MYCOLOGY -

VIROLOGY
Marc Holden Maranan Micosa, RMT,MSMTc,MLS(ASCPi)cm
College of Medical Technology
DEMATIACEIOUS (MELANIZED) MOLDS
• characterized by dark coloration
• ability to produce melanin
• superficial and subcutaneous mycoses
• Involve the skin and subcutaneous tissue
• ubiquitous
• saprophytes and plant pathogens
• Humans and animals - accidental hosts after traumatic
inoculation
• slow-growing dematiaceous mold (7-10 days)
• rapid-growing dematiaceous molds ( less than 7 days )
Epidemiology and Pathogenesis
Superficial Infections
• Tinea nigra –skin infection caused by Hortaea werneckii
• halophilic species
• blackish brown, macular patches (palm of the hand or the sole of
the foot)
• Lesions - silver nitrate staining
• Black piedra - infection of the hair, scalp, and pubic hair
• caused by the dematiaceous fungus Piedraia hortae.
• Neoscytalidium dimidiatum(plant pathogen)
• skin and nails that may lead to hyperkeratosis
• tropical areas (Africa, Asia, and Latin America)
• Chaetothyriales - superficial infections
• Cyphellophora spp., Phialophora europaea, and Knufia epidermidis
• mild cutaneous skin infections or nail infections
Mycetoma
• chronic granulomatous infection
• lower extremities and other parts of the body
• characterized by swelling, purplish discoloration, tumorlike
deformities , and multiple sinus tracts that drain purulent
material containing yellow, white, red, or black granules
• May involve bone, muscle, or other contiguous tissue
• tropical and subtropical regions
• Two types
• Actinomycotic (bacterial) -Nocardia, Actinomadura, and
Streptomyces spp
• Eumycotic (fungal) - fungi that have septate hyphae
• white grain mycetomas or black grain mycetomas
Etiologic agents of eumycotic mycetoma
• Pseudallescheria boydii and Acremonium spp
• white grain mycetomas
• most common etiologic agent in the United States is P. boydii
• Exophiala jeanselmei, Curvularia spp., Cladophialoph- ora bantiana,
Trematosphaeria grisea (previously Madurella grisea), and
Madurella spp.
• black grain mycetomas
• Madurella mycetomatis is the most common fungal agent
associated with mycetoma
• tropical regions
• saprophytic and commonly found in soil, standing water, and
sewage
• acquire infections through traumatic implantation of the organism
Chromoblastomycosis
• chronic fungal infection acquired through traumatic inoculation
• characterized by the development of a papule at the site of the traumatic insult
to form warty or tumorlike lesions characterized as resembling cauliflower
capable of spreading through the lymphatic system
• feet and legs but may involve the head, face, neck, and other body surfaces.
• sclerotic bodies - copper-colored, septate cells that appear to be dividing by
binary fission and resemble copper pennies
• hyperplasia of the epidermal layer of the skin
• Fungal brain abscess - cerebral chromoblastomycosis
• occur in tropical and subtropical areas
• agricultural workers do not wear protective clothing
• Cladophialophora carrionii, Fonsecaea pedrosoi, and Phialophora verrucosa
Phaeohyphomycosis
• infection caused by a dematiaceous organism
• molds; brownish, yeastlike cells; pseudohyphae; and hyphae,
• may be subcutaneous, localized, or systemic
• phaeohyphomycotic cysts, progressive soft tissue infection,
brain abscess, sinusitis, endocarditis, mycotic keratitis,
pulmonary infection, and systemic infection
• include headache, neurologic manifestations, and seizures
Most common fungal isolates associated with neurologic
manifestations:

• ladophialophora bantiana, Rhinocladiella mackenziei,

Verruconis gallopava, and Exophiala dermatitidis

Also commonly associated with phaeophyomycosis

• Alternaria, Exserohilum, Bipolaris, Exophiala jeanselmei, E.
dermatitidis, Exophiala spinifera, C. bantiana, and Curvularia
spp.
Pathogenesis and Spectrum of Disease

•superficial infections (e.g., skin and hair)

to emergent, rapidly progressive, and
often fatal disease (e.g., brain abscess)
Mycetoma
• Bacterial:
• Nocardia,Actinomadura,andStreptomycesspp.
• White grain mycetoma:
• P. boydii and Acremonium and Fusarium spp.
• Black grain mycetoma:
• Madurella spp., Exophiala jeanselmei, and Curvularia spp.
• Chromoblastomycosis:
• Cladophialophora, Phialophora, and Fonsecaea spp.
• Phaeohyphomycosis:
• E. jeanselmei; E. dermatitidis; and Curvularia, Bipolaris, Alternaria, and Exserohilum spp.
• Sinusitis:
• Alternaria, Bipolaris, Exserohilum, and Curvularia spp.
• Mycotic keratitis and endophthalmitis:
• E. dermatitidis and Bipolaris and Curvularia spp.
• Brain abscess:
• C. bantiana, E. dermatitidis, and Bipolaris spp.
Common Isolated Dematiaceous Fungi
Laboratory Diagnosis

• Specimen Collection, Transport, and Processing

• Refer from previous topics
Direct Detection Method
• Stains
• calcofluor white or fluorescent microscopy
• Fontana-Masson stain, 10% silver nitrate and ammonium hydroxide,
stains fungal elements brown to black in a red background
• detection of melanin granule

