REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY
REVIEW NOTES IN DIAGNOSTIC
MYCOLOGY & VIROLOGY
By: Nathaniel B. Rañon Jr., RMT
∞∞∞ MEDICAL & DIAGNOSTIC MYCOLOGY ∞∞∞
A. OVERVIEW & INTRODUCTION
1. Definition
a. MYCOLOGY is the study of the eukaryotic fungi,
including yeasts, moulds, and mushroom.
b. MEDICAL MYCOLOGY is the study of fungi that
produce pathology or disease in humans and animals
including their ecology and epidemiology
c. DIAGNOSTIC MYCOLOGY is the study of the
general characteristics, pathology & pathogenesis,
and laboratory tests & methods of medically important
fungi. that aids in their identification and treatment
2. General Key Characteristics
a. Gram Staining Reaction: Fungi stains gram positive
b. Eukaryotic organisms – have a true nucleus
bounded by a nuclear membrane. Also have
endoplasmic reticulum for protein synthesis &
mitochondria for energy production
c. They lack CHLOROPHYLL & absorbs nutrients
d. Motility: Non motile
e. Cell Wall: definite cell wall that contains complex
carbohydrates such as chitin, mannan, glucan, &
chitosan
f. Growth Requirements
1. Nutritional & Environmental Requirements
 Aerobe: require OXYGEN to grow
 Heterotrophic: require organic nutrients as a
source of energy. They utilize enzyme systems
to derive energy from organic matter. Based on
substrate that they utilize for energy source,
they are classified as follows:
o Saprophytes: live on dead organic matter
o Parasites: live on living organisms
 Temperature: Most fungi grow between 25-
300C. Some dimorphic fungi grow best @ 370C.
Any fungus capable of growing at 370C should
be considered as pathogenic.
 Incubation Period: Some yeasts grow
overnight (18-240C). Saprophytes are fast
growers (several days). Generally cultures are
held at least four (4) weeks before reporting
negative or no growth. Exceptions are as
follows:
o Paracoccidioides basiliensis: 4-5 weeks
o Histoplasma capsulatum: 10 weeks
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g. Growth Phases
1. Yeast Phase: unicellular form that exhibits creamy
colonial growth that resembles bacterial colonies
which grows best @ 35-370C
 Mould Phase: multicellular form which grows as
cottony mycelia mass in culture media which
grows best @ 250C
 Dimorphic Fungi: fungi that has both a yeast
and mould phase
h. Basic Structures
1. Hypha (hypahe; singular) microscopic units of
fungi which are tube-like filaments.
 Hyphae based on presence of crosswalls
o Septate: divided by cross walls
o Aseptate: continuous without cross walls;
also known as COENOCYTIC fungi
2. Mycelia (mycelium; singular) intertwining mass
or mat of hypha which forms the vegetative portion
of the fungi. It is composed of the following parts:
 Thallus: vegetative portion of the fungi that
absorbs water & nutrients. It grows in or on the
substrate (culture media)
 Aerial: reproductive portion of the fungi that
contains or bears the reproductive structures of
the fungi conidia or spores that extends above
the agar surface
o Reproductive structures of fungi
 Conidia: asexual spores which are
produced singly or multiply in longs chains
or clusters
 Spores: sexual spores which are
produced and contained in special sexual
structures
i. Types of Fungi Based on Reproduction
1. Sexual Reproduction: requires formation of
special structures that contain sexual spores so
that fertilization or nuclear fission can occur. In
sexual reproduction, meiosis (reduction of two
fertile cells), followed by merging of the cells, and
nuclear fusion occur.
 Perfect Fungi: exhibits a sexual phase of
reproduction
o Types of Sexual Spores
 Ascospores: formed in a sac-like
structure known as ASCUS (e.g.
Ascomycetes)
 Basidiospores: contained in a club-
shaped structure called as BASIDIUM
(e.g. Basidiomycetes)
 Zygospores: involve the fusion of two
identical cells arising from the same
hyphae (e.g. Phycometes)
 Oospores: involves the fusion of cells
from two separate, non-identical hyphae
2
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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY
. Asexual Reproduction: requires formation of
asexual spores from the mycelium. It involves only
mitosis, with nuclear & cytoplasmic division.
 Imperfect Fungi (Fungi Imperfecti): exhibits
an asexual phase of reproduction
o Types of Conidia Formation
(Conidiogenesis)
 Blastic Conidiogenesis: parent cell
enlarges, a septum forms, and the
enlarged portion splits off to form a
daughter cell
 Thallic Conidiogenesis: the septum
forms first, and new growth beyond the
septum becomes the daughter cell
o Types of Asexual Spores
 Conidia: asexual spores that are borne in
various ways either singly, or long chains
or clusters, from as specialized vegetative
hyphae known as CONIDIOPHORES
which may branch into secondary
segments, known as PHIALIDES which
the produce the conidia. Some fungi can
produce conidia in two different sizes:
 Macroconidia: large, usually septate,
and sometimes exhibits oval, club, or
spindle shaped appearance. It may be
THICK or THIN walled; and may have a
SMOOTH or SPINY (ECHINULATE)
surface.
 Microconidia: small & unicellular with
round, elliptical, or piriform (pear) shape.
Two types are:
 Sessile Microconidia: borne
directly on the hyphae
 Pedunculate Microconidia: borne
on the end of a short conidiophore
 Blastoconidia (Blastospores): develops
as the daughter cells buds off from the
mother cell and is pinched off eventually.
Blastoconidia are exhibited by yeasts,
such as Candida spp. Blastoconidia may
elongate and align in an end to end
manner called as PSEUDOHYPHAE.
 NOTE: Pseudohyphae can be
differentiated from a TRUE HYPHAE
because pseudohyphae are
constricted at the septum.
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 Chlamydoconidia (Chlamydospores):
thick walled, resistant, resting spores
produced by “rounding up” and
enlargement of the terminal hyphal cells.
The new spores germinate into a new
organism as soon as the environment
becomes favorable. Three types of
chlamydoconidia may occur depending on
the location of where it formed:
 Terminal: forms at the hyphal tip
 Sessile: forms at the hyphal sides
 Intercalary: forms within the hyphal
strand
 Arthroconidia (Arthrospores): results
from the simple fragmentation of the
mycelium at the septum into cylinder-
shaped or cask-shaped spores. They are
thick walled; and may adjacent or alternate
in arrangement. In the alternate
arrangement, empty spaces appear
between each arthrospores known as
DISJUNCTOR CELLS.
 NOTE: Useful identifying
characteristic of the dimorphic fungus
Coccidiodes immitis and the yeast,
Geotrichum candidium
 Sporangiospores: asexual spores
contained in a sac-like structure called as
SPORANGIA that are produced terminally.
Sporulation occurs when the sporangial
wall burst. This type of asexual spores is
unique to a group of fungi, Zygomycetes.
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C. LABORATORY IDENTIFICATION METHODS

