DIAGNOSIS OF FUNGAL/MYCOTIC INFECTIONS

General diagnosis of fungal diseases:

Clinical signs and appearance of the
lesion may suggest fungal infection
Radiographs (systemic fungal infection)
Wet mount of tissue/mucus with KOH 1%
Wet mount of portions of teased from
fungal colonies and mounted in
lactophenol cotton blue

2
DIAGNOSIS OF FUNGAL/MYCOTIC DISEASES……

Tissue section or clinical materials

stains with Periodic Acid-Schiff (PAS)
Isolate and characterize the fungal
cells by culture on sabouraud glucose
agar or blood agar/ Brain heart
infusion agar
Susceptibility testing
Histological – routine stain H&E,
direct immunofluorescence
3
DIAGNOSIS OF FUNGAL/MYCOTIC DISEASES……
Serological tests: May provide rapid
means for diagnosis of fungal infection
Monitoring of infection progression
Patient response to therapy
The methods include CFT,
Immunodiffusion tests, ELISA
DIAGNOSIS OF FUNGAL/MYCOTIC DISEASES……
Immunological methods:
Cryptococcal antigen test
Histoplasma antigen test

Molecular methods
Direct detection

Identification

Strain typing

Biochemical tests (metabolites (D-

arabinitol) & (cell wall components
(ß-glucan)
Common laboratory procedures in diagnosis of fungal
infection
Procedure Purpose
Wet mount of tissue or Strong alkali degrades
mucus-containing specimens tissues and mucus and
in 1% KOH permits visualization of
fungi
Wet mounts of portions Permits observation of
teased from fungal colonies fungal morphology and
and mounted in lactophenol presence of spores. Kills
cotton blue fungi and provides good
contrast
Tissue sections and clinical Both PAS and silver stain
material stained with fungal cell walls to give
periodic-acid-Schiff (PAS) or good contrast with
methanamine silver stains background in tissue
Common laboratory procedures in diagnosis of fungal
infection
Procedure Purpose
Subouraud glucose agar Low pH of the medium and
(SDA) for culture, incubation RT incubation favour growth
at room temperature (RT) for of fungi over bacteria.
up to 6 weeks Antibiotics like
chloramphenicol may be
added to discourage bacterial
growth
Blood agar or brain heart Various fungi grow at 37ºC.
infusion agar at 37ºC for a The yeast phase of domorphic
week fungi grow on these media at
37ºC
Slide culture with inoculated Permits observation of
blocks of SDA on glass slide relatively undisturbed fungal
covered with a cover slip and growth especially in
incubated in a moist chamber identification of fruiting
Laboratory Methods in Medical Mycology
A. Collection, handling and processing of
clinical mycology specimens
1. Importance
2. Collection - usually by physician or nursing
staff
a. Skin - cleaned with 70% alcohol to
remove dirt, oil and surface saprophytes
b. Nails - cleaned same as for skin. Usually
clipped; need to be finely minced before
innoculating to media
Laboratory Methods in Medical Mycology………….
c. Hair - obtained from edge of infected
area of scalp,. Use a Wood's lamp
(fluorescence) to help locate infected hair.
Hair can be obtained by plucking,
brushing, or with a sticky tape.

d. Body fluids - normal sterile collection

procedures
Laboratory Methods in Medical Mycology………….
3. Preparation of specimens for immediate
transport to laboratory

a. Hair & nails sent in a dry envelope, inside

proper container.

b. Other specimens are usually sent frozen

or on dry ice.
Laboratory Methods in Medical Mycology………….

c. Packaging - biohazard regulations. Any

growing cultures must be on tube media
(not plates). Aluminum screw-capped
mailing tube with outer cardboard mailing
tube.

d. Inside labeling information: patient ID,

specimen source, suspected organism.

e. Outside labeling information: must state

Laboratory Methods in Medical Mycology………….

