Mycoviro prelim

MLS 302 C - MICROBIOLOGY 3
MYCOLOGY
VIROLOGY

PRELIMS OUTLINE
• General characteristics of fungi
• Morphology of fungi
• Classification of fungi
• Laboratory Diagnosis of Fungal
Diseases
• Classification of Fungal Diseases

REFERENCES
• Jawetz, Melnick & Adelberg’s Medical
Microbiology
• Clinical Mycology by Dismukes, et al.
• Bailey & Scott’s Diagnostic Microbiology
• Clinical Microbiology Made Ridiculously
Simple

LECTURE 1: OBJECTIVES
• 1. Define terms used in mycology
• 2. Discuss the general
characteristics of fungi
• 3. Describe the morphology and
structure of fungi in general
• 4. State the medical importance of
the cellular structures of fungi

FUNGAL
CHARACTERISTIC
• Eukaryotic
• Facultative anaerobic/strictly aerobic
• Chemotrophic; nutrition by absorption
• Non-photosynthetic; achlorophyllos
• pH 5-6 (acidic)
• High sugar conc’n. favors growth

 Widely
distributed in
nature
 Saprophytic

Penicillium notatum
Tolypocladium inflatum

TERMINOLOGY
• Mykos (Gk): Mushroom
• Mycology: Study of fungi
• Mycoses: diseases caused by fungi
• Dimorphism: ability to grow as yeast at
37°C and as mold at room
temp.
• Hyaline fungi: non-pigmented fungi
• Dematiaceous fungi: pigment-producing fungi

Morphologic Classification
of FUNGI
 YEAST
 MOLD
 DIMORPHIC

YEAST
Unicellular fungi
• MICROSCOPIC
 Oval to round
 Budding
 Bud=Blastospore
 Pseudohyphae

• MACROSCOPIC:
Pasty, opaque, cream-colored colonies

• MONOMORPHIC
YEAST:
 Candida
albicans
 Cryptococcus
neoformans
 Geotrichum
candidum

MOLD
 Multicellular
 Hypha [2-10 µm]
 MYCELIA
 Vegetative /
Thallus
 Reproductive /
Aerial

 MACROSCOPIC:
Cottony, wooly,
velvety, granular,
filamentous
 MONOMORPHIC
MOLD:
• Microsporum
• Epidermophyton
floccosum
• Trichophyton

 PIGMENTATION:
observed from the reverse

A. EXISTENCE OF SEPTA
 CLASSIFICATION OF HYPHAE

In Medically Important Fungi:
3 Types Of Hyphae
1. Coenocytic- sparsely
septated
2. Dematiaceous fungi -
dark and pigmented
septate hyphae
3. Hyaline molds: septate,
non-pigmented hyphae

DIMORPHIC FUNGI
Thermal dimorphism (a group of pathogenic fungi)
Mold form: 25ºC to 30ºC
Yeast form 35ºC to 37ºC

DIMORPHIC FUNGI
• Sporothrix schenckii
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Penicillium marneffei
Subcutaneous
Systemic
Opportunistic

CAPSULE
Structure: Polysaccharide
Functions: Antiphagocytic factor
Cryptococcus neoformans (encapsulated yeast)

CELL WALL
• Antigenic
• Multilayered
a. polysaccharides
 poorly degraded by host
 activate CF; provokes inflammatory rxn
 Induce immune hypersensitivity
b. proteins and glycoproteins

CELL WALL
Functions:
 Provides shape, rigidity & strength
 protection from osmotic shock
 mediates attachment to host cells

Major Polysaccharides Of Fungal
Cell Wall
POLYMER MONOMER
Chitin N-acetyl glucosamine
Chitosan D-Glucosamine
Cellulose D-Glucose
-Glucan D-Glucose
-Glucan D-Glucose
Mannan D-Mannose
The type & amount of polysaccharide vary
from one fungal species to another.

CELLULAR MEMBRANE
Bilayered Structure:
1. Phospholipids
-phosphatidylcholine
-phosphatidylethanolamine
2. Sterols
- ergosterol
- zymosterol

CELLULAR MEMBRANE
Functions:
 Protects cytoplasm
 regulates intake & secretion of
solutes
 facilitates capsule & cell wall
synthesis

• CYTOPLASM
Nucleus, nuclear membrane, nucleolus,
ER, mitochondria, vacuoles

LECTURE 2: OBJECTIVES
• Classify fungi
– according to methods of reproduction
– type of spores they produce.

