LooLhed appearance aL Lhe slde of Lhe parenL cell.
Thallic Foims
PCLC1PALLlC - Lhe whole parenL cell ls lnvolved ln daughLer
cell developmenL
A. MAC8CCCnlulA - large, sepLaLe, oval shaped, splndle
shaped or club shaped. May be Lhlck or Lhln walled, splny
(echlnulaLe) or smooLh wall surface.
Thallic Foims
8. CPLAM?uCCCnlulA
- Lhlck walled, reslsLanL resung spores produced
by roundlng up and enlargemenL of Lhe Lermlnal hyphal
cells
Thallic Foims
A81P8lC - daughLer cell fragmenLs wlLhln Lhe hyphal sLrand
before dlsperslon
A. A81P8CCCnlulA - recLangular/ barrel shaped conldla,
derlved from Lhe fragmenLauon of Lhe mycellum aL Lhe
sepLum. SeparaLed wlLhln Lhe parenL hypha before belng
dlspersed.
-presence of dys[uncLor cells glvlng a checkered
appearance
Sexual Repiouuction
luSlCn of nuCLLl of Lwo Cpposlng MaLchlng SLralns
1P8LL ulS1lnC1 PASLS
A. lasmogamy - Paplold uonor Cell (+) peneLraLes Lhe
cyLoplasm of 8eclplenL Cell (-)
8. karyogamy - fuslon of Lwo haplold nuclel Lo form a
zygoLe
C. Melosls - glves rlse Lo haplold nucleus (sexual spores)

Sexual Spoies
A. ASCCSC8LS
- spores enclosed ln an ASCuS followlng karyogamy
3)%:%#$;) < =$-(*./ >:?@ := #)%:);:$')
1ypes of Ascocarps
1. ApoLheclum - cup shaped ascl are produced lnslde a
cup
2. ClelsLoLheclum - ascocarp ls enclosed (no openlng)
3. CymnoLheclum - slmllar Lo clelsLoLheclum excepL Lhe
ouLer wall of Lhe ascocarp are loosely organlzed, ascl are
released Lhrough Lhe wall openlngs.
4. AscosLroma - ascl are produced ln locules (cavlues) ln
hard masses of supporung hypha called sLroma.
3. erlLheclum - ask shaped wlLh an openlng where
ascospores are released
Ascospoies
Sexual Spoies
8. 8ASlulCSC8LS - spores are formed lnslde a basldlum (club
shaped reproducuve sLrucLure)
C. Z?CCSC8LS - Lhlck walled spores formed by fuslon of 2
hyphal sLrands
u. CCSC8LS - fuslon of cells from 2 separaLe non-ldenucal
hypha

GROUP

CHARACTERISTICS

EXAMPLE
Zygomycetes Sexual reproduction results in a zygospore
Asexual reproduction occurs via sporangia
Vegetative hyphae are sparsely septate
Rhizopus
Absidia
Mucor
Pilobolus
Ascomycetes Sexual reproduction involves a sac or ascus
in which karyogamy and meiosis occur
producing ascospores
Asexual reproduction is via conidia
(arthroconidia, blastosconidia)
Molds have septate hyphae
Aspergillus
Histoplasma
Trichophyton
Penicillium

Basidiomycetes Sexual reproduction results in four progeny
basidiospores supported by a club-shaped
basidium
Hyphae have complex septa
Mushrooms (Amanita)
Crytococcus neoformans
Deuteromycetes An artificial grouping of the imperfect
fungi for which a teleomorph or sexual
reproduction has not been discovered
Anamorphic state is characterized by
a s e x u a l c o n i d i a , a r t h r o s p o r e s ,
blastospores, chlamydospores
Coccidioides immitis
Paracoccidioides brasiliensis
Candida albicans
NEBICAL NYC0L0uY
lunCAL ulSLASLS
M?CCSLS - mycouc lnfecuons
racucal Classlcauon of lungl
A. Superclal or CuLaneous Mycoses
8. SubcuLaneous Mycoses
C. SysLemlc Mycoses
u. CpporLunlsuc Mycoses
NEBICAL NYC0L0uY
MLCPAnlSMS Cl lunCAL A1PCCLnLCl1?
A. MycoLoxlns - exoLoxlns produced by fungl
3 Ma[or Croups
1. AaLoxlns - produced by Aspetqlllos fovos, can cause
llver damage ln anlmals, hepauc carclnoma ln humans,
AaLoxln 81(mosL Loxlc)
2. tqot AlkololJs - produced by clovlceps potpoteo
lnfecung gralns llke rye
- cooses etqousm ot 5t. Aotbooys llte (qooqteooos ot
coovolslve type)
- oseJ lo obstettlcs to coottoct otetloe smootb moscles
3. sychoLroplcs