****Fontana-Masson stain is useful to detect melanization that may

appear as hyaline molds using light microscopy
Superficial Infections

• tinea nigra may show dematiaceous hyphae and small

budding yeast cells and/or hyphal fragments
• black piedra - potassium hydroxide that is gently heated for
nodules composed of cemented mycelium
• mature nodules reveals oval asci, containing two to eight
aseptate ascospores, 19 to 55 mm long by 4 to 8 mm in
diameter. The asci are spindle-shaped and have a
filament at each pole.
• Sinusitis associated with fungal infections typically reveal
dense masses of pigmented, branched and septate hyphae
Chromoblastomycosis
•10% potassium hydroxide (KOH) show
muriform cells (aggregation of dark brown
cells that resemble stones in a stone wall)
or clerotic bodies, which are rounded,
brown, 4 to 10 mm in diameter, and have
fission planes. They resemble copper
pennies
Mycetoma and Phaeohyphomycosis
• yellowish brown, septate to moniliform hyphae (string of beads),
with or without budding yeast cells present.
• P. boydii reveal them to be white to yellow and 0.2 to 2 mm in
diameter
• loosely arranged, intertwined septate hyaline hyphae
cemented together
• methenamine silver stain used to detect fungal elements in
tissues stains fungi black, which makes determining whether they
are hyaline septate or dematiaceous septate molds impossible
• Fontana-Masson stain, which stains the melanin and melanin- like
pigments
• Culture - final confirmation
Serologic Testing

• not useful for the diagnosis of invasive dematiaceous fungal

disease
Molecular Methods

•Nucleic acid amplification assays are not

routinely used
•Amplification tests have been developed (DNA)
•Nucleic acid–based sequencing of ribosomal
genes may be used for the identification of
fungal isolates
Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry

• successfully used to identify clinically relevant fungal

isolates including yeasts and molds
Cultivation

• should be interpreted in conjunction with the results

of the direct examination
Superficial Infections
•H. werneckii
•causative agent of
tinea nigra
•recovered on common
fungal media
•grows very slowly
•olive to black, shiny,
and yeastlike (initial)
•usually grow within 2
to 3 weeks
• P.hortae
• Causative agent of black piedra
• culture medium lacking cycloheximide
• very slow-growing, appear dark brown to black, and produce aerial mycelium
• Cyphellophora
• slender, curved, one- to three- septate conidia
• produced on collarettes
• Cultures are typically melanized without budding cells
• Exophilia
• black yeast
• demonstrates a high degree of morphologic variability
• Colonies initially appear moist and then become wooly or velvety
• conidia are produced from narrow scars or extensions referred to as annelidic
• capable of growth at 40°C
• fail to assimilate nitrate
• Phialophora
• phialides (flask-shaped) and have no budding cell
• Neoscytalidium dimidiatum
• rapidly growing black arthroconidia
Mycetoma
• White Grain Mycetoma
• P. boydii grows rapidly(5-10days)
• begins as a white, fluffy colony that changes in several weeks to a brownish gray (the so-called
“mousy gray”) colony
• Acremonium spp. that cause mycetomas, such as Acremonium falciforme, grow slowly and
produce gray to brown colonies
• Black Grain Mycetoma
• Madurella spp. and E. jeanselmei
• slow growing, unlike colonies of Curvularia spp.
• Madurella spp. vary from white (during the early phases of growth) to olive- brown; a brown
diffusible pigment is characteristic of this fungus
• Colonies of E. jeanselmei appear yeastlike and darkly pigmented (olive to black), but in time
develop a more velvety appearance with the production of aerial hyphae
• Curvularia spp. produce a fluffy or downy, olive-gray to black colony, and growth is rapid
• T. grisea
• forms slow- growing, velvety colonies that appear smooth or radially furrowed and dark gray or olive brown to black. The
reverse side of the colonies appears black. The hyphae are septate and nonsporulating
Chromoblastomycosis

• Cladophialophora, Phialophora spp., and Fonsecaea

spp., are all dematiaceous
• slow growing and produce heaped-up, slightly folded,
darkly pigmented colonies with a gray to olive to black
and velvety or suedelike appearance.
Phaeohyphomycosis
• rapidly growing dematiaceous
• identification relies on microscopic examination
• Alternaria spp.- rapidly growing, fluffy, and gray to gray-brown or gray- green
• Curvularia spp. produce rapidly growing colonies that resemble those of Alternaria spp
• Bipolaris spp. pro- duce colonies that are gray-green to dark brown and slightly powdery, as do
Exserohilum spp
• E. jeanselmei and E. dermatitidis grow slowly (7-21 days) and initially produce shiny, black, yeastlike
colonies.
• E. dermatitidis often is mucoid and may be brown, compared with E. jeanselmei, but the two
organisms are very similar in appearance
• become filamentous and velvety with age as a result of the production of mycelium
• E. spinifera produces large, stiff conidiophores
• Cladophialophora bantiana produces long, poorly branched conidial chains
• Rhinocladiella mackenziei produces pale brown conidiophores with elongated conidia on denticles
(projection or peg) and may produce exophiala-like budding cells in culture
• Verruconis gallopava produces a rusty- brown to olive colony with one- to three-septate condia on
small denticles
Approach to Identification
Chromoblastomycosis:
Cladosporium,Phialophora, and
Fonsecaea spp.
Pleurostomophora richardsiae (previously Phialophora richardsiae)

• produces phialides with a flattened collarette

• Conidia are produced endogenously and occur in
clusters at the tip of the phialide
Phialophora spp.: P. verrucosa

•produces phialides, each with a distinct cup-

or flask-shaped collarette
Cladophialophora (Cladophialophora carrionii)

•sporulation with long chains of elliptical

conidia (2-3 mm 3 4-5 mm) borne from
erect, tall, branching conidiophores
Fonsecaea spp.

