MYCOLOGY AND VIROLOGY (LECTURE)

WEEK 2: CLINICAL MYCOLOGY

3RD YEAR – 2ND SEMESTER
FEBRUARY 2, 2023

FUNGAL IDENTIFICATION o ANTIFUNGAL DRUGS THAT TARGET CELL

MEMBRANE: IMIDAZOLES, TRIAZOLES, AND
METHODS AND STRATEGIES POLYENES
• Mycosis – refers to infections caused by Fungi (sg. Fungus) • Non-photosynthetic; heterotrophic
o HISTORICALLY, THE FUNGI WERE REGARDED AS o HETEROTROPHIC – DEVOID OF CHLOROPHYLL
RELATIVELY INSIGNIFICANT CAUSES OF INFECTION. o THEY GET NUTRIENTS THROUGH ABSORPTION.
LIKEWISE, MOST SPECIES OF FUNGI ARE DISCOVERED • Reproduce by means of spores, asexually or sexually
FIRST ON THEIR BENEFICIAL BACKGROUND TO o SPORES ARE THE REPRODUCTING
MANKIND, ESPECIALLY ON FOOD INDUSTRY (EX. REPRODUCTIVE STRUCTURES OF FUNGI.
YEAST). • Grow best at neutral pH, moist environment
o BENEFITS OF FUNGI IN MEDICINE: ANTIBIOTICS
(PENICILLIN), CULTURE (PAGPAPATUBO NG MGA ?) TAXONOMY
o FUNGI BECAME SIGNIFICANT WHEN THERE WERE A • Vast array of organisms: mushrooms, rust and smuts, molds and
LOT OF EXTERNAL STRESSES DONE mildews, and yeasts.
ENVIRONMENTALLY TOWARDS US SO BUMABABA • Categorized into 4 well-established phyla:
YUNG HOST DEFENSES. AS A RESULT, INFECTION o Each phyla has a general characteristics.
CAUSED BY FUNGI OCCURS, WHICH IS CALLED
MYCOSIS/MYCOSES. 1. Zygomycota (Glomeromycota)
▪ Major factors responsible for the increase in the number of o ALL ORGANISMS UNDER THESE PHYLA CONTAINS A
fungal infections are alterations in the host (especially in the CHARACTERISTIC STRUCTURE CALLED SPARSELY
host’s defenses): SEPTATE HYPHAE.
a. Increase number of immunocompromised individual o HYPHAE – ACTS LIKE THE BRANCHING STEMS OF
(immunosuppressed, serious underlying conditions) FUNGI.
b. Other predisposing factors o MEANS OF REPRODUCTION: ASEXUAL AND SEXUAL
• Complex surgical procedures o ASEXUAL STRUCTURE: SPORANGIOSPORES
o PHYSICAL ENTRY OF FUNGAL ELEMENTS o SEXUAL STRUCTURE: ZYGOSPORES
THROUGH BREAKING OF THE PHYSICAL o STRUCTURE – A SPECIFIC PART OF FUNGI THAT IS
BARRIER OBSERVED MICROSCOPICALLY.
• Antibacterial therapy • Mucorales
o KAPAG SUMOBRA YUNG ANTIBACTERIAL • Lichtheimia (formerly Absidia), Cunninghamella, Mucor,
THERAPY, YUNG MGA BACTERIA NAGIGING Rhizomucor, Rhizopus, Syncephalastrum
RESISTANT AND THEN MAGTHA-THRIVE
• Entomophthorales
NAMAN YUNG FUNGI SA KATAWAN
• Basidiobolus, Conidiobolus
▪ Humans become accidental hosts for fungi and are relatively
2. Ascomycota
resistant to infections cause by this microorganisms
o THIS HAS A VERY DIVERSE GENUS SPECIES OF
• FUNGI
FUNGI.
▪ Kingdom: Fungi
o IT EXHIBITS TRUE SEPTATE HYPHAE.
▪ Eukaryotic (generally)
o MEANS OF REPRODUCTION: ASEXUAL AND SEXUAL
▪ Saprophytic (ever present in the environment)
o ASEXUAL STRUCTURE: CONIDIA
▪ Obligate/facultative aerobes
o SEXUAL STRUCTURE: ASCOSPORES
o OBLIGATE AEROBES – THEY THRIVE IN THE
o FILAMENTOUS FORM OR MOLD FORM:
PRESENCE OF OXYGEN
ASCOMYCETES
▪ Co-evolved with Animalia and Plantae except:
o SINCE ASCOMYCOTA IS VERY DIVERSE, MAY
• Most are non-motile NALALAMAN PA SILANG SEXUAL AND ASEXUAL
• Have cell wall (chitin and glucans) FORMS NA IBA PA SA STRUCTURE, WHICH IS SEEN
o THESE TWO CHARACTERISTICS – CHITIN AND MICROSCOPICALLY. WHEN WE SAY FORM, THE
GLUCANS – ARE IMPORTANT IN TERMS OF GENUS AND SPECIE OF THE FUNGI IS CHANGED
ANTIFUNGAL DRUGS. ANTIFUNGAL DRUGS DEPENDING ON ITS REPRODUCTIVE CYCLE.
MAINLY TARGET THIS PART OF FUNGI, THE CELL o TELEOMORPHS (SEXUAL FORM) ARE FUNGI THAT
WALL AND CELL MEMBRANE. EXIST INTO ANOTHER SEXUAL FORM. EXAMPLE (NOT
o ECHINOCANDINS, AN ANTIFUNGAL DRUG, FACTUAL): CLADIOSPORIUM EXISTS INTO ITS SEXUAL
TARGETS THE CELL WALL OF THE FUNGI. FORM, ENDOMYCES.