Neuical Nycology
8. PypersensluvlLy
- due Lo repeaLed exposure Lo fungal spores and
consequenL lg or sensluzed lymphocyLe producuon. May cause
allerglc rhlnlus, asLhma
C. 1lssue uamage

LAB0RAT0RY BIAuN0SIS of
Fungal Biseases
Macroscoplc
Mlcroscoplc SLudy
8lochemlcal 8eacuons
Serologlcal 1esLs


Specimen Collection anu
Banuling
SAlL1?: Class ll 8lologlcal SafeLy CablneL Should be uSLu!!
eLrl-ulshes are noL recommended, screw Lop
Lubes preferred
CCLLLC1lCn:
SLerlle (Asepuc 1echnlque should be Cbserved)
CollecL adequaLe and approprlaLe speclmens
SubmlL and rocess lmmedlaLely Lo avold overgrowLh
Specimen Banuling anu
Collection
CCMMCn SLClMLnS
1. Palr - collecL halr samples by cllpplng or plucklng from Lhe
base of Lhe halr sha, large masses of halr ls noL necessary
- Wood's Lamp - may be used Lo ldenufy lnfecLed halr
(uoresce)

Specimen Banuling anu
Collection
2. Skln - samples are scraped from Lhe :-&'$ '?/' of Lhe
surface leslon.
- dlslnfecL Lhe skln wlLh 70 alcohol
- kCP weL mounL musL be prepared Lo break ussue
debrls
3. nalls - submlued as scraplngs or cumngs or Lhe whole nall
- dlslnfecL Lhe skln wlLh 70 alcohol
- kCP weL mounL for deeper scraplngs
4. Abscess and SubcuLaneous lnfecuon - obLaln Lhe exudaLe
3. 8esplraLory Speclmens - spuLum (deep cough collecuon) ,
less vlscous samples can be lnoculaLed dlrecLly.
Specimen Banuling anu
Collection
6. 8lood - can be collecLed ln braln hearL lnfuslon, lsolaLor 1ube (lysls
cenLrlfugauon sysLem), Sepuchek (blphaslc medla),
7. 8uy CoaL - for dlagnosls of PlsLoplasmosls
8. 8one Marrow- heparlnlzed, can be lnoculaLed aL Lhe bedslde
9. CSl - 3
rd
Lube ls senL Lo Lhe Mlcroblology Secuon, for ctyptococcos
oeofotmoos Jetecuoo.
- CenLrlfuge Lo obLaln a more concenLraLed speclmen, lLrauon
can also be done
- for lndla lnk rep and CulLure lnoculauon
10. Lye- corneal scraplngs, eye dlscharge
11. urogenlLal and lecal Speclmens - may grow yeasLs, cenLrlfuge
urlne speclmens rsL Lhen lnoculaLe onLo medla. llrsL volded mornlng
speclmen preferred.
12. 1lssues - should be mlnced or grlnded
BIRECT NICR0SC0PIC EXAN
8CvluLS 8Alu 8eporL Lo Lhe uocLor
Can glve clues Lo Lhe genus of Lhe organlsm
rovlde evldence of lnfecuon even lL ls negauve ln culLures
1. WeL reparauon
- SlmplesL, uslng sLerlle nSS (1-2drops), used Lo observe
yeasL, hyphae and pseudohyphae, lacks conLrasL,
dlmculLy ln dlerenuaung and lu of fungal elemenLs
2. use of 10-20 kCP
- for lnlual examlnauon of keraunlzed ussue
- kCP dlssolves keraun Lo vlsuallzed fungal
elemenLs
2.kCP - add small amounL of Lhe speclmen Lo 1 drop of kCP
- press cover sllp, warmlng Lhe sllde can be done Lo
hasLen clearlng
-glycerol can also be added Lo prevenL Lhe soluuon from
crysLalllzlng
- allow Lo sLand for 20 mlnuLes Lo clear
-AdvanLage: Palr samples can be examlned lf lnfecuon ls
endoLhrlx or ecLoLhrlx
varlauons of kCP preparauon
1. 8lue-black lnk/meLhylene blue
-2parLs kCP: 1 parL of lnk
- SLand for 3-10 mlnuLes
- PeaL and add uMSC (peneLraung agenL excepL for halr
and skln)
2. uMSC - peneLraung agenL Lo speed Lhe clearlng process
3. Calcoour WhlLe - blnds Lo chlun and cellulose and uoresce
under Wood's lamp, (Apple Creen or WhlLe Color)
- besL sLaln for deLecung vlable fungal elemenLs
4. Add lndla lnk
8hlzopus spp. Showlng fragmenLed poruons of sepLaLe hyphae of
varylng slze
/01 '*&.2 () '%3#3*
lacLophenol couon blue or anlllne blue
4+(#+5 #.%&
%2&%.2.#-(6

