MYCOLOGY
Introduction
Mykes (Greek word) : Mushroom
Fungi are eukaryotic protista; differ from bacteria and
other prokaryotes.
1. Cell walls containing chitin (rigidity & support),
mannan & other polysaccharides
2. Cytoplasmic membrane contains ergosterols
3. Possess true nuclei with nuclear membrane & paired
chromosomes.
4. Divide asexually, sexually or by both
5. Unicellular or multicellular
Introduction
Simplest fungus :- Unicellular budding yeast
Hypha :- Elongation of apical cell produces
a tubular, thread like structure called hypha
Mycelium :- Tangled mass of hyphae is
called mycelium. Fungi producing mycelia
are called molds or filamentous fungi.
Hyphae may be septate or non-septate
CLASSIFICATION
Depending on cell morphology
1. Yeasts
2. Yeast like fungi
3. Molds
4. Dimorphic fungi
1. Yeasts
Unicellular fungi which reproduce
by budding
On culture - produce smooth,
creamy colonies.
E.g. Cryptococcus neoformans
(capsulated yeast)
2. Yeast like fungi
Grow partly as yeasts and partly as elongated cells
resembling hyphae which are called pseudohyphae
e.g. Candida albicans
3. Molds/ Filamentous fungi
Form true mycelia &
reproduce by formation of
different types of spores.
Vegetative/ aerial hyphae
e.g. Rhizopus, mucor
4. Dimorphic fungi
Occur in 2 forms
Molds (Filaments) – 250 C (soil)
Yeasts – 370 C (in host tissue)
Most fungi causing systemic infections are dimorphic:
Histoplasma capsulatum
Blastomyces dermatidis
Paracoccidioides brasiliensis
Coccidioides immitis
Penicillium marneffei
Sporothrix schenkii
Reproduction in fungi
Sexual - formation of zygospores, ascospores or
basidiospores
Asexual reproduction – budding or fission
Asexual spores are formed on or in specialized
structures.
Vary in size, shape & color but these
characteristics are constant for a particular
species.
Reproduction in fungi
Micro conidia -
Small, single celled
Macro conidia –
Large, single or
many celled
Systematic classification
Based on sexual spore formation: 4 classes
1. Zygomycetes
2. Ascomycetes
3. Basidiomycetes
4. Deuteromycetes (fungi imperfectii)
1. Zygomycetes
Lower fungi
Broad, nonseptate hyphae
Asexual spores -
Sporangiospores: present within
a swollen sac- like structure
called Sporangium
1. Zygomycetes
Sexual spores - Zygospore: a
resting, thick walled cell in
between hyphae
e.g. Rhizopus, Mucor
2. Ascomycetes
Includes both yeasts & filamentous fungi
Narrow, septate hyphae
Asexual spores are called conidia borne on
conidiophore
2. Ascomycetes
Sexual spores called ascospores
are present within a sac like
structure called Ascus.
Several asci may be seen within
a fruiting body as seen in
Penicillium, Aspergillus
Each ascus has 4 to 8
ascospores.
3. Basidiomycetes
Sexual fusion results in
the formation of a club
shaped organ called
base or basidium
which bear spores
called basidiospores
4. Deuteromycetes or Fungi
imperfectii
Group of fungi whose sexual phases are not
identified.
Grow as molds as well as yeasts.
Asexual stage – conidia
e.g. Candida, Cryptococcus
Vegetative Structures of Fungi
Arthrospores – formed by
segmentation &
condensation of hyphae
Chlamydospores – thick
walled resting spores e.g.
C.albicans
Fungal Infections/ Mycoses
Superficial mycoses:
2 types: surface and cutaneous mycoses
Skin, hair & nails.