• Have cell membrane (ergosterol) o ASEXUAL FORM: ANAMORPH
MYCVLEC@311 | PRELIMS | RGG
 o SYNANOMORPH – WHEN FUNGI HAVE MANY • Penicillium notatum, Aspergillus niger, Trichophyton sp.,
ASEXUAL FORMS OR ANAMORPHS (EX. Tolypocladium inflatum, other spp. of Penicillium
PSEUDALLESHERIA BOYDII IS A SYNANOMORPH HAS
TWO ANAMORPHS, NAMELY SCEDOSPORIUM GENERAL CHARACTERIZATION OF FUNGI
APIOSPERMUM AND GRAPHIUM SP.) • have two basic growth forms based on the appearance of
• Capnodiales colonies formed: yeasts and molds
• Cladosporium, Piedraia hortae
• Saccharomycetales A. Yeast (spherule/tissue state)
• Endomyces (Geotrichum sp.), Kluyveromyces (Candida • appears as moist, creamy, opaque/pasty bacterial-like
pseudotropicalis), Candida, Geotrichum colonies (like S. aureus but it has larger colonies) on
• Hypocreales media
• Fusarium, Scopulariopsis, Sepedonium, Trichoderma • generally, forms in vivo and at 35-37°C w/ inc. CO2
• Microascales • microscopic: spherical/ellipsoid single cells; 3-15 μm
• Pseudallesheria boydii, Scedosporium prolificans, diameter
Sporothrix • reproduce by budding or fission
• Chaetothyriales o BUDDING – MATURATION OF THE BUD TO AN
• Cladophialophora, Exophiala, Fonsecaea, Phialophora INDEPENDENT DAUGHTER CELL
• Eurotiales (BLASTOCONIDIUM)
• Emericella (Aspergillus nidulans), Aspergillus, o PSEUDOHYPHAE – ELONGATED AND NOT
Paecilomyces, Penicillium DETACHED DAUGHTER CELL DURING
• Onygenales (PLEASE TAKE NOTE DAW) BUDDING.
o MOST OF ITS SPECIES OF FUNGI INCLUDES o FISSION – TWO CELLS OF EQUAL SIZE ARE
DIMORPHIC FUNGI AND DERMATOPHYTES. FORMED
DIMORPHIC FUNGI ARE HIGHLY PATHOGENIC B. Molds (filamentous state)
SINCE THEY CAN EXIST ON OUR BODY. • produce fluffy, cottony, woolly, or powdery colonies due to
DERMATOPHYTES ARE SET OF FUNGI THAT the formation of mycelia.
CAUSES MAINLY SUPERFICIAL OR CUTANEOUS • Mycelia – mass of intertwined multicellular branching
MYCOSIS. cylindrical tubules called hyphae
• Ajellomyces (Histoplasma capsulatum, Blastomyces a) Aerial hyphae – EXTENDS ABOVE THE
dermatitidis), Arthroderma (Trichophyton sp., SURFACE; IT SUPPORTS THE REPRODUCTIVE
Microsporum sp.), Chrysosporium, Coccidioides, STRUCTURES OF THE FUNGI (SPORES, WHICH
Epidermophyton, Histoplasma, Microsporum, IS A SEEDLIKE STRUCTURE); RESPONSIBLE
Paracoccidioides, Trichophyton FOR A DISTINCT CHARACTERISTICS LIKE
3. Basidiomycota COLOR AND TEXTURE OF THE SURFACE SIDE
o GENERALLY, THEY ARE PLANT PATHOGENS. THEY OF THE COLONY FOR IDENTIFICATION.
RARELY CAUSE DISEASE IN HUMANS. b) Vegetative hyphae – EXTENDS DOWNWARD TO
o REPRODUCTIVE STRUCTURE: SEXUAL – THE MEDIUM; ABSORBS NUTRIENTS COMING
BASIDIOSPORES FROM THE MEDIUM; THEY ANCHOR THE
o BASIDIA – SUPPORTING STRUCTURE OF COLONY ON THE MEDIUM; ROOTLIKE
BASIDIOSPORES STRUCTURE OF THE FUNGI; RESPONSIBLE OF
• Filobasidiales THE REVERSE SIDE CHARACTERISTICS OF
• Filobasidium (Cryptococcus neoformans), THE COLONY.
Malassezia, Trichosporon, Cryptococcus • forms when grown at room temperature (22-30°C) at
o CRYPTOCOCCUS NEOFORMANS ambient air
TELEOMORPHIC/SEXUAL FORM CALLED o AMBIENT AIR – MAS MATAAS YUNG OXYGEN
FILOBASIDIELLA LEVEL
o BASIDIOMYCOTA INCLUDES MUSHROOMS, • microscopic: hyphae are elongated, tubular, branching;
SMUTS, AND ? YUNG MALAKING CUP NG (hyphae) with/without: septation and pigmentation.
MUSHROOM IS CALLED THE BASIDIUM • Septation
4. Deuteromycota (Fungi Imperfecti) o SEPTA – POINT OF SEPTATION OR
o IT IS CALLED FUNGI IMPERFECTI OR IMPERFECT GROWTH.
FUNGI BECAUSE IT LACKS SEXUAL REPRODUCTIVE a. Septate hyphae – SHOWS FREQUENT
CYCLE. THEY HAVE NO SEXUAL STRUCTURE. CROSS-WALLS OCCURRING
o ASEXUAL STRUCTURE: CONIDIA PERPENDICULAR ON THE OUTER WALLS;
o THEY ARE CALLED IMPERFECT FUNGI BECAUSE PANTAY-PANTAY
SPECIES UNDER DEUTEROMYCOTA ARE NOT b. Sparsely septate (Aseptate) hyphae – FEW
CLOSELY RELATED TO EACH OTHER. THAT’S WHY, CROSS-WALLS AT IRREGULAR INTERVALS
THEY ARE CALLED POLYPHYLETIC. • Pigmentation
o POLYPHYLETIC – MANY PHYLA; THEY ARE NOT a. Hyaline (moniliaceous) – HYPHAE THAT
CLOSELY RELATED; THEY DO NOT SHARE COMMON ARE NONPIGMENTED OR LIGHTLY
ANCESTRY. PIGMENTED.

