Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Enclosed electric incinerator
• Eliminates the hazards of open gas flames
SPECIMEN COLLECTION,
PROCESSING, AND Use of Petri dishes is hazardous; use screw-top tubes
to prevent aerosols.
LABORATORY DIAGNOSIS
OF FUNGI SPECIMEN COLLECTION, HANDLING, AND
TRANSPORT
Mr. Charlie Cruz, RMT, ASCP
Common specimen types
• Respiratory secretions, hair, skin, nails,
TOPIC OUTLINE tissue, blood, bone marrow, CSF

I. Laboratory Safety Issues Use antimicrobial-containing media to inhibit bacterial

II. Specimen Collection, Handling, and growth.
Transport
1. Hair
III. Predominant Culture Sites for Recovery
➢ A Wood lamp emits UV light >365 nm
of Etiologic Agents
and can be useful in identifying
IV. Major Clinical Syndromes and
infected hairs.
Commonly Associated Fungal Pathogens ➢ The Wood light can be shown on the
V. Guideline for Identification of Fungal scalp.
Isolates ➢ Sterile forceps should be used to pull
VI. Direct Examinaiton of Specimens affected hair.
VII. Staining Characteristics of Fungi ➢ Hairs are placed directly into a sterile
VIII. Isolation Methods Petri Dish then inoculated onto
i. Summary of Primary Fungal fungal medium and incubated at 22
Culture Media to 30° C.
IX. Fungal Identification
i. Immunodiagnosis of Fungal 2. Skins and nails
➢ Area is cleaned with 70% alcohol
Disease
before sampling.
ii. Molecular Identification
➢ Outer edge of a surface legion is
X. Identification of Yeast scraped (skin); nails are cut or
i. Miscellaneous Tests for Yeasts removed.
XI. Identification of Molds ➢ A KOH wet mount is prepared with
i. Miscellaneous Tests for Molds some of the sample collected.
XII. Antifungal Susceptibility ❖ KOH breaks down tissue
i. Antifungal Agents making it easier to view
ii. Antifungal Susceptibility Testing fungal hyphae.
➢ Sample material is inoculated directly
onto the agar.
LABORATORY SAFETY ISSUES
3. Blood and bone marrow
Class II biosafety cabinet ➢ Lysis centrifugation system
• Reduces personal exposure against fungal ❖ Release organisms from cells
conidia ➢ Other systems

Class III biosafety cabinet (risk group 3) 4. CSF (and other sterile fluids)
• Coccidioides immitis/posadasii ➢ Concentrate via centrifugation before
• H. capsulatum inoculation.

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College of Medical Laboratory Science

MYCOVIRO
➢ One drop for India ink or MAJOR CLINICAL SYNDROMES AND
cryptococcal antigen test for C. COMMONLY ASSOCIATED FUNGAL
neoformans PATHOGENS
➢ Plated directly (sterile)

5. Abscess, wound, or tissue

➢ Mince and grind tissue
➢ Plate directly or perform other tests

6. Respiratory specimens
➢ Plated directly or digested with
mucolytic prior to plating

7. Urogenital or fecal specimens

➢ Culture (yeast will grow on routine
culture media)
➢ If urine, first voided morning urine
❖ Centrifuge to sediment

PREDOMINANT CULTURE SITES FOR

RECOVERY OF ETIOLOGIC AGENTS

Bon
Respir Blo e Tis Sk Mu Bo
Infection atory od Mar sue in cus ne
row
Blastomycosi
+ + +
s GUIDELINE FOR IDENTIFICATION OF FUNGAL
Histoplasmos ISOLATES
+ + + +
is
Coccidioidom
+ + +
ycosis
Paracoccidioi
+ + +
domycosis
Sporotrichosi
+ + + +
s
Chromoblast
+ +
omycosis
Eumycotic
+ +
mycetoma
Phaeohypho
+ +
mycosis
Organisms may be recovered from multiple sites in
disseminated infections.

