Seminar

Introduction
General features of fungi
Morphology / structure of the fungi
Types of mycosis
Specimen collection
Classification of diagnostic methods:
Microscopy & staining
Culture techniques

Biochemical tests
Serological identification
Special methods

Candida
2

The fungi are saprophytic (derives nourishment

from dead organic matter) and parasitic eukaryotic
organisms.
Although formerly considered to be plants, they
are now generally assigned their own
kingdom, Mycota.

They resemble plants because they have rigid cell

walls and are non motile.

Unlike plants, the fungi lack chlorophyll and are

unable to photosynthesize. The fungi also lack the
multicellular complexity and organization of most
animals.

Historically, the fungi were regarded as relatively

insignificant causes of infection.

It is now well documented that the fungi are the

common cause of infection, particularly in
immunocompromised patients.

Opportunistic invasive fungal infections remain an

important cause of morbidity and mortality.

The most common fungi that cause disease in transplant

recipients and other immunocompromised patients are
Candida and Aspergillus species
5

Fungi exhibit two basic structural forms:

YEAST- these are unicellular with spherical or
ovoid bodies
MOULD - are multicellular organisms.

YEAST

MOULD
6

YEASTS:
Yeasts are unicellular organisms that are round to oval
and range in size from 2 to 60m.

The microscopic morphologic features usually appear

similar for diff. genera and are not particularly helpful in
their separation.

MOLDS (MOULDS):
The mold (or mould) form of growth refers to the
production of multicellular, filamentous colonies.

These colonies consist basically of branching cylindrical

tubules varying in diameter from 2 to 10 mm and
termed hyphae.
7

Hyphal growth occurs by apical elongation.

The mass of intertwined hyphae that

accumulates during active growth in the mold
form is called a mycelium.

Germ Tube
8

 GENERAL FEATURES OF FUNGI:

Fungi seen in the clinical laboratory can generally be

separated into two groups based on the appearance of
colonies formed.

The yeasts produce moist, creamy opaque or pasty

colonies on media,

Whereas the filamentous fungi or molds produce

fluffy, cottony, wooly or powdery colonies.

Several pathogenic species of fungi that exhibit

either yeast (or yeast like) and filamentous form are
referred to as being dimorphic.

The dimorphism is temperature dependent; the

fungi are designated as thermally dimorphic

10

Dimorphic fungi have the ability to grow vegetatively

at 25C as moulds and at 37C as yeasts, spherules or
enlarging endospores.

These fungi include :

Blastomyces dermatitidis,
Histoplasma capsulatum,
Paracoccidioides brasiliensis,
Coccidioides immitis,
Penicillium marneffei,
Sporothrix schenckii.

11

 In many species of fungi, individual cells are

separated by cross-wall, or septa, such fungi

are described as septate.

12

The septa are incomplete, however, and pores

allow adjacent cytoplasm to mix.

In other fungal species, the cells have no septa, and

the cytoplasm and organelles of neighboring cells
mingle freely. These fungi are said to
be coenocytic.

The common bread mould Rhizopus stolonifer is

coenocytic, while the blue-green mould that
produces penicillin, penicillin notatum, is septate
13

The hypha is the morphological unit of fungus

and is visible only with the aid of a microscope.

Hyphae have broad diversity of forms and many

hyphae are highly branched with reproductive
structures.

A thick mass of hyphae is called a mycelium. This

mass is usually large

14

The study of fungi is called mycology, and a

person who studies fungi is called a mycologist.

15

Based on normal habitat ,classified as

anthropophilic (humans),
zoophilic (animals)
geophilic (soil)

16

The superficial mycosis:

These are infections limited to the outermost layers of

the skin, the nails and hair, and the mucous membranes.

The principal infections in this group are the

dermatophytoses and superficial forms of candidosis.

These diseases affect millions of individuals worldwide,

but are readily diagnosed and usually respond well to
treatment.
17

The subcutaneous mycosis:

These are infections involving the dermis, subcutaneous

tissues and adjacent bone.

These infections are usually acquired as a result of the

traumatic implantation of organisms that grow as
saprobes in the soil and on decomposing vegetation.

These infections are most frequently encountered

among the rural populations of the tropical and
subtropical regions of the world, where individuals go
barefoot and wear the minimum of clothing.
18

The disease may remain localized at the site of

implantation or spread to adjacent tissue.