B. SPECIMEN COLLECTION, HANDLING, & PROCESSING
1. Microscopic Techniques
1. General Considerations
a. Always observe sterile or aseptic technique to avoid a. Saline Mount: quick & simple method to
contamination visualize budding yeasts, hyphae, &
b. Quantity should be sufficient pseudohyphae. Lack of contrast is a major
c. Specimen should properly labeled with complete disadvantage making it difficult to appreciate
information that matches the information written on the fungal elements microscopically.
request form
d. Specimen should be collected directly from the
infected or affected site
e. Transport and process specimen without delay to
avoid overgrowth of bacterial or fungal contaminants
which may prevent the recovery of the fungal pathogen
of interest
2. Specific Considerations
a. Blood: collected in Brain Heart Infusion (BHI) with
Sodium Polyanethol Sulfonate (SPS) anticoagulant. 5
mL of blood to 20 mL of broth should be observed. The
use of BUPONT ISOLATOR TUBES for transport &
processing enhances the fungal recovery from these
specimens b. Potassium Hydroxide (KOH) Mount: rapid &
b. Bone Marrow: 0.5 mL marrow collected in Brain Heart simple technique that dissolves keratin in hair,
Infusion (BHI) with Heparin anticoagulant. nails, or skin samples which may obscure the
c. Cerebrospinal Fluid (CSF): collect 2mL or more in fungal elements. Although ca also result to poor
sterile vials or tubes. Centrifugation & membrane contract like saline mount.
filtration is recommended. If volume is less than 2mL,  Used to visualize budding yeasts, hyphae, &
prepare smears & plates from uncentrifuged specimen. spherules.
d. Cutaneous Specimens  Can also be used to examine hair to
1. Hair: plucked by roots using sterile forceps. Select determine whether hair is infected within the
hairs that fluoresce or are broken and scaly shaft (ENDOTHRIX INFECTION) or hair is
2. Nails: Cleanse first with 70% Alcohol. Then scrape infected outside the hair shaft (ECTOTHRIX
the discolored or hyperkeratotic areas. May also be INFECTION)
collected using a sterile nail cutter. Place in sterile petri
plate.
3. Skin: Cleanse first with 70% Alcohol. Then scrape
outer edge of ring in cases of ringworm; or scrape area
of active infection if there’s no ring. Place in sterile petri
plate.
e. Mucocutaneous Specimens: Scrape plaque with
tongue depressor if Candida is suspected. Transport in
sterile saline
f. Subcutaneous tissues; lesions & abscesses:
Performa biopsy or needle aspiration. Tissues must be
minced or ground. Place scrapings of lesion in a sterile
saline for transport. Anaerobic processing method
should be used if Actinomyces is suspected. c. Calcoflour White Stain: used as a brightening
g. Respiratory Tract Specimens (Sputum, Bronchial agent since calcoflour white BINDS to CHITIN
Washings, Transtracheal Aspirates): Sputum should in the fungal cell wall which provides excellent
be collected early morning, deep cough for three (3) contrast over a dark background when
consecutive days. Washings & aspirates should be visualized using a fluorescent microscope.
collected using sterile saline.
h. Throat Swab: Collect specimen by depressing tongue
using a tongue depressor, then scrape off or collect
material using two (2) sterile swabs. Duplicate
specimen should be collected.
i. Urine: First morning voided midstream clean catched
urine sample should be collected in a sterile container.
Catheterized specimen is also acceptable. Centrifuge
the sample, and use sediment for smear preparation
and plating. Process within two (2) hours to avoid
bacterial overgrowth; or refrigerate if there will be
delay.
j. Vaginal or Cervical: Collected using duplicate swab
then placed in transport culture media or broth

