MYCOLOGY AND VIROLOGY

STUDY GUIDE
(Prepared by: Dr. J. Bandalan)

REFERENCES:
Bailey and Scott’s Diagnostic Microbiology 12th ed by Forbes, et al
Clinical Diagnosis and Laboratory Management by John Bernard Henry
Medical Microbiology 25th ed by Jawetz
Microbiology by Zinsser
Introduction to Microbiology 9th ed by Tortora
Fungal Diseases in the Orient by Glenn Bulmer

MYCOLOGY
 Derived from the term Mykos (mushroom)
 Study of fungi which include molds and yeasts
General Characteristics
 Less than 300 species of fungi cause human or animal diseases
 Natural habitat are water, soil and decaying organic matter
 Variable size and shape
 Single-celled or multi-celled
 Eukaryotic organisms, each fungal cell has at least a membrane-bound nucleus and
organelles
 Exist as parasites or saprophytes
 Reproduction: Asexual, Sexual, Parasexual
 Nutritional requirements
o Chemoheterotrophs - obtain their nutrients by absorbing chemicals found in the
environment
o Fungi survive by secreting enzymes that degrade organic substrates into soluble
nutrients
Fungi differ from bacteria
 Fungi grow best with a pH of 5
 Almost all molds are aerobic; most yeasts are facultative anaerobes
 Most fungi are more resistant to osmotic pressure than bacteria
 Fungi can grow on substances with a very low moisture content
 Fungi require less nitrogen than bacteria for growth
 Fungi are often capable of metabolizing complex carbohydrates, such as lignin (a
component of wood), that most bacteria cannot use for nutrients
Harmful effects of fungi
 Contaminants of food and therefore are important causes of spoilage of food
 Cause human disease in 3 general ways
o Fungous allergies
o Mycotoxicoses: aflatoxin (liver carcinogen) and amatoxins (liver cirrhosis)
o Mycoses
 Beneficial effects of fungi
o Yeasts (Saccharomyces)
o Preparation of vaccines (Hepatitis B vaccine)
o Sources of drugs (Penicillin, Statins, Antianxiety drugs))
o Higher fungi, mostly basidiomycetes may be eaten directly as mushrooms

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 o Saprophytic fungi in soil produce degradative enzymes which are essential for
the biologic recycling of organic matter
Morphology of Fungi
 Yeasts
o Unicellular growth of fungi
o Spherical or ellipsoidal (3 – 15 micra)
o Most reproduce by budding (forming blastoconidia), few by binary fission
o Some reproduce buds that characteristically fail to detach and become
elongated, producing a chain of elongated yeast cells that resemble hyphae and
are called pseudohyphae
o Colonies are pasty, opaque, 0.3 – 5 mm in diameter
o Few species produce pigments but most are cream-colored
o Yeasts have similar microscopic and colonial characteristics and therefore
species identification is based on various biochemical tests
 Molds
o Produce multicellular, filamentous colonies
o Irregular & dry colonies consisting basically of branching cylindrical tubules
varying in diameter from 2 – 10 micra, called hyphae
o Hyphal growth occurs by apical elongation
o Each part of the hypha is capable of growth, and when a fragment breaks off, it
can elongate to form a new hypha
o Hyphae grow to form a filamentous mass of intertwining strands called a
mycelium, which is visible to the naked eye
o 2 parts of hyphae
 Vegetative portion or thallus, which grows in or on a substrate and
absorbs water and nutrients
 Reproductive or aerial portion, which project above the surface of the
agar medium and contains the fruiting bodies that produce the
reproductive structures such as spores
o Hyphae are classified as
 Septate if with cross-walls which divide the hyphae into distinct
uninucleate cell-like units
 Nonseptate or coenocytic if cross-walls are absent and appear as long
continuous cells with many nuclei
 Zygomycetes: Mucor, Rhizopus, Absidia
o Hyphae can either be
 Dematiaceous if fungi produce melanin-like pigments are dark-colored
 Agents causing Chromoblastomycosis: Phialophora, Exophiala,
Curvularia, Alternaria
 Hyaline if fungal structures are colorless

Dimorphism
 The ability of some fungi, most notably the pathogenic species, to grow in 2 forms under
different environmental (particularly thermal) conditions
 Grow as
o Yeast form at 37 C
o Mold form at room temperature
 Dimorphic fungi
o Sporothrix schenckii
o Coccidioides immitis
o Histoplasma capsulatum
o Blastomyces dermatitidis
o Paracoccidioides braziliense

Subcellular Structures

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  Capsule of Cryptococcus neoformans has antiphagocytic properties and is associated
with virulence
 Cell wall
o Comprise 15-30 % of the dry weight of fungus
o Generally thicker in yeasts than in molds
o Appears highly refractile under light microscope
o 80% or more of the cell wall is carbohydrate
 Major component is chitin (N-acetylglucosamine)
 Varying amounts of glucan, cellulose and mannan
o 10% of the cell wall is composed of protein and glycoprotein
o Fungal cell wall is poorly stained with routine H and E
o Helpful fungal cell wall stains are
 Periodic acid Schiff
 Methenamine silver stain
 Calcofluor white is a fluorescent stain
 Gram stain is useful for Candida
 Cell membrane
o Bilayered membrane composed of several phospholipids
o Contain sterols which are essential for the viability of fungi
o Principal fungal sterols are ergosterol and zymosterol

Reproduction
 Asexual
o Conidia
 Chlamydospore or chlamydoconidia
 Thick-walled, resistant, resting spores
 Produced by rounding up and enlargement of hyphal segments
 Candida albicans
 Blastospore or Blastoconidia
 Develop as daughter cell buds off from parent cell and is pinched
off
 Candida & Cryptococcus
 Pseudohyphae
 Candida
 Arthrospores or arthroconidia
 Formed by fragmentation of the septate hyphae into single
rectangular or barrel-shaped thick-walled spores
 Coccidioides immitis
 Macroconidia and microconidia
 Macroconidia
 Large, multiseptate, club, oval, spindle-shaped
 Cell wall is smooth or echinulate
 Microconidia
 Small, unicellular
 Round, elliptical. pyriform, tear-shaped
 Dermatophytes
 Microsporum
 Large multicellular macroconidia; microconidia
rarely produced
 Trichophyton
 Predominant forms are microconidia;
macroconidia uncommon
 Do not fluoresce on Wood’s lamp
 Epidermophyton

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  Macroconidia only; microconidia not produced
 Clusters of 2-4 macroconidia
 Phialides (Secondary branches)
 Aspergillus
 End of conidiophore expand into a large vesicle where
short phialides (sterigma) project from which arise the
chains of conidia
 In the tissues, appear as septated hyphae with
dichotomous branching (45 degree-angle branching)
 Penicillium
 Brush-like conidiophores that give rise to phialides from
which chain of conidia arise
 Phialophora
 Flask-shaped phialides with cup-shaped collarettes and
clusters of conidia at the end
 Annellids (Exophiala)
 Conidiophores are cylindrical with tapered tip and ringed
by clusters of conidia

o Sporangiospore
 Asexual spores contained in a sac-like or sporangium
 Unique among Zygomycetes
 Nonseptate hyphae
 Rhizopus, Mucor Absidia

 Sexual
o Requires formation of specialized structures so that fertilization or nuclear fusion
can occur
o Meiosis occurs with reduction division of 2 fertile cells, followed by merging of the
cells and nuclear fusion
o A fungal sexual spore results from sexual reproduction
o 3 phases
 Plasmogamy
 A haploid nucleus of a donor cell (+) penetrates the cytoplasm of
a recipient cell (-)
 Karyogamy
 The (+) and (-) nuclei fuse to form a diploid zygote nucleus
 Meiosis
 The diploid nucleus gives rise to haploid nuclei (sexual spores),
some of which may be genetic recombinants
o Types of sexual spores
 Ascospores – contained in a sac-like ascus
 Zygospores - fusion of 2 identical cells arising from the same hypha
 Oospores - fusion of cells from 2 separate non-identical hyphae
 Basidiospores - contained in a club-shaped basidium

 Parasexual
o Sequence of events that culminates in genetic exchange via mitotic
recombination
o A laboratory tool for the genetic analysis of many imperfect fungi
 Teleomorphs
o The sexual stage of a fungus
 Anamorphs

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 o The asexual stage of a fungus

Fungi Imperfecti
 Fungi that do not exhibit sexual phase
 Most fungi of medical importance are imperfect fungi

Collection and Processing Of Clinical Specimens For Fungal Studies

General Guidelines
 Use sterile collection method and sterile instruments
 Specimen should be collected from actual infection site to avoid normal flora-
contaminating bacteria
 Adequate quantity
 Accurate and complete label of the specimen and it will be helpful if data will include the
suspected diagnosis
 Prompt delivery of the specimen to the laboratory to avoid the danger of overgrowth of
bacterial or fungal contaminants

Specimen Collection
 general guidelines for specimen collection for fungal infections are very similar to those
for bacterial infections
o use of sterile collection method and sterile instruments
o specimen should be collected from actual infection site to avoid normal flora-
contaminating bacteria
o adequate quantity
o accurate and complete label of the specimen and it will be helpful if data will
include the suspected diagnosis
o prompt delivery of the specimen to the laboratory to avoid the danger of
overgrowth of bacterial or fungal contaminants

Collection and Processing

 Blood
o absence of normal fungal flora
o collect by venipuncture, 5 ml blood to Brain heart infusion (BHI) broth
 Bone marrow
o absence of normal fungal flora
o aspirate 0.5 ml marrow, then to BHI broth
 Cerebrospinal fluid
o absence of normal fungal flora
o collect in sterile tubes by lumbar puncture
o if volume is more than 2 ml, centrifuge and use sediments for slides and plating
o if less than 2 ml, use uncentrifuged specimen
 Cutaneous
o Hair
 have contaminants
 pluck by roots by sterile forceps, select hair that fluoresce or are broken
and scaly
o Nail
 no normal flora
 clean nails with 70% alcohol, scrape the discolored areas, discard the
outer layer and collect inner infected nail
 may also collect using sterile nail clipper

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  place nail in a sterile petri dish, cut into small pieces
 examine microscopically using KOH
o Skin
 few Candida and contaminants
 clean skin with 70% alcohol, scrape outer edge of ring in case of
suspected ringworm, or scrape area of active infection
 examine microscopically using KOH
 Mucocutaneous
o no normal fungal flora
o collect by scraping plaque with sterile tongue depressor especially if Candida is
suspected
 Subcutaneous tissue: lesions and abscesses
o no normal fungal flora
o perform needle aspiration or biopsy
o examine for the presence of granules
o tissue must be minced or ground
 Sputum, bronchial washings, transtracheal aspirate
o no normal fungal flora
o sputum collected early morning on 3 consecutive days to enhance recovery
o avoid 24-hour collections due to contamination and overgrowth
o collect washings using a sterile saline
 Throat
o few yeast, few contaminants
o collect with 2 sterile swabs, scrape off and collect with sterile tongue depressor if
Candida is suspected
 Urine
o no normal fungal flora if collected clean-catch midstream or catheterized
specimen
o collect clean-catch midstream of first morning urine and void into a sterile
container
o place catheterized specimen in sterile container
o avoid 24-hour collection
o centrifuge and use sediment for microscopic exam and plating
o process within 2 hours or refrigerate to avoid bacterial overgrowth
o colony count of > 100,000/ml especially of Candida is significant
 Vaginal, cervical
o few to moderate colonies of Candida
o collect 2 swabs, prepare slide from one swab for microscopic exam with KOH
and plate the 2nd swab