More widespread dissemination of the infection,

through the blood or lymphatics, is uncommon,
and usually occurs only if the host is in some way
debilitated or immunocompromised.

19

The systemic mycoses:

These are infections that usually originate in the

lungs, but may spread to many other organs.

These infections are most commonly acquired as a

result of inhaling spores of organisms that grow as
saprobes in the soil or on decomposing organic
matter, or as pathogens on plants.

20

FUNGUS

Blastomyces dermatidis
2. Coccidioides immitis
3. Histoplasma capsulatum
4. Pneumocystis carinii
5. Candida albicans
6. Cryptococccus neoformans
7. Dermatophytes
(Trichophyton,
Epidermophyton)
1.

INFECTION

Blastomycosis
San Joaquin valley fever
Histoplasmosis
Pneumocystis pneumonia
Thrush, vaginitis,candidiasis.
Cryptococcosis
Tinea (ringworm, athletes
foot)
21

Mycosis

Oral manifestation

Blastomycosis

Tiny ulcerations on oral mucosa

(Gilchrist Disease)
(Darling Disease)

Nodular , ulcerative or vegetative lesions on buccal mucosa,

gingiva, tongue, palate & lips.

Cryptococcosis

Non specific single or multiple ulcers

Histoplasmosis

(Torulosis )

Coccidiomycosis
(San Joaquin valley fever)

Candidiasis
(Candidosis)
Mucormycosis

Non specific Proliferative granulomatous & ulcerated lesions in

oral mucosa.
Oral thrush, Actinic chelitis

Reddish-black turbinate, with sputum & nasal discharge.

Necrosis of paranasal sinuses & orbital floor.

Sporotrichosis

Non specific ulceration of oral, nasal & pharyngeal mucosa,

with regional lymphadenopathy

Rhinosporidiosis

Warts or red soft polypoid growth of tumor like nature in oral

mucosa
22

1.
2.
3.
4.

1.

2.

1.
2.
3.
4.

PRIMARY PATHOGENS
Histoplasma capsulatum
Blastomyces dermatidis
Coccidioides immitis
Paracoccidioides brasilienses
PATHOGENS WITH INTERMEDIATE VIRULENCE
Spoprothrix schenkii
Dermatophytes
SECONDARY PATHOGENS
Cryptococcus neoformans
Candida
Aspergillus
Mucorales (Rhizopus, mucor, absidia)

23

COLLECTION AND TRANSPORT OF THE CLINICAL

SPECIMEN:
The diagnosis of fungal infections is dependent
entirely on the selection and collection of an
appropriate clinical specimen for culture.

24

SPECIMENS INCLUDE

Skin scrapings.
Hair and nails.
Respiratory tract secretions.
Cerebrospinal spinal fluid.
Blood.
Smears from Mouth and vagina.
Urine.
Pus.
Ocular specimen.
Tissue.
Bone marrow.
25

1- Microscopy
2- Culture techniques

3- Biochemical reactions
4- Serological identification:
5- Special Tests
26

27

 MICROSCOPY AND STAINS

Gram Stain
Acid fast stain
Potassium Hydroxide (KOH)
Calcofluor white stain
Grocott Methenamine silver stain
PAS stain
Wright stain
India Ink
Methylene blue
Acridine orange stain
Giemsa stain

28

Gram stain:

Is most commonly performed on most clinical specimens

submitted for bacteriology & will detect most fungi , if
present.
Eg: cryptococcus .
These stains define the structure so that morphologic criteria
can be useful for identification.

29

 Principle: The difference in composition

between gram- positive cell wall, which

contain thick peptidoglycan with numerous
teichoic acid cross-linkages & gram negative
cell walls, which, contain thinner amount of
peptidoglycan accounts for gram staining
difference between two groups .

30

The extensive techoic acid cross

links contribute to the ability of
gram-positive organisms to
resist alcohol decolorization.

Gram-negative organism allow

the crystal voilet stain to wash
out with the decolourizing step
& may appear colourless after
this step & appear pink after
takin counterstain.

31

Gram stain is usually a poor stain to use when

examining a specimen for a fungus.

Gram stain may be used when examining

smears of Candida, Malassezia, and Sporothrix
but should not be relied upon to demonstrate
the yeast of the other dimorphic fungi.