REPRODUCTION
A. SEXUAL REPRODUCTION
 opposite gametes mate to form a zygote
 Sexual spores
3 phases of sexual reproduction:
1. Plasmogamy – fusion of opposite but
compatible mating types
2. Karyogamy – fusion of 2 nuclei to form a
diploid nucleus
3. By meiosis, diploid nucleus gives rise to haploid
spores

SEXUAL SPORES
ZYGOSPORE
• Enclosed in a thick wall
• Rhizopus, Mucor

ASCOSPORE
 Produced in ascus
(sac-like); 2-8
spores/ascus
 Nucleic fusion of
morphologically
dissimilar cells
 Histoplasma
capsulatum

BASIDIOSPORE
formed externally on a
base pedestal called
Basidium
 mushroom

ZOOSPORE
Fusion of cells from
2 diff. hyphae
OOSPORES
• thick-walled, long-
surviving spore
Phytophthora
capsici

SPORE GERMINATION
 Fruiting body : Principal structure
 asexual spores

CONIDIOSPORE
•Unicellular / multicellular
•Produced in a chain at the
end of conidiophore
•Penicillium
•Aspergillus
ASEXUAL SPORES

A. Arthrospore
•fragmented septate
hyphae
• single, slightly
thickened cells
•Coccidioides
•Geotrichum

 bud
Candida
Cryptococcus
B.
Blastospore
C. Chlamydospore
• Thick walled
• formed along the
periphery or tip of the
hyphae

SPORANGIOSPORE
•developed in a sac
(sporangium)
•Attached to a specialized
hyphae (sporangiophore)

C. PARASEXUAL REPRODUCTION
 Set of events that lead to genetic
exchange via mitotic recombination
 Initiated by formation of a heterokaryon
(thallus w/ 2 haploid nuclei of 2 diff.
genotypes)
 for genetic analysis of imperfect fungi

FUNGI-TAXONOMIC
CLASSIFICATION
• Depends primarily on the type of
sexual spore
Phylum -mycota
Class -mycetes
Order -ales
Family -ceae
Genus
Species

Fungi-Taxonomic classification
SEXUAL SPORE CLASS
Zygospore---------- Zygomycetes
Basidiospore--------Basidiomycetes
Ascospore---------- Ascomycetes
Zoospores---------- Chytridiomycetes
None/Unknown---- Deuteromycetes
(“Fungi Imperfecti”)

ZYGOMYCOTA
sporangium fungi / common
molds
molds & blights
(Rhizopus stolonifer)
 coenocytic
 rhizoids

ASCOMYCOTA
• sac fungi
• Reproduction:
–Conidiospores
–Ascospores
–Budding
• Ascocarp
• Ascus

BASIDIOMYCOTA
• club fungi
• Some are used as
food; others cause crop
damage
• Seldom reproduce
asexually

• Basidiocarp
• annulus
• basidia
• Volva

DEUTEROMYCOTA
 Asexual spores: conidia of various types
 Septate hyphae
 most are yeast & molds, some dimorphic

LECTURE 3:
OBJECTIVES
Discuss the laboratory
methods used in the
diagnosis of fungal diseases
as to:
1. proper collection, handling,
transport and disposal of
specimen
2. culture media to use
3. methods of identification

SPECIMEN COLLECTION
• Aseptic technique
• Collect spx from actual infxn site
• Adequate quantity
• Accurate label; Prompt delivery

CLINICAL SPECIMEN
Blood 5 ml to BHI broth
Bone
Marrow
Aspirate 0.5ml to BHI
CSF Lumbar tap
 Vol. is >2ml: centri, smear,
culture
Vol. is <2ml: use uncentri spx.