Mild but chronic disease
Deep mycoses:
2 types: subcutaneous & systemic mycoses
Caused by soil saprophytes
Infection is accidental
Range from a symptomatic infection to fatal disease
Superficial: Surface mycoses
Live exclusively on dead surfaces of skin and
its appendages
No contact with living tissue, hence no
inflammatory response
1. Tinea versicolor
2. Tinea nigra
3. Piedra
Superficial: Cutaneous mycoses
Cornified layer of skin & its appendages
Contact with living tissue, hence inflammatory
& allergic responses seen
1. Dermatophytes – skin, hair & nails
2. 3 genera –
Trichophyton
Microsporum
Epidermophyton
Deep mycoses
Subcutaneous Systemic mycoses
mycoses 1. Cryptococcoses
1. Mycotic Mycetoma 2. Blastomycosis
2. Chromoblastomycoses 3. Coccidioidomycoses
3. Sporotrichosis 4. Histoplasmoses
4. Rhinosporidiosis
LABORATORY
DIAGNOSIS OF
FUNGAL DISEASES
23
Specimen Collection
Depends on site of infection - skin scraping, hair, nail,
sputum, etc.
Systemic mycoses - blood sample, Cerebrospinal fluid
(CSF), etc
24
Microscopy
Fungal elements can be detected in the clinical specimens
by direct microscopic examination of material from the
lesion.
25
Microscopy (Cont..)
Potassium hydroxide (KOH)
preparation
Keratinized tissue specimens
treated with 10% KOH digests
keratin fungal hyphae clearly
seen
20–40% KOH - nail & hair 26
Microscopy (Cont..)
Potassium hydroxide (KOH) preparation (Cont..):
Biopsy specimens - dissolved in 10% KOH in a test tube
and examined after overnight incubation
Glycerol (10%) can be added to prevent drying
DMSO (dimethyl sulfoxide) - help in tissue digestion
27
Microscopy (Cont..)
Gram stain:
Useful in identifying the yeasts (e.g.
Cryptococcus) and yeast like fungi (e.g.
Candida).
They appear as gram-positive budding
yeast cells
India ink and nigrosin stains:
Used as negative stains for
demonstration of capsule of
Cryptococcus neoformans
28
Microscopy (Cont..)
Calcofluor white stain:
More sensitive than other
stains.
Binds to cellulose and chitin of
fungal cell wall and fluoresce
under UV light
29
Microscopy (Cont..)
Histopathological stains
For demonstrating fungal elements
from biopsy tissues
Periodic acid schiff (PAS) stain:
PAS positive fungi appear
magenta/deep pink, whereas the
nuclei stain blue
PAS which stains only the live
fungi
30
Microscopy (Cont..)
Histopathological stains
(Cont..)
Masson fontana stain: for
pigmented (or pheoid) fungi
Hematoxylin and Eosin stain
Mucicarmine stain: for
staining the carminophilic cell
wall of Cryptococcus and 31
Microscopy (Cont..)
Histopathological stains (Cont..)
Gomori methenamine silver
(GMS) stain: Alternative to PAS
Stains both live and dead
fungi
Stains the polysaccharide
component of the cell wall
Fungi appear black &
background pale green color
32
Microscopy (Cont..)
Lactophenol cotton blue (LPCB): It is
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.
33
Culture
Fungal culture is frequently performed for isolation and
correct identification of the fungi.
34
Culture Media
Sabouraud’s dextrose agar (SDA):
Most commonly used medium
Contains peptone (1%), dextrose (4%)
and pH of 5.6
May not support some pathogenic fungi
Neutral SDA (Emmons’ modification):
Differs from original SDA in having
neopeptone (1%) and dextrose (2%)
and pH of 7.2
35
Culture Media (Cont..)
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
36
Culture Media (Cont..)
Niger seed agar and bird seed agar: selective growth
of Cryptococcus
CHROMagar Candida medium: selective as well as
differential medium for speciation of Candida.
37
Culture Condition
Temperature: Most of the fungi grow well at 25–30°C except the
dimorphic fungi that grow at both 25°C and 37°C
BOD incubators (biological oxygen demand): capable of
maintaining low temperature
Incubation time: 2–3 weeks
Antibiotics - cycloheximide (actidione), chloramphenicol and
gentamicin - added to the culture media to inhibit bacterial growth
38
Culture Identification
Correct identification of the fungus is based on the
macroscopic appearance of the colonies grown on culture
and microscopic appearance (LPCB mount of colonies).
39
Macroscopic Appearance of the
Colony
Rate of growth:
Rapid growth (<5 days): seen in saprophytes, yeasts
and agents of opportunistic mycoses
Slow growth (1–4 weeks): observed in dermatophytes,
agents of subcutaneous and systemic mycoses.
