Intro to Mycology, Yeasts

fungi vs bacteria

fungi:

eukaryote, uni/multicellular heterotrophic, cell wall (chitin, glucan, mannans), asexual/sexual reproduction

bacteria:

prokaryote, unicellular, heterotrophic/autotrophic, cell wall (peptidoglycans), asexual repro

fungal cell wall

cell wall = mannoprotein, glucan, chitin

cell membrane = ergosterol

fungal forms: yeast

unicellular

asexual repro via budding

colony: creamy, dry, mucoid

fungal forms: mold

multicellular hyphae

asexual/sexual repro by spores

colony: fuzzy or powdery

fungi names

anamorph? telemorph?

anamorph = asexual (often observed in med mycology)

telemorph = sexual

conidiogenesis

blastic

thallic

blastic = produces spores from conidiogenous cell (holoblastic, enteroblastic)

thallic = “ “ from existing hyphal structure (arthoconidia, aleurioconidia)

dimorphic fungi

thermodimorphic - exist as 2 forms:

30C: mold/free living form in nature or lab temp

37C: changes to yeast/parasitic form in tissue or lab

pseudohyphae

tubular str that result from incomplete budding of yeast cells

hyphae types

most common in mold

septate: cross wall

non-septate: no cross wall

pauciseptate: rare cross walls (zygomyces)

blastoconidia (yeast)

repro yeast str, forms along pseudohyphae, hyphae, or singly

conidia types (mold)

repro mold str

form at ends of conidiogenesis cell

→ micro- & macroconidia

direct fungal exam (DFE)

calcofluor white stain → binds to chitin & cellulose

histological stains for fungi

Gomori Methenamine silver stain = gray/black (easiest histo stain)

…

non-blood fungi cultures set up (primary media)

IMA - chloramphenicol

BHI

BHI/SB - non-inhibitory

BHI G&C - gentamicin, chlora

BHI CC&G - ““ cycloheximide

Mycosel - cycloheximide & chloramphenicol (for dermatophytes)

CHROMagar Candida - for mixed Candida infns

blood fungi cultures

only upon special request, otherwise, routine blood culture bottles used

secondary fungi media

sabouraud dextrose agar - non-inhib

PDA: potato dextrose - induce sporulation (molds)

PFA: potato flake

yeast extract

BHI

corn meal agar (CMA) - to ID common yeast

fungi culture incubation

37C: BHI, CHROM

30C: BHI CC&G, IMA, Mycosel

fungal bloods: 42 days

regular: 2x/wk for 4 weeks

tools to ID fungi (macroscopic)

growth rate, colonial morph, texture, pigment (front & back)

tools to ID fungi (microscopic)

lactophenol aniline blue slide: scotch tape method

tease mount

slide culture

fungi culture workup

mold isolated: ID to genus level for all sources

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yeast isolated:

resp source: r/o Cryptococcus, ID upon request

nonresp: if Candida, ID to sp level; if non-Candida, ID to genus level

Antifungal Susceptibility testing

mold: upon request, long TAT

yeast:

done for all BLOOD cultures for Candidas p

sterile body fluids, and SIS cultures

only in house for Candida

non-invasive Candidiasis

mucocutaneous: oral cavity (thrush), trachea, bronchi, gastric

cutaneous: moist skin areas (armpit, groin), diaper rash in neonates

invasive Candidiasis

enters bloodstream → disseminates

catheter related (m/c)

acute disseminated

chronic disseminated Candidaisis - immunocompromised

deep organ

DFE note:

pseudohyphae

true hyphae & arthroconidia

number of buds & attachment

CHROM vs corn meal agar

CHROM - good for mixed Candida infns

CMA - see microscopic morph of yeast

Candida albicans

chrom: green

CMA: clusters of blastoconidia & single terminal chlamydospores

feet on SBA! (not always)

Candida dubliniensis

chrom: darker green

CMA: clusters of blastoconidia & pairs/clusters of terminal chlamydospores

Candida glabrata

chrom: smooth w mauve center

CMA: blastoconidia only, no pseudohyphae

rapid trehalose +

Candida tropicalis

chrom: metallic blue

CMA: blastoconidia arise sparsely along pseudohyphae (no chlamydospores)

Candida krusei

chrom: rose pink center w white edges, dry

CMA: blastoconidia arise only from septa of hyphae, matchstick (septate or pseudoseptate)