32

Acid-fast staining is useful for detecting Nocardia

species and for differentiating them from other aerobic
actinomycetes.
Some of the filaments stain red with carbol- fuschin
staining, while others may appear blue because of
counterstaining effect.
The slides are then examined under a bright field
microscope with oil immersion objective

33

Fungi lacking cell walls fortified

with mycolic acids cannot resist
decolourization with acid
alcohol and are categorized as
being non-acid fast,
On H&E, all fungi show pink
cytoplasm, blue nuclei and no
colouration of the wall.
Eg:, Blastomyces and Histoplas
ma can be appear red and be
acid-fast staining.

34

PAS Staining:

Periodic acidSchiff (PAS) is a staining method used to

detect polysaccharides such as glycogen, and
mucosubstances such as glycoproteins, glycolipids and
mucins in tissues.

Periodic Acid-Schiff Stain is used to detect yeast cells

and fungal hyphae in tissues.

Periodic acid (5%) hydrolyzes the cell wall aldehydes,

which then combine with the modified Schiff reagent.
35

The cell walls of fungi stain magenta.

This only works on living fungi;

Procedure is complex, most laboratories have replaced it with

the calcofluor white stain

Result: Cell wall------pink magenta

Background-------green

36

GROCOTT METHENAMINE SILVER

It is primarily used for the detection of fungal elements in
tissues.
Grocott's methenamine silver stain (GMS) will stain both living
and dead fungal organisms.
result :
Fungi --------------black
Background--------pale green.
charcoal gray--------innerparts of hyphae.

37

PRINCIPLE:
The mucopolysaccharide components of the
fungal cell wall are oxidized to release aldehyde
groups.

The aldehyde groups then react with the silver

nitrate, reducing it to a metallic silver, rendering
them visible.

38

Binds to cellulose and chitin in the cell walls of fungi,

including yeast cells, hyphae, pseudohyphae, and
spherules.

Rapid and specific.

The dye can be mixed with 10% KOH so that
mammalian cells can be dissolved, thus facilitating
visualization of fungal elements.

39

Fungi, Pneumocystis spp., and Acanthamoeba spp.

appear green or white against a dark background
when the stained slide is examined under UV
illumination.

40

Recommended for mounting and staining yeast and

molds.
Formulated with lactophenol, which serves as a
mounting fluid, and cotton blue.

Organisms suspended in the stain are killed due to the

presence of phenol.

The high concentration of the phenol deactivates lytic

cellular enzymes thus the cells do not lyse.
41

Cotton blue is an acid dye that stains the chitin

present in the cell walls of fungi .

42

Differential staining of basophilic and acidophilic material.

Polychromatic
Mixture of
*methylene blue
*azure B (from the oxidation of methylene blue)
*and eosin Y
*in methanol.

43

Like Giemsa Stain, it is primarily used for the

differentiation of intracellular and extracellular
circulating blood parasites.
Eg. Histoplasma. Pneumocystis

44

Methylene blue is another contrasting dye

commonly used in the laboratory, primarily for
detection of bacteria and fungi.

It can be mixed with potassium hydroxide and used

to examine skin scrapings for fungal elements.

45

INDIA INK

India ink is useful for indicating the presence or

absence of extra cellular polysaccharide capsules of
fungal cells.

The technique is particularly helpful for detecting

Cryptococcus neoformans in CSF.

Because India ink serves as a negative stain, the

encapsulated yeast cells can readily be detected
against the dark back ground.
46

The ink should be free from artifacts or granular

carbon particles to ensure a good preparation.

Appearance of a distinctive halo surrounding the

encapsulated fungi.

47

KOH SOLUTION
The KOH Solution is useful in detecting fungal elements in thick
mucoid material.
In specimens containing keratinous material, such as skin scales,
nails, or hair.
A 10 to 15% solution used to dissolve cellular and organic debris
and facilitate detection of fungal elements.

48

Action
Proteinacious host cell components are partially
digested by alkali while the fungal cell wall remains
intact (although fungal elements will dissolve after
exposure for a few days).

Ink (e.g., permanent blue-black Parker Super Quick Ink)

can be added as a contrasting agent to aid in the
detection of fungi.

49

ACRIDINE ORANGE STAIN

Acridine orange is a fluorescent stain.