Identification Methods
 3 methods: 1) microscopic 2) culture 3) serologic test

Microscopic Methods
 direct microscopic methods must always be confirmed with cultures or antigen
testing, if available
 can sometimes establish a diagnosis or narrow the etiologic possibilities
o Wet mount
 KOH preparation
o used to dissolve keratin in skin, hair or nail specimens
o keratin may obscure the fungal elements in these specimens
o OH acts as clearing agent, it eliminates debris and makes fungal
elements prominent
o one drop of KOH is added to the specimen, cover with cover slip

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 o allow specimen to clear, approximately 20 minutes or may warm
slide gently
o examine microscopically
o disadvantage is poor contrast
 Lactophenol cotton blue
o lactic acid preserve the fungal structures
o phenol is a killing agent
o cotton blue color fungal structures
o visualize microscopic fungal morphology by imparting a blue color to
the cell wall
o add one drop of lactophenol cotton blue to the specimen, coverslip is
applied, examine microscopically
 India ink preparation
o used to identify the capsule of the yeast Cryptococcus neoformans
o CSF can be directly examined by adding one drop of fluid to one
drop India ink, examined microscopically and capsule appear as
clear halos against a dark background
 Fluorescence
o Calcofluor white stain
 calcofluor binds to chitin in the fungal cell wall giving a
brilliant fluorescence under the fluorescent microscope
o Wood’s lamp (UV light)
 infected hair and skin will fluoresce when examined in the
dark
o Staining
 Gram stain
o fungi stain (+) but often stain poorly
o useful for Candida
 Giemsa or Wright’s stain
o used for the detection of intracellular Histoplasma capsulatum in
blood smears, lymph nodes, lung, liver, or bone marrow
o the organisms appear as small, oval yeast cell staining light to dark
blue
 Periodic acid-Schiff (PAS)
o stains the hyphae of molds and also some yeasts
o stains red
 Methenamine silver stain
o stain cell wall black
 Papanicolaou method
o better demonstrate Blastomyces dermatitidis than wet mounts

Culture Procedures
 media for cultivation of fungi must include a source of nitrogen, such as nitrite, nitrate,
amino acids or urea, and a carbon source, which is usually glucose
 vitamins, minerals and other supplements may also be added including antimicrobial
agents
o the addition of cyclohexamide inhibit saprophytic contaminating fungi
o the addition of chloramphenicol inhibit growth of bacteria
 cultures should be incubated at room temperature or preferably at 30 C for 30 days,
examined 3x a week, before reporting as negative
o some laboratories prefer to grow 2 sets of cultures, one at room and the other at
37 C
 aerobic, prefer moisture and increased humidity

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  agar plates are preferred because of large surface area and provide better aeration of
cultures, however plates are discouraged because of safety considerations; screw-
capped tubes are more easily stored and less hazardous to handle, disadvantages
include poor isolation of colonies, reduced surface area for culturing, and tendency to
promote anaerobiosis

Culture Media
 Primary isolation media should include media with or without blood and media with or
without cyclohexamide; all media should contain antibacterial agents
o Sabouraud dextrose agar (SDA)
 general isolation medium which contains glucose and peptone
 for primary isolation of saprophytic and pathogenic fungi
 may contain cyclohexamide alone or chlorampenicol alone or both may
be present
 if both are present, SDA-CC, it is commercially available as Mycosel or
Mycobiotic
 SDA-CC is for recovery of pathogenic fungi; bacteria and saprophytic
fungi are inhibited
o Dermatophyte test medium
 recovery of dermatophytes from hair, skin and nail specimens
 dermatophytes produce alkaline metabolites, which raise the pH and
change the color of the indicator from yellow to red
 useful for screening only
o Brain-heart infusion (BHI) medium
 most useful in the isolation of pathogenic fungi from sterile specimens
 can be supplemented with blood (BHI biphasic blood culture bottles) is
useful for the recovery of fungi from blood or bone marrow
 cyclohexamide and chloramphenicol may be incorporated into the
medium (BHI with antibiotics); useful for the isolation of pathogenic fungi
exclusive of dermatophytes; useful for specimens contaminated with
bacteria or saprophytic fungi
 Differential test media are used to differentiate various groups or species of fungi
o Birdseed (niger seed) agar
 isolation of Cryptococcus neoformans, which produces phenol oxidase,
breaking down the medium and resulting in the production of brown to
black colonies in 4 – 7 days
o Cornmeal agar with Tween 80
 stimulation of chlamydospore formation in Candida species
 useful for species differentiation of Candida
o Cottonseed agar
 conversion of mold phase of Blastomyces dermatitidis to yeast phase
o Potato dextrose agar
 Pigment production by Trichophyton rubrum
o Czapek agar
 Recovery and differential identification of Aspergillus spp
o Rice medium
 Identification of Microsporum audouinii

Macroscopic Examination of Colonies: criteria for describing colonies include

 rate of growth
 topography
o best observed on the reverse side since it may be obscured by the aerial hyphae
o may be flat, heaped, or folded
o rugose topography contains deep furrows that radiate from the center
o umbonate colonies are elevated in the center

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 o colonies may also be wrinkled or verrucose
 texture
o best observed in a cross section; it is usually related to the length of the aerial
hyphae
o dense, high aerial mycelia produce a cottony or woolly texture
o dense, low aerial mycelia produce a velvety or silky texture
o flat, rough, crumbly colonies are described as powdery or granular
o yeast typically produce glabrous or smooth colonies that are wet, waxy, creamy,
or pasty since no significant mycelia are produced
 pigmentation
o surface pigmentation and pigmentation on the reverse side are also important
gross colonial characteristics
o description of color must be specific

Microscopic Examination of Growth

 used to observe conidia and spores for identification
 methods include
o tease mount
 for the rapid examination of conidia, spores, and other microscopic
fungal structures
 with the use of a bent dissecting needle or wire, remove a small portion
of the colony from the agar surface; place a drop of lactophenol cotton
blue (LPCB) on the microscopic slide and place the culture into the stain,
then either
 place a cover slip over the culture, and using a pencil eraser,
press down gently to disperse the mycelium
 by using 2 dissecting needles, gently tease apart the mycelium
and then add the cover slip
 observe microscopically under low and high power magnification
 disadvantage: preservation of the original arrangement of spores may be
disrupted due to teasing or pressure applied on the cover slip

o slide culture
 this is the most accurate method to preserve and observe fungal
morphology
 it requires more skill and time than the tease mount but allows the fungus
to be preserved in its original state
 procedure
 cover the bottom of the Petri plate with filter paper
 place a bent glass rod in the Petri plate
 place a clean microscopic slide onto the bent glass rod
 by using a sterile scalpel, cut a 2 mm deep section of agar that
measures approximately 1 x 1 mm
 transfer the block of agar to the microscope slide
 using either a sterile wire needle, inoculate the fungus into the
center of the 4 sides of the agar square
 gently place a coverslip over the block
 add approximately 1.5 – 2.0 ml sterile water to the bottom of
Petri plate
 incubate at room temperature
 examine periodically for growth, growth usually seen on the
slide’s surface and underneath the coverslip
 when growth is visible, remove the coverslip and place on a
microscopic slide with LPCB, examine under low and high power

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  do not make slide culture of the following (too hazardous):
Histoplasma, Coccidioides, Blastomyces, Sporothrix,
Paracoccidioides
o cellophane tape mount
 involves the application of double-sticky tape or cellophane tape looped
back on itself to the surface of the fungal colony
 aerial hyphae will adhere to the tape, then tape on a slide containing
LPCB
o other methods
 hair baiting technique
 fill Petri plate with soil, make holes on soil
 cut hair into small pieces and place them into the holes
 incubate at room temp and examine regularly for growth

Biochemical Tests
 carbohydrate assimilation test for yeast
o provide a definite identification for yeast and yeastlike organisms
o based on the ability of yeast to utilize a particular carbohydrate
o determined by using a carbohydrate-free (nitrogen-based) agar and filter paper
disks that are impregnated with various carbohydrates
o growth around the disk indicates the yeast can utilize that carbohydrate and is a
positive result
 carbohydrate fermentation test
o carbohydrate agar inoculated with the yeast, if carbohydrate is fermented there is
change of color of the media
 rapid urease test for yeast
o production of urease enzyme is useful in the preliminary identification of
Cryptococcus neoformans, which is urease (+)
o urease converts urea to ammonia and carbon dioxide, which produces an
alkaline environment in the medium
o pink to purple color is positive reaction

Serologic Tests
 performed to supplement microscopic or culture methods for identification
 methods include: agglutination, precipitation (immunodiffusion), complement-
fixation, IF, ELISA
 antigen detection
o may be hampered by cross-reacting components
o very useful test is the Cryptococcal antigen test for Cryptococcus neoformans
which detect the polysaccharide antigen from the CSF, utilize latex agglutination
 antibody detection
o relies on correct timing of blood specimens
o antibody detection for histoplasmosis, blastomycosis, coccidioidomycosis,
sporotrichosis can detect active infections, but cross-reacting antigens and false-
negative results are problems
Laboratory Safety Considerations
 it is necessary that all mold cultures and clinical specimens be handled in a
biological safety cabinet with no exceptions
 cultures of organisms suspected of being pathogens should be sealed with tape to
prevent laboratory contamination and should be autoclaved as soon as definitive
identification of the organism is made

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CLASSIFICATION OF FUNGI
 Based on Morphology
 Based on Major Site of Involvement (Clinical Classification)

FUNGAL AGENTS CAUSING SUPERFICIAL MYCOSES

 Cause noninvasive infection involving the stratum corneum layer of the skin, hair and
nails

Pityriasis versicolor (Tinea versicolor)