4. Appropriate processing of specimen to

recover fungus

a. Skin, nails, & hair - direct exam following

KOH preparation
Laboratory Methods in Medical Mycology………….

b. Body fluids
(1) CSF - centrifuged; examine sediment
microscopically, inoculate media

(2) Pleural fluid, sputum, and bronchial

aspiration - specimen must be fresh as
saprophytes would overgrow pathogens
such as H. capsulatum.
Specimens may be refrigerated up to 2
hours.
Laboratory Methods in Medical Mycology………….

(3) Gastric washings - same as for pleural

fluids

(4) Genito-urinary specimens - first morning

specimen preferred; centrifuge

(5) Blood/bone marrow - generally inoculated

directly to BHI broth and BHI slant.
- Extra specimen should be inoculated to
other fungal media.
Laboratory Methods in Medical Mycology………….

(6) Wound abscess or drainage - should be

cultured anaerobically, especially if
actinomycosis is suspected.

(7) Tissue specimens - examine for pus,

caseous material or granules; mince
aseptically , can use small amount of
sterile saline and the supernatant also
inoculated.
Laboratory Methods in Medical Mycology………….

B. Direct examination of specimens

1. Direct exam required on any

biological material sent to lab for
fungus culture. Look for spores,
hyphae, mycelial elements, budding
yeast, mycotic granules.
Laboratory Methods in Medical Mycology………….

2. Wet mount prep - good for yeast;

examination is done in natural environment,
so loss of fragile structure is minimal.

3. KOH prep - Potassium hydroxide; done on

skin scrapings, hail, nails, sputum, vaginal
specimens, etc.
The KOH digests and clears the specimen’s
tissue cells, mucous, etc., so fungal elements
can be seen.
Laboratory Methods in Medical Mycology………….

C. Stains
1. Lactophenol Cotton Blue (LPCB) - very
popular for quick evaluation of fungal
structures; will stain the chitin in cell walls
of fungi.

2. Periodic Acid - Schiff Stain (PAS) - stains

certain polysaccharide in the cell walls of
fungi.
Fungi stain pink-red with blue nuclei.
Laboratory Methods in Medical Mycology………….

C. Stain………..
3. Gomori Methenamine Silver Stain - silver nitrate
outlines fungi in black due to the silver
precipitating on the fungi cell wall.
The internal parts of hyphae are deep rose to
black, and the background is light green.

4. Gridley Stain - Hyphae and yeast stain dark blue

or rose.
Tissues stain deep blue and background is yellow.
Laboratory Methods in Medical Mycology………….
C. Stains …………………..
5. Mayer Mucicarmine Stain - will stain
capsules of Cryptococcus neoformans
deep rose.

6. Fluorescent Antibody Stain - simple,

sensitive, and extremely specific method
of detecting fungi in tissues or fluids.
Applications for many different fungal
organisms.
Laboratory Methods in Medical Mycology………….
C. Stains …………………..
7. Papanicolaou Stain - good for initial
differentiation of dimorphic fungi.
Works well on sputum smears.

8. Gram Stain - generally fungi are Gram positive;

Actinomyces and Nocardia are Gram variable.

9. Modified Acid-Fast Stain - used to differentiate

the acid-fast Nocardia from other aerobic
Actinomyces.
Laboratory Methods in Medical Mycology………….
C. Stains …………………..
10. Giemsa Stain - used for blood and bone
marrow specimens.
Histoplasma capsulatum is an intra cellular
organism, which appears as small oval to
pear-shaped yeast-like cells with crescent
shaped red-stained protoplasm surrounded
by clear halo in segmented neutrophils.