•Conidial heads with sympodial arrangement of

conidia are seen, with primary conidia giving rise
to secondary conidia
Phaeohyphomycosis: Alternaria, Bipolaris,
Cladophialophora, Curvularia, Exophiala,
Exserohilum, and Phialophora spp.
Alternaria spp.

•hyphae are septate and golden-brown

pigmented; conidiophores are simple
but sometimes branched.
Conidiophores bear a chain of large,
brown conidia resembling a drumstick
and contain both horizontal and
longitudinal septa
Bipolaris spp.
• Hyphae are dematiaceous and septate
• Conidiophores- bent (geniculate) at the locations
where conidia are attached;
• conidia are arranged sympodically and are oblong to
fusoid.
• The hilum protrudes slightly
• Germ tubes are formed at one or both ends, parallel
to the long axis of the conidium, when the fungus is
incubated in water at 25°C for up to 24 hours
Cladophialophoraspp.
• Hyphae are septate and brown
• Conidiophores are long, branched, and give rise to
branching chains of darkly pigmented, budding conidia.
• Conidia usually are single-celled and exhibit prominent
attachment scars (dysjunctors)
• The cells that produce the branch points are often referred
to as shield cells
• commonly fails to reveal chains of conidia on wet mounts,
because conidia are so easily dislodged
Curvularia spp.

•hyphae are dematiaceous and septate

•Conidiophores are geniculate
•Conidia are arranged sympodically and are
golden-brown, multicelled, and curved, with
a central swollen cell
•end cells are lighter in color than the
swollen cell
Exophiala spp.

•Exophiala species E. jeanselmei and E.

dermatitidis
• are considered here; although other species exist, they
are recovered far less commonly in the clinical laboratory
•dematiaceous, yeastlike cells
•The microscopic features of young colonies
of Exophilia spp.
• exhibit dematiaceous, yeastlike cells
• Feltlike, filamentous colonies produce
dematiaceous hyphae and conidiophores that are
cylindrical and have a tapered tip
• Annellations may be visible at the tip, and clusters
of oval to round conidia are apparent
Exserohilum spp.
•Hyphae are septate and dematiaceous.
Conidiophores are geniculate, and conidia are
produced sympodically. Conidia are elongate,
ellipsoid to fusoid, and exhibit a prominent
hilum that is truncated and protruding
•The conidia are multicellular, have
perpendicular septa, and usually contain five
to nine septa.
Antifungal Susceptibilities

• Amphotericin B and the azoles have demonstrated

clinical effectiveness against infections with melanized
fungi
• Triazoles, posaconazole, and voriconazole have a
broad spectrum of activity against most of these fungi
• ***Occasional treatment failure of mycetoma has
been associated with the use of voriconazole
Midterm: Pre-quiz #1
1. It is characterized by dark
coloration and has ability to
produce melanin.
2. Stain that is useful to detect
melanization that may appear as
hyaline molds using light microscopy
3. It is the most common
fungal agent associated
with mycetoma
4. Used to detect fungal elements in
tissues stains fungi black, which makes
determining whether they are hyaline
septate or dematiaceous septate
molds impossible.
5. causative agent of tinea nigra ,
recovered on common fungal
media, grows very slowly , olive to
black, shiny, and yeastlike (initial)
and usually grow within 2 to 3
weeks
Give the disease associated:

6. P. boydii and Acremonium and Fusarium spp.

7. Madurella spp., Exophiala jeanselmei, and
Curvularia spp.
8. Cladophialophora, Phialophora, and
Fonsecaea spp.
9. E. jeanselmei; E. dermatitidis; and Curvularia,
Bipolaris, Alternaria, and Exserohilum spp.
10. Identify the organism:
Opportunistic Atypical Fungus:

Pneumocystis jirovecii
•1999 - causes a pneumonia in
immunocompromised humans (pneumocystis
pneumonia (PCP) )
•opportunistic, atypical fungus that infects
immunocompromised hosts and mostly manifests
as PCP
•was believed to be a trypanosome
•its morphology is similar to that of protozoa
•responds to antiprotozoal drugs but not to
antifungal drugs in patients with pneumocystosis
•exists as three forms in its life cycle:
• trophic form (trophozoite)
• sporozoite (precyst),
• ascus (cyst)-diagnostic form
•differs from other fungi (cell membrane contains
cholesterol rather than ergosterol)
•contains only one or two copies of the small
ribosomal subunit gene
•DNA sequence analysis of the small ribosomal
subunit gene has disclosed a greater sequence
homology with the fungi than with the protozoa
Epidemiology
•worldwide distribution
•most commonly presents as pneumonia in an
immunocompromised host
•transmitted person-to-person via airborne
particles
•Most children by age 2 to 4 years have antibodies
•Pneumocystis DNA was present in 24 of 72 infants
(nasopharyngeal specimens )
•seroconversion occurred in 85% of infants by 20
months of age
•most common opportunistic infection among
those with HIV or AIDS in the United States
•introduction of highly active antiretroviral therapy
(HAART) for patients with HIV has reduced the
incidence of disease
Pathogenesis and Spectrum of Disease