 A chronic and nonirritating superficial infection of the stratum corneum
 Characterized by brownish scaly areas on light-skinned and lighter areas on dark-skinned
persons
 Found on the trunk, arms, shoulders, face and neck
 Etiologic Agent: Malassezia furfur (Pityrosporum orbiculare)
o Lipophilic and grows in areas where sebum and skin oil accumulates
o Seen in warmer environments and is associated with poor personal hygiene
o Direct microscopic examination of skin scrapings using 10% KOH
 Clusters of spherical cells and short unbranched hyphae, “spaghetti
and meat balls” appearance
o Culture is not necessary but grow on SDA with olive oil in 2-4 days at room
temperature
 Creamy, yeast-like colonies
Tinea Nigra (Tinea Nigra Palmaris)
 Superficial, chronic infection of palms of hands
 Characterized by black to brown scaly patches
 Etiologic Agent: Hortaea (Exophiala) werneckii
o Saprophyte, dematiaceous fungi
o Microscopic examination
 Brown-pigmented, branched, septated hyphae and budding yeast cells;
older colonies show annellids
o Culture
 On SDA at room temperature: black, yeast colonies which later grow as
olive-green mycelium
Black Piedra
 Hard, brown-black crusts on the outside of the hair shaft
 Primarily involve the scalp hair, rarely axillary and pubic hair
 Etiologic Agent: Piedraia hortae
o Dematiaceous fungi
o Microscopic examination
 Direct examination of portions of hair with nodules: dark, septate thick-
walled hyphae with swellings
o Culture
 SDA grow black to dark brown heaped colonies
White Piedra
 Produces light brown, soft nodules especially on the beard or mustache
 Etiologic Agent: Trichosporon beigelii
o Microscopic
 Direct wet mount with KOH show hyaline hyphae and numerous
rectangular arthroconidia, occasional blastoconidia in chains or clusters
o Culture
 SDA grow as cream-colored, wrinkled colonies

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 FUNGAL AGENTS CAUSING CUTANEOUS MYCOSES OR
DERMATOPHYTOSES

 An infection of the skin, hair, or nails by any member of the keratinophilic fungi called
dermatophytes
 Dermatophytes parasitize the nonliving, cornified layer of the skin
 Secrete keratinases which are proteolytic enzymes that digest keratin
 Classified according to its habitat: zoophilic, anthropophilic, geophilic
 3 genera associated with human infection: Identified on the basis of their colonial
appearance and microscopic morphology

 Microsporum : tend to produce large, spindle-shaped multicellular

macroconidia with echinulate walls, produced singly from short
conidiophore or directly from hyphae; microconidia rarely produced
 Microsporum audouinii
 Anthropophilic; infect hair and scalp
 Important cause of tinea capitis
 Colonies grow in 2 weeks: cream, tan, light brown
velvety colony; reverse side is salmon pink or orange
brown
 Microscopic: rare bizarre-shaped macroconidia, thick-
walled, club or spindle-shaped, multiseptate with rough
surface; usually seen are terminalor intercalary
chlamydospore; microconidia rare
 Microsporum canis
 Zoophilic; infect; hair and scalp
 Colonies grow in 1 week: white or buff granular or
cottony with bright yellow periphery; reverse is lemon
yellow or yellow-orange
 Microscopic: thick-walled, rough-walled, spindle-shaped,
multiseptate macroconidia with a curved tip; rare
microconidia
 Microsporum gypseum
 Geophilic
 Rare cause of human or animal infection
 Colonies grow rapidly as flat irregularly fringed with a
coarse powdery surface, buff or cinnamon color; reverse
is orange to brownish
 Microscopic: macroconidia ellipsoidal with rounded ends,
multiseptate with rough surface; rare or absent
microconidia

 Trichophyton
 Predominant forms are microconidia which are spherical, tear-
shaped, or clavate; develop from the side of the hyphae usually
in grape-like clusters
 Macroconidia uncommon, when present they are smooth-walled,
elongate, pencil-shaped, multicellular
 Do not fluoresce under Wood’s light
 Calcofluor white or KOH preparation reveals hyaline septate
hyphae and/or arthroconidia
 Direct microscopic examination of infected hairs

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  Hair shaft filled with masses of large (4-7u) arthroconidia
in chains, endothrix invasion
 Hair with external masses of spores that ensheath hair
shaft, ectothrix hair invasion
 Trichophyton mentagrophyts
 White granular and fluffy colonies; occasionally light
yellow periphery in younger cultures; reverse buff to
reddish brown
 Microscopic: spiral hyphae; numerous small spherical
microconidia in grape-like clusters
 Trichophyton rubrum
 White downy to pink granular, rugal folds are common;
cherry red color is best observed on the reverse side
and only after 3-4 weeks incubation
 Microscopic: small teardrop-shaped microconidia

 Trichophyton tonsurans
 White. tan to yellow or rust, suede-like to powdery,
wrinkled with heaped or sunken center; reverse yellow to
tan to rust red
 Microscopic: balloon-shaped microconidia
 Trichophyton verrucosum
 Glabrous to velvety white colonies, rare strains produce
yellow -brown color; reverse yellow
 Microscopic: rare microconidia but when present they
are large teardrop-shaped; many chlamydospores in
chain ; antler hyphae
 Trichophyton schoenleinii
 Irregularly heaped, smooth white to cream colony with
radiating grooves; reverse white
 Many antler-type hyphae seen

 Epidermophyton
 Produces macroconidia which are multicelled club-shaped,
smooth- walled, either singly or in groups of 2 to 3
 Microconidia are not produced
 Epidermophyton floccosum
 Center of colony tends to be folded and is khaki green,
periphery is yellow; reverse yellowish brown
 Macroconidia in clusters of 2-3

Hair Skin Nail

Microsporum + + -
Trichophyton + + +
Epidermophyton - + +

 Dermatophytosis (Tinea or ringworm)

 Tinea capitis – scalp
 Tinea barbae – beard or mustache (Barber’s itch)
 Tinea corporis – body
 Tinea cruris – inguinal area or groin (Jock itch)

13
  Tinea pedis – athlete’s foot
 Tinea ungium – nail (Onychomycosis)

SKIN DISEASE LOCATION OF LESIONS FUNGI MOST FREQUENTLY

RESPONSIBLE
Tinea corporis Nonhairy, smooth skin T. rubrum, E. floccosum
(ringworm)
Tinea pedis Interdigital spaces on feet T. rubrum, T. mentagrophytes, E.
(athlete’s foot) floccosum
Tinea cruris Groin T. rubrum, T. mentagrophytes, E.
(groin itch) floccosum
Tinea barbae Beard hair T. mentagrophytes, T. rubrum, T.
(barber’s itch) verrucosum
Tinea ungium Nail T. rubrum, T. mentagrophytes, E.
(onychomycosis) floccosum
Dermatophytid Usually sides and flexor No fungi present in lesion. May
Id reaction) aspects of fingers. Palm. become secondarily infected with
Any site of the body. bacteria

Anthropophilic species
 Epidermophyton floccosum
 Trichophyton mentagrophytes
 Trichophyton rubrum
 Trichophyton tonsurans
Geophilic species
 Microsporum gypseum
Zoophilic species
 Microsporum gypseum (dogs and cats)
 Microsporum gallinae (fowl)
 Microsporum nanum (pigs)
 Trichophyton equinum (horses)
 Trichophyton verrucosum (cattles)

FUNGAL AGENTS CAUSING SUBCUTANEOUS MYCOSES

Subcutaneous Mycoses
 Caused by exogenous fungi that normally reside in nature, mostly in soil and
vegetations
 Portal of entry
 skin or subcutaneous tissue by traumatic inoculation with contaminated
material
 Cause chronic infections
 Types
 Sporotrichosis
 Mycetoma
 Chromoblastomycosis

14
  Phaeohyphomycosis
Sporotrichosis
 Chronic infection of the subcutaneous tissues and lymphatics
 Acquired through trauma (thorns or splinters) usually to the hand, arm or leg
 Occupational hazard for farmers, miners, gardeners, florists
 “Rose gardener’s disease”
 Primary lesion begins as a small, nonhealing ulcer, commonly in the index finger
or the back of the hand
 Later becoming nodular lesions
 Involves the lymphatic vessels and lymph nodes draining the region
 Etiologic Agent: Sporothrix schenckii
o Dimorphic fungi
o Specimens for diagnosis: aspirated pus from nodules, swabs, scrapings,
biopsy tissue
o Macroscopic
 Rapidly growing (3-5 days), white, pasty, moist colony that later
becomes brown, black, wrinkled or leathery
o Microscopic
 Mycelial form (room temp): narrow, septate hyphae with pyriform
conidia arranged singly or in a flowerette
 Yeast form (37C): small, elliptoid, budding, cigar-shaped yeasts
Mycetoma
 Other names: Madura foot or Maduromycosis
 Traumatic inoculation with several saprophytic fungi, commonly involving the
lower extremities but may occur in any part of the body
 Chronic infection characterized by swelling, purplish discoloration, tumor-like
deformities of subcutaneous tissue
 Multiple sinus tracts that drain pus containing yellow, white, red or black granules
 2 types
o Actinomycotic (bacterial)
 Actinomycetes (Actinomadura, Streptomyces, Nocardia)
o Eumycotic (fungal)
 White grain mycetoma
 Pseudoallescheria boydii – most common
 Acremonium
 Fusarium

 Black grain mycetoma

 Madurella mycetomatis
 Exophialla jaenselmi
 Curvularia
o Pseudoallescheria boydii
 Ascomycota group
 Soil, standing water and sewage
 Humans acquire infection by traumatic inoculation into the skin
and subcutaneous tissues
 Clinical specimens: granules from the lesions

 Macroscopic

15
 Rapidly growing (5-10 days), initial growth as a white fluffy
colony
 Changes after several weeks to brownish-gray mycelium,
reverse of colony is black
 Microscopic
 Asexual form (anamorph): golden brown elliptic, single-
celled conidia borne singly from the tips of conidiophores
 Sexual form (teleomorp): brown sac-like cleistotheca
containing asci and ascospores
o Acremonium
 Hyaline hyphae
 Produces simple, unbranched, erect conidiophores
 Conidia arranged in masses at the tip of conidiophores

o Madurella mycetomatis
 Characteristic is a brown diffusible pigment
 Long tapering phialides with colarettes

Chromoblastomycosis (Chromomycosis)
 Traumatic inoculation into the subcutaneous tissue
 Chronic infection producing warty or cauliflower-like or tumor-like lesions mostly
in the lower extremities
 Etiologic agents
o Cladosporium (Cladosporium carrionii)
o Phialophora (Phialophora verrucosa)
o Fonsecaea (Fonsecaea pedrosoi)
 Macroscopic
o All grow slowly and produce heaped-up and slightly folded, darkly
pigmented colonies with a gray to black velvety colonies; reverse side of
colonies is jet black
 Microscopic
o Cladosporium: chains of budding blastoconidia borne from branching
conidiophores
o Phialophora: short flask-shaped phialides with collarette
o Fonsecaea: conidial heads with sympodial arrangement of conidia,
primary conidia giving rise to secondary or tertiary conidia
o Sclerotic Bodies
 Characteristic histologic findings in tissues with
chromoblastomycosis
 Copper-colored, septate cells that appear to be dividing
Phaeohyphomycosis
 Infections include subcutaneous abscesses, brain abscess, sinusitis,
endocarditis, pulmonary and systemic infections
 Etiologic Agents
o Exophiala jaenselmei, E. dermatitidis
o Alternaria
o Bipolaris
o Drechslera
o Curvularia
 Exophiala