For the detection of bacteria and fungi in clinical
specimens.
Acridine orange stains nucleic acid, changing dyes
optical characteristics so that it will fluoresce bright
orange under UV-light.
The dye intercalates into nucleic acid in both the native
and denatured states.

50

Neutral pH: bacteria, fungi and cellular material (e.g.,

leukocytes, squamous epithelial cells) stain red-orange.

Acid pH (pH 4.0), bacteria and fungi remain red-orange

but background material stains green-yellow..

Rapid stain, particularly useful in thick or purulent

specimens.

51

GIEMSA STAIN

Giemsa Stain, combines methylene blue and eosin.

Typically used by hematology laboratories for demonstrating
the differences of nuclei and cytoplasmic features of the blood
cell components.
Also used for detection of blood parasites.
Eg. Histoplasma, pneumocystis.
Intracellular yeasts and inclusions typically stain blue
(basophilic)

52

Special stains called brightners/

whiteners bind to carbohydrate
on the fungal surface and
flouresce.
(Tinopal for Candida)

53

Culture media

54

Culture media used for fungal growth:

The most commonly used cultures media used for
fungal growth are as follows:
Malt Extract Broth
Sabouraud agar
Mycological Agar
Hay infusion agar
Soy Peptone Yeast Extract
Potato dextrose agar
Agar
Potato Dextrose Broth Water Agar (WA)
Antibiotic Agar (AA)
Yeast Agar
Acidified Cornmeal Agar
Yeast Broth
(ACMA)
Mycological Agar
Cornmeal Agar (CMA)
Potato Carrot Agar (PCA)
Malt extract agar

55

Common media for primary fungal isolation include

Sabouraud dextrose agar and brain-heart infusion
agar, either in petri dishes or screwtop tubes.

Sabouraud dextrose agar

brain-heart infusion
56

The media may be enriched with 5% to 10% sheep

blood to support the growth of certain fungi.

Media generally contain a source of carbon,

nitrogen and vitamins. Glucose (dextrose) is the
most widely utilizable carbon source, and hence is
the most commonly used in growth media.

Fructose and mannose are the next most commonly

utilized sugars by fungi and are found in media from
natural sources
57

Mostly natural media based on materials such

as cornmeal, carrots, hay, potatoes, oatmeal,
soil, etc. Are used.

Semi-synthetic media, containing both natural

ingredients and defined components include
Malt Extract Agar, Malt Agar.

Synthetic or defined media which contain

precise amounts of a carbon source, vitamins
and minerals is less commonly used.
58

Water Agar (WA)--use for isolating fungi from surface-sterilized

substrates.

Antibiotic Agar (AA)--use for isolating fungi from substrates not

readily surface-sterilized, or to clean up a culture contaminated
with bacteria.

Acidified Cornmeal Agar (ACMA)--use for isolating fungi from

substrates that are likely to be contaminated with bacteria. as the
acidity inhibits bacteria and the medium supports the growth of a
wide range of fungi.

Cornmeal Agar (CMA)--use for growing a wide range of fungi,

particularly members of the Fungi imperfecti; provides a good
balance of mycelial growth and sporulation.
59

Potato Carrot Agar (PCA)--considered a relatively

weak medium somewhat comparable to CMA, good
for some Fungi imperfecti.

Malt Agar (MA)--lacks peptone, and is useful for

culturing many Ascomycota; sporulation in some
species is inhibited by peptone.

Malt Extract Agar (MEA)--a good growth medium for

soil fungi, fungi isolated from wood, basidiomycetes,
etc. An all-purpose type of medium.

60

 Potato Dextrose Agar (PDA)--a relatively rich

medium for growing a wide range of fungi.

Potato Dextrose-Yeast Extract Agar (PDYA)---

good for growing cultures derived from

mushrooms.

61

62

CARBOHYDRATE FERMENTATION

Yeasts are able to metabolize some foods, but not

others. In order for an organism to make use of a
potential source of food, it must be capable of
transporting the food into its cells.

It have the proper enzymes capable of breaking the

foods chemical bonds in a useful way.

63

Sugars are vital to all living organisms.

Yeast is capable of using some, but not all sugars

as a food source.

2% of suger is added to basal media.

Yeast ferments suger into CO2 & ethanol.

64

CO2 is collected in durhams tube, & ethanol as a

byproduct of the fermentation. Shows positive
result.