• After P. jirovecii is inhaled, the trophic form of the pathogen is

believed to adhere to type I pneumocytes
• replicate extra- cellularly while bathed in alveolar lining fluid
• technique does not provide direct staining of the organisms
• Methenamine silver or another fungal stain - identify the cyst
form of the organism in lung tissue
• interstitial mononuclear inflammatory response
• interstitial plasma cell pneumonia (before)
Symptoms of PCP
• nonproductive cough
• low-grade fever
• dyspnea
• chest tightness
• night sweats
• Extrapulmonary infection - 0.6% to 3% of postmortem
samples
• Extrapulmonary cysts
• Multiple sites of infection typically indicate a more rapid
disease progression and fatal outcome
Laboratory Diagnosis
Specimen Collection and Transport
•Respiratory specimens
•bronchoalveolar lavage (BAL) - best
•Sputum specimen - induced sputum
• tracheal aspirates, pleural fluid, transbronchial biopsy, or
cellular material from bronchial brushings
•Nasopharyngeal and oropharyngeal samples
•high sensitivity and specificity (nucleic acid–
based testing)
•extrapulmonary pneumocystis
• biopsy of the infected organ and histologic staining
Direct Detection Methods
Stains

• Diagnosis
• based on the clinical presentation
• radiographic studies
• direct or pathologic examination of respiratory
samples
• biopsy material
• flexible-walled trophic forms are the predominant
morphology of the organism- difficult to visualize
• discernible in Giemsa-stained material
• Giemsa stains
• nuclei of all the various life cycle stages - reddish purple with a light blue
cytoplasm
• firm-walled cyst
• Cysts are more easily recognized than the trophic form
• calcofluor white, methenamine silver, and
immunofluorescent staining
•Cysts
•spherical to concave
• uniform in size (4-7 mm in diameter)
•do not bud
• contain distinctive intracystic bodies
Four most common staining methods

• immunofluorescent staining (Merifluor

Pneumocystis; Meridian Bioscience, Cincinnati, OH)
• Monofluo Pneumocysitis jirovecii IFA (Bio-Rad,
Hercules, CA)
• Calcofluor white staining (Fungifluor; Polysciences,
Warington, PA)
• Methenamine silver staining (GMS and DiffQuick;
Baxter Scientific, McGaw Park, IL)
•immunofluorescent method - greater
sensitivity than the other three but a
smaller negative predictive value
•confirmatory method should be
performed
Direct Detection of (1-3)-Beta-D-glucan

• used to successfully diagnose infections

• Fungitell Assay (Associates of Cape Cod, Falmouth, MA) uses
patient serum

• *****It is important to use additional diagnostic information

and confirmatory testing
Molecular Methods

•nucleic acid amplification assays

•real-time polymerase chain reaction (PCR)
methods

•results must be directly correlated with the

patient’s history and clinical presentation
Cultivation

•very difficult to cultivate outside the lung;

therefore routine culture methods are not
performed
Serologic Testing

•not useful for the diagnosis of

pneumocystosis
The Yeast
GENERA AND SPECIES TO BE CONSIDERED

• Blastoschizomyces spp
• Candida spp.
• Cryptococcus spp.
• Pseudozyma spp.
• Rhodotorula spp.
• Saccharomyces cerevisiae
• Sporobolomyces spp.
• Trichosporon spp.
• Malassezia spp.
General Characteristics

• eukaryotic
• unicellular
• round to oval
• 2 to 60 um
• microscopic morphologic features have limited usefulness
• microscopic morphology on cornmeal agar is useful when considered in
conjunction with the biophysical profile
• moist, creamy colonies
• produce a capsule resulting in a shiny or mucoid colonial appearance
• may produce bright pigments or appear hyaline or melanized
Epidemiology
• Yeast - ubiquitous
• particularly susceptible to infection
Blastoschizomyces Spp.

• white to cream-colored
• moist colonies that may have radiating margins
• produces hyphae, pseudohyphae, and annelloconidia
• found in regions where the summers are hot and dry
with warm, wet winters
• primarily identified in immunocompromised patients
Candida spp.

• most commonly encountered opportunistic fungal

infections
• fourth most common cause of hospital- acquired
bloodstream infections
• C. albicans is the most commonly isolated yeast (60%
to 70% of yeast infections)
Cryptococcus spp.

• divided into the two species and five serotypes

• Serotype A - C. neoformans var. grubii
• Serotype D - C. neoformans var. neoformans
• Serotypes B and C - independent species C. gattii
• Canavanine-glycine-bromothymol blue (CCB) agar has been
recommended for the differentia- tion of serotypes A/D and
B/C
Malassezia, Pseudozyma, Rhodotorula,
Saccharomyces, Sporobolomyces, and
Trichosporon spp.
Pathogenesis and Spectrum of Disease
Blastoschizomyces spp.

• emerging pathogen that is often isolated from

patients with leukemia, renal transplants, ambulatory
dialysis, and osteomyelitis
Candida albicans
• Candidiasis
• oroesophageal candidiasis, intertriginous candidiasis (in which
skin folds are involved), paronychia, onychomycosis, respiratory
infections, vulvovaginitis, thrush, pulmonary infection, eye
infection, endocarditis, meningitis, fungemia or candidemia, or
disseminated infection
• Thrush, an infection of the mucous membranes in the mouth, is
considered a localized infection
• newborns, patients with human immunodeficiency virus (HIV)
infection, individuals with diabetes, Creamy patches or colonies
appear on the tongue and mucous membranes
• and patients undergoing chemotherapy
Non-albicans Candida

• once believed to not cause disease

• C. glabrata to become resistant to common antifungal
drugs such as fluconazole and echinocandins
• endocarditis, meningitis, and disseminated disease
• C. tropicalis has been shown to be prevalent in patients
with hematologic malignancies
• C. parapsilosis is the primary cause of fungemia in the
neonatal intensive care unit (NICU)
• second most commonly isolated
Cryptococcus neoformans

• Cryptococcosis is an acute, subacute, or chronic fungal

infection that has several manifestations
• C. neoformans var. grubii, C. neoformans var. neoformans,
and C. gattii
• major human pathogens
• C. neoformans
• very characteristic polysaccharide capsule
• C. gattii
• modulate the host’s immune system by reducing the
inflammatory response or evading the immune system
completely
Malassezia spp.