16
 o Macroscopic
 Grow slowly (7-21 days) and initially grows black yeast-like
colonies; as colonies age they become filamentous, velvety, gray
to black
o Microscopic
 Pale brown conidiophores that form cylindrical annellids, hyaline
conidia gather at its tip

 Alternaria
o Macroscopic
 Grow rapidly and appear fluffy, gray to black colonies
o Microscopic
 Hyphae are septate and golden brown, conidiophores are simple
sometimes branched which bear a chain of large brown conidia
resembling a drumstick
 Bipolaris
o Macroscopic
 Rapid grower, gray-green to dark brown powdery colonies
o Microscopic
 Hyphae are dematiaceous and septate; conidiophores are bent
(geniculate) at the ends where conidia are attached; conidia are
oblong and multicelled
 Drechsela
o Septate hyphae and darkly pigmented
o Conidiophores are geniculate
o Conidia are sparse

 Curvularia
o Macroscopic
 Rapid growing, most are fluffy, gray to black colonies
o Microscopic
 Hyphae are dematiaceous and septate; conidiophores are
geniculate at the ends where conidia are attached; conidia are
multicelled, curved with a central swollen cell

FUNGAL AGENTS CAUSING SYSTEMIC AND OPPORTUNISTIC MYCOSES

 Primary Systemic Mycoses

o Coccidioidomycosis
o Histoplasmosis
o Blastomycosis
o Paracoccidioidomycosis
o Generally caused by dimorphic fungi
 Opportunistic Mycoses
o Candidiasis, systemic
o Cryptococcosis

17
 o Aspergillosis
o Mucormycosis
 Identified by
o Growth characteristics
o Colonial morphology
o Microscopic characteristics of conidia and hyphae
o Conversion of mycelial to yeast phase
o Antigen testing
o Nucleic acid probes
 Transmission
o Inhalation of fungal spores
o Lead initially to pulmonary infection which may be symptomatic or
asymptomatic
o Dissemination to other body sites can occur

Coccidioidomycosis
o Acquired through inhalation of the infective arthroconidia
o Approximately 60% are asymptomatic and self-limited respiratory tract infections
o Infection may become disseminated to visceral organs, meninges, bone, skin,
lymph nodes and subcutaneous tissue
o Etiologic agent: Coccidioides immitis
o Clinical specimens
o Sputum, tissues or body fluids
o Direct microscopic examination from clinical specimens
 Nonbudding, thick-walled spherule, 20-200 u in diameter
containing either granular material or numerous small nonbudding
spores
o Macroscopic
 Colonies appear after 3-21 days, delicate fluffy white which turn
tan or brown with age
o Microscopic
 Mycelial phase: septate, branched hyphae that produce thick-
walled barrel-shaped, rectangular arthroconidia that alternate
with empty alternate cells
 Yeast phase: large, round, thick-walled spherules with
endospores observed in tissues and direct examination
o Other nonvirulent fungi that resemble C. immitis microscopically may be
found in the environment and may produce hyphae that may dissociate
into arthroconidia
 Geotrichum
 Trichosporon
o Considered as the most infectious of all fungi
o Extreme caution should be observed in handling cultures of this organism
o Safety Precautions in Handling C. immitis Cultures
 If culture plates are used, they should be handled only in a
biological safety cabinet (BSC)
 Cultures should be sealed in tape if the specimen is suspected of
containing C. immitis
 The use of cotton-plugged tubes is discouraged and screw-
capped tubes are preferred

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  All microscopic preparations for examination should be performed
inside a BSC
 Cultures should be autoclaved as soon as the final identification of
C. immitis is made

Histoplasmosis
o A chronic granulomatous infection that is primary and begins in the lungs and
may produce cavitary lesions that resemble tuberculosis
o Infection may disseminate to the lymphatic tissue, liver, spleen, kidneys,
meninges
o Involvement of the heart is becoming more common especially in
immunocompromised patients
o Acquired by inhalation of infective conidia from the environment
o 95% of infection are asymptomatic and self-limited
o Considered as the most prevalent pulmonary mycosis of humans and animals
o Etiologic Agent: Histoplasma capsulatum
o Direct microscopic examination
 Difficult to visualize in the sputum and other tissues
 May be detected in Wright or Giemsa-stained smears of the bone
marrow
 Rarely seen in the peripheral blood
 Small, round to oval yeast cells 2-5 u in diameter usually found
intracellularly within macrophages
o Macroscopic
 Slow growing requires 2-4 weeks
 SDA: white to brown mold with fine fluffy texture; white, yellow or
tan reverse side
 BHI: moist, white to cream heaped colony
o Microscopic
 Mycelial phase: septate hyphae with large spherical or pyriform
tuberculate macroconidia; some produce small round smooth
microconidia
 Yeast phase: small budding yeast cells 2-5 u usually
intracellular within macrophages

Blastomycosis
o Chronic suppurative and granulomatous infection which involve the lungs and
spread to the skin and bones
o Acquired through inhalation of the conidia and hyphal fragments
o Etiologic Agent: Blastomyces dermatitidis
o Direct microscopic examination of tissues or body fluids
 large, spherical, thick-walled yeast cells 8-15 u usually with a
single bud that is connected to its parent cell by a broad base
o Macroscopic
 Growth rate is 7-21 days
 SDA: colony at first white, waxy, yeast-like and later becoming
cottony with white aerial mycelium; turns tan to brown with age
 BHI with blood: cream to tan, waxy. wrinkled colonies
o Microscopic
 Mycelial phase: delicate, septate hyphae with round or pyriform
conidia borne singly on conidiophores resembling “lollipops”

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  Yeast phase: thick-walled, large yeast cells with a single bud
on a broad base

Paracoccidioidomycosis
o Chronic granulomatous infection that begins as a primary pulmonary infection
o Usually asymptomatic but may disseminate to produce ulcerative lesions of
mucous membranes of the nasal cavity, oral, gingival and conjunctiva
o Etiologic Agent: Paracoccidioides brasiliensis
o Direct microscopic examination of sputum, tissues or scrapings of
ulcerative lesions
 Large, round or oval yeast cells, 8-40 u, producing multiple buds
and each is attached to the parent cell by a narrow base
 “Mariner’s wheel”
o Macroscopic: very slow-growing 21-28 days
 SDA: white, glabrous, leathery colony which turns tan-brown with
age
 BA: cream to tan, moist, wrinkled colony which turns waxy with
age
o Microscopic
 Mycelial phase: small, septate, branched hyphae with intercalary
and terminal chlamydospores; few piriform microconidia
 Yeast phase: large, round to oval, thick-walled yeast cells (8-40 u)
with multiple buds with a narrow base (mariner’s wheel)

Candidiasis
o Most frequently encountered fungal infection
o Etiologic agents
o Candida albicans – most frequent
o C. tropicalis
o C. parapsilosis
o C. glabrata
o Candida albicans and other species are part of the normal flora, seen in the
oropharynx, GIT, GUT, skin
o Infections are believed to be endogenous in origin
o Candida Infections In Normal And Immunocompromised Hosts
o Intertriginous candidiasis (skin folds)
o Onychomychosis and paronychia
o Mucocutaneous junction of lips
o Oral thrush
o Vulvovaginitis
o Pulmonary infection
o Eye infections
o Endocarditis
o Meningitis
o Fungemia and disseminated infection
 Most commonly seen in immunocompromised patients
 Onychomycosis and esophagitis caused by Candida albicans are
very common in AIDS patients
o Predisposing Factors For Candidiasis
 Alteration in the normal skin and mucous membrane barriers
 Prolonged antibiotic administration

20
  Use of immunosuppressive drugs
 Diseases of the immune system
o Candida
 Direct microscopic examination of clinical specimens
 Budding yeast cells (blastoconidia 2-4 u)
 Pseudohyphae
 These structures are strongly Gram (+)
 Macroscopic
 Candida species cannot be differentiated based on
colonial appearance
 Colonies are produced within 24-48 hours
 Raised, cream-colored, opaque, 1-2 mm; after several
days on agar medium hyphae may be observed
 Candida albicans
 Identified microscopically by production of germ tubes and
chlamydospores
 Germ tube is definitive identification of Candida albicans
 Germ Tube Test
o Germ tube is a hypha-like extension of the yeast
cells with no constriction at the point of origin
o Candida albicans will form germ tubes when
incubated with serum at 37C for a few hours
o Procedure
 Pipette 0.5 ml sterile serum into a test tube
 Inoculate the tube with small amount of
organism to be tested
 Incubate at 37C for 1-3 hours
 Place a drop of the suspension on a slide,
apply a cover slip, examine microscopically
 Cornmeal Agar with Tween 80
o Stimulates conidiation
o Used to distinguish the various species of Candida
and other yeasts through examination of hyphae,
blastoconidia, chlamydospores. and arthroconidia
o Tween 80 is added to reduce the surface tension to
allow conidiation
o Procedure
 Obtain an isolated colony from the primary
culture media
 Inoculate a plate of cornmeal agar
containing 1% Tween 80 and trypan blue by
making 3 parallel cuts about ½ inch apart at
a 45º angle to the culture medium
 Incubate at room temperature for 48 hours
 After 48 hours, remove and examine the
areas where cuts into the agar were made
for the presence of blastoconidia,
arthroconidia, pseudohyphae,
chlamydospore
 Expected results

21
  C. albicans will produce
chlamydospores and clusters of
blastoconidia arranged at regular
intervals along pseudohyphae

Cryptococcosis
 A subacute or chronic fungal infection that has several manifestations
 Disseminated disease with or without meningitis in immunocompromised patients
 Meningitis occur occur in 2/3 of patients with disseminated infection
 Disseminated infection is becoming very common in patients with AIDS
 Exist as saprophyte in nature
 Often found associated with pigeon, bat, or bird droppings as well as decaying
vegetations, fruit, plants
 Acquired through inhalation
 Primarily affecting lungs, then disseminate to meninges and other sites
 Etiologic Agent: Cryptococcus neoformans
o Direct microscopic examination
 Spherical, single or multiple budding, thick-walled yeast cell
surrounded by a wide, refractile polysaccharide capsule
 India ink preparation of CSF has been widely used (capsule
unstained)
 Replaced by latex agglutination for detection of
cryptococcal antigen
o Macroscopic
 Colonies appear in 1-5 days as smooth, white to tan, mucoid,
gelatin-like colonies (soap-bubble)
 Brown-black colonies on Niger seed agar

Differentiation of Some Yeast Forms:

Organism Capsule Germ Blastoco Arthroco Chlamyd

Tube nidia nidia oconidia
C. albicans - + + - +
C. tropicalis - - + - V
C. parapsilosis - - + - -
C. glabrata - - + - -
C. neoformans + - + - -
Geotrichum - - - + -
Trichosporon - - + + -

Organism G M S L Urease Nitrate

Reduction
C. albicans + + - - - -
C. tropicalis + + + - - -
C. parapsilosis + - - - - -
C. glabrata + - - - - -