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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY
d. India Ink or Nigrosin Preparation: used to
visualize a clear halo around capsulated
organisms such as Cryptococcus neoformans in
CSF sample. This technique is already replaced
by DIRECT ANTIGEN TESTING for the
Cryptococcal Capsular Protein (CALAS®) since
WBCs may be mistaken as yeasts or capsules
& in other instances, the organism may be
capsule negative especially for immunodeficient
patients (AIDS).
e. Lactophenol Cotton Blue (LPCB): used to
visualize fungal structure by staining chitin in the
fungal cell wall color BLUE. Also useful in
staining tease preparation (wet mount) or slide
cultures.
 Phenol: kills other organisms (bacteria)
 Lactic Acid: preserves fungal elements
 Cotton Blue: stains the chitin in the fungal
cell wall blue.
f. Hucker Modification of Gram Stain:
Recommended for mycology since fungi stain
gram positive but sometimes may stain pale
lavender due to fungal capsule which prevents
the stain from penetrating and staining the
organism. Fungi are 2-3X bigger than Gram +
Cocci & 2-3X wider than Gram + Bacilli.
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g. Giemsa or Wright Stain: used to visualize
intracellular Histoplasma capsulatum in blood
smears, lymph nodes, lung, liver, or bone
marrow. The organism appears as small, oval
yeast cell staining light to dark blue.
Cryptococcus neoformans also stains well using
this method.
h. Periodic Acid-Schiff (PAS): used to stain the
hypha of moulds and some yeasts. Periodic
Acid oxidizes the OH- in the cell wall CHO to
form aldehydes which reacts with the basic
fuchsin dye to form a pink-purple complex. A
counterstain (fast green) can be used to provide
contrast
i. Methenamine-Silver Nitrate Stain: useful for
screening of clinical specimens for presence of
fungal elements which stains black. Fungi
appear outlined in black against a pale-black-
ground. GOMORI METHENAMINE-SILVER
NITRATE STAIN is a modification of this
method used for histological examination of
specimens.
j. Other Special Fungal Stains
1. Gridley Stain: Hyphae & stain stain dark blue
or rose. Tissues stain deep blue &
background is yellow.
2. Papanicolaou Stain: good for initial
differentiation of dimorphic fungi; works well in
sputum smears
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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY
3. Mayer Mucicarmine Stain: stains the
capsule of Cryptococcus neoformans deep
rose.
2. Culture Media
a. Primary Isolation Media
1. Saboraud Dextrose Agar (SDA or Sab-
Dex): a general isolation medium which
contains peptone & glucose; has a pH @ 5.6
which inhibits most bacteria but will allow
fungal contaminants to grow. Histopasma
capsulatum fails to grow in this medium.
2. Saboraud Dextrose-Cycloheximide &
Chloramphenicol (SDA-CC): same as SDA
as described above but with the addition of
the following antibiotics:
 Cycloheximide: inhibits many
saprophytic fungi & contaminating fungi
including Cryptococcus neoformans,
Candida spp. & some Aspergillus spp.
 Chloramphenicol: inhibits most
bacteria
 NOTE: Also available commercially
as MYCOSEL or MYCOBIOTIC
3. Brain Heart Infusion (BHI): is a rich
medium that is optimum for the recovery of
systemic fungi such as Histoplasma
capsulatum. BHI can also be added with
blood, cycloheximide, & chlorampenicol.
4. Dermatophyte Test Medium (DTM): can be
substituted for SDA-CC for the recovery of
dermatophytes from specimens
contaminated with fungi.
b. Differential or Special Culture Media
1. Potato Dextrose Agar (PDA): used as a
subculture medium rather than a primary
isolation medium. This medium enhances
the sporulation & pigmentation of fungi.
2. Corn Meal Tween 80 Agar (CMT 80): used
for the demonstration of blastoconidia,
pseudohyphae, arthroconidia, and
chlamydospores in Candida spp. and some
other yeasts.
3. Dermatophyte Test Medium (DTM): used
for the selective growth of dermatophytes.
This medium will turn from yellow to red
within 14 days incubation at room
temperature if dermatphytes are growing. It
should be checked once every week for 1
month.
4. Birdseed Agar (Niger Seed Agar): used to
isolate Cryptococcus neoformans from
contaminated cultures which grows brown to
black colonies in 4 to 7 days.
5. Caffeic Acid Agar: Cryptococcus
neoformans grows black colonies in this
medium when protected from light due to
melanin production.
6. CottonSeed Agar, KT Medium, & Kelley
Agar: used to covert dimorphic fungus
Blastomycetes dermatitidis from mycelia to
yeast form.
7. Modified Converse Lequid Medium
(Levine): used to promote the spherule
production by Coccidioides immitis.
8. Christensen UREA Slant: used to detect
urease production by turning the slant to
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pinkish purple color after 48 hours
incubation.
 UREASE POSITIVE (+): Trichosporon,
Rhodotorula, & Cryptococcus
 UREASE NRGATIVE (-): Geotrichum,
Saccharomyces, & most Candida spp.
3. Macroscopic Examination of Fungal Colonies
a. Topography: since some fungi may be free
growing which cover the entire agar, this is best
observed at the reverse side of the plate. Fungal
colonies may be FLAT, HEAPED, or FOLDED.
RUGOSE topography contains deep furrows that
radiates from the center. Some has raised or
bulging center referred to as UMBONATE. And
some may also be WRINKLED or VERRUCOSE.
b. Texture: usually related to length of the aerial
hyphae which is best observed in a cross section.
1. Cottony or Wooly: high dense aerial mycelia
2. Velvety or Silky: low dense aerial mycelia
3. Powdery or Granular: flat, rough, & crumbly
colonies
4. Glabrous or Smooth: wet, waxy, creamy, or
pasty since no significant mycelia are produced
c. Pigmentation: surface and reverse side
observation of coloration is very important.
Description of color should be specific.
4. Important Biochemical Tests for the
Identification of Yeast & Yeast-like Organisms
a. Carbohydrate (CHO) Fermentation
 Principle: growth and utilization of CHO
under anaerobic conditions as determined by
acid & gas production
 Indicator: Bromcresol Purple (BCP)
 Positive Reaction: Yellow Color for Acid
production and bubbles trapped in the
fermentation tube for gas production.
 NOTE: Observe every 48 hours for 14 days.
b. Carbohydrate (CHO) Assimilation
 Principle: the yeast ‘s ability to utilize a
particular CHO is determined by using a
CHO-free (nitrogen-based) agar and filter
paper disks that are impregnated with various
CHO. Growth around the disk indicates the
yeast can utilize the CHO.
 Positive Reaction: growth around the disk
indicates the CHO has been assimilated by
the yeast
 NOTE: Incubate plate for 24 hours at 300C.
Plates should be re-incubated for another 24
hours and read again if the growth is
insufficient.
c. Nitrogen Assimilation
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 Principle: Nitrate assimilation is defined a. Dermatophytes: group of fungi that causes TINEA
simply as the utilization of a nitrogen source (RINGWORM) which is red & scaly with distinct
by a microorganism in the presence of margin and ring-like appearance due to cord-like
oxygen. A positive reaction is indicated by the bumps underneath the skin which resembles a worm.
presence of growth or the use of a pH Upon healing, the central area clears.
indicator in the medium. The indicator method  Sources of Dermatophyte Infections (TINEA)
is a modification of the Wickerham method Type Source Representative Fungi
that was devised by Adams and Cooper. Geophilic Fungi: Soil & soil- Microsporum gypseum
free-living soil coontaminated Microsporum manum (swine
 Indicator: Bromthymol Blue (BTB)
saprophytes animals from contaminated soil)
 Positive Reaction: Green to Blue Slant due Arthrophilic Fungi: Through human Epidermophyton floccosum
to alkaline pH when nitrate is assimilated. Infects humans sources; such as Microsporum audouinii
 NOTE: Incubate aerobically with loosen caps fomites Trichophyton rubrum
at 35-370C for 24-72 hours. Zoophilic Fungi: Direct contact with Microsporum canis (dogs,
d. Germ Tube Test (Reynold’s Brande Parasitic on an infected animal cats)
animals other than Trichophyton
Phenomena): a screening procedure which is humans mentagrophytes (dogs, cats)
used for the identification and differentiation Trichophyton verrucosum
of Candida albicans from other yeasts. (cattles, horses)
 Principle: This test allows the detection of
the Germ Tubes which are the initial stage of  Common TINEA Infections & Associated
hyphae formation. These are the short, non- Organisms
septate germinating hyphae which are one Tinea Infection Description Associated Fungi
half the width and three to four times the Tinea Barbae Ringworm of the Trichophyton mentagrophytes
length of the yeast from which they originate. (usually beard & mustache Microsporum canis
zoophilic; Trichophyton verrucosum
Approximately 95-97% of Candida occupational
albicans isolate develop germ tube when hazard for farm
incubated in a proteinaceous media at 35C workers
for 2.5-3 hours. Tinea Capitis Ringworm of the Microsporum canis & audouinii
scalp, eyebrows, (Gray Patch Ringworm:
eyelashes Common in children)