11. India Ink - demonstrates the capsule of

Cryptococcus neoformans in CSF specimens.
Laboratory Methods in Medical Mycology………….
D. Fungal Culturing
1.Media introduction
a. Generally tube media is used rather than
plated media because:
(1) there is less chance for spore release into
the environment.
(2) less chance for dehydration
(3) ease of storage.
Laboratory Methods in Medical Mycology………….

b. The agar in a tube is inoculated in a

straight line. Preliminary identification is
based on differential growth patterns on
various media

2. Media (Media should be carefully

selected, and more than one media should
be used)
a.Sabouraud's dextrose agar (Sab-Dex) -
classic medium, recommended for most
studies.
Laboratory Methods in Medical Mycology………….

b. Sabouraud's dextrose agar with

chloramphenicol - chloramphenicol inhibits
bacterial growth

c. Mycosel agar - commercially produced agar

containing chloramphenicol to inhibit
bacterial growth, and cycloheximide to
inhibit saprophytic fungi and some yeasts
(including C. neoformans).
Laboratory Methods in Medical Mycology………….
Notes:
(1) Aspergillus and Scopulariopsis
(saprophytes) are opportunistic pathogens.
Cycloheximide will prevent their growth.

(2) Cryptococcus neoformans is also inhibited.

(3) Bacteria-like fungi (such as Actinomycetes)

are inhibited by chloramphenicol.
Laboratory Methods in Medical Mycology………….
2. Media………..
d. Brain heart infusion slant (BHI) - more
enriched than Sab-Dex. Used in recovery
of H. capsulatum.

e. Potato-dextrose agar (PDA) and Corn-

meal agar - are used in slide cultures; as
they induce spore formation, which
greatly aids in identification.
 Laboratory Methods in Medical Mycology………….
3. Special applications agar
a. Caffeic Acid Agar - Cryptococcus neoformans will
produce melanin resulting in black colonies.
(protect media from light)
b. Birdseed Agar - used to isolate Cryptococcus
neoformans from contaminated cultures.
c. KT Medium & Kelley Agar - used to convert
dimorphic fungus Blastomycetes dermatitidis
from mycelial to yeast form.
d. Modified Converse Liquid Medium (Levine's) -
used to promote spherule production by
Coccidioides immitis.
Laboratory Methods in Medical Mycology………….
4. Fungal growth requirements
a. Temperature - Room temperature (25-
30ºC ) for most fungi.

Notes:
(1) Nocardia sp. and some dimorphic
organisms grow best at 37ºC.
(2) Any fungus capable of growing at
37ºC, should be considered potentially
pathogenic.
Laboratory Methods in Medical Mycology………….
4. Fungal growth requirements…………………….
b. Atmosphere - True fungi are aerobic; there are a
few anaerobes among the bacteria-like fungi.
c. Time - Some yeasts grow overnight.
Saprophytes are fast growers (several days).
Generally cultures are held at least 4 weeks. *

Exceptions: Paracoccidioides brasiliensis may

require 4-5 weeks, & 10 weeks are recommended
if Histoplasma capsulatum is suspected.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification of Fungi & lab
ID
A. Inoculation
1. Plates - Inoculated like a large "S", so that
rapid growing fungi can removed.
2. Slants - Inoculated with a straight line.
Laboratory Methods in Medical Mycology………….

V. Techniques for identification of Fungi &

lab ID……..

B. Incubation
1. Aerobic (and anaerobic if Actinomycetes
are suspected)

2. Room temperature & also sometimes at

37ºC if dimorphic fungus is expected.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………
C. General considerations
1. Type of media used. Does it contain
antibiotics?
2. Growth rate & age of the culture.

a. Routine cultures are kept for 4 weeks &

should be examined every other day.
b. Most systemic pathogens require 10 days to
2 weeks, while saprophytic fungi grow
usually grow within 1 week.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………

D. Colony Morphology (macroscopic features)

1. Surface topography - Some fungal colonies
may be free growing, covering the entire
surface of agar in a particular manner;
others grow in a restricted manner.