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 b. Phaeoid (dematiaceous) – DARKLY FUNGAL REPRODUCTION
PIGMENTED HYPHAE DUE TO THE A. Sexual (perfect)
PRESENCE OF MELANIN; DARK BROWN TO ▪ Requires joining of 2 compatible nuclei, followed by
ALMOST BLACK meiosis
▪ Types of spores:
OTHER HYPHAE MORPHOLOGY o SPORES ARE THE REPRODUCTIVE
1. Favic Chandelier – resembles antler of deer (antler hyphae) STRUCTURES AND TYPICALLY, THEY ARE
ex: Trichophyton schoenleinii (DERMATOPHYTE) USUALLY DORMANT, READILY DISPERSED, AND
2. Spiral – not as common; spirally coiled hyphae (corkscrew) MORE RESISTANT TO ADVERSE CONDITIONS.
ex: Trichophyton mentagrophytes o PARA SILANG MGA BUTO NA KAPAG
3. Pectinate - short, unilateral projections from the hyphae that NAKAHANAP NG CERTAIN CONDITIONS TO
resemble a broken comb GROW DUN SILA MAGTTHRIVE.
ex: Microsporum audouinii 1. Zygospores – spores produced from the fusion of 2
4. Nodular - enlargement in the mycelium that consists of knotted identical hyphae
and twisted hyphae o EXAMPLE: SA ISANG SPECIE; MUCOR AND
ex: Microsporum canis MUCOR NAGDIDIKIT SILA
5. Racquet – resembles tennis racquets or badminton racquets 2. Oospores – spores produced from fusion of 2
stacked on top of one another different hyphae
ex: Epidermophyton flocossum o EXAMPLE: MUCOR AND RHIZOPUS
6. Stolon/Runners – unbranched, straight or arched aerial 3. Ascospores – spores contained in a sac-like
hyphae that extends over a long distance and connect groups structure - ASCOSCARP
of rhizoids o ON ASCOMYCOTA
ex: Absidia, Rhizopus o GROUPS OF ASCOSPORES ARE
7. Rhizoids - lateral outgrowths of intracellular hyphae specially NAKABALOT SA ISANG STRUCTURE
modified for absorption of nutrients CALLED THE ASCI/ASCUS. USUALLY,
ex: Order Mucorales GROUPS OF 4-8 ASCOSPORES ARE
o ROOT-LIKE STRUCTURES CONTAINED IN ONE ASCI.
o FOR STOLON/RUNNERS AND RHIZOIDS, THEY ARE ONLY o SOME SPECIES OF ASCOMYCOTA,
SEEN ON ZYGOMYCOTA PHYLA. NAGPPRODUCE PA NG ISANG BALOT.
BINABALUTAN NAMAN NYA YUNG
DIMORPHISM AND POLYMORPHISM ASCOCARP. ANG TAWAG NAMAN DITO AY
• Dimorphic fungi – can exist in two forms (yeast or mold); CLEISTOTHECIUM, WHICH IS A SAC-LIKE
dependent on growth conditions STRUCTURE THAT ENVELOPS OR TOTALLY
o EITHER YEAST OR MOLD LANG; DI SYA PWEDE ENCLOSES THE ASCOCARPS.
MAGING YEAST AND MOLD SA ISANG FORM 4. Basidiospores – spores contained in a club-shaped
o DEPENDING ON THE GROWTH CONDITION structure – BASIDIUM OR BASIDIOSPORES
ESPECIALLY ON THERMALLY DEPENDENT. o FUSION OF TWO COMPATIBLE NUCLEI OR
ASCOSPORES OR BASIDIOSPORES.
• Medically significant dimorphic fungi:
B. Asexual (imperfect)
o THESE ARE THE CAUSATIVE AGENT OF SYSTEMIC
▪ Only involves division of the nucleus and cytoplasm
MYCOSES. THEY ARE THERMALY DIMORPHIC
o IT ONLY INVOLVES PRODUCTION OF ASEXUAL
MEANING THEY CREATE FORMS BASED ON
STRUCTURES – CONIDIA AND
TEMPERATURE EXCEPT ON COCCIDIOIDES IMMITIS,
SPORANGIOSPORES
WHICH IS NOT THERMALLY DIMORPHIC.
▪ Are the only fungal group that produces conidia;
✓ Blastomyces dermatitidis
sporangiospores for Order Mucorales
✓ Coccidioides immitis
o CONIDIA: ASCOMYCOTA AND
✓ Histoplasma capsulatum var. capsulatum
DEUTEROMYCOTA; SEEN IN SEPTATED FUNGI
✓ Paracoccidioides brasiliensis
o NOTE THE TYPE OR COLONY ARRANGEMENT
✓ Sporothrix schenckii
OF CONIDIA TO IDENTIFY THE ORGANISM
✓ Penicillium marneffei
▪ Types of conidia:
• Polymorphic fungi – have more than one independent/spore
1. Arthroconidia – simplest type of sporulation arising
form in its life cycle; not temperature dependent
from the fragmentation of fertile vegetative hyphae
o EXIST IN TWO BASIC FORMS ON BOTH SITUATION:
through septation points
BOTH YEAST AND MOLD SA ISANG MEDIA
o ARTHRO – ARTICULATIONS: POINT OF
o IT IS NOT DEPENDENT ON TEMPERATURE OR
ATTACHMENT NG BAWAT CONIDIA
GROWTH CONDITION
o ONCE IT IS FRAGMENTED, NAHAHATI SYA
Ex: Exophiala spp. (YUNG YEAST FORM NYA
NAGKAKAROON NG SPACES, WHICH IS
NAOOBSERVE TYPICALLY KAPAG NAGGGROW THEN KAPAG
CALLED THE SEPTATION POINTS,
TUMANDA YUNG CULTURE NAGIGING MOLD
SPECIFICALLY TERMED AS DISJUNCTOR
CELLS, WHICH ARE EMPTY SPACES OF
SEPTATION POINTS.
o ITS CELLS ARE LIKE WINE BARRELS.