Trichophyton tonsurans (Black-

dot Ringworm: Endothrix
infection)

Trichophyton mentagrophytes
(Inflammatory Ringworm:
Ectothrix Infection)

Tinea Corporis Ringworm of the Trichophyton rubrum

body; especially Trichophyton tonsurans
the trunk) Epidermophyton floccosum
Tinea Cruris Ringworm of the Trichophyton rubrum
(Common groin Trichophyton mentagrophytes
Infection in Epidermophyton floccosum
athletes, military
personnel, etc.)
Tinea favosa Ringworm of the Trichophyton schoenleinii
e. Serological Tests: may be performed to hair follicle at its
supplement microscopic and culture methods. base
 Crytotococcal Antigen Latex Agglutination Tinea manuum Ringworm of the Trichophyton rubrum (most
System (CALAS®) detects cryptococcal hands, palms, & frequent cause)
between fingers
polysaccharide in body fluids such as CSF.
Tinea pedis Ringworm of the Trichophyton rubrum
This method uses latex particles coated with (Common foot, or “athlete’s Trichophyton mentagrophytes
anticryptococcal globulin to detect infection found foot” Epidermophyton floccosum
cryptococcal antigen. on soles of the
 Other Serological Tests includes: feet & between
toes
o Immunodiffusion characterized by
o Complement Fixation itching & scales)
o Immunoflourescence Tinea unguium Ringworm of the Trichophyton rubrum
o Counterimmunoelectrophoresis nails Trichophyton mentagrophytes
(Onychomycosis) Epidermophyton floccosum

D. FUNGAL INFECTIONS (MYCOSES)  Tinea Versicolor (Pityriasis Verisicolor):

characteristized by superficial brownish or
1. SUPERFICIAL MYCOSES: Non-invasive fungal scaly areas on light-skinned individuals &
infections of the outermost layer of the skin (stratum irregular patches of unpigmented or
corneum). It may also involve the hair and the nails. untanned skin on dark-skinned individuals
Although most infections are mild & superficial, others o Causative Agent: Malassezia furfur
are more severe which involve the dermis evoking an o Common in warm, humid environments
immune response leading to pain & ulcerative lesions. including the tropics. Also in person with
excess perspiration & oily skin