65

This test is used to detect presence of urease

enzyme produced by different Candida species.

Christensens urea agar slants are used.

Conversion of the yellow slope to pink or red is

considered positive.

A negative test is reported when there is no colour

change observed
66

Urease produced by the organisms split urea into

ammonia and CO2.
Urease positive pink colour
Urease negative yellow colour

67

Fungi has ability to break down milk protein

called casein into small peptides & amino acids.

The hydrolytic reaction creates a clear zone

around the cell as casein protein is converted to
soluble & transparent end products.

68

There is no reagent or indicator in the agar. A

zone of clearing around the growth area
identifies the presence of the enzyme caseinase

69

70

Available tests
Antibodies

Immunodiffusion
Radioallergosorbent Test (RAST)

Antigens

Radioimmunoassay (RIA)
Exoantigen test

Antibodies and antigens

Complement fixation

Enzyme-linked immunosorbent assay (ELISA)

71

Immunodiffusion is a diagnostic test which

involves diffusion through a substance such as agar.

Immunodiffusion

o Radial Immunodiffusion (Mancini method).

o Ouchterlony Double Diffusion

72

Radial Immunodiffusion
- A single diffusion technique where Ab is put into gel

and Ag is measured by the size of a precipitin ring

formed when it diffused out in all directions from a
well cut into the gel.

Ouchterlony Double Diffusion

- Both Ab and Ag diffuse from wells into a gel medium.

73

 Ag is added to an
antibody rich media.
The two continue to
react until the zone of
equivalence is
reached.
The area of ring is a
measure of the Ag
concentration.

74

 Method
Ab in gel
Ag in a well

Interpretation
Diameter of ring is
proportional to the
concentration

75

Both antigen and

antibody can diffuse
independently.

76

Wells are cut in the gel

Reactants are added in the well

Incubate (12-48 hrs) in a moist chamber

Precipitin lines will form

(where the moving front of the antigen meets antibody)
77

Fusion of lines at their

junction to form an arc
- Serologic identity /
presence of common epitope
Crossed lines
- Demonstrates 2 separate
reactions
- Compared antigens shared no
common epitopes
Fusion of 2 lines with spur
- Partial identity

78

Radioallergosorbent Test (RAST)

IgE is the antibody associated with Type I allergic response.

The RAST is a radioimmunoassay test to detect

specific IgE antibodies to suspected or known allergens for the
purpose of guiding a diagnosis about allergy.

The suspected allergen is bound to an insoluble material and the

patient's serum is added.

If the serum contains antibodies to the allergen, those antibodies

will bind to the allergen.
79

 Radiolabelled anti-human IgE antibody is

added where it binds to those IgE antibodies

already bound to the insoluble material.
The unbound anti-human IgE antibodies are

washed away. The amount of radioactivity is

proportional to the serum IgE for the allergen.

80

The RAST is scored on a scale from 0 to 6

RAST rating

IgE level (IU/ml)

comment

< 0.35

ABSENT OR UNDETECTABLE
ALLERGEN SPECIFIC IgE

0.35 0.69

LOW LEVEL OF ALLERGEN

SPECIFIC IgE

0.70 3.49

MODERATE LEVEL OF
ALLERGEN SPECIFIC IgE

3.50 17.49

HIGH LEVEL OF ALLERGEN

SPECIFIC IgE

17.50 49.99

VERY HIGH LEVEL OF

ALLERGEN SPECIFIC IgE

50.00 100.00

ULTRA HIGH LEVEL OF

ALLERGEN SPECIFIC IgE

> 100.00

EXTREMELY HIGH LEVEL OF

ALLERGEN SPECIFIC IgE

81

Radio immuno assay

A technique used to measure the concentration of hormones, Drugs, enzymes,
viruses, bacterial antigens and other organic substances of biological interest
found in Blood, Tissues and other biological fluids

Principle: Uses an immune reaction

[Antigen Antibody reaction] to estimate a
ligand
Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab*
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc is inversely related to
radioactivity of radiolabelled ligand.
[Ag : ligand to be measured ; Ag*
radiolabelled ligand]
82

83

Advantages
Highly specific: Immune reactions are specific
High sensitivity : Immune reactions are sensitive

Disadvantages

Radiation hazards: Uses radio labelled reagents

Requires specially trained persons
Labs require special license to handle radioactive material
Requires special arrangements for
Requisition, storage of radioactive material
radioactive waste disposal.