• tinea versicolor
• skin infection characterized by superficial, brownish, scaly areas on
light-skinned individuals and lighter areas on dark-skinned people
Pseudozyma spp.

• environmentally associated plant pathogens that appear as beige to

tan, moist, wrinkled colonies on routine laboratory media
Rhodotorula spp.

• resemble Cryptococcus spp., appearing as round, oval-shaped,

budding yeasts that produce capsules
• normal resident microbiota of the human skin
• typically be recovered in shower and bathtub grout, shower curtains,
and toothbrushes
Saccharomyces cerevisiae

• common yeast that is used in baking and the

preparation of a variety of food products
• isolated from cases of thrush, vulvovaginitis, and BSIs
Sporobolomyces spp.

• appear as salmon to pink on routine laboratory media

• ubiquitous throughout the environment
Trichosporon spp.

• Trichosporonosis
• primarily associated with human disease almost exclusively
in immunocompromised patients, particularly those with
leukemia
• White piedra - uncommon fungal infection of
immunocompetent patients
Laboratory Diagnosis

• Specimen Collection, Transport, and Processing

• Refer from our previous discussion
Stains

• Candida spp. - budding yeast cells (blastoconidia) 2 to 4 mm

in diameter and/or pseudohyphae showing regular points of
constriction / resembling links of sausage
Antifungal Susceptibility Testing,
Therapy, and Prevention
• Cryptococcus spp.(C. neoformans)
• India ink preparation has been the most widely used
method
• spherical, single-or multiple-budding, thick-walled yeast 2
to 15 mm in diameter
• surrounded by a wide, refractile polysaccharide capsule
Malassezia spp.

• often detected through direct microscopic

examination of skin scrapings
• oval- or bottle-shaped cells that exhibit monopolar
budding in the presence of a cell wall with a septum at
the site of the bud scar
• morphology is commonly described as “spaghetti and
meatballs
Trichosporon spp.

• reveals hyaline hyphae, numerous round to rectangular

arthroconidia, and occasionally a few blastoconidia
• Hyaline hyphae 2 to 4 mm wide and arthroconidia
Antigen Detection
• may be detectable using a common fungal cell wall antigen,
beta-1,3-glucan
Molecular Methods

• Nucleic acid amplification tests (NAATs) have been

developed for a variety of yeast species
• Real-time polymerase chain reaction (PCR) methods
are now commercially available
• sensitivity and specificity from 95% to 92%
• Multiplex PCR method is much faster and more
sensitive than the current phenotypic tests
Cultivation

• Candida spp. - produce smooth, creamy white colonies, but

some produce dry, wrinkled, dull colonies
• Cryptococcus spp. - cultured on routine fungal culture media
without cycloheximide / smooth, white to tan colony that may be
mucoid to creamy
• Trichosporon spp. - most are cream-colored, heaped, dry to
moist, and wrinkled. Some may appear white, dry, and powdery
• Malassezia spp. - are creamy and white to off-white /
infrequently cultured in the clinical laboratory
Approach to Identification
Candida spp.

• most commonly identified by the utilization of specific

substrates and the fermentation or assimilation of particular
carbohydrates
• C. albicans may be identified by the production of germ
tubes or chlamydoconidia
Germ Tube Test

• most generally accepted and economical method used in the

clinical laboratory to identify yeasts
• hyphal-like extensions that are produced without a
constriction at the point of origin from the yeast cell
Cryptococcus neoformans - typically made by
identifying the encapsulated yeast in the spinal fluid
using India ink
- presumptive identification of C. neoformans may
be based on rapid urease production and failure to
utilize inorganic nitrate
-Final identification of C. neoformans usually is
based on substrate utilization pat- terns and pigment
production on niger seed (thistle or birdseed) agar
Serologic Testing

• The use of serologic techniques for the detection of

the cryptococcal polysaccharide capsule
glucuronoxylomannan (GXM) may be completed using
latex agglutination or enzyme-linked immunosorbent
assay (ELISA)
Rapid Urease Test

• useful tool for screening for urease-producing yeasts

recovered from respiratory secretions and other clinical
specimens
Rapid Trehalose Test

• used for presumptive identification of C. glabrata

• designed to provide information that helps the
physician select the appropriate antifungal agent to
treat a specific infection
• Clinical Laboratory Standards Institute (CLSI) sets the
standards for antifungal susceptibility testing
Antifungal Therapy and Prevention

• Polyene Macrolide Antifungals

• Azole Antifungal Drugs
• Echinocandins
• Allylamines
Polyene Macrolide Antifungals