2. Surface texture -examples: cottony or wooly

(floccose), granular, chalky, velvety,
powdery, silky, glabrous (smooth, creamy),
waxy, etc.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………

D. Colony Morphology (macroscopic features)

………

3. Pigmentation - Fungi may be colorless or

brightly colored.
Color may be on fungus itself, on its
sporulating apparatus, on the agar, or on the
bottom of the colony (reverse pigmentation).
4. Mycelium
a. Vegetative mycelium - provides nutrition
b. Aerial mycelium - reproductive
Laboratory Methods in Medical Mycology………….
V. Techniques for
identification…………………

E. Microscopic evaluation
1. Methods
a. Teased Preparation
b. Slide Culture Techniques - best as it gives
undisturbed microscopic morphology.
c. Transparent Tape Preparation
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………

2. A review of terms associated with the

microscopic features is advisable.
a. Hyphae structure. Hyphae (plural); hypha
(singular)
(1) Septate vs. non-septate (aseptate)
(2) Dematiaceous vs. hyaline
b. Spore bearing structures
c. Spores - Many terms addressing
reproduction are provided.
Laboratory Methods in Medical Mycology………….

V. Techniques for
identification…………………

3. Biochemical studies - generally

used to ID yeast and yeast-like
organisms.

a. Carbohydrate fermentation
Laboratory Methods in Medical Mycology………….
(1) Growth and utilization of a carbohydrate
under anaerobic conditions as determined
by acid and gas production.

(2) Specimen is inoculated beneath broth so

that it is completely covered.
Bromocresol purple is the indicator.
Acid production turns purple to yellow.
Gas is detected by appearance of bubbles
trapped in the fermentation tube.
Observe every 48 hrs for 14 days.
Laboratory Methods in Medical Mycology………….
V. Techniques for
identification…………………
b. Carbohydrate assimilation
(1) Ability to utilize a carbohydrate as sole
source of carbon.
(2) Bromocresol purple indicator turns from
purple to yellow.
(3) Tubes unchanged (as determined by
comparing to a blank tube) by 10 days are
negative.
Laboratory Methods in Medical Mycology………….
V. Techniques for
identification…………………

c. Nitrogen assimilation

(1) Utilizes 3 tubes with differing sources of

nitrogen.
Bromthymol blue is the indicator (blue to
yellow is positive).
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………
d. Growth on specific agars
(1) Christensen's urea agar
(a) Urea is hydrolyzed by some yeast to form
ammonia (pH increases) which turns media
from yellow to dark pink.
(2) Caffeic acid medium (protect media from
light)
(a) Production of melanin by Cryptococcus
neoformans resulting in black colonies.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………
4. Other tests
a. Germ tube - Candidia albicans & Candidia
stellatoidea produce germ tubes when
incubated in a protein medium.
b. Demonstration of chlamydospores - Yeast is
inoculated by jabbing appropriate agar
(Cornmeal with tween 80) and observed every
24 hours for 3 days for chlamydospore
production.
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………
5. Hypersensitivity (seromycology; skin tests)
and serological tests
a. Skin tests - demonstrates T-cell immunity
(cellular) to a fungus
These tests may be performed in
Histoplasmosis, Candidiasis, Sporotrichosis,
Coccidioidomycosis, Blastomycosis,
Paracoccidiodomycosis and dermatophytosis
Laboratory Methods in Medical Mycology………….
V. Techniques for identification…………………
b. Serological tests - demonstrates B-cell
(humoral) immunity to a fungus; sera should
be drawn in pairs (acute and convalescent).
(1) Complement fixation
(2) Agglutination tests
(3) Precipitin tests
(4) Immunofluorescence
(5) Immunodiffusion techniques
(6) ELISA
(3) Counterimmunoelectrophoresis
Laboratory Methods in Medical Mycology………….
6. Antifungal susceptibility testing -
some progress has be made in the
attempt to standardize susceptibility
testing as a means in evaluating
unusual fungal isolates, determining
the cause(s) of patient relapse and
treatment failure.
Laboratory Methods in Medical Mycology………….

7. Determining serum concentration of

antifungal agents - can be very
important for patients with renal or
liver problems or in cases where poor
absorption is a concern.
HPLC has proven to be an excellent
method due to its accuracy and ability
to analyze more than one drug in a
specimen
Laboratory Methods in Medical Mycology………….