84

Exoantigen tests for the immunoidentification of

fungal pathogens are playing a new and significant role
in the diagnostic laboratory.

Properly performed and controlled exoantigen tests

lead to rapid, accurate identification of cultures of
many fungal pathogens.

The tests are particularly valuable in identifying

dimorphic pathogens that are difficult to convert or
with atypical cultures.
85

Specific antibodies developed against particular

mycelial antigens will react in a gel
immunodiffusion precipitin test.

The mold form of dimorphic fungi can be

identified definatly by an antigen- antibody
reaction.

86

Exoantigen immunodiffusion plate showing positive

identification of Histoplasma capsulatum. Note H and M bands
of identification; EX = culture filtrate; H = Histoplasma antibody
and antigen, C = Coccidioides antibody and antigen; B
= Blastomyces antibody and antigen.
87

The ability of antigen-antibody complexes to fix

complement is the basis of CFT.

To detect the fixation of complement, Sheep RBC

coated with amboceptor is used as the indicator
system

Very sensitive method to detect Ags & Abs.

88

89

Antigen+Test serum
(Contains Ab)
+ Complement

- Complement fixed

+ Hemolytic system

- No hemolysis, +ve CFT

Antigen+Test serum
(Contains no Ab)
+ Complement

- Complement not fixed

+ Hemolytic system

- Hemolysis, -ve CFT

90

The enzyme-linked immunosorbent

assay (ELISA) has become one of the
most widely used serological tests for
antibody or antigen detection.

This test involves the linking of

various label enzymes to either
antigens or antibodies.

Enzymes used in ELISA include Alkaline Phosphatase, Peroxidase

and Galactosidase.

91

 For Ab detection,

Ag coated wells used

If Ab present, it binds to Ag
Add goat anti-human

Ig antibody conjugated with

enzyme
Add a substrate. Enzyme

acts & colour produced

92

 For Ag detection, wells

coated with Sp. Ab.

Ag in specimen binds to

coated Ab.
Add enzyme conjugated

antiserum.
Add substrate.

Colour produced
93

 Ag coated wells
Two specific Abs,

(enzyme conj.& test Ab)

added simultaneously
More specific test Abs
attach to Ag
Substrate added
No colour since enzyme
not available
Positive test- no colour
94

95

A number of dermatophytes have the ability to

penetrate hair in vitro. The test is particularly
useful in distinguishing atypical isolates of
Trichophyton mentagrophytes from T. rubrum.

Procedure
1.Place several sterile hairs in a sterile glass Petri
dish. Hair from a blond child is preferred.
2.Add 25 ml of sterile water and 0.1 ml of sterile
yeast extract to the Petri dish.

96

c. Transfer a small amount of the fungus colony to the hairs in the

Petri dish.
d. Incubate at 25 C and examine weekly for up to 4 weeks.
e. Examine hairs by placing a few hairs in a drop of lactophenol on a
microscope slide. Place a cover glass over the preparation and
examine microscopically.
It will be necessary to examine several hairs before concluding a
negative result.

3. Results
a. Positive - Presence of perforations (conical or wedge-like holes) in
the hair.
b. Negative - Absence of perforations in the hair.
97

Thermotolerance is a useful characteristic that can be

used as an aid in the identification of several medically
important moulds. Thermotolerance of some medically
important fungi include:

Fungus Upper Growth Limits C

Aspergillus fumigatus 48-50 C
C. carrionii 35-36 C
Fonsecaea pedrosoi 38 C
Rhizomucor pusillus 45-55 C
Trichophyton mentagrophytes 37 C
Wangiella dermatitidis 40 C
Xylohypha bantiana 42-43 C
98

Nucleic acid probes can identify microorganisms

more rapidly than traditional culture.

Culture identification of fungi is based upon DNA

probes that are complimentary to species
specific ribosomal RNA.

The fungal cells are lysed by sonication, heat

killed & exposed to DNA that has been labelled
with a chemiluminescent tag.
100

The labelled DNA combines with the organisms

ribosomal RNA to form a stable DNA/RNA
hybrid.

Complex is measured in luminometer.

101

Cannot be used on fresh clinical specimens.

It is to be used for culture identification only.