• most commonly used antifungal agents

• Amphotericin B -commonly infused intravenously to treat deep-
seated fungal infections; produced by the actinomycete
Streptomyces nodosus
• invasive aspergillosis)
• Candida spp.,
• Cryptococcus spp.,
• members of the Mucorales
• Nystatin -produced by Streptomyces noursei
• used locally to treat oral or vulvovaginal candidiasis
• Griseofulvin -produced by a species of Penicillium
• used to treat dermatophytoses
• 5-Fluorocytosine (Flucytosine)
• used in combination therapy for treating infections by Candida
spp. and Cryptococcus spp.
Azole Antifungal Drugs
• Clotrimazole and Miconazole
• topical or intravaginal application
• useful in mild cases of dermatophytosis
• Fluconazole
• excellent activity against most Candida spp. and Cryptococcus spp.
• Ketoconazole
• useful in mild cases of paracoccidioi- domycosis and is an alternative to amphotericin B
for infections caused by Blastomyces or Histoplasma spp.
• Itraconazole
• effective in cases of aspergillosis, sporotrichosis, cryptococcosis, and onychomycosis
• Voriconazole
• useful activity against some Fusarium strains
• strains of C. glabrata and some strains of other Candida spp. that are fluconazole-
resistant
• Posaconazole
• effective against dermatophytes, including Candida spp., Aspergillus spp. C.
neoformans, and Mucorales
Echinocandins

•effective against Candida spp., including strains

that are resistant to fluconazole.
•used for the prophylaxis and treatment of
Candida spp. infections in adult and pediatric
patients
•ineffective against fungi that lack 1,3-beta-D-
glucan, including C. neoformans, Trichosporon
spp., Fusarium spp., and the mucoraceous molds
Allylamines

• Terbinafine (Lamisil) and Naftifine

• used in dermatophyte infection
• Selenium Sulfide
• antifungal activity against Malassezia furfur
• Potassium Iodide
• therapy of choice for cutaneous/ lymphatic sporotrichosis
“You're never too young to
make a difference. Hindi
kayo bata lang. Pilipino
kayong may kakayahan
iangat ang kapwa.”
--MARIA LEONOR ROBREDO
On AdDU's Social Sciences Week VerSSatility: Resilience in Adversity
 MYCOLOGY -
VIROLOGY
Marc Holden Maranan Micosa, RMT,MSMTc,MLS(ASCPi)cm
College of Medical Technology
 OVERVIEW OF THE
METHODS AND
STRATEGIES IN VIROLOGY
• viral disease exists in ancient records - 23 BC
• Eschunna Code of ancient Mesopotamia
• “the bite of mad dogs to affect disease on humans”
• Homer, author of the Iliad, characterizes Hector as
“rabid.”
• Aristotle’s work, The Natural History of Man – 4TH
CENTURY B.C
• “madness” in dogs that “causes them to become very
irritable and all mammals they bite become diseased.”
• refer to the rabies virus (through infected animal’s
saliva)
•Retroviruses - herpes viruses and
papillomaviruses
VIRUS
•submicroscopic
•obligate intracellular parasite
•among the smallest of all infectious agents
•animal, plant, bacterial cell
•found in every ecosystem
•incapable of replication without a living host
•Much is still unknown about the origins of viral agents
•transmission of an animal virus to a human
•(SARS)
•West Nile
•influenza A H5 viruses
•2009 H1N1 virus aka “swine flu”
•SARS-CoV-2 virus aka COVID-19 (believed
to be bat origin) – Wuhan, China (2019)
•influenza virus - deadliest viruses - 1700s in Italy
•“influence” of miasma (bad air)
• pandemic – emergence worldwide with prolonged human-to-human
transmission
• The genetic changes in viral genomes may result from
• antigenic shift (major changes that result in novel viral antigens)
• antigenic drift (minor changes that occur infrequently)
• Spanish flu pandemic –one of the most deadly influenza (1918-1919)
• novel influenza virus of avian origin
• more than 50 million deaths worldwide
• young and healthy individuals
• Vaccination (immunization)
• valuable tool in the control
• yellow fever and rabies
• influenza, acquired immunodeficiency syndrome (AIDS), and hepatitis
(unsuccessful)
• Since 1988 - 50 new viruses have been identified
General Characteristics
• Virus particles (virions)
• inner nucleic acid core (either DNA or RNA)
• Capsid-protein coat that surrounds and protects the nucleic acid
• lipid-containing envelope (for larger virus)
• transmitted by
• direct contact- respiratory and sexual
• parenteral contact
• Viruses that do not have an envelope are often referred to as “naked”
viruses
• very resistant to environmental factors
• transmitted by the fecal-oral route
• Many viruses have glycoprotein spikes
• nucleocapsid -nucleic acid genome surrounded by a symmetric protein
coat
• nucleic acid genome - encode the proteins required for viral
penetration, transmission, and replication
• viral genome structure - determines the mechanism for viral
replication
• (+) sense–strand RNA, (–) sense– strand RNA, and DNA
genomes
• may be single- or double-stranded molecules
• viral capsid - protects the viral genome and is re- sponsible for
the tropism to specific cell types in naked viruses
• typically are composed of repeating structural subunits
(capsomeres)
•most common capsid structures
•helical
•irregularly shaped capsids
•helical form
•spiral-shaped
• icosahedral structure
•cubical and have 20 flat sides
•Viruses that cause disease in humans range from
approximately 20 to 300 nm
•cannot be detected with a light microscope
(even the largest)
•Less than ¼ the size of staph.
•electron microscope in the 1930s were viruses
visualized
Virus Taxonomy
• determined by the International Committee on Taxonomy
of Viruses (ICTV) of the Virology Division of the
International Union of Microbiological Societies
• divided into categories: six orders (name ending in -virales),
87 families (-viridae), 19 subfamilies (-virinae), 348 genera
(-virus), and 2290 species.
• information related to host range, transmission, disease
pathology, antigenicity, and viral particle properties, such
as size, envelope, capsid structure, physical properties,
genome type, and configuration
Three basic properties:
• (1) viral morphology;
• (2) method of replication, including genome organization
(whether the genome is RNA or DNA and single- or double-
stranded);
• (3) presence or absence of a lipid envelope
Viral Replication
•INFECTIOUS CYCLE
• Attachment
• Penetration
• Uncoating
• Macromolecular synthesis
• Viral assembly
• Release
Attachment