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 o ASEXUAL REPRODUCTION ONLY MICROCONIDIA
INVOLVES DIVISION OF THE NUCLEUS AND • forms when a whole hyphal element converts into a single-
CYTOPLASM NG HYPHAL/HYPHAE celled conidium
ELEMENT. o NAGFRAFRAGMENT OR NABABASAG
Ex: Dermatophytes, Geotrichium sp., Coccidioides • typically, round-to-oval or club-shaped
immitis
o COCCIDIOIDES IMMITIS IS SPORANGIOSPORES
DISTINGUISHED BY ITS FORMATION
• only seen in the phylum Zygomycota (Mucorales)
OF DISJUNCTOR CELLS.
• formed in sparsely septate fungi
2. Chlamydoconidia – round, thick-walled spores
• situated inside a specialized sac called sporangium
formed directly from the differentiation of hyphae
o COLUMELLA – SITUATES THE SPORANGIUM
o ALSO CALLED AS SURVIVAL CONIDIA
o SPORANGIOPHORE – SUPPORTS COLUMELLA
BECAUSE THESE ARE RESISTANT
(BASTA PAG -PHORE, IT IS A SUPPORTING
RESTING SPORES, WHICH PRODUCE BY
STRUCTURE); CONNECTED TO STOLON AND
ROUNDING UP AND ENLARGEMENT OF
RHIZOIDS
THE CELLS OF THE HYPHAE.
Types:
a) Intercalary – conidia within the hyphae CLINICAL CLASSIFICATION OF FUNGI
b) Sessile – conidia on the side of hyphae A. Superficial (Cutaneous) Mycoses
c) Terminal – conidia on the end of hyphae • involves hair, skin and nails without direct invasion of
3. Blastoconidia – conidia that is form as a result of deeper tissue
budding • All infect keratinized tissues
o SEEN IN CANDIDA SPP. o DEAD CELLS
Ex: Candida sp., Cladosporium • agents of ringworms (tinea) and athlete’s foot
4. Poroconidia – Conidia formed by being pushed A.1. Superficial mycoses
through a small pore in the parent cell or pore at the • confined to the outermost layer of skin and hair
top of parent cell (epidermis only)
o A SPECIAL TYPE OF CONIDIA • caused by non-dermatophytic tinea (tinea
Ex: Bipolaris sp. versicolor, tinea nigra, white and black piedra)
5. Phialoconidia – asexual structures that are formed o NON-DERMATOPHYTIC IS ALSO CALLED
from a phialide DERMATOMYCOSIS
o NADIDISTINGUISH LANG SYA BASED ON o NO IMMUNE RESPONSE
THE CONIDIOGENOUS CELL. A.2. Cutaneous mycoses
o CONIDIOGENOUS CELLS – PRODUCE THIS • cause destruction of the keratin layers of the skin,
TYPE CONIDIA hair and nails (epidermis and dermis)
o NADIDISTINGUISH YUNG DIFFERENT • cause mostly by dermatophytes (Trichophyton,
TYPES OF CONIDIGENOUS CELLS BASED Epidermophyton, Microsporum); or yeasts (Candida
ON ITS SHAPE MICROSCOPICALLY. spp.)
o PHIALIDE – A CONIDIOGENOUS CELL THAT • Dermatophytosis (dermatophytic tinea) is based on
IS SHAPED AS VASE/TUBE-SHAPE THAT the localization of inf.
PRODUCES PHIALOCONIDIA B. Subcutaneous Mycoses
o CONIDIOPHORE – SUPPORTS THE • involve the deeper skin layers (hypodermis), including
PHIALIDE muscle, connective tissue, and bone; rarely spread to
Ex: Penicillium sp., Aspergillus sp. distant organs
6. Annelloconidia – asexual structures that are • often caused by traumatic inoculation of the fungus
formed from an annelide • fungal infections include: sporotrichosis,
o ANNELLIDE – BOWLING SHAPED chromoblastomycosis, eumycotic mycetoma,
CONIDIOGENOUS SHAPE, WHICH phaeohyphomycosis, subcutaneous zygomycosis
PRODUCES ANNELLOCONIDIA C. Systemic Mycoses
o ANNELLOPHORE – SUPPORTS THE • invade the deeper tissues and spread throughout the
ANNELLIDE body and bloodstream into the organs (liver, lungs, brain)
Ex: Scopulariopsis o THE MOST COMMON TARGETED ORGAN IS THE
LUNGS BECAUSE THE MOST COMMON MOT IS
MACROCONIDIA INHALATION.
