Blakes mycology part 2 Flashcards
Identification of fungi
Molds are usually identified regardless of the source
Molds that fail to sporulate may be reported but not identified if dimorphism has been ruled out
Usually identified based on macroscopic & microscopic morphology
Asexual reproduction
Conidiophore vesicle—> Metulae—-> Phialides —–> Mature conidiophore —-> Conidia
Vegetative growth
Germling—> Foot cells—-> Formation of mycelium—-> Stalk
Safety
All molds must be handled in
plates should be
yeast are handled on
All molds must be handled in Class II biological safety cabinet
Plates should be sealed
Yeasts can be handled on benchtop
Mold identification
Growth rate
Colonial morphology
Microscopic morphology
Preparation for microscopic observations
Tape preparation
Wet mount (tease prep)
Microslide culture
Lactophenol cotton (or aniline) blue
Tease mount and tape prep
Cultivation
Incubate at blank
common media
Mycosel has what
Incubate at room temperature unless looking for yeast phase
Common media: Sabouraud dextrose, potato flake, BHI, mycosel (cycloheximide)
dermatophyte test medium
Deferential media
Differential: cottonseed conversion, urea,
trichophyton agars 1-7
Corn meal agar
Typical media selections
All-purpose: SABS or potato-based (PFA, PDA)
Selective: Mycosel (contains cycloheximide)*
Enriched: BHI (for Histoplasma)
Some Cryptococcus and Aspergillus are inhibited by
some Cryptococcus and Aspergillus inhibited by cycloheximide
Penicillium marneffei
from specimen
-Gram stain from blood culture bottle showing hyphae (mold form that exists at room temperature). Since blood cultures are incubated at 37 degrees Celsius, you would not expect to see hyphae unless the bottle had been left at room temperature for some time.
-Gram stain from blood culture showing yeast forms. This is what you would expect to see if the bottle was placed into the incubator with minimal delay.
Trichophyton species
The most common species are
both can produce a
The most common species are T. rubrum and T. mentagrophytes
-pigment
T. rubrum has a
Red pigment
T.mentagrophytes can range from
red to orange to reddish brown.
T. mentagrophytes are
T. mentagrophytes is urease positive and can penetrate hair.
Hair perforation test for
T. mentagrophytes
T.rubrum is
Urease negative
Macroconidium for Microsporum species
Large thick walled and divided into many cells by transverse septa. Tend to be spindle or boat-shaped
Microconidia for Microsporum species
Relatively few or absent if present they are tear-shaped and bome singly on the hyphae
Trichophyton species Macroconidium
Few or absent in some species. If present, they are elongated and cigar or pencil shaped. The walls are thin and smooth. Divided by septa into 3-8 cells
Microconidia in Trichophyton species
Usually numerous and bome singly along the hyphae or in grape like clusters
Direct detection of fungi
Calcofluor white
KOH
Histology stains
Many fungi are diagnosed
note the Aspergillus fumigatus
-Many fungal infections are diagnosed microscopically before culture by visualizing fungal elements.
-Note the Aspergillus fumigatus infection seen in the following slides. The specimen was pleural fluid.
Other direct detection methods
NAT
Nucleic acid amplification
not available for most fungi
NAT for Histoplasma, Blastomyces, Coccidioides: culture confirmation
Serodiagnosis
Useful for systemic fungi
False-negatives: immunocompromised
False-positives: cross-reactivity
Complement fixation and immunodiffusion should be used in conjunction with antibody detection
EXO antigent test
Immunodiffusion
Yeast general charateristics
Unicellular organisms that
reproduce asexually by budding
(Blastoconidia) or sexually.
Easy to see microscopically, but unable to identify to species level.
Microscopic analysis
Certain environmental stimuli can produce different morphologies that provide useful information for physician
Candida albicans features
Germ tube
Pseudohyphae
Chlamydospores
Cryptococcus spp
Capsule
Structures seen in yeast
Blastoconidia
Chlamydospores and pseudohyphae are most common in Candida albicans
Agents of yeast infection
The most commonly seen/significant isolates are:
Candida albicans (and other species)
Cryptococcus neoformans and C. gattii
Trichosporon
Malessezia furfur
Candida
most common
other species
is a what
extended antibiotic use and chemo
Candida: most common yeast isolated
C. albicans most common species
Other species: C. glabrata, C. tropicalis, C. parapsilosis, C. krusei
Candida is normal flora
Extended antibiotic use and chemotherapy can lead to the overgrowth of yeast and infection
Candida conditions
Intertriginous candidiasis
(cutaneous)
Chronic paronychia
Candidial Onychomycosis
Perleche
Candida albicans
Common Candida Infections
Thrush
Vulvovaginitis
Pulmonary infections
Eye infection
Endocarditis
Meningitis
Fungemia
Candida laboratory diagnosis
Gram stains
Germ tubes/pseudohyphae
Chlamydospores (chlamydoconidia)
Culture
Germ tube procedure
Inoculate a small amount of yeast cells into
about 0.5 mL serum.