40
Macroscopic Appearance of the
Colony (Cont..)
Pigmentation: seen on the reverse side of the culture
media
Texture: refers to how the colony would have felt if
allowed to touch. It may be of various types such as -
glabrous (waxy/leathery), velvety, yeast like, cottony or
granular/powdery.
41
Colony topography
Microscopic Appearance of Fungi
Teased mount: Identification is based on :
Nature of hyphae (septate or aseptate, hyaline or phaeoid,
narrow or wide)
Type of sporulation (conidia or sporangia)
Slide culture: in situ microscopic appearance of the
fungal colony
42
Microscopic Appearance of Fungi
(Cont..)
43
Microscopic Appearance of Fungi
(Cont..)
Cellophane tape mount:
Impressions taken by placing the cellophane tape on the
colonies LPCB mount is made
Easy to perform than slide culture and in-situ fungal
morphology maintained
44
Other Methods of Identification
For Candida: Germ tube test, Dalmau plate culture,
carbohydrate fermentation and carbohydrate
assimilation tests are done
For dermatophytes: Hair perforation test,
dermatophyte test medium and dermatophyte
identification medium are used
Urease test can be done for the fungi that produce
urease enzyme, e.g. Cryptococcus
45
Immunological Methods
Antibody detection - ELISA, immunodiffusion test,
agglutination test, and complement fixation test (CFT)
Antigen detection - latex agglutination test for
cryptococcal antigen from CSF
Immunohistochemistry: detecting antigens (e.g.
proteins) on cells of a tissue section
46
Automations
Automated identification system such as MALDI-TOF and
VITEK are revolutionary in accurate identification of yeasts
and to some extent molds.
47
Molecular Methods
Polymerase chain reaction (PCR) and its modifications -
multiplex PCR, nested PCR and the most advanced real
time PCR
DNA sequencing methods.
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OVERVIEW OF
FUNGAL INFECTIONS
49
SUPERFICIAL
MYCOSES
50
SUPERFICIAL MYCOSES
Tinea versicolor:
Caused by lipophilic fungus Malassezia furfur -
characterized by flat-round scaly hypopigmented to
hyperpigmented patches of skin.
Tinea nigra:
Characterized by painless, black, nonscaly patches present
on palm and sole; caused by - black-colored yeast like
fungus Hortaea werneckii.
51
SUPERFICIAL MYCOSES
(Cont..)
Piedra:
Characterized by nodule formation on hair shaft - either
black in color (caused by Trichosporon beigelii) or white in
color (caused by Piedraia hortae).
52
SUPERFICIAL MYCOSES
(Cont..)
Dermatophytoses (or tinea or ringworm):
Most common superficial mycoses affecting skin, hair and
nail - caused by a group of related fungi (called
dermatophytes). These include:
Trichophyton species: Infect skin, hair and nail
Microsporum species: Infect skin and hair
53
Epidermophyton species: Infect skin and nail.
SUBCUTANEOUS
MYCOSES
54
SUBCUTANEOUS MYCOSES
Mycetoma:
Also known as Maduramycosis or Madura foot - chronic,
slowly progressive granulomatous infection of the skin and
subcutaneous tissues
Clinically manifested - triad - swelling, discharging sinuses
and presence of granules in the discharge.
Two types – Eumycetoma and Actinomycetoma 55
SUBCUTANEOUS MYCOSES
(Cont..)
Sporotrichosis :
Also known as Rose Gardner’s disease - caused by a
thermally dimorphic fungus, Sporothrix schenckii.
Presents as subcutaneous noduloulcerative lesions.
56
SUBCUTANEOUS MYCOSES
(Cont..)
Chromoblastomycosis:
Refers to slow growing chronic subcutaneous lesions
caused by group of dematiaceous or phaeoid fungi (i.e.
darkly pigmented fungi).
Produce a characteristic morphology called sclerotic body.
Agents include: Fonsecaea, Phialophora Cladosporium and
Rhinocladiella. 57
SUBCUTANEOUS MYCOSES
(Cont..)
Rhinosporidiosis:
Caused by Rhinosporidium seeberi.
Produces large friable polyps in the nose (most common
site), conjunctiva and rarely in other sites.