• Direct culture
• Biphasic culture bottle
• Membrane filtration
• Lysis centrifugation

• Sputum
• Bronchial
washings
• Tracheal
aspirate
 Collect sputum in a.m. for 3
days
No 24 hr. collections
sterile NSS for washings
Throat
 + few yeast & contaminants
 Use 2 sterile swabs
 Use tongue depressor if ?
candida

Urine  Midstream, clean-catch,
catheter
 Centri, smear, culture
(sediments)
Process in 2hrs or refrigerate
>100,000/ml is significant
Vaginal &
cervical
discharges
 +few to moderate Candida
colonies
2 swabs: for KOH & culture

CUTANEOUS
a. Hair  May contain contaminants
 Pluck hair by roots (forceps)
 Select hair that fluoresce, broken,
scaly
b. Nail  70% alc., scrape discolored areas,
collect inner infected nail
 Nail clippings: cut into small pcs.
 w/KOH
c. Skin  Few Candida & contaminants
 scrape infected area
 w/KOH

Muco-
cutaneous
Scrape plaque w/ TD
(Candida)
Subcutaneous Lesions, abscesses,
tissues
Needle aspiration or
biopsy
Check for granules

LABORATORY DIAGNOSIS OF
FUNGAL INFECTIONS
• Direct microscopic examination
• Culture
• Serology
• others

MICROSCOPIC METHOD
WET MOUNT
A. 10-20% KOH
 destroy keratin layer
 OH
o clears debris
o makes fungi
prominent

B. Lactophenol Cotton
Blue (Aman’s medium)
 Lactic acid
 Phenol
 Cotton blue

C. India Ink
 capsule of C.
neoformans
 Capsules
(clear halo)
against dark
background

D. Calcofluor White +
KOH
 For dermatophytes
 Not suitable for
Pneumocystis carinii
 Binds with chitin in cell
wall
 under Wood’s light/UV
light
 apple-green/ bluish-
white fluorescence

II. STAIN PREPARATION
•
TISSUE
SECTION
•Grocott’s
•Griffith’s
•Gomori’s
• Methenamine
Ag*
•PAS**
* Stains cell wall
black
** Stains hyphae of
molds & some
yeast
PIGMENTED
TISSUE
•Fontana
Tribondeau
MALIGNAN
T
CELLS
•PAPS •better demo of
B. dermatitidis
than wet mounts

BONE
MARROW
•Giemsa
•Wright’s
•Detects intracellular
H. capsulatum in
bld. smears
•Orgs. appear as
small, oval yeast cells
(light-dark blue)
GENERA
L TYPE
•Kinyoun’s AFS
•Hucker’s GS
• fungi are GS + but
poorly stained
• Useful for Candida

PAS
Grocott/Gomori/Methenamine
Silver Stain
Wright’s H&E

CULTURE METHOD
REQUIREMENTS
Media must include:
•Amino acids or urea (N), Glucose (C), etc.
• Antimicrobial agents (cyclohexamide &
chloramphenicol)
 Aerobic environment;  humidity & moisture

TYPE ADVANTAGES DISADVANTAGES
AGAR
PLATES
• Better aeration
• Large surface area
for better isolation
• Ease of handling
• Easily dehydrates (use
40 ml agar)
• biological safety
cabinet
• Hazardous to handle
SCREW-
CAPPED
TUBE
•Easy storage
•Less space for
incubation
•More easily handled
•Less hazardous
•Lower dehydration
rates
•Poor colony isolation
• reduced surface area
•Promote anaerobiosis

CULTURE MEDIA
PRIMARY CULTURE
 SDA – Sabouraud Dextrose Agar ; general isolation
 BHI – useful for pathogenic fungi from sterile
spx.
 SDA/BHI w/ antibiotics

SELECTIVE CULTURE MEDIUM
 corn meal agar
 corn meal agar w/ Tween 80 –
stimulates chlamydospore formation (Candida)
 CZAPEC agar – routine media for Aspergillus &
Penicillium
 Cottonseed agar - conversion of B. dermatitidis
mold phase to yeast phase
 Niger seed/ Birdseed agar – C. neoformans ;
brown– black colonies
 Rice agar
 Urea agar

III. CULTURE METHOD: Primary culture
Medium
1. Sabouraud Dextrose Agar (SDA)

III. CULTURE METHOD: Primary culture Medium
2. Brain Heart infusion Agar
3. SABHI + antibiotics
– Recovery of saprobic and pathogenic fungi

III. CULTURE METHOD: Primary
culture Medium
4. SDA/BHI w/ antibiotics
• Recovery of pathogenic fungi exclusive of
dermatophytes

III. CULTURE METHOD: selective medium
1. Corn meal agar
2. Corn meal agar w/ Tween 80

III. CULTURE METHOD: Selective medium
3. Czapek agar
– Differential ID of Aspergillus spp.

III. CULTURE METHOD: Selective medium
4. Urea agar
– detection of C. neoformans
– differentiate T. mentagrophytes vs T. rubrum
– detection of Trichosporon spp.