showlng agar
posluoned under
coversllp before uslng
pressure Lo dlsperse
growLh

7&2)(2*.6+& () .
8&# *(36#
showlng lnoculauon of agar plug
9-+2(',-:& +3,#32&
CLher ulrecL reparauons
1. Clemsa/WrlghL's SLaln -for dlagnosls of PlsLoplasmosls
2. LacLophenol Couon 8lue (Aman) - blue color
3. lndla lnk/nlgrosln - capsule demonsLrauon
4. AS - presence of fungal hypha (purpllsh red)
3. Cram SLaln (Pucker Modlcauon)
6. Comorl MeLhenamlne Sllver
7. Acrldlne Crange
8. Masson lonLana SLaln
P
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Figure 61-6 Sporangiophores of Rhizopus spp. supporting sporangia that contain
sporangiospores. Rhizoids arise from the hyphae near the origin of the sporangio-
phores. (Lactophenol cotton blue stain, 100.)
Superimposed dyes in stained smears, wet preps, or histologic sections may
partially obscure the color, which can be more obvious in unstained prepa-
rations. Those fungi that lack dark hyphal pigmentation are referred to as
hyaline (clear or colorless). This term may not fully reect the fungal
species appearance, because in some cases a light pigmentation may
produce colored colonies and asexual reproductive structures of some
hyaline moulds may have green, brown, or black pigments that impart a
color to the surface of the colony once the reproductive structures have
formed. The true appearance of these moulds usually can be discerned by
observing the back of the colony, which maintains a light coloration. In
contrast, both the front (obverse) and the back (reverse) of dematiaceous
colonies usually demonstrate the dark pigment.
REPRODUCTIVE STRUCTURES
The primary means for identication of mould fungi is by characterization
of asexual reproductive structures. For yeast, phenotypic studies are the
mainstay in identication, with asexual reproductive structures serving as
ancillary clues in the identication process. The two principal asexual
structures are spores (which may also be present as sexual structures) and
conidia. Asexual spores (called sporangiospores) are produced by cleavage
within an encompassing structure called a sporangium (Fig. 61-6). Conidia
(singular, conidium) are much more diverse and form by differentiation
from the tip or side of a fertile hypha, such as a conidiophore, or by hyphal
differentiation. Unfortunately, interchangeable use of the terms conidium
and spore in the literature has led to confusion. Sporulation and spores
often are used as general terms for asexual reproduction, and the term
spore is sometimes used when conidium would have been more accurate.
The principal means of asexual reproduction in yeast is by the forma-
tion of blastoconidia (i.e., budding). A bud starts as a softening of the cell
wall of the mother cell, followed by expansion of the cell wall (blown out)
and migration of nucleus and cytoplasm to the swollen area. A septum seals
the boundary between daughter and parent cells (Fig. 61-7). If separation
does not occur, a pseudohypha results.
The portions of the vegetative mycelium that differentiate into conidia
are referred to as conidiogenous cells. Specialized hyphae that support the
conidia are termed conidiophores, which may be the conidiogenous cell
itself arising from the vegetative mycelium or may be a supporting hypha.
In Aspergillus spp., the conidiophore, which is aseptate, enlarges at the tip
to form a swollen vesicle (Fig. 61-8). Conidiogenous cells, now termed
phialides, arise from the vesicle to support chains of conidia. Some species
of Aspergillus produce a row of phialides, which occur on a row of sterile
cells called metulae, with the conidia arising from the distal phialides. The
Zygomycetes produce structures called sporangiophores, which support
the sporangium with enclosed sporangiospores (see Fig. 61-6). An exten-
sion of the apex of the sporangiophore into the sporangium is termed the
columella.
Thallic conidiogenesis is a process in which the conidium does not
develop until a septum is formed between the conidium and the parent
cell. The conidium originates from the whole of the parent cell. The most
important human pathogens that exhibit thallic conidiogenesis are the
dermatophytes and the dimorphic fungi in the Coccidioides spp. As
Figure 61-7 Budding and nonbudding yeast cells detected in a blood culture
bottle sample. (Gram stain, 1000.)
Figure 61-8 Fruiting head of Aspergillus fumigatus. The conidiophore is swollen
at the tip to form a vesicle, and phialides arise from the upper half of the vesicle
with chains of conidia present that align parallel to the long axis of the conidio-
phore. (Lactophenol cotton blue stain, 400.)
Figure 61-9 Arthroconidia of Coccidioides species. Alternating barrel-shaped
arthroconidia are separated by thin-walled, empty disjunctor cells within portions
of the hyphae. (Lactophenol cotton blue stain, 400.)
conidiogenesis progresses in these species, barrel-shaped conidia called
arthroconidia are produced; these fragment easily and are disseminated
with little difculty, resulting in the high degree of infectivity demon-
strated by these important human pathogens (Fig. 61-9). The thallic
conidia of the dermatophytes are separated by size into two types: large
septate macroconidia (Fig. 61-10) and small, one-celled microconidia that
are simpler structures (Fig. 61-11).
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(LPCB) stain. If diagnostic structures are not observed, incubation can be
continued and the process repeated. The traditional method used in
observing mould morphology is to tease the mycelium apart with inoculat-
ing needles and examine the teased hyphae with LPCB stain.
Occasionally, a slide culture may be necessary to preserve easily dis-
rupted conidial structures in their original relationships. The classic
approach involves cutting a square of an appropriate agar medium (usually
Sabouraud dextrose or potato dextrose agar), which is suspended on a glass
slide and overlaid with a coverslip. The slide is supported by glass rods in
a Petri dish, to which sterile water is added for maintenance of humidity.
The coverslip subsequently can be removed after a few days incubation,
placed in a drop of LPCB, and observed for undisturbed reproductive
structures.
When a mould isolate is suspected of being a dimorphic fungus (e.g.,
growth on cycloheximide-containing medium), a slide culture should not
be performed, and a cellophane tape test or teased preparation should be
examined only after the preparation has been sealed in a biosafety cabinet
certied for use. Lactophenol cotton blue is fungicidal, but sealing the
coverslip with nail polish before observation provides additional protec-
tion. Alternatively, the culture may be ooded with 10% formalin (4%
formaldehyde solution) and incubated at room temperature overnight