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College of Medical Laboratory Science

MYCOVIRO
DIRECT EXAMINATIONS OF SPECIMENS

KOH preparation
• 10% to 20% solution of KOH
➢ Some may contain dimethyl sulfoxide
(DMSO) and a stain
• Equal parts sample to KOH
• Cover-slipped and heated gently then cooled C. neoformans: India ink preparation at (a) low & (b) high
for 15 minutes magnification show spherical, narrow-budding yeasts with thick
capsule.
• Purpose of KOH
➢ Dissolve the keratin and skin layers
to visualize dermatophytes (hair,
skin, nails, tissues)

KOH with calcofluor white

• Dye binds to cellulose or polysaccharides
present in chitin.
➢ Apple-green or blue-white in UV light

India ink
• Black background stain
➢ Visualize capsules (looks like halos)
of yeast or bacteria
• Presumptive ID of C. neoformans in CSF but Bone marrow stained with Giemsa stain.
low sensitivity

Tissue
stains STAINING CHARACTERISTICS OF FUNGI
• Periodic acid–Schiff (PAS)
• Gomori methenamine-silver (GMS) Color of
• Hematoxylin and eosin (H&E) Background
Stain Fungal
• Giemsa Color
Element
➢ Detection of H. capsulatum in blood Periodic acid–
or bone marrow Magenta Pink or green
Schiff
• Fontana-Masson Gomori
methenamine Black Green
silver
Purple-to-blue
yeast with
Giemsa Pink to purple
clear halo
(capsule)
Yeast with
India ink clear halo Black
(calsule)
KOH Refractile Clear
KOH–
calcofluor Fluorescent Dark
Fungal hyphae stained with calcofluor white, viewed under UV white
light. Masson-
Brown Pink to purple
Fontana

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College of Medical Laboratory Science

MYCOVIRO
ISOLATION METHODS The yeast form of
dimorphic fungi and
Culture media other organisms grow.
SDA
• Sabouraud dextrose agar (SDA) (with or
without antimicrobials) Dermatophytes grow
• Potato dextrose agar (PDA) poorly.
• Brain–heart infusion (BHI) agar enriched Yeasts, such as
with blood and antimicrobials Cryptococcus, grow
• Plates or tubed media well.
➢ Tubes safer to handle and less
susceptible to drying The yeast form of
BHI agar with blood Histoplasma capsulatum
Incubation takes up some of the
• Room temperature or 30° C heme pigment in the
➢ Dimorphic at 37° C medium and becomes
• Maintain 4 to 6 weeks. light tan, with a grainy,
• Examine twice a week for growth. wrinkled texture.
• Incubation characteristics should be *The antibiotics generally used are cycloheximide and
recorded. chloramphenicol.

Summary of Primary Fungal Culture Media Fungal

Principle Purpose
medium
Expected Growth Acid pH & high
Medium
Results dextrose
At 22oC concentration
General-
Initial isolation of inhibits
purpose
pathogens and Sabouraud bacterial
medium for
saprobes. dextrose agar growth but
cultivation or
SDA (SDA) permits growth
isolation of
Dimorphic fungi may of fungi; some
many fungi.
exhibit their mycelial formulations
phase. also contain
Saprobes generally antibiotics.
inhibited on this Nutrient-rich
medium. medium
containing Selective
SDA with antibiotics*
Dermatophytes and chloramphenico isolation of
most of the fungi Inhibitory l & sometimes fungi from
considered primary mold agar gentamicin or specimens that
pathogens grow. (IMA) ciprofloxacin; may contain
Initial isolation of chloramphenico commensal
BHI agar pathogens and l suppresses bacterial flora.
saprobes. the growth of
BHI agar with Recovery of pathogenic many bacteria.
antibiotics fungi.
Initial isolation of
Inhibitory mold agar pathogens except
dermatophytes.
Primary recovery of
Cycloheximide
dermatophytes.
At 37oC

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College of Medical Laboratory Science