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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY
o LIPOPHILIC: requires lipid for growth;
hence usually seen where sebum and
skin oil accumulates.
o Body Sites Affected: skin of the chest,
back, & upper arms
o Specimen: Skin scrapings which
flouresces under Wood’s Light
o Microscopic Characteristics: tight
clusters of spherical, budding, yeast-like
cells with short unbranched hyphae or
hyphal fragments resembling “Sphagetti
& Meatballs” appearance
o Culture Media: SDA with olive oil or
sterile mineral oil in 2 to 4 days @ 300C
incubation.
o Colonial Characteristics: smooth,
cream-colored yeast-like colonies which
turns to tan to brown with dull
appearance as it ages.
 Tinea Nigra Palmaris: characterized by a
single brown to black scaly patch with a
distinct border.
o Causative Agent: Hortae werneckii
(formerly known as Phaeoannellomyces
werneckii and Cladosporium werneckii)
o Common in tropical countries, usually
seen in Africa, Asia, South & Central
America; rarely found in the US.
o Microscopic Characteristics: brown
septate, branching hyphae, and
elongated budding yeast cells.
o Culture Media: SDA for 7 days
incubation @ RT
o Colonial Characteristics: gray, shiny,
moist yeast-like cells with an olive-green
mycelium. Colonies become olive green
to black upon aging.
 Black Piedra characterized by a hard
brown-black crusts on the outside of the hair
shaft, usually on the scalp.
o Causative Agent: Piedraia hortae
o Common in tropical climates of South
America, Africa, & Eastern Asia
o Microscopic Characteristics: direct
examination of infected hair shaft will
show hard black nodules and
ascospores. There are 2-8 spindle
shaped spores per cell.
o Culture Media: SDA for 7 days
incubation @ RT
o Colonial Characteristics: velvety,
green-black heaped colonies
 White Piedra characterized by light brown,
soft nodules on the beard , axillae, scalp, or
mustache, which are less firmly attached
than those of black piedra
o Causative Agent: Trichosporon beigelii
(also known as Trichosporon cutaneum)
o Common in tropical climates of South
America, Africa, & Eastern Asia
o Microscopic Characteristics: direct
examination of infected hair shaft will
show light-colored septate hyphae &
round to rectangular arthoroconidia
o Culture Media: SDA for 5 days
incubation @ RT
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o Colonial Characteristics: velvety,
green-black heaped colonies
o Colonial Characteristics: moist, white to
creamcolored colonies which becomes
dar yellow to gray & wrinkled with age.
2. SUBCUTANEOUS MYCOSES: Usually a result of
traumatic skin puncture from thorns or vegetation
contaminated with fungi which may be saprobes in the
soil or as plant pathogens. Host immune response is
evident from phagocytes. The infection may spread to
other sites and can bceome chronic. There are four (4)
major groups of subcurtaneous fungal infections:
a. Chromoblastomycosis: puncture of trauma to the
skin leads to chronic, non-healing, hard, warty, tumor-
like lesions
o Causative Agent: Dematiacious Fungi: group of
dark, slow-growing fungi that are found on
vegetation and in te soil.
 Fonsacaea pedrosoi – most common agent
 Phialophora verrucosa
 Cladosporium carrionii
 Wangiella dermatitidis
o Although prevalent worldwide, it is most often
seen in the topics.
o Body Sites Affected: most frequently involves the
feet & lower legs
o Microscopic Characteristics: Tissue biopsy
shows sclerotic bodies that are copper-colored
septate cells
o Colonial Characteristics: heaped, folded, and
darkly pigmented (gray, olive, or black), colonies
with a velvety black underside
b. Mycetoma: chronic granulomatous infection of the
cutaneous and subcutaneous tissues and bones with
tumor like deformities of the subcutaneous tissues
with abscess, draining sinuses, granulomatous puss.
Yellow, red, white, or black granules may be present
in the puss
o Causative Agent
 Actinomycotic Mycetoma: aerobic
Actinomyces such as nocardia, Actinomadura,
Streptomyces
 Eumycotic Mycetoma: Pseudoallescheria,
Aspergillus, Oxiphiala, Acremonium, Curvularia,
Madurella
c. Phaeohyphomycosis: refers to any infection caused
by any dematiaceous fungi excluding
chromoblastomycosis and mycetoma
d. Sporotrichosis: most often occurs from a skin
trauma caused by finger prick from thorny plants,
such as roses.
o Causative Agent: Sporothrix schenckii
(Dimorphic)
o Worldwide prevalence; rose gardeners are mostly
susceptible
o Body Sites Affected: may remain local or
cutaneous but usually subcutaneous. Local &
regional lymphadenopathy also may occur, & may
disseminate. Inhalation may also result to
pulmonary infection
o Microscopic Characteristics: from colonies:
 Mould Phase: thin septate, branching, hyaline
hyphae. Oval, elliptical, or pyriform conidia are
seen. The conidia gather to form flower petals
or a “ROSETTE” appearance
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 Yeast Phase: oval, spherical, or fusiform yeast
cells, which may produce multiple buds. May
be seen inside PMNs in tissue biopsy as
“ASTEROID BODY” which is a small, snail-
shaped yeast cell with a single bud and a
narrow attachment to the mother cell.
o Culture Media: BHI with or without blood at 370C
incubation
o Colonial Characteristics: soft, white, cream-to-
tan-colored colonies
3. SYSTEMIC MYCOSES (DIMORPHIC FUNGI)
Important Characteristics Description
Organism Blastomyces dermatitidis
Clinical Significance Blastomycosis (North American
Blastomycosis or Gilchrist’s
Disease)
Prevalence Most often found in the
southeastern US; area south of
Ohio & Mississippi River
Culture Growth Rate 7-28 days
Microscopic Appearance Mycelial Phase: delicate, septate
hyphae with round or pyriform
conidia borne singly on
conidiophores or directly from
hyphae resembling “LOLLIPOPS”
Yeast Phase: Thick walled, large
yeast cells with single bud on a
broad base; broad isthmus at
constriction
Important Characteristics Description
Organism Coccidioides immitis
Clinical Significance Coccidioidomycosis (Desert or San
Joaquin Valley Fever)
Prevalence High Incidents in the San Joaquin
Valley in California
Culture Growth Rate 5-14 days; growth may occur as
early as 3-5 days’ Arthroconidia
may need 1-2 weeks to form
Microscopic Appearance Mycelial Phase: Coarse, septate
hypahe that produce thick walled,
barrel-shaped, rectangular
arthroconidia that alternate with
empty disjunctor cells
Yeast Phase: Large, round, thick
walled spherules with endospores
observed in tissues; not a true
yeast
Important Characteristics Description
Organism Histoplasma capsulatum
Clinical Significance Histoplasmosis Darling’s Disease)
Prevalence Endemic in Ohio & Mississippi
River Valleys and Appalachian
Mountains
Culture Growth Rate Slow growing; usually requires 2-4
weeks @ 250C or 300C
Microscopic Appearance Mycelial Phase: Septate hyphae
with round to pyriform microconidia
on short branches or directly from
hyphal stalk; later large, round,
thick walled knobby tuberculate
macroconidia forms
Page 8 of 13
Yeast Phase: Small budding,
round to oval yeast cells;
intracellular to mononuclear cells
with Giemsa or Wright Stain
Important Characteristics Description
Organism Paracoccidioides brasiliensis
Clinical Significance Paracoccidioidomycosis
Prevalence South & Central America & Mexico;
especially in Brazil, Colombia, &
Venezuela
Culture Growth Rate Very slow growing; usually requires
21-28 days
Microscopic Appearance Mycelial Phase: Small, septate,
branched hyphae with intercalary &
terminal chlamydoconidia; few
pyriform microconidia
Yeast Phase: Large round thick
walled yeast cells with multiple
buds that attach to the mother cell
by narrow constrictions, resembles
a “SHIP’s WHEEL” or
:MARINER’S WHEEL”
4. OPPORTUNISTIC MYCOSES
Important Characteristics Description
Organism Candida spp.
Clinical Significance Candidiasis
Oral: thrush
Vaginal: vulvovaginitis
Cutaneous: Diaper rash, Nail
(Onychomycosis), Cuticle
(Paronychomycosis)
Systemic: Endorcarditis, meningitis,
pulmonary infections, fungemia
Culture Growth Rate BAP & SDA in 48-72 hours @ 35-
370C
Colonial Characteristics White to cream or tan colored
colonies with pasty or creamy
appearance resembles bacterial
colonies; may have spider like
projections
Key Lab Tests  Germ Tube Test POSITIVE
 Urease & Nitrate Reduction.
NEGATIVE
 Produce chlamydioconidia on
Cornmeal Agar when incubated
at RT for 24-48 hours
Important Characteristics Description
Organism Cryptococcus neoformans
Clinical Significance Cryptococcosis
Route of Infection Contact with bat, pigeon, , & bird
droppings
Colonial Characteristics Mucoid colonies, which are cream,
tan, or pink in color
Key Lab Tests  Brown to black colonies in Bird
(Niger) Seed Agar
 UREASE & Phenol oxidase
POSITIVE
 Nitrate Reduction Negative;
 Assimilation of INOSITOL
 India Ink: capsulated yeast
cells
 Serological Tests: CALAS +
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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY Page 9 of 13