• occurs when a whole hyphal element converts into a multicelled • caused by thermally dimorphic fungi
conidium o HIGHLY PATHOGENIC
• may be club-shaped, oval, elongated, curved, have a thick or o SERIOUS INFECTIONS; MAHIRAP NA GAMUTN
thin wall, be smooth or spiny, or form on the side of the • serious infections include histoplasmosis,
condiophore (sessile); have septations blastomycosis, coccidioidomycosis and
o NAGKAKAROON NG SEPTATION KASI NAGBUBUO paracoccidioidomycosis
BUO YUNG CELLS

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D. Opportunistic Mycoses SPECIMEN COLLECTION & PROCESSING
• are infections cause by ubiquitous, low virulence fungi • All specimens should be collected using aseptic technique
which requires the host’s defenses to be lowered before it specially from normally sterile sites
is established o CSF IS STERILE
• infections include candidiasis, cryptococcosis, o URINE IS NOT STERILE BECAUSE CANDIDA IS A PART
aspergillosis, mucormycoses, talaromycosis OF THE MICROBIOTA OF THE URINARY TRACT
(penicilliosis), otomycosis, keratomycosis, and • Specimen volume should be adequate
pneumocystis pneumonia • Should be transported and processed as soon as possible (<2
hrs)
PATHOGENESIS AND SPECTRUM OF DISEASE OF FUNGI o BECAUSE THEY ARE SLOW GROWERS
• Fungal infection is caused by either primary pathogens or • If transport should be delayed, refrigerate specimen for a short
opportunistic pathogens time
• Infections cause by primary pathogen: usually occur in ** except for skin, hair, nails, and CSF
immunocompetent host • Proper transporting condition and techniques should be strictly
• infections cause by opportunistic pathogen: occurs in followed
immunocompromised hosts
• virulence factors: RESPIRATORY TRACT SECRETIONS
a. The organism’s size (with inhalation, the organism must be
• Most common specimens collected for culture
small enough to reach the alveoli)
o SPUTUM, BRONCHIAL WASHINGS,
b. The organism’s ability to grow at 37°C at a neutral Ph
BRONCHOALVEOLAR LAVAGE, AND TRACHEAL
o FUNGI THAT CAN EXIST IN YEAST FORM
ASPIRATIONS
c. Conversion of the dimorphic fungi from the mycelial form
• Cultured on non-selective media and media with antibiotics
into the corresponding yeast or spherule form in the host
➢ Sputum
o INHALED AS MOLLD TAPOS NAGIGING YEAST SA
o MOST COMMON
KATAWAN; HIGHLY VIRULENT
• obtained from a deep cough early morning or using
d. Toxin production
nebulizer (induced sputum)
Fungal Pathogen Putative Virulence Factor
Aspergillus spp. Elastase-serine protease • N-acetyl-L-cysteine added if sample is
Proteases viscous/mucoid.
Toxins (gliotoxin, fumagillin, *For thick tracheal aspirate, use Dacron swab for
helvolic acid) inoculation
Elastase-metalloprotease • For direct (microscopic) examination – KOH wet mount
Aspartic acid proteinase
• Common fungal agents:
Aflatoxin
Catalase ▪ Candida albicans, Aspergillus, Rhizopus, Penicillium,
Lysine biosynthesis Histoplasma capsulatum, Blastomyces dermatitidis,
p-aminobenzoic acid synthesis and Coccidioides immitis
Blastomyces spp. Cell wall alpha-1,3-glucan
BAD-1 an adhesion and DERMATOLOGIC SPECIMENS
immune modulator
Coccidioides spp. Extracellular proteinases • Often collected when dermatophytes are suspected
Cryptococcus neoformans Capsule • most dermatologic (and tissue) specimens are treated with 10-
complex Phenoloxidase melanin 20% KOH before microscopic exam
synthesis o KOH ACTS LIKE A CLEARING AGENT WHICH
Varietal differences DISSOLVES THE KERATIN.
Dematiaceous fungi Phenoloxidase melanin • before collection, area must be cleansed with 70% alcohol
synthesis
• remaining specimens are inoculated on proper culture media
Histoplasma capsulatum Cell wall alpha-1,3-glucan
Intracellular growth • Common fungal agents:
Thermotolerance - Aspergillus,Trichophyton, Epidermophyton, Microsporum,
CBP, binds calcium Blastomyces dermatitidis, Candida
Paracoccidioides brasiliensis Estrogen-binding proteins ➢ Skin
Cell wall components • collected through scrapings (skin scrapings)
Beta-glucan
• area is cleansed first with 70% alcohol
Alpha-1,3-glucan
Sporothrix spp. Thermotolerance Extracellular • collect skin samples by scraping outer edge of a surface
enzymes lesion by sterile scalpel blade or microscope slide
• transport on a sterile petri dish or paper
TRANSMISSION, SPECIMEN COLLECTION, HANDLING AND • proceed to direct examination and culturing
TRANSPORT • Common fungal agents:
• Fungal infection occurs after introduction of spores either by: - Candida, Trichophyton, Epidermophyton,
a. Inhalation Microsporum, Blastomyces dermatitidis
b. Direct contact to skin and bodily surface ➢ Hair
c. Accidental entry – through the skin (cut, wound, injection); • collected by plucking(hair pluckings) or cutting affected
ingestion sections

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 • Wood’s lamp = emits UV light >365 nm that helps in - All dimorphic fungi, the zygomycetes, Aspergillus,
identifying infected hairs Scedosporium, Fusarium, Candida albicans, C.