MYCOVIRO
It is frequently
The
used to target
nonselective
dermatophytes
Nutrient-rich formulation is a
or thermally
medium general-
dimorphic
containing purpose
fungi.
brain/meat medium used
infusion, in the Presumptive ID
peptome & cultivation or for C. albicans
Chloramphenol
dextrose. isolation of
inhibits
bacteria, yeasts C. albicans:
bacteria.
& molds. light to medium
Brain heart green colonies
Chromogenic
infusion agar When CHROMagar
mix selects for
(BHI) supplemeted C. tropicalis:
yeasts &
with dark blue
differentiated
chloramphenic colonies
by color
ol &
formation.
Chloramphenic gentamicin, it is C. kusei: flat,
ol & gentamicin used for pink colonies
can be added selective
for selectivity. isolation of Specialized fungal culture media
fungi from Medium Purpose
specimens that Promotion of characteristic
may contain reproductive structures &
commensal pigmentation useful for
bacterial flora. Cornmeal or potato morphologic identification of
Cycloheximide Selective dextrose agar mold isolates, when general
inhibits the isolation of purpose media prove
growth of many slow-growing inadequate for a particular
saphrophytic pathogenic isolate.
fungi, while fungi that may Promotion of characteristic
permitting be overgown structures (e.g.:
growth of most by rapidly chlamydospores,
(but not all) growing Cornmeal or rice arthroconidia) useful for
pathogenic saprophytic agar with Tween 80 morphologic identification of
fungi. fungi. yeast isolates, when more
Notably, routine identification
Cycloheximide
cycloheximide methods prove inadequate.
-containing
also inhibits the Sabouraud
media Isolation/cultivation of
growth of dextrose, agar,
certain Malssezia spp., all of which
Dixon medium, or
Chloramphenic pathogenic (except M. pachydermatis)
Leeming-Notman
ol & gentamicin fungi including require lipid
medium overlaid
can be added to C. neofromans, supplementation for growth.
with sterile olive oil
inhibit bacteria. many Candida Differentiation between
spp., species of Trichophyton,
Aspergillus spp. Trichophyton agars which can be difficult to
& the speciate based on
zygomycetes, morphology alone.
among others. Bird seed (Niger Demonstration of phenol
seed) agar oxidase activity in

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College of Medical Laboratory Science

MYCOVIRO
Cryptococcus neoformans Topography
isolates. • Verrucose
➢ Furrowed or convoluted
• Umbonate
FUNGAL IDENTIFICATION ➢ Slightly raised in the center
• Rugose
• It typically takes a group of tests to ➢ Furrows radiate out from the center
sufficiently identify fungi.
• Typically start by determining if the isolate is
a yeast or a mold.
➢ Molds do not always produce
structures to facilitate ID.
• Molecular and proteomic tests can be used
with traditional procedures.
➢ DNA hybridization, MALDI-TOF, PCR

Macroscopic
• Colony growth time
• Color
• Texture
• Pigment on reverse of colony

Texture
• Cottony
➢ Loose, high aerial mycelium
• Velvety
Microscopic
➢ Low aerial mycelium
• Septate versus sparsely septate hyphae
• Glabrous
• Hyaline or dematiaceous hyphae
➢ Smooth with no aerial mycelium
• Fruiting structures
• Granular
• Types, size, shape, and arrangement of
➢ Dense and powdery
conidia
• Wooly
➢ High aerial mycelium that is slightly
Tease mount
matted down
• Remove part of colony
• Mount in drop of lactophenol aniline (cotton)
blue
• Tease apart the colony (teasing needle) and
overlay with cover slip to disperse
• Examine at ×100 or ×400

Cellophane tape preparation

• Press cellophane tape gently but firmly to
surface of the colony.
• Press one side of tape to slide with drop of
LPCB.
• Stretch tape across slide, lowering it into the
stain.
• Pull taught and affix other side of tape,
avoiding air bubbles.

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College of Medical Laboratory Science

MYCOVIRO
Scedosporium species
Aspergillus species
Calmodulin Fusarium species
Scedosporium species
Chitin synthase Dermatophyte species
Cytochrome b Candida albicans
Pneumocystis jirovecii
Dihydrofolate reductase
(carinii)
Elongation factor (EF-
Fusarium species
1α gene)
Mitochondrial rRNA Pneumocystis jirovecii
rDNA complex (5S Candida albicans
gene) Pneumocystis jirovecii
rDNA complex (18S
Multiple
Slide culture gene)
• Mostly used to create permanent slides rDNA complex (28S Aspergillus species
• Grow fungus in plug of cornmeal or potato gene) Candida species
dextrose agar with cover slip on top. rDNA complex (IGS
Aspergillus species
• LPCB stain the cover slip after growth gene)
➢ Preserve slide with clear nail polish rDNA complex (ITS
Multiple
around cover slip if desired gene)
RNA polymerase (RPB1
Fusarium species
and RPB2)
IGS: intergenic spacer region
ITS: internal transcribed spacer region
rDNA: ribosomal DNA

IDENTIFICATION OF YEAST

• Colony morphology
• Microscopic morphology
• Physiologic studies
Immunodiagnosis of Fungal Disease • Chromogenic agars
• Rapid commercial identification tests or
Skin test reactivity to fungal antigens panels
• Uses antigen extract • Nucleic acid based methods
• Only valuable if patient has a history of a • MALDI-TOF MS
nonreactive skin test

Antibody detection assays

• Most common is probably double-
immunodiffusion
• Individuals can have detectable antibodies
but not active infection.