before the mould is manipulated.
BIOCHEMICAL IDENTIFICATION
Biochemical tests are at the heart of identication schemes for yeast and
occasionally are useful for identication of moulds. A number of rapid tests
for the presumptive identication of yeasts are described in Table 61-9.
Biochemical characterization of yeasts may be accomplished by study
of fermentation or assimilation patterns. Assimilation testing, which is used
more extensively in the laboratory, assesses the ability of an isolate to use
a carbohydrate as the sole source of carbon needed for growth, or of nitrate
as the sole source of nitrogen. Numerous commercial identication
systems with varying incubation times from 472 hours are available and
have become the mainstay for yeast identication. These systems include
the API 20C AUX system and the VITEK 2 System (both from bio-
Mrieux, Hazelwood, Mo.), the MicroScan system (Siemens Healthcare
Diagnostics, West Sacramento, Calif.), the UniYeastTek system (Remel
Laboratories, Lenexa, Kan.), and the RapID Yeast Plus System (Innovative
Diagnostics Systems, Norcross, Ga.). All of these systems perform well, as
judged by reports in the literature and by prociency testing surveys of the
College of American Pathologists (Fenn, 1994; Riddle, 1994; Crist, 1996;
Bernal, 1998; Espinel-Ingroff, 1998; Ramani, 1998; Hata, 2007). However,
because no one system is known to be 100% accurate for identication of
yeast species, a combination of methods should be considered, especially
when rare species are recognized (Pincus, 2007).
In addition to fermentation and assimilation studies, stimulation of
growth by biochemical compounds is a secondary test used in the differ-
entiation of certain Trichophyton spp. (Weitzman, 1983). Inclusion of ino-
sitol and thiamine in various combinations into agar media (Trichophyton
agars) allows assessment of growth-stimulating properties. The endpoint
of the test, relative growth in comparison with a basal medium, is subjec-
tive, and both positive and negative controls should be included.
The test for urease production in cryptococci is of general utility in the
clinical laboratory to differentiate these species from Candida spp., particu-
larly in respiratory specimens (Canteros, 1996). C. neoformans is a pulmo-
nary and systemic pathogen, whereas Candida spp. are frequent inhabitants
of the upper airways but uncommon causes of primary pneumonia. Urease,
however, may be produced by other nonpathogenic species of Cryptococcus,
by Rhodotorula spp., and by some isolates of Trichosporon spp. and C. krusei.
Yeast Morphology
The germ tube test is an important initial step in the identication of yeast
isolates. Germ tubes, which are elongated, nger-like extensions from a
yeast cell, represent the beginnings of a true hypha (Fig. 61-16). This
structure can be differentiated from pseudohyphae by the lack of a con-
striction at the junction of germ tube and yeast cell and by the parallel cell
walls in the germ tube. True germ tubes are formed by both C. albicans
and Candida dubliniensis after growth in serum at 37 C for no longer than
4 hours. In many laboratories with well-trained staff, the germ tube in
combination with results of morphology on CM-T80 agar is considered
conrmatory for the identication of C. albicans/dubliniensis.
The traditional germ tube test involves inoculation of a tube of serum
(Table 61-7). After observation of the isolate for germ tubes at 37 C,
incubation is continued for subsequent study of hyphal morphology and
chlamydoconidia formation at 2530 C. Thus, all the information neces-
sary for rapid identication can be collected in one procedure. The
CM-T80 agar may be substituted, but regardless of the medium used,
incubation conditions must be carefully controlled and the test monitored
with controls to achieve good results. The traditional Dalmau technique
for demonstration of chlamydoconidia on cornmeal agar is detailed in
Table 61-8 (McGinnis, 1980).
Mould Morphology
The simplest method for examination of moulds is the cellophane tape
mount, using clear tape and staining with lactophenol cotton (aniline) blue
Figure 61-16 Germ tubes have extended from the yeast cells of Candida albicans.
No constriction is seen at the junction of yeast cell and germ tube. The walls of the
germ tube are parallel. The yeast was incubated for 2 hours at 37 C in serum.
(Gram stain, 400.)
TABLE 61-7
Serum Germ Tube Test
1. Aseptically transfer several colonies of yeast to a 12 75-mm test tube
containing approximately 0.5 mL of serum (human, fetal calf, bovine, or
rabbit).
2. Incubate the tube at 35 C for up to 3 hours.
3. Place one drop of the mixture on a clean glass slide and coverslip.
4. Examine under high dry (400) magnication and reduced light for the
presence of germ tubes.
TABLE 61-8
Yeast Morphology Test
1. Streak a light inoculum of yeast onto a section of a cornmeal agar con-
taining Tween 80.
2. Coverslip the area inoculated.
3. Incubate at 2530 C for 2472 hours.
4. Observe under 100 and 400 magnication using reduced light for
morphologic structures.
TABLE 61-9
Rapid Testing for Presumptive Identication of Yeast Following
Colonial Formation
Urease production Cryptococcus neoformans
Germ tube production Candida albicans/dubliniensis
Pseudohyphae present Candida species
Chlamydoconidia present Candida albicans/dubliniensis
Lipid growth requirement Malassezia furfur species complex
Red colonial pigmentation Rhodotorula species
Ascospore formation Saccharomyces cerevisiae
Trehalose assimilation Candida glabrata
F0NuAL C0LT0RE
8lMA8? CuL1u8L MLulA
1. Sabouraud's uexLrose Agar - general purpose lsolauon
medlum. pP 3.6 lnhlblLs bacLerlal growLh
2. oLaLo llake Agar - encourage growLh of
reproducuve sLrucLures
3. nuLrluonally oor Medla - used Lo sumulaLe
producuon of reproducuve sLrucLure
- ulluLe Pay lnfuslon Agar, Soll LxLracL Agar, 2
WaLer Agar
4. Mycosel or Mycoblouc - for dermaLophyLes
- conLalns SuA, cyclohexlmlde, chloramphenlcol
3. uermaLophyLe 1esL Medlum - for Mlcrosporum,
LpldermophyLon, 1rlchophyLon
F0NuAL C0LT0RE
ulllL8Ln1lAL MLulA
1. Cornmeal Agar - wlLh 1 glucose, Lo dlerenuaLe
1tlcbopbytoo tobtom ooJ 1tlcbopbytoo meotoqtopbytes
2. Czapek's - for Asperglllus
3. 8lrdseed/nlgerseed/SLalb's - for CrypLococcus
(brownblack colonles due Lo producuon of phenol oxldase),
uses LhlsLle (Culzoua seeds)
4. Couonseed - converLs mold phase of 8lasLomyces Lo
yeasL phase
5. klce MeJlom - fot lJeoufcouoo of Mlctospotom ooJoooll
6. 1tlcbopbytoo oqots - fot Jl[eteououoo of
1tlcbopbytoo
7. nlLraLe 8educuon Agar - for conrms nlLraLe reducuon
of CrypLococcus
F0NuAL C0LT0RE
8. urea Agar - deLecuon of urease producuon of C. neoformans
and dlerenuaLes 1. menLagrophyLes (+) from 1. rubrum (-)
9. ?easL lermenLauon 8roLh
10. ?easL Asslmllauon Medla