o INFECTED SITES FLUORESCE neoformans
• affected hair are pulled using sterile forceps and are
placed on sterile petri dish UROGENITAL AND FECAL SPECIMEN
• microscopic: KOH prep • Usually requested occasionally for yeast identification
a. Ectothrix – fungal invasion outside of the hair shaft o KUNG GUSTO MALAMAN NG DOCTOR KUNG MAY
b. Endothrix – fungal invasion inside of the hair shaft OVERGROWTH NG NORMAL FLORA NA FUNGI
• Common fungal agents: USUALLY FOR DIABETIC PATIENTS
- Microsporum, Trichophyton • Urine samples submitted for fungal culture should be
➢ Nails processed as soon as possible
• Submitted as nail clippings, scrapings or as a whole nail • Early morning urine should be submitted; centrifuged before
• area is cleansed with 70% alcohol processing
• deep scrapings are necessary to perform KOH prep and • Vaginal swabs should be kept moist in sterile tubes
inoculating media • Fecal specimen is usually submitted for yeast overgrowth
• Common fungal agents: • Common fungal agents:
- Aspergillus, Epidermophyton,Trichophyton - Candida albicans, Candida glabrata

CORNEAL SCRAPINGS/VITREOUS HUMOR LABORATORY TECHNIQUES FOR FUNGAL IDENTIFICATION

• Done by a physician • Different laboratory techniques exist that aids on identifying
• Corneal scrapings placed directly onto slides and inoculated in fungi base on its specific characteristics
media in either a C or X-shaped pattern • Methods of identification include:
• Vitreous humor aspirate should be concentrated by a. Phenotypic – microscopic, macroscopic
centrifugation and sediments be used for smears and culture b. Biochemical
• Common fungal agents: o TINETEST YUNG METABOLISM NG FUNGI
- Fusarium, Aspergillus, Candida, Penicillium, c. Serologic
Trichosporon, Macrophoma sp., Phialophora verrucosa, d. Molecular (not well established)
Culvularia lunata
MACROSCOPIC IDENTIFICATION
BLOOD AND BONE MARROW • Involves isolation and observation of fungi through culture
• Blood should undergo centrifugation before culturing. • Includes gross morphologic traits: color, texture, & growth rate
Isolator Tube = a lysis centrifugation system; most sensitive • Fungi have minimal nutritional requirements compared to
method for the recovery of some fungi in blood bacteria
• Heparinized bone marrow spx. should be plated directly at • Fungal growth requires:
bedside. - Suitable organic source/substrate
• Blood culture bottles are not recommended for bone marrow. - Favorable range of temperature and Ph (NEUTRAL)
• Common fungal agents: - Adequate moisture
- Candida spp., Blastomyces dermatitidis, Histoplasma • Growth rate usually: 3 – 28 days or longer
capsulatum, and Cryptococcus neoformans o BEFORE I-REPORT AS NEGATIVE FOR CULTURE,
WAIT MUNA NG 21-30 DAYS
CEREBROSPINAL FLUID o ACTIVELY GROWING: 1-3 DAYS
• Centrifuged before processing o INTERMIDIATE: 5-9 DAYS
o SLOW GROWERS:11-21 DAYS
• Portion of the concentrate is used for microscopic (india ink
o THINGS TO NOTE (MACROSCOPIC) : 1) NUMBER OF
prep) or serological (latex agglutination for Cryptococcus) test
DAYS UNTIL FIRST VISIBLE GROWTH, 2) YEAST OR
• Remaining sample is filtered and cultured
MOLD, 3) TYPE OF MEDIA THE FUNGUS GROWS, 4)
• Should not be refrigerated
TEMPERATURE REQUIREMENT, AND 5) COLONY
• Common fungal agents:
MORPHOLOGY
- Cryptococcus neoformans, Candida spp., Histoplasma
capsulatum, and Coccidioides immitis
CULTURAL CHARACTERISTICS OF FUNGI
A. Colony Texture
TISSUE, ABSCESS AND WOUND EXUDATES
• Basically describes the height of aerial hyphae
• Abscess and exudates should be examined for granules before
➢ Cottony/Woolly – very high, dense mycelium; fluffy
planting
colonies
o GRANULES ARE EVIDENT ON THE NAKED EYE.
➢ Velvety/Sued-like – aerial hyphae are lower; resemble
PARANG BUO-BUONG PLEMA OR MUKHANG KANIN.
velvet/velour fabrics
DINUDUROG SYA KASI ANDUN YUNG FUNGAL
➢ Granular/Sugary – aerial hyphae are flat, dry, rough,
ELEMENTS.
crumbly, or flourlike; colonies sometimes contains granules
• Tissue samples should be gently minced before inoculation;
➢ Glabrous/Waxy – no aerial hyphae observed; colony are
*do not grind unless sufficient sample is submitted
shiny, waxlike, mucoid w/ smooth surface; sometimes
• Common fungal agents: moist, creamy, buttery, with “bread-like” odor
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B. Colony Morphology ▪ Enhances recovery of Blastomyces and Histoplasma
• Includes colony: capsulatum from contaminated specimens
▪ Topography and shape on Surface side: top
agar of agar DIFFERENTIAL MEDIA
▪ Specific color Reverse side: 1. Potato Dextrose Agar (PDA)
I. Colony Topography underside of agar ▪ Enhance conidia production
- Rugose, Verrucose, ▪ Enhances pigment development of Trichophyton rubrum
Umbonate (like a button/snap), Flat, Heaped, Folded, 2. Birdseed (Nigerseed) Agar
Striated, Wrinkled, Lacy ▪ For identification of Cryptococcus neoformans and
o RUGOSE: WRINKLED OR FOLDED Cryptococcus gattii
o VERRUCOSE: MOUNTAINOUS OR ▪ Brown pigmentation due to C. neoformans and C. gattii
CONVOLUTED (PATONG-PATONG) production of phenol oxidase, metabolizing caffeic acid
o UMBONATE: THICK OR SLIGHT POINT OF (COMPONENT OF THE BIRDSEED AGAR)
ELEVATION (USUALLY BUTTON SHAPE) 3. Cottonseed Agar
II. Colony Shape/Form ▪ Allows conversion of Blastomyces sp. From mold to yeast
- Circular, Irregular, Filamentous, Rhizoid form within 3 days
o FILAMENTOUS – ROOTLIKE 4. Czapek’s Agar
III. Colony Color ▪ For differential identification of Aspergillus sp.
- Brown, Black, Light Green, Olive Green, Dark Green, 5. Rice Medium
Pink, White, Cream, Off-White, Beige, Tan, Yellow, ▪ Differentiates and identifies M. audouinii to M. canis
Green With White Fringe etc. ▪ M. canis (grows well with a yellow pigment)
▪ M. audouinii (no growth)
CULTURAL CHARACTERISTICS OF FUNGI 6. Cornmeal Tween 80 Agar
• compose of primary recovery(non-selective) and differential ▪ Identification of C. albicans by chlamydospore formation
media ▪ Addition of trypan blue provides contrasting background
• tubed media are safer; however, plated media are more easily for observing morphologic features if yeasts
manipulated hence are more frequently used 7. Christensen’s Urea Agar
**except when Coccidioides immitis is suspected: should ▪ For identification of Cryptococcus, Trichosporon, and
always be placed on tubed media Rhodotorula sp.