22
 C. neoformans - - - - + -
Geotrichum - - - - - -
Trichosporon - - - - + -

o Biochemical Tests (Review previous notes for procedure)

o Rapid urease test
o Carbohydrate utilization test
o Carbohydrate assimilation test

Aspergillosis
o An opportunistic infection causing disseminated infection in immunocompromised
patients
o Also cause various other infections including invasive lung infection, pulmonary
fungus ball (tangled mass of hyphae), allergic pulmonary aspegillosis,
onychomycosis, keratitis
o Acquired through inhalation
o Etiologic Agents: Aspergillus fumigates
o Direct microscopic examination
 Septate hyphae that usually show dichotomous branching (45°
angle branching)
o Macroscopic
 Rapidly growing mold (2-6 days) producing fluffy to granular, white
to blue green colonies
o Microscopic
 Branching septate hyphae that terminate in conidiophore that
expands into a large spherical vesicle with phialides from which
chains of conidia arise

Zygomycosis (Mucormycosis)
o Less common cause of infection as compared to Aspergillus
o Acquired by inhalation
o Rhinocerebral infection involving nasal mucosa, palate, sinuses and brain
o Immunocompromised patients like those with diabetes mellitus and with
immunosuppressive drugs are at greater risk
o Etiologic Agents: Zygomycetes
o Mucor
o Rhizopus
o Absidia
 Direct microscopic examination of tissue specimens or exudates
 Branching non-septate hyphae
 Macroscopic
 Fluff, white to gray to brown colonies covering the surface
of the agar within 24-95 hours, grayish hypha with brown to
black sporangia
 Microscopic
 Zygomycetes produce large ribbon-like hyphae that are
irregular in diameter and non-septate
 Sac-like sporangia containing sporangiospores are borne
at the tip of sporangiophore

23
  Sporangiophores are connected to each other by stolons
where root-like structures called rhizoids are attached
 Mucor
 Sporangiophores the tip of which have sporangia filled with
sporangiospores
 No rhizoids
 Rhizopus
 Has unbranched sporangiophores with rhizoids that
appear at the point at which the stolon arises
 Absidia
 Similar to Rhizopus except
 Branched sporangiophores
 Sporangiophores arise between nodes from which rhizoids
are formed

Penicillium
 When clinically significant, clinical manifestations include bronchopulmonary,
endocarditis, cutaneous ulcers of extremities
 Macroscopic
o Colonies with shades of green, blue-green, white, pink , other colors,
powdery colonies
 Microscopic (25⁰C)
o Hyaline and septate hyphae, brush-like conidiophores
o Conidiophores produce metulae from which phialides producing chains of
conidia arise

 Penicillium marneffei
o Emerging dimorphic pathogen endemic in Southeast Asia particularly
People’s Republic of China
o Commonly infects immunocompromised host
o Cause cutaneous of mucocutaneous infection or a progressive
disseminated and frequently fatal infection
o Transmission is unknown but the bamboo rat has been implicated
o At 37⁰C, oval yeast-like cells (2-6 u) with septa are seen

Fusarium
 Infections becoming more common especially in immunocompromised patients
 Common environmental flora
 Cause mycotic keratitis after traumatic implantation into the cornea
 Other infections: sinusitis, wound (burn) infections, allergic fungal sinusitis,
respiratory tract infections
 Macroscopic
o Grow rapidly (2-5 days) , fluffy to cottony and may appear pink, purple,
yellow, or green
 Microscopic
o Hyphae are small and septate
o Large sickle- or boat-shaped macroconidia; single-celled microconidia
ma be produced

24
Pneumocystis jeroveci (Atypical Fungus)
 Opportunistic atypical fungus that is a common cause of pneumonia in
immunocompromised hosts
 Differ from fungi
o Cell membrane contains cholesterol
o Trophozoite and cyst (octonucleate cyst)
 Direct detection methods
o Stains
 Calcofluor white, methenamine silver, IF
o Monoclonal antibodies
o PCR
 Does not grow in routine culture

End of Mycology

25
 VIROLOGY

 Virology
 Study of viruses
 Dmitri Iwanowski (1892)
 First noted the filtrable organism, the tobacco mosaic virus (TMV)
 Wendell Stanley (1935)
 Confirmed that the filtrable organism is TMV
Origin of Viruses
• Derived from DNA or RNA nucleic acid components of host cells that have
acquired the ability to exist independently
• May be degenerate forms of intracellular parasites
Classification of Viruses
(Animal Viruses)
 DNA Viruses
Poxviridae
Herpesviridae
Adenoviridae
Papovaviridae
Papillomaviridae
Polyomaviridae
Hepadnaviridae
Parvoviridae
 RNA Viruses
Picornaviridae Filoviridae
Caliciviridae Paramyxoviridae
Togaviridae Orthomyxoviridae
Flaviviridae Bunyaviridae
Coronaviridae Arenaviridae
Reoviridae Rhabdoviridae
Astroviridae Retroviridae

Characteristics of Viruses
Structure
• Nucleic acid: either DNA or RNA
 Viral genome: for viral replication
 Single-stranded (SS) or double-stranded (DS)
 DNA viruses are DS except for Parvovirus
 RNA viruses are SS except for Reovirus
• Protein coat or capsid
 Composed of capsomeres
 Virus: Nucleocapsid or Naked virus
 Viral envelope
 Lipid bilayer consisting of
a) matrix proteins
b) glycoproteins
 Virus: Enveloped virus
 Generalities
 DNA viruses are enveloped except for Parvovirus,
Papillomavirus, Polyomavirus, Adenovirus

26
  RNA viruses are enveloped except for Picornavirus,
Calicivirus, Reovirus , Astrovirus
Size
 Unit of measurement: Nanometer (nm)
 E/M; vary from 10 – 300 nm
 Smallest
 Parvovirus (22 nm)
 Picornavirus (28 nm)
 Largest
 Poxvirus (225 – 300 nm)
 Paramyxoviridae (150-300 nm)
 Filoviridae (80x1000 nm)
Shape (Capsid Symmetry)
 Icosahedral
 Spherical or cubic
 Helical
 Rod-shaped
 Complex symmetry
 Brick-shaped Poxvirus
 Tadpole-shaped
 Typical of bacteriophages
Multiplication
 Obligate intracellular organism
 Multiplication: Viral replication
 Adsorption (Attachment): have specific receptors on host cell
membranes
 Penetration: receptor-mediated endocytosis and membrane fusion
 Uncoating: physical separation of nucleic acid from its protein coat;
mediated by cellular enzymes
 Synthetic phase: early period and late period
 Inhibition of host cell DNA
 Early: synthesis of nucleic acids
 Late: synthesis of protein components
 Location of viral genome replication is characteristic for each virus
 DNA viruses, except Poxvirus, replicate in the nucleus of
the host cell
 Most RNA viruses replicate in the cytoplasm of the host
cell except for retroviruses and Influenza virus, in which
part of their replicative cycle occur in the nucleus of host
cell
 Nucleic acid: positive (+) or negative (-) sense
 Positive-sense
 Isolated RNA genomes which functions as mRNA
within the infected cells; are infectious
 Picornavirus and Togavirus
 Negative-sense
 Isolated RNA is noninfectious, virions usually carry
RNA polymerase that transcribes genomic RNA to
several complementary strands which serve as
mRNA
 Rhabdovirus and Retroviridae

27
  Assembly or Maturation
 Newly formed nucleic acids are enclosed by capsids
 Release: 2 mechanisms
 Rupture or lysis: for naked viruses
 Budding: for enveloped viruses
Atypical Viruses
 Defective viruses
 requires a helper virus (Delta Agent or Hepatitis D virus)
 Pseudovirions
 contain host cell DNA enclosed within capsid; can infect cells but cannot
replicate
 Viroids
 consist of single molecule of circular RNA without capsid; cause plant
disease
Prions
 Infectious particles composed of abnormal proteins (PrP)
 Abundant in neurons
 Cause of transmissible spongiform encephalopathy
 Humans: Kuru, Creutzfeldt-Jakob Disease
 Animals: Scrapie (goats and sheeps), Bovine spongiform encephalopathy
(Mad cow disease)

Cytopathic Effect (CPE)

 Cellular morphologic changes in the virus-infected host cells
 Cellular changes are observable on light microscopy
 Cellular changes include
 Cell death or lysis
 Fusion of virus-infected cells to form syncytia ( multinucleated giant cell
formation)
 Measles - Warthin-Finkeldey syncytia formation
 Inclusion body formation: discrete areas in the host cell nucleus or
cytoplasm containing viral proteins or particles
 Rabies - Negri body inclusion
 Cytomegalovirus - Giant cell with “owl’s eye”
 Smallpox - Guarnieri body
 Herpes simplex - Cowdry type A
 Molluscum contagiosum - Henderson-Patterson body or
Molluscum body :
 Koilocytosis in HPV infection
 Transformation of host cells

Viral Interference
 In host cells infected with more than one virus, one virus may inhibit the
multiplication of the other virus (virus-induced inhibition of viral replication)
 Mechanisms
 First virus either block or destroy the receptors
 One virus may compete for components needed for replication
(polymerase)
 Production of interferon (IFN)

28
  Glycoproteins produced by many cells in the body in response to
inducers, the primary inducer: viruses
 RNA viruses are better interferon inducer than DNA viruses
 Species specific; not virus specific
 Antiviral mechanism
 In uninfected cells, INF induce the formation of antiviral
protein which alters the cell’s protein synthetic mechanism
and therefore inhibit viral replication
 Examples: synthetase (degrades viral mRNA); protein
kinase (inhibits protein synthesis)
 Types
 Alpha
 Derived from lymphocytes; have antiviral effect
 Beta
 Derived from fibroblasts, macrophages, epithelial
cells; have antiviral effect
 Gamma
 Derived from T lymphocytes; immunoregulatory
function

Antiviral Agents
 Analogues of Ribonucleosides and Deoxyribonucleosides: Inhibit viral RNA and
DNA synthesis
 Acyclovir: highly specific for Herpes simplex and Varicella-Zoster virus;
less active against CMV and EBV
 Ganciclovir: powerful inhibitor of HSV multiplication; the best inhibitor of
CMV
 Zidovudine (AZT): inhibitor of retrovirus reverse transcriptase
 Ribavirin: treatment in aerosol form of infants with severe respiratory
syncytial virus infections
 Amantadine
 Interfere with the earliest stages of viral replication, during penetration
and uncoating
 Inhibit Influenza A virus multiplication
 Interferon
 Antiviral, immunoregulatory, antiproliferative
 Antitumor drug
 for hepatitis infections

Resistance/Sensitivity to Chemical and Physical Agents

 Ether Sensitivity
 Used to distinguish those viruses that possess envelope from
those that do not
 Enveloped viruses are generally sensitive
 Naked viruses are resistant
 Formaldehyde
 Destroy viral infectivity by reacting with nucleic acid
 Viruses with single-stranded genomes are inactivated much more
readily than double-stranded genome