lnCu8A1lCn of lunCAL CuL1u8LS
-Speclmens should be lncubaLed up Lo a monLh (4weeks) and
examlned perlodlcally before reporung as negauve.
- MosL lungl grow opumally aL 30C
- ?easLs usually grow wlLhln 1-3 days whlle PlsLoplasma may
requlre 10-12 weeks growLh
- eLrl-dlshes should be sealed wlLh paramn or scoLch Lape Lo
enhance humldlLy
F0NuAL C0LT0RES
8LSL8vA1lCn of CuL1u8LS
1. SLorage ln WaLer - spores and conldla are washed wlLh
sLerlle waLer and placed ln vlals. ?easL culLure can be
Lransferred dlrecLly Lo sLerlle waLer ln small vlals and sealed
and sLored aL 81.
2. lreezlng - aL -70C and placed ln vlals, paLhogenlc culLures
should be placed ln crushproof meLal shlpplng conLalners
before freezlng.
3. Mlneral Cll - overlald onLo culLures, cap Lhe Lube ughLly and
sLored aL 81.
4. lreeze urylng
Nacioscopic Examination
lCMLn1
Cbserve Lhe reverse and obverse (surface) plgmenL of Lhe culLure
1ake noLe lf lL ls dlused or conned ln an area
uemauaceous -dark ollve green Lo darkbrown Lo black plgmenL
Pyallne - clear/colorless or pasLel
1Lx1u8L - besL observed ln cross secuon, relaLed Lo aerlal
hypha and number of conldla/spores
Colony textuie
Clabrous
leaLhery or waxy
llule lf any aerlal mycellum
velveLy
resembles plush or velveL fabrlc or suede
have shorL aerlal hyphae, few conldla or sopres
yeasLllke
resembles colonles of coagulase-negauve sLaphylococcl
bacLerla llke"
yeasLs appear dryer and duller
no aerlal mycella
couony
develop when colonles produce long aerlal hyphae
granular
fungl LhaL conldlaLe or sporulaLe heavlly, powdery
Nacioscopic Examination
1CCC8AP? - how Lhe colony surface ls arranged, besL
observed on Lhe reverse slde
1. llaL - common, form ls emclenL and requlres no exLra
eorL or enzymes from Lhe fungus
2. 8ugose - radlal grooves or deep furrows LhaL radlaLe
from Lhe cenLer, llke spokes of a blcycle wheel"
3. lolded - random folds, may be long, shorL, parallel or
aL rlghL angles
4. CraLerlform - leasL common, cenLral depresslon
surrounded by a ralsed edge
3. verrucose - warL llke or wlLh rough knobs, wrlnkled
convoluLed
6. Cerebrlform - braln llke
7. umbonaLe - buuon llke cenLral elevauon
C0L0NIES (Flat)
Folueu