• primary and differential agars can be made selective by o UREASE TEST
addition of antibiotics: cyclohexamide, chloramphenicol, and
gentamycin MICROSCOPIC EXAMINATION
o CYCLOHEXAMIDE – INHIBIT SAPROPHYTIC FUNGI • Involves microscopic morphologic features which provides the
o CHLORAMPHENICOL – INHIBITS GRAM POSITIVE most definitive means of identification
AND GRAM NEGATIVE BACTERIA • Provides a rapid report to the physician
o GETAMYCIN – INHIBITS GRAM NEGATIVE BACTERIA • Provide a clue to the genus of the organism
• Microscopic characteristics that should be observed:
PRIMARY RECOVERY MEDIA ▪ Septation (septate or sparsely septate hyphae)
1. Sabouraud Dextrose Agar (SDA) ▪ Pigmentation (hyaline or phaeoid)
▪ General-purpose, nutritionally poor, mildly selective for ▪ Reproductive (fruiting) structures
fungi; no longer commonly used ▪ Types, size, shape & arrangement of conidia
▪ Has an acidic pH (5.6)
▪ Modified SDA (Emmons) – neutral pH; better fungi growth DIRECT MICROSCOPIC EXAMINATION OF SPECIMENS
2. Sabouraud Dextrose-Brain Heart Infusion Agar (SABHI) • involves direct microscopic observation of fungal materials
▪ Meduim of choice for initial isolation • Might provide evidence of infection despite negative cultures
▪ Enhances growth of pathogenic fungi; non-selective • Includes wet mount preparations:
▪ Can be made selective by antibiotics 1. Potassium Hydroxide (KOH) Preparation
3. Brain Heart Infusion Agar with Blood (BHIB) - Useful for rapidly detecting fungal elements
▪ Used to grow most fungi, especially those from sterile body embedded within skin, hair, nails and tissue
sites - Acts by clearing specimen by dissolving keratin,
▪ Can be made selective by antibiotics leaving fungi readily visible - recommended method
4. Mycosel Agar - Common conc. used: 10-20% KOH
▪ Primary recovery for dermatophytes; inhibits bacteria and 2. Saline Wet Mount Preparation
saprophytic fungi - Quickly performed and cost effective
5. Inhibitory Mold Agar (IMA) - Specimen must be fresh; not all elements are visible
▪ Especially formulated to recover the cyclohexamide- with this prep.
sensitive Cryptococcus - Performed to quantitate epithelial cells and white
6. Dermatophyte Test Medium (DTM) blood cells and to include the presence of
▪ Recommended as screening medium for dermatophytes Trichomonas vaginalis in vaginal specimens
▪ Contains phenol red (pH indicator) 3. Stain Wet Mounts
7. Yeast-extract Phosphate Agar (YPA) I. Lactophenol Cotton Blue (LPCB) Wet Mount
▪ Exclusive for dermatophytes
7
 ➢ Most widely used method of staining and - It can be preserved indefinitely
observing fungi
➢ Components: MISCELLANEOUS TESTS (MOLDS)
✓ Lactic acid – preserves structures and clears 1. Hair Perforation Test
cellular debris ▪ Differentiates Trichophyton sp. and Microsporum sp. by
✓ Phenol – kills any viable cells (including the their capability to penetrate hair shafts or not
fungi itself) ▪ Sterile 5-10 mm hair fragments are floated on sterile water
✓ Aniline blue (Cotton blue) – stains chitin supplemented with a few drops of sterile 10% yeast
(fungal cell wall) extract.
o PWEDENG I-KEEP PAG LPCB GAMIT ▪ Conidia or hyphae of suspected dermatophyte is
II. India Ink Wet Mount inoculated onto the water surface
➢ Primarily use for the detection (presence of ▪ Hair is weekly removed for 1 month and microscopically
capsules) of Cryptococcus neoformans in CSF checked for perforation
III. Calcofluor White Stain ▪ Result: positive = presence of penetration pegs on hair
➢ Believe to be superior among other methods shafts (MAY BUTAS)
➢ Stain is taken up by chitin and allows the fungi to negative = hair shaft is still intact
produce chalk-white or apple-green fluorescence 2. Urease Test
under a fluorescence microscope ▪ Differentiates T. mentagrophytes from T. rubrum by urease
➢ Can be mixed with KOH prep. production
▪ Tubes of Christensen’s urea agar are lightly inoculated
OTHER STAINS USED with the organism and held for 5-days at room temperature
1. Gram Stain ▪ Result: positive urease = peach to bright fuchsia color of
▪ Fungi stains as gram positive; Actinomyces and Nocardia medium
stains variably negative urease = no color change
2. Modified Acid-Fast stain T. mentagrophytes – positive within 5 days
▪ Stains partially-acid fast, fungus-like bacteria Actinomyces T. rubrum – negative within 5 days or gives positive rxn >5
and Nocardia (appears red against blue background) days
3. Tissue Stains o PAG UMABOT NA NG 10 DAYS BAKA DI SYA T.
▪ Periodic Acid-Schiff stain (PAS) RUBRUM
o PINK TO RED WITH BLUE NUCLEI 3. Thiamine requirement
▪ Gomori Methenamine Silver stain ▪ Single most useful nutritional test for dermatophytes
o BLACK IN LIGHT GREEN BACKGROUND ▪ Tubes of media with and without thiamine are inoculated
▪ Papaniculaou stain with a tiny, medium-free portion of the colony and observed
o PINK TO BLUE for growth after 10 to 14 days
▪ Giemsa stain ▪ Result: positive = with growth; negative = no growth
o PINK 4. Trichophyton Agars
▪ 7 different Trichophyton agars – used to determine the
DIRECT MICROSCOPIC EXAMINATION OF CULTURES nutritional requirements of the Trichophyton spp.