29
  Minimal adverse effect on antigenicity of proteins
 Radiation
 Ultraviolet, x-ray, high-energy particles inactivate viruses
 Infectivity is the most radiosensitive because replication requires
expression of the entire genetic contents
 Heat sensitivity
 Icosahedral viruses are more stable, losing infectivity after several
hours at 37°C
 Enveloped viruses are more heat labile, rapidly dropping in titer at
37°C
 Viral infectivity generally destroyed by heating at 50-60°C for 30
minutes (except hepatitis B and polyomaviruses)

Specimen Collection For Viral Studies

• Specimens for detection of viruses should be collected as early as possible
• Viruses may no longer be present as early as 2 days after the appearance of the
symptoms
Throat or Nasopharyngeal Swab or Aspirate
• Nasopharayngeal aspirates are superior to swabs for recovering viruses
– Nasopharyngeal secretions are collected by inserting a swab through the
nostril or by using a bulb syringe with 3-5 ml buffered saline
– Preferred for detection of RSV, influenza, parainfluenza, rhinovirus
Throat Swab
• Done by rubbing inflamed or purulent areas of the posterior pharynx with a dry,
sterile swab (avoid touching other unaffected areas)
• Acceptable for recovering enteroviruses, adenoviruses, herpes simplex virus
Bronchial and Bronchoalveolar Washing
• Wash and aspirated fluid collected during bronchoscopy are excellent specimens
for detection of viruses infecting the lower respiratory tract
• Influenza virus and adenoviruses
Rectal Swabs And Stool Specimens
• Fecal specimens are used to detect rotavirus, enteric adenoviruses and
enteroviruses (do not grow in cell cultures)
– Collection of 5-10 ml freshly passed diarrheic stools
– Rectal swab may be acceptable and is collected by inserting a swab 3-5
cm into rectum to obtain feces
Urine
• CMV, mumps, rubella, measles, polyomavirus and adenoviruses
• Virus recovery may be increased by processing multiple (2-3) specimens
• Best specimen is at least 10 ml of a clean-voided, first morning urine
Skin And Mucous Membrane Lesions
• Herpes simplex virus, Varicella-Zoster virus, Enteroviruses, can be detected in
vesicular lesions of the skin and mucous membranes
– Once the vesicle has ulcerated and crusted, detection of the virus
becomes difficult
– Tzanck Smear
• Small drop of vesicle fluid must first be aspirated using a
tuberculin syringe to be used for viral or bacterial culture
• Carefully unroof the vesicle; roof folded back

30
 • Press a clean glass slide against the base of the ulcer making an
impression smear
• Slide is sent to the laboratory for modified Giemsa
Sterile Body Fluids Other Than Blood
• CSF, pericardial, pleural and peritoneal fluids may contain enteroviruses, herpes
simplex virus, influenza virus or cytomegalovirus
• Specimens are collected aseptically by the physician
Blood
• Viral culture of blood is primarily used to detect CMV
– Occasionally HSV, VZV, enteroviruses, adenoviruses are encountered
• A 3-5 ml anticoagulated blood (EDTA, citrate or heparin) collected in a vacutainer
tube
– 2 ml specimen is acceptable for pediatric patients
Bone Marrow
• Collected by aspiration
• Bone marrow for culture should be added to sterile tube with anticoagulant
Tissue Specimens
• Tissue specimens are especially useful for detecting viruses that commonly
infect the lungs, brain, and GIT
• Collected by biopsy
Serum For Antibody Testing
• Acute and convalescent serum specimens may be needed to detect antibody
against specific viruses
• Acute specimens should be collected as soon as possible after the appearance
of the symptoms
• Convalescent serum collected 2-3 weeks after the acute serum
• Appropriate specimen is 3-5 ml serum

Specimen Transport, Storage, Processing

Specimens For Viral Culture
• Should not be allowed to stand at room temperature or higher temp
• Specimens should be placed in ice and transported to the lab at once
• If delay is unavoidable, should be refrigerated
– Storage up to 5 days, 4°C
– 6 or more days, at -20°C or preferably at -70°C
– Specimens for freezing should first be diluted or emulsified in viral
transport medium
• Blood for viral culture, is transported in a sterile tube containing anticoagulant
– Clotted blood are unacceptable
– Should be processed immediately; if delay, hold specimen at 2-8°C
– Processing must occur within 12-24 hours
• Blood
– Detection of most viruses is best accomplished by separating and
culturing leukocytes
– One of the best is a leukocyte preparation using Polymorphprep
(mixture of sodium metrizoate and dextran)
• mononuclear and polymorphonuclear cells are isolated from red
blood cells in a one-step procedure
• Serum can be stored for
– Hours or days at 4°C or for
– Weeks or months at -20°C or below before testing

31
 • Many types of specimens are collected by swabs
– Cotton, rayon, dacron (preferred)
• Once collected, it is recommended that specimens on swab must be emulsified
in viral transport medium before transporting to the laboratory
Viral Transport Media
• Used to transport small volume of fluid specimens, small tissues, scrapings and
swabs
• Contain proteins such as serum, albumin, or gelatin to stabilize the virus and
antimicrobials to prevent the overgrowth of bacteria and fungi
• Examples: Stuart, Amies, Leibovitz-Emory, Hanks balanced salt solution (HBSS),
Eagle’s tissue culture medium
Processing
• Processing should occur in a biological safety cabinet or behind a protective
glass shield on the countertop
• Each specimen for viral studies should be accompanied by a request form which
contain the following
• Patient identification, age, sex, source of specimen, clinical history or
virus suspected, date and time collected

Detection of Viruses in Clinical Specimens

Methods of Detecting Viruses
• Cytology and Histology
• Detection of viral inclusions and syncytia formation
• Histologic examination of tissues stained with H and E to detect
inclusions of CMV, Rabies, HPV, Molluscum contagiosum virus
• Cytology stains
• Papanicolaou (Pap) smear
• Giemsa stain
• Detection of Herpes simplex and Varicella-Zoster inclusion bodies
• Tzanck test
• Electron Microscopy
• Most helpful in detecting viruses that do not grow readily in cell cultures
• Immune E/M
• May be used to detect the following viruses
• Viruses causing gastroenteritis (Rotavirus, Norwalk agent,
Adenovirus) and encephalitis (Herpes simplex, Measles, JCV)
• Detection of Viral Antigens
• Most useful in detect ing RSV, Influenza and Parainfluenza viruses,
Herpes simplex, VZV, CMV and Rabies virus
• Immunofluorescent antibody methods
• Enzyme immunoassay methods
• Radioimmunoassay
• Latex agglutination
• Molecular Detection
• Nucleic acid probes
• Polymerase chain reaction
• Cultivation
• Embryonated egg
• Cell Cultures
• Originate as few cells and grow into a monolayer on the sides of
glass or plastic tubes

32
 • Cells are continuously immersed in cell culture medium
• Once inoculated with specimen, cell cultures are incubated for 1-4
weeks (depending on the viruses suspected), at 35°C
• Kinds
• Primary cell lines
• Those that have been passed only once or twice
since harvesting; further passage result in
decreased susceptibility to viral infection
• Examples
• Primary monkey kidney cell (PMK)
• Human embryonic kidney (HEK)
• Low-passage (diploid) cell lines
• Cell cultures that remain virus-sensitive through 20-
50 passages
• Examples
• Human diploid fibroblast cells (HDF) derived
from human kidney and lung fibroblasts
• WI-38 and MRC-5 both derived from human
embryonic lung
• Continuous cell lines
• Cells can be passed and remain sensitive to virus
infection indefinitely
• Examples
• Hep-2 cells (Human epidermoid carcinoma
cells)
• HeLa cells (Cervical carcinoma cells)

** A cell culture becomes a cell line once it has been passed or

subcultured in
• Identification of viruses detected on cell cultures are based on the following
– Noting the cell type that support viral replication
– Time of detection of cytopathic effect (CPE)
– Morphology of CPE

Quantitation of Cell Culture CPE

Quantitation Interpretation

Negative Uninfected monolayer

Equivocal (+/-) Atypical alteration of monolayer
involving few cells
1+ 1% - 25% monolayer exhibit CPE
2+ 25%-50% monolayer exhibit CPE
3+ 50%-75% monolayer exhibit CPE
4+ 75%-100% monolayer exhibit CPE

Shell Vial Culture

• A rapid modification of conventional cell culture
• Quick identification of the virus because the infected cell monolayer is
stained for viral antigen soon after infection before the development of
CPE
• Stain used is usually fluorescein-labeled virus specific antibody

33
 • Procedure
• Shell vial culture tube with a round cover slip at the bottom of the
tube
• Add growth medium
• Add appropriate cells
• During incubation a cell monolayer is formed on top of the cover
slip
• Inoculate with unknown virus
• Advantage: its speed; most viruses are detected within 24 hours
Other Methods of Virus Quantitation
• Virus particles directly counted through E/M
• Nucleic acid-based assay through PCR
• LD50 or ID50
• Assay for infectious virus by plaque assay
– Cell monolayers or chorioallantoic membrane
• Viral Serology
• Serological detection of antibodies to a virus is an indirect evidence of
viral infection
• Primarily used for evaluating immune status and to diagnose viral
infections in situations in which virus cannot be cultivated
• Within 1-2 weeks after a primary viral infection, virus-specific IgM begin to
appear; followed a few days later by IgG
• IgM level peaks in 3-6 weeks and drop to undetectable levels in 2-3
months
• IgG levels peak in 4-12 weeks and remain for several months, some for
life
• Diagnosis of active infection
• Detection of virus-specific IgM in acute phase serum sample taken
at least 10-14 days after the onset of illness
• Detection of a four-fold rise in antibody titer between acute and
convalescent sera (taken 2-3 weeks after acute serum sample)

VIROLOGY
 Virology
 Study of viruses
 Dmitri Iwanowski (1892)
 First noted the filtrable organism, the tobacco mosaic virus (TMV)
 Wendell Stanley (1935)
 Confirmed that the filtrable organism is TMV
Origin of Viruses
• May be derived from DNA or RNA nucleic acid components of host cells that
have acquired the ability to exist independently
• May be degenerate forms of intracellular parasites

Classification of Viruses
(Animal Viruses)
 DNA Viruses
Poxviridae

34
 Herpesviridae
Adenoviridae
Papovaviridae
Papillomaviridae
Polyomaviridae
Hepadnaviridae
Parvoviridae
 RNA Viruses
Picornaviridae Filoviridae
Caliciviridae Paramyxoviridae
Togaviridae Orthomyxoviridae
Flaviviridae Bunyaviridae
Coronaviridae Arenaviridae
Reoviridae Rhabdoviridae
Astroviridae Retroviridae