Rugose
Ciateiifoim
Ceiebiifoim
veiiucose
Nacioscopic Examination
C8CW1P 8A1L
- 8apld Crowers - less Lhan 3 days (Saprobes)
- lnLermedlaLe Crowers - 6-10 days (CpporLunlsuc lungl
and uermaLophyLes)
- Slow Crowers - 11 or more days or up Lo 8 weeks
(SysLemlc and CpporLunlsuc)
0thei Tests
CL8M 1u8L 1LS1
- small amounL of lsolaLed yeasL colony plus serum or
plasma, lncubaLe aL 37C for 2-3 hours
- a drop of suspenslon ls examlned mlcroscoplcally
- (+) Cerm 1ube ln cooJlJo olblcoos
- Cetm 1obes ote bypbol llke exteosloos of yeost cells
ptoJoceJ wltboot o coosttlcuoo ot tbe polot of otlqlo

0thei Tests
SL8uM CuL1u8L - serum lncubaLed wlLh yeasL cell aL 37C for
2-3 hours
noLe:
-lf ?easLs are only seen - (negauve - noL Candlda)
-lf Lhere are yeasL cells and hypha (lL ls Candlda)
- lf yeasLs, hyphae and chlamydoconldla and germ Lubes
are presenL lL ls cooJlJo olblcoos or cooJlJo Joblloleosls
P
A
R
T