Culture method: selective
5. Niger seed/
Birdseed agar
– Identification of C.
neoformans
– Phenoloxidase

Culture method: selective
6. Cottonseed conversion agar
– conversion of B. dermatitidis from mold to yeast
form
7.Rice agar
– ID of M. audouini

IV. GERM TUBE TEST
• Candida albicans
• 0.5 mL rabbit serum +
a colony
• incubate for 2 ½ - 3
hours @ 37°C
• (+) Germ tube
formation

V. Hair baiting/perforation
• T. mentagrophytes
• Fill petri dish w/ soil then make
holes on soil
• Cut hair into small pcs. & place
in holes
• Incubate @ RT
• Examine regularly for growth.

VII. REDUCTION OF NITRATE TO NITRITE
• Cryptococcus neoformans

BIOCHEMICAL TEST
CHO Assimilation Test For Yeast
 (+) growth around disk
Reduction Of Nitrate To Nitrite
 C. neoformans
Rapid Urease Test
 For preliminary ID of C. neoformans

EXAMINATION OF FUNGAL GROWTH
MACROSCOPIC
• Rate of growth
• Topography – flat, heaped, folded, rugose,
wrinkled
• Texture – cottony, velvety, powdery, creamy or
pasty
• Pigmentation – surface & reverse side

Rate of Growth
• Rapid or slow grower
• Specify days or weeks

Topography
• Flat, heaped or folded
• Rugose: deep furrows that radiate from
the center
• Umbonate: elevated in the center
• Wrinkled or verrucose

Texture
• Cottony or woolly
• Velvety or silky
• Powdery or granular
• Moist, creamy, pasty

Pigmentation
• Surface pigmentation and pigmentation
on the reverse side
• Color description is very specific

EXAMINATION OF FUNGAL GROWTH
MICROSCOPIC (to observe spores & conidia)
• TEASE MOUNT – rapid method
- 1 gtt LPCB + sample
• SLIDE CULTURE
• CELLOPHANE TAPE MOUNT – scotch tape
mtd.

Tease Mount (double layer tape
prep)

SLIDE CULTURE
• Most accurate method to preserve & observe fungi
• Allows fungi to be preserved in orig. state
• Requires more skill & time than tease mount
Not for:
•Histoplasma,
•Coccidioides,
•Blastomyces,
•Sporothrix,
•Paracoccidioides

SEROLOGICAL TESTS
• Used to supplement microscopic &
culture mtds.
• Methods:
– Agglutination
– Precipitation
– Complement Fixation (CF)
– ImmunoFluorescence (IF)
– Enzyme Linked Immunosorbent Assay
(ELISA)

SEROLOGICAL TESTS
Antigen Detection
 May hamper cross
reacting components
 Cryptococcal Ag test
–detects PS Ag from
CSF (latex agglut’n.)

Antibody Detection
 Relies on correct timing of spx collection
 Detects active infxns. for Histoplasmosis,
Blastomycosis, coccidioidomycosis,
sporothricosis
 May be hampered by cross reacting
antigens

Classification of Fungal
Diseases
• Superficial
• Cutaneous
• Subcutaneous
• True systemic (endemic)
• Opportunistic

Superficial mycoses
Disease Etiological
Agent
Symptoms Identification of organism
Pityriasis
versicolor
Malassezia
furfur
hypopigment
ed macules
"spaghetti and meatballs"
appearance of organism in
skin scrapings
Tinea
nigra
Exophiala
werneckii
black
macules
black, 2-celled oval yeast
in skin scrapings
Black
piedra
Piedraia
hortai
black nodule
on hair shaft
black nodule on hair shaft
composed of spore sacs and
spores
White
piedra
Trichosporum
beigelii
creme-
colored
nodules on
white nodule on hair shaft
composed of mycelia that
fragment into arthrospores

Superficial Mycoses
• Pityriasis versicolor
– Chronic mild infxn of
stratum corneum
– Cosmetic problem
• Discrete, serpentine,
hypo- or hyperpigmented
spot on the skin (chest,
upper back arms or
abdomen)
• Enlarge and coalesce