Characteristics of Viruses
Structure
• Nucleic acid: either DNA or RNA
 Viral genome: for viral replication
 Single-stranded (SS) or double-stranded (DS)
 DNA viruses are DS except for Parvovirus
 RNA viruses are SS except for Reovirus
• Protein coat or capsid
 composed of capsomeres
 Virus: Nucleocapsid or Naked virus or Nonenveloped virus
 Viral envelope
 Lipid bilayer consisting of
a) matrix proteins
b) glycoproteins
 Virus: Enveloped virus
 Generalities:
 DNA viruses are enveloped except for Parvovirus,
Papovavirus (Papillomavirus and Polyomavirus), Adenovirus
 RNA viruses are enveloped except for Picornavirus,
Calicivirus, Reovirus, Astrovirus

Size
 Unit of measurement: Nanometer (nm)
 E/M; vary from 10 – 300 nm
 Smallest
 Parvovirus (22 nm)
 Picornavirus (28 nm)
 Largest
 Poxvirus (225 – 300 nm)
 Paramyxoviridae (150-300 nm)
 Filoviridae (80x1000 nm)

Shape (Capsid Symmetry)

 Icosahedral
 Spherical or cubic

35
  Helical
 Rod-shaped
 Complex symmetry
 Brick-shaped Poxvirus
 Tadpole-shaped
 Typical of bacteriophages

Multiplication
 Viral replication: Steps
 Adsorption (Attachment): have specific receptors on host cell
membranes
 Penetration: receptor-mediated endocytosis and membrane fusion
 Uncoating: physical separation of nucleic acid from its protein coat;
mediated by cellular enzymes
 Synthetic phase: early period and late period
 Inhibition of host cell DNA
 Early: synthesis of nucleic acids
 Late: synthesis of protein components
 Location of viral genome replication is characteristic for each virus
 DNA viruses, except Poxvirus, replicate in the nucleus of
the host cell
 Most RNA viruses replicate in the cytoplasm of the host
cell except for retroviruses and Influenza virus, in which
part of their replicative cycle occur in the nucleus of host
cell
 Nucleic acid: positive (+) or negative (-) sense
 Positive-sense
 Isolated RNA genomes which functions as mRNA
within the infected cells; are infectious
 Picornavirus and Togavirus
 Negative-sense
 Isolated RNA is noninfectious, virions usually carry
RNA polymerase that transcribes genomic RNA to
several complementary strands which serve as
mRNA
 Rhabdovirus and Retroviridae
 Assembly or Maturation
 Newly formed nucleic acids are enclosed by capsids
 Release: 2 mechanisms
 Rupture or lysis: for naked viruses
 Budding: for enveloped viruses

Atypical Viruses
 Defective viruses
 requires a helper virus (Delta Agent or Hepatitis D virus)
 Pseudovirions
 contain host cell DNA enclosed within capsid; can infect cells but cannot
replicate
 Viroids
 consist of single molecule of circular RNA without capsid; cause plant
disease

36
Prions
 Infectious particles composed of abnormal proteins (PrP); no viral component
 Abundant in neurons
 Most resistant infectious agent
 Cause of transmissible spongiform encephalopathy
 Humans: Kuru, Creutzfeldt-Jakob Disease
 Animals: Scrapie (goats and sheeps), Bovine spongiform encephalopathy
(Mad cow disease)

Cytopathic Effect (CPE)

 Cellular morphologic changes in the virus-infected host cells
 Cellular changes are observable on light microscopy
 Cellular changes include
 Cell death or lysis
 Fusion of virus-infected cells to form syncytia ( multinucleated giant cell
formation)
 Measles - Warthin-Finkeldey syncytia formation
 Inclusion body formation: discrete areas in the host cell nucleus or
cytoplasm containing viral proteins or particles
 Rabies - Negri body inclusion
 Cytomegalovirus - Giant cell with “owl’s eye”
 Smallpox - Guarnieri body
 Herpes simplex - Cowdry type A
 Molluscum contagiosum - Henderson-Patterson body or
Molluscum body
 Koilocytosis in HPV infection
 Transformation of host cells

Viral Interference
 In host cells infected with more than one virus, one virus may inhibit the
multiplication of the other virus (virus-induced inhibition of viral replication)
 Mechanisms
 First virus either block or destroy the receptors
 One virus may compete for components needed for replication
(polymerase)
 Production of interferon (IFN)
 Glycoproteins produced by many cells in the body in response to
inducers, the primary inducer: viruses
 RNA viruses are better interferon inducer than DNA viruses
 Species specific; not virus specific
 Antiviral mechanism
 In uninfected cells, INF induce the formation of antiviral
protein which alters the cell’s protein synthetic mechanism
and therefore inhibit viral replication
 Examples: synthetase (degrades viral mRNA); protein
kinase (inhibits protein synthesis)
 Types
 Alpha
 Derived from lymphocytes; have antiviral effect
 Beta

37
 
Derived from fibroblasts, macrophages, epithelial
cells; have antiviral effect
 Gamma
 Derived from T lymphocytes; immunoregulatory
function

Antiviral Agents
 Analogues of Ribonucleosides and Deoxyribonucleosides: Inhibit viral RNA and
DNA synthesis
 Acyclovir: highly specific for Herpes simplex and Varicella-Zoster virus;
less active against CMV and EBV
 Ganciclovir: powerful inhibitor of HSV multiplication; the best inhibitor of
CMV
 Zidovudine (AZT): inhibitor of retrovirus reverse transcriptase
 Ribavirin: treatment in aerosol form of infants with severe respiratory
syncytial virus infections
 Amantadine
 interfere with the earliest stages of viral replication, during penetration
and uncoating
 inhibit Influenza A virus multiplication
 Interferon
 antiviral, immunoregulatory, antiproliferative
 antitumor drug
 for hepatitis infections

Resistance/Sensitivity to Chemical and Physical Agents

 Ether Sensitivity
 Used to distinguish those viruses that possess envelope from
those that do not
 Enveloped viruses are generally sensitive
 Naked viruses are resistant
 Formaldehyde
 Destroy viral infectivity by reacting with nucleic acid
 Viruses with single-stranded genomes are inactivated much more
readily than double-stranded genome
 Minimal adverse effect on antigenicity of proteins
 Radiation
 Ultraviolet, x-ray, high-energy particles inactivate viruses
 Infectivity is the most radiosensitive because replication requires
expression of the entire genetic contents
 Heat sensitivity
 Icosahedral viruses are more stable, losing infectivity after several
hours at 37°C
 Enveloped viruses are more heat labile, rapidly dropping in titer at
37°C
 Viral infectivity generally destroyed by heating at 50-60°C for 30
minutes (except hepatitis B and polyomaviruses)

Specimen Collection For Viral Studies

• Specimens for detection of viruses should be collected as early as possible

38
 • Viruses may no longer be present as early as 2 days after the appearance of the
symptoms
Throat or Nasopharyngeal Swab or Aspirate
• Nasopharayngeal aspirates are superior to swabs for recovering viruses
– Nasopharyngeal secretions are collected by inserting a swab through the
nostril or by using a bulb syringe with 3-5 ml buffered saline
– Preferred for detection of RSV, influenza, parainfluenza, rhinovirus
Throat Swab
• Done by rubbing inflamed or purulent areas of the posterior pharynx with a dry,
sterile swab (avoid touching other unaffected areas)
• Acceptable for recovering enteroviruses, adenoviruses, herpes simplex virus
Bronchial and Bronchoalveolar Washing
• Wash and aspirated fluid collected during bronchoscopy are excellent specimens
for detection of viruses infecting the lower respiratory tract
• Influenza virus and adenoviruses
Rectal Swabs And Stool Specimens
• Fecal specimens are used to detect rotavirus, enteric adenoviruses and
enteroviruses (do not grow in cell cultures)
– Collection of 5-10 ml freshly passed diarrheic stools
– Rectal swab may be acceptable and is collected by inserting a swab 3-5
cm into rectum to obtain feces
Urine
• CMV, mumps, rubella, measles, polyomavirus and adenoviruses
• Virus recovery may be increased by processing multiple (2-3) specimens
• Best specimen is at least 10 ml of a clean-voided, first morning urine
Skin And Mucous Membrane Lesions
• Herpes simplex virus, Varicella-Zoster virus, Enteroviruses, can be detected in
vesicular lesions of the skin and mucous membranes
– Once the vesicle has ulcerated and crusted, detection of the virus
becomes difficult
– Tzanck Smear
• Small drop of vesicle fluid must first be aspirated using a
tuberculin syringe to be used for viral or bacterial culture
• Carefully unroof the vesicle; roof folded back
• Press a clean glass slide against the base of the ulcer making an
impression smear
• Slide is sent to the laboratory for modified Giemsa
Sterile Body Fluids Other Than Blood
• CSF, pericardial, pleural and peritoneal fluids may contain enteroviruses, herpes
simplex virus, influenza virus or cytomegalovirus
• Specimens are collected aseptically by the physician
Blood
• Viral culture of blood is primarily used to detect CMV
– Occasionally HSV, VZV, enteroviruses, adenoviruses are encountered
• A 3-5 ml anticoagulated blood (EDTA, citrate or heparin) collected in a vacutainer
tube
– 2 ml specimen is acceptable for pediatric patients
Bone Marrow
• Collected by aspiration
• Bone marrow for culture should be added to sterile tube with anticoagulant
Tissue Specimens

39
 • Tissue specimens are especially useful for detecting viruses that commonly
infect the lungs, brain, and GIT
• Collected by biopsy
Serum For Antibody Testing
• Acute and convalescent serum specimens may be needed to detect antibody
against specific viruses
• Acute specimens should be collected as soon as possible after the appearance
of the symptoms
• Convalescent serum collected 2-3 weeks after the acute serum
• Appropriate specimen is 3-5 ml serum

Specimen Transport, Storage, Processing

Specimens For Viral Culture
• Should not be allowed to stand at room temperature or higher temp
• Specimens should be placed in ice and transported to the lab at once
• If delay is unavoidable, should be refrigerated
– Storage up to 5 days, 4°C
– 6 or more days, at -20°C or preferably at -70°C
– Specimens for freezing should first be diluted or emulsified in viral
transport medium
• Blood for viral culture, is transported in a sterile tube containing anticoagulant
– Clotted blood are unacceptable
– Should be processed immediately; if delay, hold specimen at 2-8°C
– Processing must occur within 12-24 hours
• Blood
– Detection of most viruses is best accomplished by separating and
culturing leukocytes
– One of the best is a leukocyte preparation using Polymorphprep
(mixture of sodium metrizoate and dextran)
• mononuclear and polymorphonuclear cells are isolated from red
blood cells in a one-step procedure
• Serum can be stored for
– Hours or days at 4°C or for
– Weeks or months at -20°C or below before testing
• Many types of specimens are collected by swabs
– Cotton, rayon, dacron (preferred)
• Once collected, it is recommended that specimens on swab must be emulsified
in viral transport medium before transporting to the laboratory
Viral Transport Media
• Used to transport small volume of fluid specimens, small tissues, scrapings and
swabs
• Contain proteins such as serum, albumin, or gelatin to stabilize the virus and
antimicrobials to prevent the overgrowth of bacteria and fungi
• Examples: Stuart, Amies, Leibovitz-Emory, Hanks balanced salt solution (HBSS),
Eagle’s tissue culture medium
Processing
• Processing should occur in a biological safety cabinet or behind a protective
glass shield on the countertop
• Each specimen for viral studies should be accompanied by a request form which
contain the following