7
1169
classied within the phylum Basidiomycota, they are capable of producing
basidiospores under the right circumstances. This teleomorphic (sexual)
stage of cryptococci occurs only when appropriate mating types are
crossed; thus this stage is not generally recognized in the laboratory. Sexual
reproduction in the genera Cryptococcus, however, leads to increasing
genetic diversity with the potential to produce strains that are hyper-
virulent and show increased antifungal resistance (Huston, 2009).
Risk Factors
Immunosuppressive therapy or disease is a risk factor for cryptococcosis
(Huston, 2009). Before the appearance of HIV infection, 30%50% of
patients with cryptococcal infection were immunologically normal, as
measured by available parameters. Risk factors for these patients include
neoplasia, diabetes mellitus, immunosuppressive therapy, and immuno-
logic disease. The introduction of the HIV-infected patient dramatically
increased the number of cases of cryptococcosis, and although advances
have been made in antiretroviral therapy, in antifungal treatment, and in
intracranial pressure management in these patients, Cryptococcus continues
to have a high rate of mortality (Sajadi, 2009). An estimate of the global
burden of cryptococcal meningitis nds the numbers of cases and deaths
to be very high within areas of sub-Saharan Africa, where there is a high
incidence of HIV-infected people (Park, 2009). Cryptococcosis also
remains a signicant opportunistic infection in solid organ transplant
recipients (Singh, 2008).
Clinical Disease
Primary cryptococcal disease generally occurs in the lungs following inha-
lation of the fungus from the environment. This disease can remain local-
ized or can disseminate by hematogenous spread to other tissues, most
frequently the central nervous system. The severity of the disease is depen-
dent on the hosts immune response, with severe disease most frequent in
immunologically compromised patients. Practical guidelines for the man-
agement of cryptococcal diseases was recently published by the Infectious
Diseases Society of America (Perfect, 2010).
Respiratory Tract
Cryptococcal infection of the respiratory tract exhibits a wide variety of
presentations (Jarvis, 2008; Shirley, 2009). Immunologically competent
patients may exhibit no symptoms despite the presence of cryptococci in
the lower respiratory tract, and the infection may be diffuse or localized,
to include the formation of coin lesions that usually do not calcify. Immu-
nocompromised patients on the other hand may have extensive infection
that often is accompanied by other infectious agents, particularly Pneumo-
cystis (carinii) jiroveci or cytomegalovirus. Extrapulmonary disease may
appear weeks after a pulmonary infection has been documented.
Skin Lesions
Skin lesions usually result from hematogenous dissemination from the
respiratory tract in immunocompromised patients (Christianson, 2003).
These lesions present as single or multiple papules, which enlarge and
ulcerate, producing a thin exudate that contains the yeast. Primary cutane-
ous manifestations of the disease are rare but may also be reported in
immunocompetent individuals (Revenga, 2002).
Bone and Joint Infection
Bone and joint infection may occur usually as a result of dissemination
from the respiratory tract (Liu, 1998). Osteolytic lesions are produced,
with abscesses formed in adjacent soft tissue that contain a thin exudate
with large numbers of cryptococci. Less commonly, joint spaces are
involved.
Central Nervous System Infection
Cryptococcal meningitis is the most frequent and most serious focus of
disseminated cryptococcal infection (Satishchandra, 2007; Dorneanu,
2008; Patel, 2009). Onset of this disease may be acute, or presentation may
be insidious and progression torpid. Headache and changes in mental
status and personality often dominate the clinical picture. Basilar menin-
gitis, involvement of the cranial nerves, and invasion of the underlying
cortex result in hydrocephalus and decreased visual acuity. Fever, if present,
usually is of low grade, and typical signs of acute meningeal irritation, such
as stiff neck and Kernigs and Brudzinskis signs, are often absent.
Pathology of Cryptococcal Infection
The histologic response depends on the degree of encapsulation of the
infecting cryptococcal strain. Most commonly, little or no inammatory
blood culture systems detect most clinically signicant yeast isolates
(Reimer, 1997). Tissue specimens, scrapings, and swabs from the mouth
or vagina should be inoculated onto primary fungal isolation media with
and without cycloheximide. The presence of lamentous extensions from
the edges of the colony (feet) is a macroscopic indication that pseudohy-
phae are being produced (see Fig. 61-1). C. glabrata (formerly Torulopsis
glabrata ) and Cryptococcus spp. do not form pseudohyphae in vitro, and
some other Candida spp., such as C. lusitaniae and Candida guilliermondii,
also may not form pseudohyphae.
The extent of the mycologic evaluation depends on the clinical setting
and the specimen type. Candida spp. are frequently isolated from the
respiratory and urinary tracts; however, interpretation of a nding of
Candida spp. in these areas is difcult. Complete identication of isolates
from these sites should be accomplished only selectively after consultation
with the responsible clinician.
A preliminary report of C. albicans/dubliniensis may be issued if the germ
tube test is positive (see Fig. 61-16). Additional study of yeast morphology
using cornmeal agar to conrm the presence of chlamydoconidia can
facilitate identication of C. albicans/dubliniensis within 2448 hours (Fig.
61-17). Because both C. albicans and C. dubliniensis are germ tube positive,
and because they have a high degree of phenotypic similarity, distinguish-
ing between these two species has been difcult. However, strict adherence
to detail when it comes to growth at 42 C, the production of abundant
chlamydoconidia, and the sugar assimilation pattern can be used to dif-
ferentiate between them (Campanha, 2005; Ells, 2009).
When germ tubes and chlamydoconidia are not demonstrated, a pre-
liminary or presumptive identication of Candida spp. can be made only
if pseudohyphae are present and arthroconidia are absent. Although con-
rmed species identication under this circumstance requires the use of
assimilation tests, ancillary morphologic observations can be used to speed
up the identication process (see Table 61-10). For instance, a rapid assimi-
lation trehalose test procedure has been suggested by the CLSI (document
M35-A2) for the identication of C. glabrata, a species that has emerged
as a common cause of invasive disease with known resistance to standard
antifungal therapy (Clinical Laboratory Standard Institute, 2008a).
THE GENUS CRYPTOCOCCUS
The Cryptococcus spp. complex consists of two species: C. neoformans and
Cryptococcus gattii (formerly called C. neoformans var. gattii). These species
are known to cause systemic infection in both immunocompetent and
immunocompromised individuals (Bovers, 2008; Ma, 2009). The environ-
mental reservoir for C. neoformans (teleomorph, Filobasidiella neoformans) is
primarily pigeon guano, and infections caused by this organism occur
worldwide. C. gattii on the other hand is found predominantly in tropical
and subtropical areas, especially those associated with eucalyptus trees, and
infection appears to be limited in distribution, primarily to northern Aus-
tralia and Papua New Guinea (Huston, 2009). However, recent infections
have been noted in Vancouver Island and surrounding areas, and a high
rate of mortality has been associated with these infections (Kidd, 2007;
MacDougall, 2007; Bartlett, 2008; Dixit, 2009). Because both species are
Figure 61-17 Chlamydoconidia produced by Candida albicans. These thick-
walled asexual reproductive structures that occur most commonly at the ends
of pseudohyphae are characteristic for this species and for Candida dubliniensis.
(Cornmeal agar plate, 400.)
0thei Tests
Palr 8alung 1esL - for uermaLophyLes slnce Lhey are
keraunophlllc, dermaLophyLes wlll grow selecuvely on lL.
Palr eneLrauon 1esL - ln-vlLro LesL Lo dlsungulsh 1.
menLagrophyLes from 1. rubrum
- no eneLrauon aer 1 monLh = 1. rubrum
- WlLh v shaped peneLrauon = 1. menLagrophyLes
0thei tests
8apld urease LesL - for urease produclng yeasLs recovered
from resplraLory speclmens and oLher speclmens
(+) plnk Lo purple color aer 2 days
(-) = ctyptococcos oeofotmoos
(-) = ctyptococcos olblcoos
urease 1esLs for 1. menLagrophyLes (+) and 1. rubrum (-)
8apld nlLraLe 8educLase 1esL - for CrypLococcus (+)
Levodopa-lerrlc ClLraLe 1esL
- phenol oxldase reacLs wlLh dlhydroxyphenylalanlne ln
Lhe presence of ferrlc nlLraLe Lo form melanln
- Levodopa ls used as a subsLraLe
- CrypLococcus neoformans produces henol Cxldase (+).