102

Strain of several bacteria produces germ tubes

from their yeast cells when placed in a liquid
nutrient environment & incubated at 35C for 3
hrs.
Eg: Candida albicans.
By this method several fungi can be
differentiated from fungi, not forming germ
tube.

103

Determining the resistance of isolates to

cycloheximide (0.5 mg/ml) is useful when screening
cultures for Blastomyces dermatitidis, Coccidioides
immitis, Histoplasma capsulatum, Microsporum
spp., Paracoccidioides brasiliensis, Sporothrix
schenckii, and Trichophyton spp. Etc.

All of these fungi will grow in the presence of

cycloheximide at 30 C or less, while fungi such as
Absidia, Aspergillus, Mucor, Rhizopus,
Scedosporium, and many more are inhibited by
cycloheximide.
104

105

Candida : Candida is a genus of yeasts and is the

most common cause of fungal infections
worldwide.
Many species of candida are
harmless commensals or endosymbionts of
hosts including humans; however, when mucosal
barriers are disrupted or the immune system is
compromised they can invade and cause disease.

106

Species:
Candida albicans
Candida tropicalis
Candida parapsilosis
Candida guilliermondii
Candida kefyr
Candida krusei
Candida lusitaniae
Candida grabrata (no pseudohyphae)
107

Holmstrup and Bessermann ( 1983)

Primary candidiasis
Acute infections
Acute pseudomembranous candidiasis
Acute erythematous candidiasis

Chronic infections
Chronic pseudomembranous candidiasis
Chronic erythematous candidiasis

Chronic plaque like candidiasis

Chronic nodular candidiasis
108

Secondary candidiasis
Chronic Mucocutanous Candidiasis with
endocrinopathy
Familial CMC with endocrinopathy
Familial CMC without endocrinopathy
Idiopathic CMC with juvenile onset (<19yr)
CMC associated with thymoma
Idiopathic CMC with premature onset (>20yr)

109

Candidiasis is a fungal disease

Other names- Candidosis, Moniliasis , Thrush

Caused by Candida albicans

110

Exists in 3 forms
Pseudohyphae
Yeast
Chlamydospore

Reproduction- Asexual budding

Temperature-25-370C

111

Common inhabitant- oral cavity, GIT & Vagina

Most oppotunistic infection in world

112

Acute & Chronic diseases- tuberculosis, diabetes

mellitus & anemia

Myxedema, hypoparathyroidism, Addisons

disease

Immunodefeciency AIDS

113

Nutritional defeciency like Fe, Vitamin A, Vitamin

B6

Prolonged hopitalization for chronic illness and

debilitating diseases

Prolonged use of antibiotics, corticosteroids and

cytotoxic drugs

Radiation therapy

114

Use of intravenous tubes, catheters, heart valves

and poorly maintained dentures, heavy smoking

Old age, infancy and pregnancy

115

Candida species highly developed mechanisms

to adapt readily to changes in host environment

Shifts b/w different phenotypes in a reversible

and random fashion

Phenotypic switching involves co-ordinated

regulation of phase specific genes and a way to
adapt

116

It produces genetically altered variants at a high

rate

These differ in colony morphology, cell shape,

antigenecity and virulence

Produces a number of adhesins that mediate

adherance to host cells, candidal morphogenesis
or signaling

117

Morphogenesis
hyphal form is invasive and pathogenic, while
the yeast is the commensal nonpathogenic form
hyphae are capable of contact-sensing or
thigmotropism- facilitate the penetration
Secreted Hydrolytic Enzymes
Secrete: phospholipase , lipase ,
phosphomonoesterase , hexosaminidase , and at
least three separate aspartic proteinases

118

Aspartic proteinases (Sap proteins) fulfill

number of specialized functions during the
infective process:
digesting molecules for nutrient acquisition
digesting or distorting host cell membranes
facilitate adhesion and tissue invasion
digesting cells and molecules of the host
immune system
avoid or resist antimicrobial attack by the host.