Incubate and examine on 40x after two to four hours
Chromagar what
Candida
C.glabrata what
mauve to dark mauve- light purple color
C.krusei color
light rose to pink with a whitish border
Candida auris
emerging what
four geographic clades
-Emerging fungal pathogen (considered a global health threat)
Four geographic clades
-South Asian, South African, South American, and East Asian
Candida auris diseases
and causes what
- Can cause invasive disease
- Has caused outbreaks
Can persist on surfaces
Routine disinfectants (Quaternary Ammonium Compounds) may not be effective
Candida auris
difficult to what
- Difficult to identify by standard lab methods
No phenotypic methods easily distinguish from other Candida
Most frequently misidentified as C. haemulonii
MALDI-TOF is the best method
Candida auris is frequently what
- Frequently multi-drug resistant
90% resistant to fluconazole (in the US)
30% resistant to amphotericin B (in the US)
<5% resistant to echinocandins (in the US)
Cryptococcus neoformans is what
Saprobe in nature (pigeons)
Immunocompromised: dissemination common with or w/o meningitis
Cryptococcus infections
-Manifestation depends on host immune status
-Initially, may present as a pulmonary infection.
-May travel to the central nervous system (neurotropic) where it may be able to cross the blood-brain barrier which is facilitated by a thick polysaccharide capsule and other virulence factors such as phenyloxidase.
Disseminated infections in cryptococcus infections
Symptoms
Specimens
Patients with disseminated infection
may have painless papular skin lesions
that may ulcerate.
Symptoms are similar to bacterial meningitis
Specimens: Blood and Spinal Fluid
Cryptococcus lab diagnosis
-India ink, gram stain
-Culture; bird seed agar (niger seed)
Urease test,
-Serologic: Based on detection of antigens (not antibodies). Latex test detects capsular polysaccharide antigens, lateral flow test also available now
Caffeic acid test
Malassezia furfur
Causes tinea versicolor (pityriasis versicolor)
Superficial brownish scars
Disseminates in infants
Malassezia furfur
Rare but important agent of
Attention to medical practices when administering what
-Rare but important agent of pulmonary infection or fungemia in neonates, especially pre-term infants.
-Attention to medical practices when administering lipid-containing emulsions to infants can greatly reduce the incidence
culture of Malessezia
-Culture is usually performed in cases of suspected fumgemia (babies).
-M. furfur is a lipophilic yeast, therefore in vitro growth must be stimulated by natural oils or other fatty substances.
-The most common method used is to overlay Sabouraud’s dextrose agar containing cycloheximide with olive oil or alternatively to use a more specialized media like Dixon’s agar which contains glycerol mono-oleate (a suitable substrate for growth).
Trichosporon beigelii
-Yeast-like organism
-Immunocompromised patients/leukemia
-Usually superficial/Rarely disseminated
It also causes white piedra
Usually identify to species
from
in
patient is not
-From sterile sources
-In serious ill/compromised patients
-Patient not responding to therapy
Presumptive identification
C.albicans-
Cryptococcus-
Commercially blank
-C. albicans – germ tubes
-Cryptococcus – India Ink and antigenic typing
-Commercially manufactured kits are available
Cornmeal agar
Cornmeal agar (chlamydospores in Candida)
Sabouraud Dextrose Agar
will grow most yeast and molds
Chromagar
Chromagar (for Candida)
Niger seed
Niger seed (for Cryptococcus
Oil overlay for
Oil overlay for Malessezia with olive oil in sabouraud dextrose media
Mycosel for
Mycosel for filamentous fungi
Other ID methods
Sugar assimilation
API, RapID Yeast Plus Panel, Vitek, MALDI-TOF
Vitek
Yeast card
Antifungal agents
-Polyenes: Amphotericin B
Binds ergosterol, increases cell permeability, leading to leakage of cellular contents and cell death
-Azoles: fluconazole, itraconazole, voriconazole, posaconazole
Impair ergosterol synthesis in the cytoplasmic membrane
-Nucleoside analog – flucytosine
Disrupts pyrimidine metabolism in the cell nucleus
-Echinocandin – caspofungin and micafungin
Inhibits formation of 1,3 glucan which is part of the cell wall
-Allylamine – terbinafine
Disrupts the fungal cell membrane (squalene oxidase)
Polyenes: Amphotericin B
Binds ergosterol, increases cell permeability, leading to leakage of cellular contents and cell death
Azoles
fluconazole, itraconazole, voriconazole, posaconazole
Impair ergosterol synthesis in the cytoplasmic membrane
Nucleoside analog
flucytosine
Disrupts pyrimidine metabolism in the cell nucleus
Echinocandin
caspofungin and micafungin
Inhibits formation of 1,3 glucan which is part of the cell wall
Allylamine
– terbinafine
Disrupts the fungal cell membrane (squalene oxidase)
Susceptibility testing methods yeast
frequently performed for yeast
Usually from sterile sites and upon Physician request
IVD method, Sensititre YeastOne
Suceptibility testing for molds
molds performed at reference laboratories
Sterile site infections
Therapeutic failure
Sensititre
Yeast one
Dixon media
Media for M furfur with glycerol mono oleate
Spaghetti and meatballs