0thei tests
1hlamlne 8equlremenL - very useful for dermaLophyLes
1rlchophyLon Agars (1-7)
CrowLh on 8lce Craln - dlerenuaLes M. canls (+ growLh)
from M. audoulnll (no growLh aer 10days)
1emperaLure SLudles
Seiologic anu Antigen Tests
ComplemenL llxauon - for P. capsulaLum and oLher
dlmorphlc fungl
lmmunodluslon 1esL - for P. capsulaLum
LLlSA - Asperglllus anubodles
CrypLococcal Anugen ln CSl and Serum
CounLer lmmunoelecLrophoresls
Lxoanugen 1esLs

1?L Cl M?CCSLS

CAuSA1lvL lunCAL ACLn1S

M?CCSlS
Superclal
lnfecuons llmlLed Lo Lhe
ouLermosL dead" layers of skln
and halr
Malassezla furfur
PorLaea weneckll
1rlchosporon specles
eldrala horLae
lLyrlasls verslcolor
1lnea nlgra
WhlLe pledra
8lack pledra
CuLaneous
lnfecuons LhaL exLend deeper
lnLo Lhe epldermls as well as
lnvaslve halr and nall dlsease
(keraunlzed poruons)
Ml crosporum specl es, LrchophyLon specl es, and
LpldermophyLon occosum
Candlda alblcans and oLher candlda specles
uermaLophyLosls
Candldlasls of skln, mucosa, or nalls
SubcuLaneous
lnfecuons lnvolvlng Lhe dermls,
subcuLaneous ussues, muscles
and fascla
SporoLhrlx schenckll
hlalophora verrucosa, lonsecaea pedrosol, oLhers
seudallescherla boydll, Madurella myceLomaus, oLhers
Lxophlala, blpolarls, exserohllum, and oLhers
SporoLrlchosls
ChromoblasLomycosls
MyceLoma
haeohyphomycosls
Lndemlc (prlmary, sysLemlc)
lnfecuons LhaL orlglnaLe prlmarlly
ln Lhe lung buL may spread Lo
many organ sysLems (lymphauc,
clrculaLory)
Coccldloldes lmmlus
PlsLoplasma
capsulaLum
8lasLomycoses dermauudls
aracoccldloldes braslllensls
Coccldloldomycosls

PlsLoplasmosls
8lasLomycosls
aracoccldloldomycosls
CpporLunlsuc
lnfecuons caused by fungl LhaL
lnfecL because of compromlslng
slLuauons
Candlda alblcans and oLher candlda specles
CrypLococcus neoformans
Asperglllus fumlgaLus and oLher asperglllus specles
Specles of rhlzopus, absldla, mucor, and oLher zygomyceLes
enlcllllum marneel
SysLemlc candldlasls
CryLococcosls
Asperglllosls
Mucormycosls (zygomycosls)
enlcllllosls