V. Techniques for
identification…………………

Molecular techniques:
Newer techniques such as DNA
hybridization, PCR are useful in diagnosis
of mycoses in a shorter period as well as
detect those fungi that are difficult or
dangerous to cultivate in vitro.
Laboratory Methods in Medical Mycology………….
The DNA-based molecular methods are also
common:
Method Fungal pathogen
DNA based
methods
Southern Candida spp, Aspergillus spp,
hybridization Cryptococcus neoformans, Trichosporon
analysis (RFLP) beigilii, Histoplasma capsulatum
Restricted Candida spp, Aspergillus spp, Malassezia
endonuclease spp., Histoplasma capsulatum
analysis of
genomic DNA
Pulsed field gel Candida albicans
electrophoresis
PCR finger Candida spp, Aspergillus spp,
printing Cryptococcus neoformans, Histoplasma
Fungal species most commonly
recovered from
clinical specimens

1. Blood
Candida
Cryptococcus
Histoplasma
filamentous fungi: rarely isolated
from blood with the exception of
Fusarium
Fungal species most commonly
recovered from
clinical specimens

2. Cerebrospinal fluid
Candida
Coccidioides
Cryptococcus
Histoplasma
Fungal species most commonly
recovered from
clinical specimens
3. Pus and other exudates (abscesses,
wounds, and ulcers)
Blastomyces
Coccidioides
Cryptococcus
Fusarium
Histoplasma
Sporothrix
Fungal species most commonly
recovered from
clinical specimens
4. Respiratory secretions (sputum, bronchial
lavage, bronchial brushings, and transtracheal
aspirates)
Aspergillus Scedosporium
Blastomyces Rhizopus
Candida Sporothrix
Coccidioides
Cryptococcus
Histoplasma
Mucor
Paracoccidioides
Fungal species most commonly
recovered from
clinical specimens
5. Swabs
Aspergillus
Candida
Fusarium
Rhizopus
Fungal species most commonly
recovered from
clinical specimens
6. URINE
Candida
Cryptococcus

7. CHEST, ABDOMINAL, AND

SYNOVIAL
Aspergillus
Candida
Fungal species most commonly
recovered from
clinical specimens
8. VITREOUS
Candida

9. BONE MARROW
Candida
Cryptococcus
Histoplasma
Agents to Treat Fungal Infections
• Four main groups
– Macrolide polyene antibiotics,
– Griseofulvin,
– Synthetic azoles,
– Flucystosine
Macrolide Polyene Antibiotics
Bind to fungal membranes and cause loss of
selective permeability
Specific for fungal membranes because fungal
membranes contain ergosterol
Mostly Amphotericin B, Nystatin and pimaricin
Useful on dermatophyte infections, may result
in eye damage, not good for Fusarium sp.

Mimics lipids in some cell membranes

Griseofulvin
Especially active in certain dermatophyte
infections such as athlete’s foot

Griseofulvin synthesized by several species

of Penicillin for fungus with walls containing
chitin

Requires several months and is relatively

nephrotoxic, so only given for most
stubborn cases
Synthetic Azoles
• Broad-spectrum antifungal agents

Synthetic benzimidazole derivatives

collectively called imidazoles like:
• Ketoconazole, fluconazole, clotrimazole,
and miconazole
Synthetic Azoles…………
• Ketoconazole: orally and topically for
cutaneous mycoses, vaginal and oral
candidiasis, and some systemic mycoses

• Fluconazole: used in selected patients

for AIDS-related mycoses

• Clotrimazole and miconazole: mainly

topical ointments for infections in the
skin, mouth, and vagina
Flucystosine
Analog of the nucleotide cytosine

5-fluorocytosine (flucytosine)

Can be used to treat certain cutaneous

mycoses
Effective against Cryptococcus and Candida

Usually combined with amphotericin B for

systemic mycoses