Cross-reactivity and interlaboratory discrepancies

have been reported.

Molecular Identification

Gene Target Fungal Species

Actin Candida albicans
β-Tubulin Aspergillus species

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College of Medical Laboratory Science

MYCOVIRO
Miscellaneous Tests for Yeasts

Germ tube production

• Most important and easiest to perform
• Steps:
1. Aseptically transfer several colonies
of yeast to a 12-mm x 75-mm test
tube containing approximately o.5
mL of serum (human, fetal calf,
bovine, or rabbit).
2. Incubate the tube at 35oC for up to 3
hours.
3. Place 1 drop of the mixture on a clean
glass slide and coverslip. Germ tubes have extended from the yeast cells of Candida
4. Examine under high dry (400x) albicans. No constriction is seen at the junction of yeast cell
magnification and reduced light for and germ tube. The walls of the germ tube are parallel. The
yeast was incubated for 2 hours at 37oC in serum. (Gram
the presence of germ tubes.
stain, 400x)

Schematic Diagram Showing Germ Tube Test to Presumptively

Identify Yeasts
C. albicans of cornmeal agar.

(left) Germ tube production, (right) pseudogerm tube

production

Pseudohyphae

Carbohydrate assimilation
• Relies on the aerobic utilization of carbon or
nitrogen as the sole source for the metabolic
process.

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College of Medical Laboratory Science

MYCOVIRO
Chromogenic substrates ➢ Combines nuclear magnetic
• Based on different colors resonance spectroscopy with PCR
➢ Qualitative molecular diagnostic
Cornmeal agar method that can detect and speciate
• For recognition of blastoconidia, the 5 most common Candida spp.;
chlamydoconidia, pseudohyphae, namely, Candida albicans, C.
arthroconidia glabrata, C. parapsilosis, C.
tropicalis, and C. krusei, in
Potassium nitrate assimilation approximately 5 hours
• Positive turns the medium blue.
• Negative result turns the medium yellow. Cryptococcal antigen (CrAg)
• Potassium Nitrate Assimilation Agar contains • Indicator of infection, in serum (a component
yeast carbon base, which provides amino of blood) and in cerebrospinal fluid (CSF).
acids, vitamins, trace elements, and salts, • Detects CrAg in serum a median of 22 days
necessary to support the growth of yeast. before symptoms of meningitis develop

Temperature studies IDENTIFICATION OF MOLDS

Urease • Growth rate

• Christensen urea agar • Colony morphology
• Cryptococcus neoformans, Coccidioides • Microscopic morphology
immitis, Histoplasma capsulatum, Sporothrix • MALDI-TOF MS
schenckii, and species of Trichosporon and
Aspergillus

(1-3)-β-D-glucan detection (cell wall

component)
• Chromogenic test
• Detected in serum during invasive fungal
infections (IFI): invasive aspergillosis,
invasive candidiasis, & P. jirovecii

Galactomannan
• Component of the Aspergillus cell wall
• Diagnosis of invasive pulmonary aspergillosis
• ELISA assay has been developed.

T2 Magnetic Resonance (T2MR) assay

• For diagnosis of invasive candidiasis

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College of Medical Laboratory Science

MYCOVIRO
Miscellanous Tests for Molds Antifungal Susceptibility Testing

• Hair perforation test Clinical and Laboratory Standards Institute (CLSI)

standards
• M27-A3 for yeast testing, M38-A2 for mold
testing, M44-A for disk diffusion testing for
yeasts, and M51-A
➢ Remains limited

• Urease test
• Thiamine requirement
• Trichophyton agars
• Growth on rice grains
• Germ tube production
➢ Most important and easiest test to
perform
• Carbohydrate assimilation
➢ Relies on the aerobic utilization of
carbon or nitrogen as the sole source
for the metabolic process.
• Chromogenic substrates
➢ Based on different colors
• Cornmeal agar
➢ For recognition of blastoconidia,
chlamydoconidia, pseudohyphae,
arthroconidia

ANTIFUNGAL SUSCEPTIBILITY

Antifungal agents

• Main agent is amphotericin B.

➢ Many side effects
❖ Fever, rigors, renal
impairment, and possibly
shock
• Examples of other agents
➢ Fluconazole (FLU), itraconazole
(ITRA), posaconazole (POSA), and
voriconazole (VORI)

Misuse has resulted in the development of

resistance, most notably with C. glabrata.

Mycology and Virology 10 [Buen, M. G. V.]