• Aka adsorption
• first step
• recognition of a suitable host cell
Penetration

• virus entry
• viruses enter the host cell
• fusion of the viral envelope with the host cell
membrane
• formation multinucleated cells called syncytia
• used to determine the presence of virus in cell
cultures or stained smears of clinical specimens
Uncoating

•occurs once the virus has been internalized

•capsid is removed
•release the viral genome for delivery of the
viral DNA or RNA to its intracellular site of
replication
Macromolecular synthesis

•production of nucleic acids and protein polymer

•Viral transcription leads to the synthesis of
messenger RNA (mRNA), which encodes early
and late viral proteins
VIRAL ASSEMBLY

•process by which structural proteins, genomes,

and in some cases viral enzymes are assembled
into virus particles
•Envelopes are acquired during viral “budding”
from a host cell membrane
RELEASE

•intact virus particles occurs after cell lysis

(lytic virus) or by virus particle budding
from cytoplasmic membranes
•Each infected host cell results in as many as
100,000 virions
•1% of these may be infectious or “viable” in
the practical sense
Epidemiology

• transmitted from person to person

• respiratory , fecal-oral, and sexual contact routes
• trauma or injection with contaminated objects or needles
• tissue transplants (including blood transfusions)
• arthropod or animal bites
• during gestation (transplacental transmission)
PATHOGENESIS AND SPECTRUM OF DISEASE

• THREE CHARACTERISTIC CLINICAL PRESENTATIONS

• acute viral infection - evident signs and symptoms
• latent infection -no visible signs and symptoms, but the
virus is still present in the host cell
• lysogenic state (inserted into the host genome in a
resting state)
• maintained as a nuclear or cytoplasmic epi- some
• chronic or persistent infection - low levels of virus are
detectable and the degree of visible signs or symptoms
varies
•local (mucosal site ) à viremia à skin, salivary
glands, kidneys, brain, and other central nervous
system (CNS) tissues à symptoms
•Disease resolves when specific antibody and cell-
mediated immune mechanisms prevent
continued replication, spread of the virus, and
associated host immune responses
•Tissue is damaged as a result of lysis of virus-
infected cells or by immunopathologic
mechanisms directed against the virus that are
also destructive to neighboring tissue
• Most DNA-containing viruses
• herpes- virus group, remain latent in host tissues with no
observable signs or symptoms of disease
• Retroviruses and most DNA viruses establish a latent state
after primary infection
• In some cases, viral infection can promote transformation or
immortalization of host cells through expression of specific
viral proteins that affect the cell cycle, ultimately resulting in
dysregulation or uncontrolled cell proliferation
• Viruses with the ability to stimulate uncontrolled growth of
host cells are referred to as oncogenic viruses
• Several high-risk subtypes of human papillomaviruses (HPV)
are oncogenic
PREVENTION AND THERAPY

•Immunizations
•regular, thorough hand washing and avoiding
contact with others during episodes of evident
signs of disease
ANTIVIRAL AGENTS

• More than 70 antiviral drugs are formally licensed for

clinical use in the United States
• antiretroviral therapy (ART)
• human immunodeficiency virus (HIV) infections
VIRUSES THAT CAUSE HUMAN DISEASES

•Hundreds of viruses cause disease in humans

•comprise 4 orders, 25 families, 13 subfamilies,
and 66 genera
•For example:
•cause encephalitis
•HSV, many arboviruses, rabies virus, HIV,
measles virus
LABORATORY DIAGNOSIS

•demand for clinical virology laboratory

services has skyrocketed during the past
two decades
•conventional methods - fluorescence
microscopy and enzyme immunoassays
•detection in cell cultures
•nucleic acid amplification tests
• Laboratory scientists in a clinical virology laboratory
must be familiar with cell culture, enzyme
immunoassay, immunofluorescence methods, and
molecular methods
• laminar flow biologic safety cabinet (BSC), fluorescence
microscope, inverted bright field microscope,
refrigerated centrifuge, incubator, refrigerator and
freezer, roller drum for holding cell culture tubes during
incubation, and enzymes for molecular testing
instrumentation
• Standard precautions and Biosafety Level 2 conditions
are needed for community and most nonretroviral
laboratories.
SPECIMEN SELECTION AND COLLECTION
• depends on the specific disease syndrome, viral agents suspected, and time
of year
• should be collected as early as possible after the onset of symptomatic
disease
• Virus may no longer be present as early as 2 days after the appearance of
symptoms
• Swab specimens should not contain chemicals or other compounds that
may be toxic to cultured cells and therefore are unsuitable for viral
specimen collection
• Calcium alginate swabs interfere with PCR, the recovery of some enveloped
viruses, and fluorescent-antibody tests and therefore should not be used
Throat, Nasopharyngeal Swab, or Aspirate

•Throat swabs are acceptable for the recovery of

enteroviruses, adenoviruses, and HSV
•nasopharyngeal swab or aspirate specimens are
preferred for the detection of RSV and influenza
parainfluenza viruses
•Rhinovirus detection requires a nasal specimen
Bronchial and Bronchoalveolar Washes

•excellent specimens for detecting

viruses that infect the lower
respiratory tract, especially
influenza viruses and adenoviruses
Rectal Swabs and Stool Specimens