• Done after mature fungal cultures are observed after incubation ▪ Has different nutritional ingredients – growth is scored on
• Methods used to obtain culture material for slide prep. includes: a scale of 1 to 4.
a. Tease mount method ▪ Based on the growth pattern, identification is made
- Uses a dissecting needle to pull apart a fungal colony 5. Growth on Rice Grains
and placed onto slide ▪ Typically used for the differentiation of M. canis from M.
o DISSECTING NEEDLE – INOCULATING WIRE audouinii
- May not preserve fungal structures (conidia) well if ▪ Sterile, non-fortified rice is inoculated lightly with a portion
not careful of a colony then incubated for 10 days at room temp.
- Can be made permanent (sealed using paramount or ▪ Results: with growth – M. canis and other dermatophytes
nail polish) no growth – M. audouinii (but turns rice grain
b. Cellophane/Scotch tape method brown)
- Involve gently touching a piece of clear tape, sticky
side down, to the surface of the colony and then MISCELLANEOUS TESTS (YEASTS)
removing it 1. Germ Tube Production
- Conidial structures are retained if carefully done ▪ Most important and easiest test to perform for the
- Should be read within 30 mins and discarded; cannot identification of yeasts (Candida albicans, C. dubliniensis
be kept permanently and other Candida sp.)
c. Slide Culture method (Riddell Technique) ▪ Requires the use of serum or plasma (fetal bovine serum)
- Uses block of agar placed in a slide and inoculated o ALTERNATVE FOR FETAL BOVINE SERUM IS
with spores and then covered EXPIRED FRESH FROZEN PLASMA FROM BLOOD
o YUNG CULTURE ICCULTURE MO ULIT BANK
o TIME CONSUMING ▪ serum/plasma is inoculated by the test fungi then
- Best method as it preserves natural fungal incubated at 37°C (WITHIN 3 HOURS)
morphology well
8
 ▪ Provides only a presumptive identification because not all C. Post-analytical Phase Hazards
strains of C. albicans will be positive
▪ True germ tubes lack constriction at their bases, where PRE-ANALYTICAL PHASE HAZARDS
they attach to the mother cell • Best Practices
o PSEUDO GERM TUBE – WITH CONSTRICTION; ✓ Only trained staff (phlebotomist, nurse or medical doctor)
CANDIDA TROPICALIS should collect samples – e.g. drawing blood, fine needle
2. Carbohydrate Assimilation aspiration and punch biopsy – to avoid needlestick and
▪ Although valuable, are time- and labor-intensive prick injuries
▪ Readily performed as part of routine identification ✓ Always perform skin scrapings and nail drilling in an
protocols isolated room or area of lab to avoid creating contaminated
▪ Identify which carbohydrates a yeast can use aerobically aerosols
as a sole carbon source ✓ Staff should wear personal protective equipment (PPE),
▪ Api 20c yeast identification system (biomérieux) is such as mask, gloves, and gown, as appropriate
probably the most used
o BIOCHEMICAL TEST ANALYTICAL PHASE HAZARDS
3. Potassium Nitrate Assimilation
• Best Practices
▪ Provides valuable information for separating the clinically
✓ Every chemical/hazardous material placed in the
significant yeasts
laboratory must be accompanied with an MSDS sheet
▪ Modified KNO3 agar - rapid, easy, and accurate method to
✓ Handle all cultures, especially molds, in a Class II BSC
determine the ability of yeast to use nitrate as the sole
✓ Cultures with yeast can be read on the open bench in a
source of nitrogen
BSL-2 laboratory; if suspected dimorphic fungi = Class
▪ Results: positive KNO3 assimilation = turns medium blue
IIA2 BSC
negative KNO3 assimilation = turns medium yellow
✓ Highly pathogenic fungi must always be handled in a
▪ Control organisms: Cryptococcus albidus (positive) and C.
BSL-3 facility
albicans (negative)
o PINAPADALA SA REFERENCE LABORATORY
4. Temperature Studies
✓ Always wear gloves and practice hand hygiene when
▪ Cryptococcus spp. = weak growth at 35°C; no growth at
handling chemical and stains used for processing wet
42°C
mounts
▪ Optimal temperature for growth is about 25°C
✓ Recommended biosafety cabinet: Class II-A1 or II-A2 BSC
▪ Several Candida spp. can grow well at temp. as high as
45°C
POST-ANALYTICAL PHASE HAZARDS
▪ results: C. albicans grows at 45°C C. dubliniensis does not
grow at 45°C • Best Practices
5. Urease Test ✓ Laboratory must have a waste management plan that
▪ Yeast isolates producing urease can be detected easily identifies and categorises waste as infectious and non-
with Christensen’s urea agar infectious at the site of waste generation
▪ slants are inoculated and incubated at room temperature ✓ All infectious waste must be either autoclaved or
for 48 hours disinfected before it leaves the laboratory
▪ results: Urease positive = Cryptococcus sp., Rhodotorula ✓ If autoclaving is unavailable, then fugal culture can be
sp., Most strains of Trichosporon spp. Urease negative = decontaminated by soaking the plates and tubes overnight
All clinically encountered Candida sp. in a fresh 1:10 bleach solution
6. (1-3)-β-D-Glucan Detection
▪ assays are based on (1-3)-β-D-glucan activating the
coagulation cascade in Limulus amebocyte lysate
(material found in the North American horseshoe crab)
o SEROLOGICAL TEST
o USED FOR THERAPEUTIC MONITORING
▪ a colorogenic substrate is used so that a positive reaction
occur, producing a yellow end product that can be read
spectrophotometrically
▪ result: positive >80 pg/mL

SAFETY IN MYCOLOGY LABORATORY

• A common-sense approach to the handling of these specimens
protects the laboratory from contamination and workers from
becoming infected
• Not only confined to potentially pathogenic fungi but also to
hazardous nature of reagents, flames, glassware and
procedures
• Hazards can be divided as such:
A. Pre-analytical Phase Hazards
B. Analytical Phase Hazards
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