∞∞∞ MEDICAL & DIAGNOSTIC VIROLOGY ∞∞∞

RNA VIRUSES
A. OVERVIEW & INTRODUCTION
DOUBLE
1. General Key Characteristics SINGLE
STRANDED
STRANDED (SS)
a. Obligate intracellular organisms – require host cell (DS)

biochemical mechanism to replicate

b. lacks ribosomal RNA (rRNA) – hence can’t make positive (+) sense
negative (-)
Naked
sense
their own proteins
c. Composed of either DNA or RNA but not both
d. Very small size: Ranging from 18 nm (Parvovirus) to
Naked Enveloped Enveloped **REO
300 nm (Poxvirus); not visible by light microscopy
e. There are currently 21 viral families associated with
human infection; 14 are RNA viruses while the rest
are DNA viruses **PICORNA **TOGA BUNYA

2. Basic Viral Structure

**CALICI **FLAVI ORTHOMYXO

CORONA RHABDO

RETRO ARENA

FILO

**ICOSAHEDRAL; All the rest have helical symmetry

 IMPORTANT NOTES:
a. VIRION: virus particle o All RNA Viruses are single stranded
b. CAPSID: protein coating EXCEPT for REO VIRUS
c. VIRAL GENOME: Genetic material (RNA or DNA) o All Naked RNA Virus (REO VIRUS) is
d. ENVELOPE: outer membrane double stranded
o All NEGATIVE SENSE RNA Viruses are
3. Classification ENVELOPED