58
OPPORTUNISTIC
MYCOSES
59
OPPORTUNISTIC MYCOSES
Caused by the fungi that are normally found as human
commensals or in environment; but can act as human
pathogen in presence of opportunities such as low
immunity.
60
OPPORTUNISTIC MYCOSES
(Cont..)
Candidiasis:
Most common fungal disease in humans, affecting the skin,
mucosa, and various internal organs.
Caused by Candida, a yeast like fungus that produces
pseudohyphae.
Various species - Candida albicans, C. tropicalis, C.
parapsilosis etc.
Common in presence of various predisposing factors -
patients on steroid or immunosuppressive drugs, post-
61
transplantation, malignancy, HIV infection.
OPPORTUNISTIC MYCOSES
(Cont..)
Cryptococcosis:
Caused by a capsulated yeast - Cryptococcus neoformans -
capable of producing potentially fatal meningitis in HIV
infected people.
Negative staining of CSF - performed to demonstrate the
capsule - appears as refractile delineated clear space
surrounding the round budding yeast cells against the black
62
OPPORTUNISTIC MYCOSES
(Cont..)
Zygomycosis:
Group of life-threatening infections caused by aseptate
fungi; belonging to the phylum Zygomycota.
Include - Rhizopus, Mucor, Rhizomucor, Lichtheimia, etc.
Risk factors: diabetic ketoacidosis.
Clinical forms: Rhinocerebral mucormycosis.
63
OPPORTUNISTIC MYCOSES
(Cont..)
Zygomycosis (Cont..):
Laboratory diagnosis:
Culture on SDA - white cottony woolly colonies and LPCB
mount of the colonies showing typical broad aseptate
hyaline hyphae.
Rhizopus shows a unique root like growth arising from
hyphae called rhizoid. 64
OPPORTUNISTIC MYCOSES
(Cont..)
Aspergillosis:
Refers to the invasive and allergic diseases caused by a
hyaline mold named Aspergillus.
Important species - A. fumigatus, A. flavus and A. niger.
Risk factors: Glucocorticoid use and profound neutropenia
65
OPPORTUNISTIC MYCOSES
(Cont..)
Aspergillosis (Cont..):
Clinical forms:
Pulmonary aspergillosis – most common form.
Other forms - invasive sinusitis, cardiac, cerebral, ocular
aspergillosis.
Laboratory diagnosis:
66
SDA reveals typical narrow septate hyaline hyphae.
OPPORTUNISTIC MYCOSES
(Cont..)
Penicilliosis:
Penicillium species produce various opportunistic infections
- endophthalmitis, otomycosis, keratitis and
onychomycosis.
Penicillium marneffei - only dimorphic species of
Penicillium, which causes opportunistic infection (wart-like
skin lesions) in HIV-infected patients. 67
OPPORTUNISTIC MYCOSES
(Cont..)
Pneumocystosis:
Refers to plasma cell pneumonia due to Pneumocystis
jirovecii, which typically occurs in HIV infected patients.
68
MYCOTOXICOSES
69
MYCOTOXICOSES
Mycotoxicoses:
Occurs following consumption of food contaminated by
toxins liberated by certain fungi; e.g. aflatoxin by
Aspergillus flavus which causes carcinoma liver.
70
MYCOTOXICOSES (Cont..)
Mycetism:
Refers to toxic effects produced by, eating poisonous fleshy
fungi; usually different types of mushrooms.
71
Questions:
Q1. All are yeast or yeast like fungi except:
a. Candida
b. Trichosporon
c. Cryptococcus
d. Trichophyton
72
Questions:
Q2. Organisms that does not affect the nail:
a. Trichophyton
b. Epidermophyton
c. Microsporum
d. Candida
73
Questions:
Q3. Asteroid bodies is observed in:
a. Sporotrichosis
b. Histoplasmosis
c. Candidiasis
d. Chromoblastomycosis
74
Questions:
Q4. Tinea versicolor is caused by:
a. Trichophyton rubrum
b. Trichophyton violaceum
c. Malasezzia furfur
d. Candida albicans
75
Questions:
Q5. Azole active against mucormycoses:
a. Voriconazole
b. Fluconazole
c. Itraconazole
d. Posaconazole
76