•used to detect rotavirus, enteric

adenoviruses (serotypes 40 and 41), and
enteroviruses
•PCR or electron microscopy for detection
•rectal swab is inserted 3 to 5 cm into the
rectum and rotated against the mucosa to
obtain feces
Urine

•CMV; mumps, rubella, and measles

viruses; polyomaviruses; and
adenoviruses can be detected in urine
Skin and Mucous Membrane Lesions

• Enteroviruses, HSV, VZV, and in rare cases CMV or pox

viruse
• Tzanck smear if PCR testing is not available
• carefully unroofing the vesicle
Sterile Body Fluids Other Than Blood

• CSF and pericardial and pleural fluids,

• enteroviruses, HSV, VZV, in- fluenza viruses, or CMV
Blood

•used primarily to detect CMV

•HSV, VZV, enteroviruses, and adenovirus
occasionally may be encountered
•Heparinized, citrated, or
ethylenediaminetetraacetic acid (EDTA)
anticoagulated blood is acceptable
Bone Marrow

•should be added to a sterile tube with

anticoagulant
•Heparin, citrate, or EDTA
•nucleic acid testing
Tissue

•useful for detecting viruses that commonly

infect the lungs , brain and gastrointestinal
tract
•Fresh tissue is preferred for nucleic acid
assays
Genital Specimens

•for detection of HSV and human

papillomavirus (HPV)
•may be collected using a swab or brush and
placed in viral transport media
Serum for Antibody Testing

•Acute and convalescent serum specimens

may be needed to detect antibody to
specific viruses
SPECIMEN TRANSPORT AND STORAGE
• should be processed immediately
• Specimens for viral isolation should not be allowed to sit at room or
higher temperature
• should be kept cool (4°C) and immediately transported to the
laboratory
• If a delay in transport is unavoidable, the specimen should be
refrigerated, not frozen, until processed
• 5 days specimens are held at 4°C
• 6 days or longer should be at –20°C, preferably –70°C
• synthetic swabs, such as rayon and Dacron,
• cotton tips and wooden shafts are not recommended
• Calcium alginate is not acceptable for the culture-based detection of
HSV
•Examples of successful transport media
•Stuart’s medium
•Amie’s medium
• Leibovitz-Emory medium
•Hanks’ balanced salt solution (HBSS)
•Eagle’s tissue culture medium
•commercially available M4, M5, and universal
transport media
Specimen Processing

• Specimens for viral culture should be processed immediately

upon receipt in the laboratory
• patient identification and demographics, each specimen for
virus isolation should be accompanied by a requisition
• the source of the specimen
• clinical history or viruses suspected
• date and time of specimen collectio
• Viral specimens should be processed in a BSC whenever
possible
LABORATORY PROCESSING OF VIRAL SPECIMENS
Processing Based on Requests for Specific Viruses
• Arboviruses - Serologic tests
• Cytomegalovirus -conventional cell culture, shell vial assay, antigenemia
immunoassay, or molecular methods
• Enteroviruses - conventional cell culture and PCR
• Epstein-Barr Virus - Serologic tests
• Hepatitis Viruses -detected using serology, antigen detection, or PCR
• Herpes Simplex Virus - MRC-5 or mink lung fibroblast cell lines are
recommended, along with a continuous cell line such as A-549
• Cultures , Real-time PCR , enzyme-linked viral-induced system
• Human Immunodeficiency Virus and Other Retroviruses - serologic
methods, molecular methods (reverse transcriptase PCR [RT-PCR]) , HIV-1
enzyme-linked im- munosorbent assay (ELISA)
• anti-HIV-I and HIV-2 and HIV-1 p24 antigen immunoassay
• Influenza A and B Viruses - conventional cell culture, shell
vial culture, membrane enzyme immunoassay (EIA), direct
staining of respiratory tract secretions using FA methods and
RT-PCR (most recommended)
• Pediatric Respiratory Viruses - fluorescent staining of
respiratory secretions or rapid cell culture (shell vial)
• Gastroenteritis Viruses - Electron microscopy (EM) can be
used / Immunoassays for rotaviruses and enteric adenovirus
types 40 and 41 are commercially available / RT-PCR may be
used to detect norovirus
• TORCH - infection in newborns is appropriate during
pregnancy
• Toxoplasma, rubella, cytomegalovirus, and herpes simplex
virus
• Varicella-Zoster Virus - chickenpox (varicella) and shingles
(zoster)
• staining cells / culturing cells and vesicular fluid /PCR
testing / cytologic staining method
Virus Detection Methods

• Cytology and Histology - characteristic viral inclusion /

morphologic study of cells or tissue
• Electron Microscopy - most helpful for detecting viruses
• most useful for detecting gastroenteritis viruses that
cannot be detected by other methods
• encephalitis-causing viruses that are undetectable with
cell culture
• Immunodiagnosis (Antigen Detection) - detect viral antigen in
patient specimens
• Molecular Methods - generate results within 2 to 6 hours
• Enzyme-Linked Virus-Inducible System - uses a baby hamster kidney (BHK)
cell culture system
• Cell Culture
• Conventional Cell Culture
• Shell Vial Cell Culture - rapid modification of conventional cell culture
• Immune Status Testing - used to determine if patients have been infected
with (or vaccinated for) a virus
• Serology Panels - less helpful than using a battery of antigens to test for
antibody
Preservation and Storage of Viruses

• can be stored by freezing at –70°C or in liquid nitrogen

• Freezing at –70°C is more practical for clinical
laboratories