B. SPECIMEN COLLECTION, HANDLING & PROCESSING

DNA VIRUSES
1. General Considerations
a. For most viral infection, viral titer is highest during
the first 4 days of infection after onset of symptoms
ENVELOPED NAKED EXCEPT: Enterovirus, Adenovirus, Cytomegalovirus
(CMV)because prolonged shedding of Enterovirus &
Adenovirus into the stool and CMV into the urine
DOUBLE
STRANDED (ds)
Icosahedral occurs.
b. It is best to sample the infected site directly such as
rash or vesicles; throat swab for upper respiratory
Icosahedral Complex
DOUBLE SINGLE infection. Exception is certain CNS viral infections
STRANDED (ds) STRANDED (ss)
which may need to culture stool or throat instead of
CSF)
c. Viral transport medium is highly recommended for
HEPADNA POX PAPOVA PARVO
most specimens which contains buffered saline, a
protein stabilizer, & antibiotics (to suppress bacterial
or fungal overgrowth). Commercially available viral
HERPES ADENO transport medium includes:
 Hank’s Balanced Salt Solution
 Veal Infusion Broth
 IMPORTANT NOTES:  Sucrose-Phosphate-Glutamate Broth
o All DNA Viruses are double stranded  Leibovitz-Emory Medium
EXCEPT for PARVO VIRUS d. Collection Systems: Viral Culturettes (BD): swabs
o All Enveloped DNA Viruses are double with a viral culture medium
stranded e. Cotton, Dacron, Rayon SWAB with PLASTIC
o All Naked DNA Viruses are ICOSAHEDRAL SHAFT should be used; Never use wooden shaft
because it is toxic to viruses
f. Calcium Alginate SWABS should be avoided
because it may bind and inactivate the virus.
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REVIEW NOTES IN DIAGNOSTIC MYCOLOGY & VIROLOGY Page 10 of 13

g. Optimum Storage Temperatures: As much as 4. Molecular Diagnostic Methods

possible transport & process specimen without delay
but if can’t be avoided; store as follows:
 40C: less than 24 hours delay
 Freezing Temp 00C: more than 24 hours delay
 -700C Deep Freezing: longer delay (months)
 NOTE: -200C is not suitable for storing
specimens for virology
C. LABORATORY METHODS
1. Cytological or Histological Examinations
a. Presence of multinucleated giant cells or
cytoplasmic or nuclear inclusions may aid in
diagnosis
b. CMV may produce characteristic “OWL’S EYE”
inclusions
c. Disadvantage: Insensitive & non-specific
2. Electron Microscopy
a. Sensitive tool but not readily available in most
laboratories
b. PRINCIPLE: Clinical material is placed in a carbon-
coated grid then stained with potassium
phosphotungstate or uranylacetate. Stain
surrounds that virus, & the electron beam cannot
pass through the virus; hence virus is seen as a
light structure over a dark background.
c. The ONLY METHOD that can detect Norwalk
Agent, Astroviruses, Caliciviruses,
Coronoviruses because they can’t grow in tissue
cell lines & no available serological tests
a. Detection of viral DNA or RNA in specimens
b. Molecular Methods used in Virology
 Restriction Fragment Length Polymorphism
(RFLP)
 Gene Probes in situ Hybridization
 Polymerase Chain Reaction (PCR)
 Reverse Transcriptase PCR
 Real Time PCR
5. Serological Methods
a. detection of viral antibodies is an indirect indicator of
infection or exposure to a particular virus
b. useful when the virus will not grow in cell cultures
c. Paired sera is recommended for detection:
 Acute Phase Sample: Collected when the clinical
signs first appear
 Convalescent Phase Sample: Collected 2 to 3
weeks later, depending on the virus
d. Traditionally, a FOUR FOLD INCREASE in antibody
titer indicates a seropositive reaction and strongly
supports a diagnosis of current infection
&

3. Viral Tissue Culture

a. remains to be the “GOLD STANDARD” for the
isolation of many viruses
b. Three (3) types exist:
 Traditional cell culture using cell lines
 Shell vial centrifugation-enhanced (SVCE)
cultures
 Multi-well Microplate Cultures
c. Primary Cell Cultures
 Human Embryonic Kidney (HEK)
 Rabbit Kidney (RK)
 Primary Monkey Kidney (PMK)
 Rhesus Monkey Kidney (RMK)
 Cynomolgus Monkey Kidney (CMK)
 African Green Monkey Kidney (AGMK)

d. Heteroploid Cell Line

CELL LINE SOURCE

Vero Cell Line Kidney cells of an African Green Monkey
A549 Cells Human lung carcinoma
HELA Cell Line Human cervical carcinoma
Hep-2 Cell Line Human epithelial cells of larynx carcinoma
KB Human Nasopharyngeal Carcinoma

d. Cytopathic Effect (CPE): cellular damage or

changes cellular structure which results from viral
inoculation & incubation of tissue cell lines. CPE
includes:
 Rounding of Cells
 Forming of giant multinucleated cells
 Discoloration of cytoplasm
 Disintegration of cells

