fungaldiagnosis-200728061058.........pptx

LABORATORY
DIAGNOSIS OF FUNGAL
DISEASES
Dr. GEETANJALI DIXENA
MDS III rd year
TRIVENI DENTAL COLLEGE BILASPUR

TOPICS for discussion-
• SPECIMEN COLLECTION
• MICROSCOPY
• CULTURE
• IMMUNOLOGICAL METHODS
• TESTS FOR METABOLITES
• TESTS TO DEMONSTRATE DELAYED
HYPERSENSITIVITY
• MOLECULAR METHODS

• Depends on the site of infection such as
Skin scrapping
Hair
Nail
Sputum
Blood sample- For systemic mycoses may also be
collected.
Cerebrospinal fluid (CSF) is collected for
cryptococcal meningitis
Specimen Collection

• Fungal diagnosis
Hair Sputum & other Blood Urine CSF
Nail respi. secretion
Skin G/S India ink
G/S
KOH KOH Bac T SDA
Mannual cult.
on Biphasic media Identification culture
SDA SDA
s/c to
check
G/S
Identification s/c to check latex agglut.
LPCB of growth

• Identification
yeast mould
G/S, India ink LPCB
colony creamy white, smooth, and cottony, powdery, wolly, waxy, velvety
pasty with typical yeasty odor
mucoid creamy white
GTT
Dalmau
CHROM agar
Assimilation

Microscopy
• Direct microscopic examination of material
from the lesion.
Potassium hydroxide (KOH) preparation:
Gram stain
India ink and Nigrosin stains
Lactophenol cotton blue (LPCB)
Calcofluor white stain
Histopathological stains

Potassium hydroxide (KOH) preparation
• Skin scrapings and plucked hair samples
(Keratinized tissue specimens)
• Treated with 10% KOH which digests the
keratin material release fungal hyphae
clearly seen under the microscope.
1O% KOH used.
20-40% KOH - for Nail and Biopsy tissues that take
longer time to dissolve.
Glycerol (10%) is added to prevent drying
DMSO ( dimethyl sulfoxide)- can be added which
helps in tissue digestion.

• India ink and nigrosin stains:
 Negative stains for
demonstration of capsule of
Cryptococcus neoformans.
• Gram stain:
 identifying the yeasts (e.g.
Cryptococcus) and yeast like
fungi (e.g. Candida).
 They appear as gram-positive
budding yeast cells.
– Pseudohyphae-
 In yeast cell, the bud remains
attached to the mother cell,
elongates and undergoes
repeated budding to form chains
of elongated cells known as
pseudohyphae.

• Calcofluor white stain:
more sensitive than
other stains;
binds to cellulose and
chitin of fungal cell wall
and fluoresce under UV
light

• Demonstrate the fungal elements from biopsy tissues
(deep mycoses)
Periodic acid schiff (PAS)stain: fungi appear magenta/deep
pink, whereas the nuclei stain blue.
Gomori methenamine silver (GMS) stain:
• Alternative to PAS.
• Stains both live and dead fungi, as compared to PAS which stains
only the live fungi.
• Stains the polysaccharide component of the cell wall.
• Fungi appear black whereas the background tissue takes pale green
color.
Mucicarmine stain: staining tie carminophilic cell wall of
Cryptococcus and Rhinosporidium.
Masson fontana stain: used for pigmemed (or pheoid) fungi.
Hematoxylin and Eosin (Hand E) stain.
Histopathological stains

Culture
• Fungal culture - performed for isolation and
correct identification of the fungi.
• Culture Media
Basic media
• Sabouraud's dextrose agar (SDA):
• Neutral SDA (Emmons' modification)
• SDA with antibiotics

Culture
• SDA:
It is the most commonly used media in diagnostic
mycology. It contains
Pepone 1%
Dextrose 4%
Agar 2%
pH of 5.6.
may not support some pathogenic fungi.
• Neutral SDA (Emmons' modification):
lt differs from original SDA in having
• Neopeptone (1%)
• Dextrose (2%) and
• pH of 7.2

Culture Condition
• Temperature:
Most of the fungi grow well at 25-30°C except the
dimorphic fungi that grow at both 250
C and 370
c.
• BOD incubators (biological oxygen demand):
capable of maintaining low temperature.
• Incubation time:
Culture plates should be incubated for 2-3 weeks
• Antibiotics
added to the culture media to inhibit bacterial growth.
Cycloheximide (Actidione), chloramphenicol and
gentamicin

Culture Identification
• Macroscopic and microscopic appearances of the colonies
grown on culture.
• Macroscopic Appearance of the Colony
Rate of growth:
• Rapid growth (<5 days): in saprophytes, yeasts and agents of opportunistic
mycoses.
• Slow growth ( 1-4 weeks): in agents of subcutaneous and systemic
mycoses.
 Pigmentation:
• seen on obverse and reverse of the culture media.
 Texture:
• glabrous (waxy/leathery), velvety, yeast like or cottony or
granular/powdery.
Colony topography:
• Colony surface may be rugose (radial grooves), folded or verrucose or
cerebriform (brain-Like).