**intrinsically fluconazole-R

Candida parapsilosis & other spp

chrom: white to pink w age

CMA: highly branched curved pseudohyphae; “tumbleweed”

Candida auris

MDR, high mortality rate

90% fluconazole R

30% amphotericin R

some found to be R to all classes: azoles, polyene, echinocandins

persist on human skin, environment due to biofilm & resist standard cleaning procedures

C. auris protocol

report to CDC

ID w MALDI; Vitek can misID

germ tube test

germ tube = non-constricting tube str from yeast after 2-4 hrs of incubation in fetal bovine serum

C. albicans + in 37C & 42C

C. dubliensis + in 37C, but - in 42C

if + → presumptive ID for either C. albicans or C. dubliensis

rapid trehalose

pos rxn = yellow color after 3 hr at 42C

pos for C. glabrata

API20C

based on carb assimilation

48-72h → pos = turbidity

backup if no MALDI ID

Vitek II YST card

same as API20C but automated

MALDI-TOF

matrix assisted laser deabsorption / ionization time of flight (rapid ID)

sample plus matrix → submitted to laser → convert to heat energy → vaporizes into ions

electrical field guides ions into vacuum tube → sep it by weight & flight time → displays into peaks/spectrum → compared to database, given confidence level

AST for yeast

agar: KB, etest

broth

sensititre yeastone (Trek Dx)

for Candida spp

MIC = 50% of growth reduction, 1st blue well

pink = pos/growth

blue = neg/NG

left to right one Afx (low to high conc)

amphotericin B is vertical

intrinsic Afx resistance

amphotericin B

fluconazole

caspofungi/micafungin

amp B-R → C. lusitaniae, Malassezia furfur

fluconazole-R → C. krusei

Casp/Mica-R → Cryptococcus neoformans / gattii

Cryptococcus neoformans

#1 clin significant

found in bird poop (pigeons) & contaminated soil; infn via aerosols

immunocompromised, infants, young children, HIV

Cryptococcus gattii

#2 clin significant

found in Pacific NW, eucalyptus tree, soil in tropical/subtropical areas

causes disease in immuno- competent & compromised

• pneumonia, meningitis

• cryptococcoma: infectious granules/lesions in brain, lungs, eyes

• aggressive treatment req’d!

Crypto virulence factors

capsules

proteinase

mannoproteins

superoxide dismutase

melanin pigment

polyol metabolites

Cryptococcosis forms

CNS: MG, 100% fatal if untreated

pulmonary: pneumonia, pulmonary lesions

cutaneous: manifestation of systemic infn

disseminated

lab ID of Crypto

DFE: india ink (can be false neg due to non-capsule strains)

urease +

NO3 reduction -

phenoloxidase (L-DOPA ferric citrate test)

niger seed agar test: maroon red-brown black pigment

Crypto Ag lateral flow assay (CSF, for both sp)

why is india ink not reliable?

false neg from non-encapsulated Crypto

caffeic acid agar/niger seed

cornmeal agar morph: Crypto

no pseudohyphae

round yeast

how to distinguish Crypto neoformans from C. gattii?

canavinine-glycine-bromthymol blue agar:

pos rxn: blue color → C. gattii

MADLI TOF

DNA detection by PCR

impt to do bc treatment is more aggressive for C. gattii!

Malassezia furfur complex (lipophilic)

normal skin flora, lipophilic, can cause disease

disease: Tinea versicolor

may infect pt due to lipid rich catheter infusions, ie neonates receiving parenteral nutrition w lipids

non-lipophilic yeast Malassezia pachydermatis

dogs = host

occasionally iso from humans

doesn’t need oil

Lipophilic yeast ID

Malassezia furfur req lipids → olive oil

fungal BC (pt <1yr) → add olive oil

GST: unipolar budding yeast w collarettes, bowling pins or russian dolls

KOH prep: spaghetti & meatballs

other yeast:

Rhodotorula spp.

Saccharomyces spp.

col: smooth, mucoid, salmon color

col: moist, white

yeast-like fungi

Trichosporon spp: wrinkled, heaped, white; rectangular arthroconidia

Geotrichium spp.: dry to powdery, white, rectangular arthroconidia

Prototheca spp.

green algae that lacks chlorophyll

resembles yeast

round cells (sporangia) containing sporangiospores (endospores) no true budding

stains blotchy