M. furfur
hyperpigmented hypopigmented

Superficial mycoses
• P. versicolor
– Lipophilic yeasts
– KOH and calcouflour white
– Lesion flouresce under wood’s lamp
– Tx: selenium sulfide, topical or oral azole
– Implicated in seborrheic dermatitis or dandruff

Superficial mycoses
• Tinea nigra ( tinea
nigra palmaris)
– Asymptomatic infxn
caused by
dematiaceous fungi
Hortae (Exophiala
werneckii)
– Warm coastal regions
and among women
– Dark (brown to black)
palm discoloration

Superficial mycoses
• Tinea nigra
– Branched, septate
hyphae and budding
yeast cells w/ melanized
cell wall
– Tx: keratolytic solutions,
salicylic acid, azole
antifungal drugs

Superficial mycoses
• Piedra
– Black piedra: hard,
nodular infxn of the hair
– White piedra: larger,
softer and yellowish
nodules of the hair
– Tx: hair removal, topical
tx
– Endemic in tropical
underdeveloped
countries

Cutaneous mycoses
Dermatophytoses – (ringworm, caused by
dermatophytes) affect keratin-containing
tissues such as hair, nails, and skin.
– cellular IR may be evoked, causing pathologic
changes in the host that may be expressed in the
deeper layers of the skin
Dermatomycoses- cutaneous mycoses caused
by other fungi, most often Candida

Cutaneous mycoses
Etiological agents:
• Epidermophyton spp – affects only skin and
nails
• Trichophyton spp – can affect hair, skin, or
nails
• Microsporum spp. – usually affects only
hair or skin

Cutaneous mycoses
Disease Symptoms Identification
Tinea capitis Lesion in scalp
presence/absence and
shape of micro- and
macroconidia in
scrapings from lesion
Tinea corporis trunk, arms,
legs
Tinea manus hand
Tinea cruris "jock itch" groin
Tinea pedis"athlete's
foot"
foot
Tinea unguium Infection of nails
Ectothrix infection of hair
shaft surface mycelium and spores on
hair shaftEndothrix infection of hair
shaft interior

Clinical Manifestation: Dermatophytes

Clinical Manifestation: Dermatomycoses

Superficial/Cutaneous mycoses
Methods of diagnosis: Calcoflour white

KOH
Skin scrapings Nail scrapings

Macroconidia and Microconidia
• Macroconidia
– Large, multi-septate,
club, oval, spindle-
shaped
– Cell wall is smooth or
echinulate
• Microconidia
– Small, unicellular
– Round, elliptical.
pyriform, tear-shaped

• Microsporum
– Large multicellular
macroconidia;
microconidia rarely
produced

• Trichophyton
– Predominant forms
are microconidia;
macroconidia
uncommon
– Do not fluoresce on
Wood’s lamp

• Epidermophyton
– Macroconidia only;
microconidia not
produced

Superficial/ cutaneous mycoses:
Methods of transmission

Superficial mycoses
• Prevention and control

Subcutaneous mycoses
• fungal infections beneath the skin.
• Sporotrichosis – Sporothrix schenckii
• Chromomycosis – Fonsecaea, Phialophora,
Cladosporium

Systemic mycoses
• fungal infections deep within the body that
affect many tissues and organs.
• Coccidiodomycosis – Coccidioides immitis
• Histoplasmosis – Histoplasma capsulatum
• Blastomycosis – Blastomyces dermatitidis
• Meningitis - Cryptococcus neoformans

Systemic mycoses
• Ergot poisoning - Claviceps purpurea
• psilocybin "poisoning" - Psilocybe cubensis,
Psilocybe mexicana , Psilocybe tampanensis
and Psilocybe atlantis
• Aflatoxin poisoning - Aspergillus flavus
• Mycotoxin poisoning (phalloidin and amanitin)
- Amanita phalloides

Opportunistic mycoses
• caused by normal microbiota or fungi that are
not usually pathogenic.
• infect any tissues, however, they are usually
systemic

Opportunistic mycoses
• AIDS patients susceptible to Cryptococcus,
Pneumocystis, and Penicillium infections.
• Mucormycosis - Rhizopus and Mucor.
• Aspergillosis - Aspergillus.
• Candidiasis- C. albicans, can be dermal, oral
(thrush) or vaginal.

Mycoviro prelim

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Mycoviro prelim

Similar to Mycoviro prelim (20)

More from reena condez

More from reena condez (6)

Recently uploaded

Recently uploaded (20)

Mycoviro prelim