Microscopic Appearance of Fungi
• Teased mount:
 A bit of fungal colony is teased out from the culture tube and LPCB mount
is made on a slide
 viewed under microscope.
• ldentification is based on the following:
a. Nature of hyphae -such as septate or aseptate, hyaline or phaeoid, narrow or
wide) and
b. Type of sporularion (conidia or sporangia).
• Slide culture:
 this is a tedious procedure,
 It gives most accurate in situ microscopic appearance of the fungal colony.
 A sterile slide is placed on a bent glass rod in a sterile petri dish.
 2 square agar blocks measuring around 1 cm2
(smaller than the coverslip)
are placed on the slide.
 Bits of fungal colony are inoculated onto the margins of the agar block.
 Then the coverslip is placed on the agar block and the petri dish is
incubated ac 250
C.
 LPCB mounts are made both from the coverslip and the underneath slide

Lactophenolcottonblue (LPCB):
• Used to Study the microscopic
appearance of the fungal
isolates grown in culture.
• It contains:
Phenol- acts as disinfectant
Lactic acid- preserves the
morphology of fungi.
Glycerol- prevents drying.
Cotton blue- stains the fungal
elements blue.

• Cellophane tape mount:
This is easy to perform than slide culture
in-situ fungal morphology is also maintained.
placing the cellophane tape on the colonies
present on the surface of SDA plate,
then LPCB mount is made from the cellophane
tape.

Other Methods of Identification
• For Candida:
Germ tube rest
Dalmau plate culture
Sugar fermentation and
sugar assimilation
• For dermatophytes:
Hair perforation tests
Dermacophyte
identification medium
• For Cryptococcus
Urease test

Germ tube test:
Also called Reynolds Braude phenomenon.
Rapid method of identifying C. albicans.
It is a specific test for C. albicans but also be
positive for C. dubliniensis
Procedure-
• Few colonies are mixed with human or sheep serum
and incubated for 2 hours. Wet mount preparation is
examined under microscope.
• Germ tubes are formed, described as long tube like
projections extending from the yeast cell. It is
differentiated from pseudohyphae as there is no
constriction at the origin.

Dalmau plate culture:
• Culture on cornmeal agar / Rice starch agar at
200
c can provide clue for species
identification.
 C. albicans produces refractile, terminal thick
walled chlamydospores.

Identification
Nutritionally deficient media
• Corn meal agar and
• rice starch agar:
Enriched media
• Brain heart infusion (BHI) agar and blood agar:
• Niger seed agar and bird seed agar:
Differential/Indicator media
• CHROM agar Cadida medium:

Identification
• Corn meal agar and rice starch agar:
Nutritionally deficient media
used for stimulation of chlamydospore production.
• Brain heart infusion (BHI) agar and blood agar:
Enriched media
used for growing fastidious fungi like Cryptococcus
and Histoplasma.
• Niger seed agar and bird seed agar:
for selective growth of Cryptococcus

Identification
• CHROM agar Candida
medium:
selective as well as
differential media for
speciation of Candida.
 C. albicans- Light green
 C. dubliniensis- Dark green
 C. glabrata- Pink to purple
 C. krusei- Pink

Tests for species Identification
• CHROM agar:
Different Candida species produce different colored colonies on
CHROM agar…
 C. albicans- Light green
 C. dubliniensis- Dark green
• Growth at 450
C:
It differentiates C. albicans (grows) from C. dubliniensis (does not
grow at 45°C).
• Sugar fermentation test and sugar assimilation test
differentiate between various Candida species.
• Molecular methods –
PCR using species specific primers are useful for species
identificacion.

Immunological Methods
• Detect the antibody or antigen from serum and/or other body
fluids.
• Antibody detection
 by ELISA, immunodiffusion test, agglutination test, and
complement fixation test (CFT).
• Antigen detection:
 latex agglutination test for detecting cryptococcal antigen from CSF
• lmmunohistochemistry:
 useful in deep mycoses
 detect antigens (e.g proteins) on the cells of a tissue section
 by using fluorescent tagged antibodies that bind specifically to the
antigens.

• Tests for Metabolites
Alternate approach for the diagnosis of fungal
infections
is detection of specific fungal metabolites in body
fluids
By gas liquid chromatography.
• Tests to Demonstrate Delayed Hypersensitivity
Skin tests
hypersensitivity for Histoplasma, Blastomyces,
Coccidioides, Paracoccidioides, Dermatophyte,
Sporothrix and Candida.

Molecular Methods
• Polymerase chain reaction (PCR)
• Multiplex PCR, nested PCR and the most
advanced real time PCR and DNA sequencing
methods
• accurate identification of fungi from culture as
well as from the specimens.

fungaldiagnosis-200728061058.........pptx

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