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DNA VIRUSES
FAMILY GENUS or GROUP COMMON NAME CLINICAL SIGNIFICANCE
Adenoviridae Mastadenovirus Adenovirus Childhood Upper Respiratory Tract Infections (Rhinitis, Sore Throat, etc.)
Epidemic keratoconjunctivitis (pink-eye)
Hepadnaviridae Headnavirus Hepatitis B virus (HBV) Acute Viral Hepatitis; Fulminant Hepatitis; Chronic Hepatitis (10%);
Co-infection or superinfection with Hepatitis D Virus (HDV)
Herpesviridae
Subfamilies
Alphaherpesvirinae Simplex Virus Herpes Simplex Virus Types 1 (HSV-1) Gingivostomatitis; Herpectic keratitis of the eye; Encephalitis
Varicella Virus Herpes Simplex Virus Types 2 (HSV-2) Genital Herpes; Neonatal Herpes (one of the TORCHES organisms)
Varicella-Zoster Virus (VZV) or (HHV-3) Varicella (Chickenpox); Zoster (Shingles)
Betaherpesvirinae Cytomegalovirus Cytomegalovirus (CMV) or (HHV-5) Asymptomatic Infection (latent phase)
Congenital (part of TORCHES)
CMV Mononucleosis
Gammaherpesvirinae Lymphocryptovirus Epstein-Barr Virus (EBV) or (HHV-4) Infectious Mononucleosis (Kissing Disease); Associated with Burkitt’s B Cell Lymphoma
Human Herpesvirus Type 6 (HHV-6) Roseola (Exanthem Subitum)
Human Herpesvirus Type 7 (HHV-7) Roseola (Exanthem Subitum)
Human Herpesvirus Type 8 (HHV-8) Kaposis Sarcoma
Papillomaviridae Papillomavirus Human Papillomavirus (Wart Virus) Human Papiloma Virus (Warts)
Common Warts (types 1, 2, 4, & 7)
Genital Warts (types 6, 11, 16, & 18)
Laryngeal Warts (types 6 & 11)
Cervical Cancer (types 16 & 18)
Polyomaviridae Polyomavirus Polyomavirus strain JC & BK JC Polyomavirus: Progressive multifocal leukoencephalopathy
BK Polyomavirus: causes mild or asymptomatic infection in children
Parvoviridae Parvovirus Parvovirus strain B-19 & RA-1 Erythema Infectiosum (Fifth Disease); affects children 4 to 12 yo; “Slapped Cheek Rash”
Transient Aplastic Anemia Crisis
Poxviridae Orthopoxvirus Variola Virus (small pox virus) Small Pox
Vaccinia Virus
Molluscum Contagiosum
Monkeypox Virus

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RNA VIRUSES
FAMILY GENUS or GROUP COMMON NAME CLINICAL SIGNIFICANCE
Arenaviridae Arenavirus Lymphocytic choriomeningitis (LCM) Virus Lymphocytic choriomeningitis (LCM)
Lassa fever Virus Lassa fever Virus
Machupo Virus; Junin Virus; Sabia Virus
Bunyaviridae Bunyavirus La Crosse Virus; California Encephalitis Virus California Encephalitis
Phlebovirus Rift Valley Fever Virus Rift Valley Fever
Nairovirus Crimean-Congo Hemorrhagic Fever Virus Crimean-Congo Hemorrhagic Fever
Hantavirus Hantaan Virus Korean Hemorrhagic Fever
Caliciviridae Calicivirus Norwalk Virus; Sapporo Virus Viral Gastroenteritis; explosive but self limiting
Coronaviridae Coronavirus Coronavirus Strains Common Cold; Upper respiratory tract infection
Filoviridae Filovirus Marburg Virus Acute Viral Hemorrhagic Fever;
Ebola Virus (Hemorrhagic Fevers) High Mortality Rate (50-90%)
Flaviviridae Flavivirus Yellow Fever Virus Yellow Fever (hepatitis with jaundice)
Dengue Fever Virus Dengue Fever & Dengue Hemorrhagic Fever (DHF)
St. Louis Encephalitis Virus St. Louis Encephalitis
Japanese Encephalitis Virus Japanese Encephalitis
Hepacivirus West Nile Virus West Nile Encephalitis
Hepatitis C & G Virus Acute Viral Hepatitis; 50% will develop chronic hepatitis; 20% will develop cirrhosis
Orthomyxoviridae Orthomyxovirus Influenza Virus A, B, & C The FLU FEVER
Paramyxoviridae Paramyxovirus Paramyxoviruses Upper respiratory tract infection; Viral Pneumonia in children, elderly & immunocompromised
Mumps Virus Mumps (parotid gland swelling)
Morbilivirus Measles Virus Rubeola (Measles)
Pneumovirus Respiratory Syncytial Virus (RSV) Most common Cause of Pneumonia in infants less than 6 months of age
Picornaviridae Human Enterovirus Poliovirus Paralytic poliomyelitis
Coxsackie A & B viruses Cold, rashes, viral meningitis; Herpangina; Hand, Foot & Mouth Disease
Echoviruses Cold, rashes, viral meningitis; pericarditis
Hepatovirus Hepatitis A Virus (HAV) Infectious Hepatitis; fever, jaundice, & enlarged liver; never becomes chronic
Rhinovirus Rhinoviruses (types A, B, & C) Common Cold
Reoviridae Reovirus; Reoviruses; Viral Gastroenteritis
Orbivirus; Colorado Tick fever virus Colorado Tick fever
Rotavirus Rotaviruses Viral Gastroenteritis
Rhabdoviridae Lyssavirus Rabies Virus RABIES
Togaviridae Alphavirus Eastern, Western, & Venezuelan Encephalitis Virus Eastern, Western, & Venezuelan Equine Encephalitis
Rubivirus Rubella Virus Rubella (German Measles/3 day measles)
Retroviridae
Subfamilies
Oncornaviridae Oncornavirus Human T Cell Lymphotropic Virus Types 1 (HTLV-1) Human T Cell Lymphotropic Disease
Human T Cell Lymphotropic Virus Types 2 (HTLV-2)
Lentiviridae Lentivirus Human Immunodeficiency Virus Type 1 (HIV-1) Human Immunodeficiency Syndrome;
Human Immunodeficiency Virus Type 2 (HIV-2) Acquired Immune Deficiency Syndrome (AIDS)

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Copyright 2016 NBRJR