119

Variety of clinical manifestations

Range from mild superficial mucosal

involvement to severe, fatal disseminated form
seen in immunocompromised individuals

Classified into 2 categories

-Mucocutaneous
-Systemic

120

Mucocutaneous
oral & oropharyngeal ( thrush)
candidal oesophagitis
candidal vulvovaginitis & balanitis
intertrigo or intertriginous candidiasis

Systemic
Eyes, kidney and skin through hematogenous
spread and other visceral organisms

121

Acute candidiasis

Chronic cndidiasis

122

Gross Appearance
Soft, white, slightly
elevated plaques
resembling
milk curds
Site
Buccal mucosa, tongue,
palate, gingiva, floor of
mouth
123

Histopathology
Tangled mass of fungal hyphae

Intermingled with desquamated epithelium,

keratin, fibrin, necrotic debris,leukocytes and
bacteria

White plaque can be wiped away leaving a

normal area or an erythematous area

124

Antibiotic sore mouth under category of

erythematous candidiasis

Sequale to course of broad spectrum antibiotics

Gross :Lesions appear red or erythematous

Site - Any

125

Central Papillary Atrophy of

Tongue
Asymptomatic, symmetric
lesions of tongue
Dorsal aspect in posterior
region
Erythematous appearanceloss of filliform papillae
Strong relationship b/w
lesion and chronic smoking

126

Leukoplakia type candidiasis

Gross: Firm white, persistent plaques

Site: Lips, tongue , cheeks

Persist for years

127

Roed-Peterson- Incidence of candida is seen in

lesions of leukoplakia.

Occurrence of cytological atypia in lesions of

leukoplakia.

Cawson & Binnie definite relationship b/w

chronic candidiasis and oral epidermoid
carcinoma

128

Specimens
swab, scrapings, blood, CSF, biopsies, urine,
exudates, material from removed iv. Catheters

1.

Microscopic examination
plaque smear--- Gram stain---pseudohyphae,
budding cells.
20% KOH/ Calcoflour white: skin & nail scrapings.
129

2.

H&E
Both yeast and hyphae
superficially + deep

3.

Special stains
PAS, Gomori Methanamine Silver

130

Culture
Blood/ cornmeal/
sabourauds agar

oval, budding yeast

cells, 3-6m.
Soft cream colored
colonies with a
yeasty odor.
Sabauruods agar slant
for culture of candidiasis
131

Morphologic tests
Germ Tube Test
Filamentous extension from a yeast cell that is
about one half the width and 3-4 times the
length of the cell
1.

Small portion of an isolated colony suspended

intest tube containing 0.5ml sheep serum.

2.

Tube inoculated at 35o for no longer than 2 hrs

132

Serology:
Limited specificity and sensitivity (Ab detection)

EIA/latex agglutination(ag detection)

cell wall mannan.
beta 1,3,D glucan

No clear criteria for establishing diagnosis.

133

Nucleic acid detection:

Hybridisation
amplification
analysis

Detection of fungal metabolites

D-arabinitol (Bernard et al)

134

Seen microscopically in exfoliative and biopsy

specimens

PAS candidal hyphae and yeasts

PAS method demonstrates stains carbohydrates

contained in abundance by fungal cells walls;
organism identified as bright magenta
135

Hyphae are approx 2 microns in diameter and vary

in length and may show branching.

Hyphae accompanied by variable no. of yeasts,

squamous epithelial cells & inflammatory cells.

10% to 20% KOH may be used to evaluate

specimens for presence of fungal organisms.

KOH lyses the background of epthelial cells allowing

more resistant yeasts and hyphae.
136

Characteristic pattern of
parakeratosis, neutrophilic
microabscess, a thickned
spinous layer and chronic
inflammation of connective
tissue

Tubular hyphae embedded in

the parakeratin layer

137

138

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THANK YOU...

140

Bibliography

Bailey & Scotts Diagnostic microbiology. Twelfth

edition.
R.Ananthanarayan, CKJ paniker text book of
microbiology 6th edition.
Laboratory diagnosis on fungal infections- a review.Dr.
Shruti nayak, dr. Amarnath shenoy, dr. U. S. Krishna
nayak
Text book of microbiology by prof. C.P.Baveja.
Microbiology & microbial infections: Mycology. Topley
& Wilson. 10th edition.
141

Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,

and R. H. Yolken (eds.). Manual of clinical microbiology,
6th edition.
Medical Microbiology: A guide to microbial infections,
pathogenesis, laboratory diagnosis & control. David
Greenwood. Sixteenth edition.
Oral Candida: Clearance, Colonization, or Candidiasis?
JDR 74(5); 1995.
Candida albicans Secreted Aspartyl Proteinases in
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