40
 • Patient identification, age, sex, source of specimen, clinical history or
virus suspected, date and time collected

Detection of Viruses in Clinical Specimens

Methods of Detecting Viruses
• Cytology and Histology
• Detection of viral inclusions and syncytia formation
• Histologic examination of tissues stained with H and E to detect
inclusions of CMV, Rabies, HPV, Molluscum contagiosum virus
• Cytology stains
• apanicolaou (Pap) smear
• Giemsa stain
• Detection of Herpes simplex and Varicella-Zoster inclusion bodies
• Tzanck smear/test
• Electron Microscopy
• Most helpful in detecting viruses that do not grow readily in cell cultures
• Immune E/M
• May be used to detect the following viruses
• Viruses causing gastroenteritis (Rotavirus, Norwalk agent,
Adenovirus) and encephalitis (Herpes simplex, Measles, JCV)
• Detection of Viral Antigens
• Most useful in detect ing RSV, Influenza and Parainfluenza viruses,
Herpes simplex, VZV, CMV and Rabies virus
• Immunofluorescent antibody methods
• Enzyme immunoassay methods
• Radioimmunoassay
• Latex agglutination
• Molecular Detection
• Nucleic acid probes
• Polymerase chain reaction
• Cultivation
• Embryonated egg
• Cell Cultures
• Originate as few cells and grow into a monolayer on the sides of
glass or plastic tubes
• Cells are continuously immersed in cell culture medium
• Once inoculated with specimen, cell cultures are incubated for 1-4
weeks (depending on the viruses suspected), at 35°C
• Kinds
• Primary cell lines
• Those that have been passed only once or twice
since harvesting; further passage result in
decreased susceptibility to viral infection
• Examples
• Primary monkey kidney cell (PMK)
• Human embryonic kidney (HEK)
• Low-passage (diploid) cell lines
• Cell cultures that remain virus-sensitive through 20-
50 passages
• Examples

41
 • Human diploid fibroblast cells (HDF) derived
from human kidney and lung fibroblasts
• WI-38 and MRC-5 both derived from human
embryonic lung
• Continuous cell lines
• Cells can be passed and remain sensitive to virus
infection indefinitely
• Examples
• Hep-2 cells (Human epidermoid carcinoma
cells)
• HeLa cells (Cervical carcinoma cells)
** A cell culture becomes a cell line once it has been
passed or subcultured in
• Identification of viruses detected on cell cultures are based on the following
– Noting the cell type that support viral replication
– Time of detection of cytopathic effect (CPE)
– Morphology of CPE

Quantitation of Cell Culture CPE

Quantitation Interpretation

Negative Uninfected monolayer

Equivocal (+/-) Atypical alteration of monolayer
involving few cells
1+ 1% - 25% monolayer exhibit CPE
2+ 25%-50% monolayer exhibit CPE
3+ 50%-75% monolayer exhibit CPE
4+ 75%-100% monolayer exhibit CPE

Shell Vial Culture

• A rapid modification of conventional cell culture
• Quick identification of the virus because the infected cell monolayer is
stained for viral antigen soon after infection before the development of
CPE
• Stain used is usually fluorescein-labeled virus specific antibody
• Procedure
• Shell vial culture tube with a round cover slip at the bottom of the
tube
• Add growth medium
• Add appropriate cells
• During incubation a cell monolayer is formed on top of the cover
slip
• Inoculate with unknown virus
• Advantage: its speed; most viruses are detected within 24 hours

Other Methods of Virus Quantitation

• Virus particles directly counted through E/M
• Nucleic acid-based assay through PCR
• LD50 or ID50
• Assay for infectious virus by plaque assay

42
 – Cell monolayers or chorioallantoic membrane

• Viral Serology
• Serological detection of antibodies to a virus is an indirect evidence of
viral infection
• Primarily used for evaluating immune status and to diagnose viral
infections in situations in which virus cannot be cultivated
• Within 1-2 weeks after a primary viral infection, virus-specific IgM begin to
appear; followed a few days later by IgG
• IgM level peaks in 3-6 weeks and drop to undetectable levels in 2-3
months
• IgG levels peak in 4-12 weeks and remain for several months, some for
life
• Diagnosis of active infection
• Detection of virus-specific IgM in acute phase serum sample taken
at least 10-14 days after the onset of illness
• Detection of a four-fold rise in antibody titer between acute and
convalescent sera (taken 2-3 weeks after acute serum sample)

Tumor Viruses

DNA Tumor Viruses

 Human papillomavirus (HPV)
 Cervical carcinoma, vaginal and vulvar carcinoma, penile carcinoma
 High risk are serotypes 16 and 18; low risk are 6 and 11
 Epstein-Barr virus (EBV)
 Burkitt’s lymphoma and Nasopharyngeal carcinoma
 Hepatitis B virus
 Primary hepatocellular carcinoma
 Human Herpesvirus 8 (Kaposi sarcoma-associated herpesvirus)
 Kaposi sarcoma

RNA Tumor Viruses

 Human T cell leukemia virus (HTLV)
 Adult T cell leukemia and lymphoma
 Hepatitis C virus (HCV)
 Primary hepatocellular carcinoma
 Hepatitis D (Delta agent)
 Primary hepatocellular carcinoma

43
 DNA VIRUSES

Poxviridae
 Brick-shaped complex viruses;225-300 nm
 All members produce skin lesions
 Human pathogens: Variola or Smallpox, Monkeypox, Vaccinia, Cowpox, Orf
(contagious pustular dermatitis), Pseudocowpox (Milker’s nodule virus), Yaba
monkey tumor virus, Tanapox virus, Molluscum contagiosum

Herpesviridae
 Double-stranded DNA genome; enveloped icosahedral capsid ;180-250 nm
 General Characteristics
 Produce skin lesions
 Cause latent infection
 Oncogenic potentials

Human Herpesvirus Classification

 Alphaherpesvirus
 Herpes simplex virus (HSV) 1 and 2
 Varicella-Zoster virus (VZV)
 Betaherpesvirus
 Cytomegalovirus (CMV)
 Gammaherpesvirus
 Epstein-Barr virus (EBV)
 Human herpesvirus 6 (Exanthem subitum or Roseola
infantum)

Herpes Simplex Virus (HSV) 1 & 2

• Transmission: Direct contact with infected secretions
• Site of latency: sensory nerve ganglia
• Disease: Gingivostomatitis, pharyngitis, herpes labialis, genital infection,
neonatal, conjunctivitis, keratitis, herpetic whitlow, encephalitis, disseminated
disease
• Detection: Cell culture (HDF, MRC-5), EIA, FA stain, PCR
• Cowdry type A inclusions
• Treatment: Acyclovir
• Prevention: Avoid contact

Varicella-Zoster Virus (VZV

• Transmission: Close personal contact, especially respiratory
• Site of latency: Dorsal root ganglia

• Disease
– Primary infection: Chicken pox (Varicella)
– Reactivation: Shingles (Herpes zoster)
• Detection: Cell culture (HDF), Shell vial culture, FA stain, PCR
• Treatment: Acyclovir
• Prevention: Varicella vaccine

Epstein-Barr Virus (EBV)

• Transmission: Close contact with infected saliva

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 • Site of latency: B lymphocytes
• Disease: Infectious mononucleosis
• Detection: Serology (positive heterophile antibodies), PCR
• Oncogenic: Burkitt’s lymphoma, Nasopharyngeal carcinoma
• Treatment: Supportive
• Prevention: Avoid contact

Cytomegalovirus (CMV)
• Transmission: Close contact with infected secretions, blood transfusion,
organ transplant, transplacental
• Site of latency: White blood cells and endothelial cells
• Disease
– Asymptomatic infection, congenital disease of newborn, symptomatic
disease of immunocompromised host, heterophile-negative infectious
mononucleosis
• Diagnosis: Cell culture (HDF), Shell vial culture, FA stain, PCR
• Treatment: Ganciclovir; supportive
• Use of CMV antibody-negative blood and tissue for transfusion and
transplantation

Adenoviridae
 Double-stranded DNA genome; icosahedral capsid, non-enveloped; 70 nm
 Human Adenoviruses: 51 serotypes
 Predilection for mucosal epithelial cells of the respiratory, gastrointestinal
and conjunctiva
 Transmission: Respiratory, fecal-oral, direct contact (eye)
 Site of latency: replication in oropharynx
 Disease
 Pharyngitis, keratoconjunctivitis, Pneumonia, Hemorrhagic cystitis,
Disseminated disease, Gastroenteritis in children
 Diagnosis: Cell culture (Hep-2), EIA
 Treatment: Supportive

Papillomaviridae
• Double-stranded DNA; icosahedral, non-enveloped; 55 nm
• Human Papillomavirus (HPV): >100 serotypes
• Transmission: Direct contact, sexual transmission
• Site of latency: Epithelial tissue
• Disease: Skin, genital warts and anogenital warts, condyloma acuminata
• Diagnosis: Cytology, DNA probes
• Oncogenic: Cervical and penile carcinoma (especially HPV types 16 and 18)
• Treatment: Spontaneously disappear; surgical or chemical removal
• Prevention: Avoid contact with infected person

Polyomaviridae
• Double-stranded DNA; non-enveloped, icosahedral; 45 nm
• Human Polyomavirus
– JC virus – Progressive multifocal leukoencephalopathy (PML)
– BK virus – nephropathy in transplant patients
• Transmission: Probably direct contact with infected respiratory secretions
• Site of latency: Kidney

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 • Disease
– BKV: Reactivation in immunocompromised patients causes hemorrhagic
cystitis
– JCV: Progressive multifocal leukoencephalopathy (PML)

• Detection
– JCV by E/M or PCR
– BKV by cytology or PCR
• Treatment: Supportive
• Prevention: Avoid contact with virus

Parvoviridae
 Non-enveloped icosahedral capsid; 22 nm
 Transmission: Close contact, probably respiratory
 Parvovirus B19: Erythema infectiosum or Fifth disease, aplastic crisis in
patients with chronic hemolytic anemia and fetal infection and stillbirth
- targets the immature erythroid precursor cells
 Detection: Serology, PCR, histology
 Treatment: Supportive
 Prevention: Avoid contact

Hepadnaviridae: Hepatitis B Virus (HBV)

 Enveloped icosadedral nucleocapsids
 Partially double-stranded DNA genome and DNA polymerase
 Integrate into hepatocyte DNA and cause transformation
 HBV Structure
 42 nm Dane particle
 27 nm core (nucleocapsid)
 22 nm spherical or tubular particles are envelope components without
nucleic acids
 Hepatitis B surface antigen (HBs Ag; Australia Ag) HBs Ab
 Hepatitis B core antigen (HBc Ag) HBc Ab
 Hepatitis B e antigen (HBe Ag) HBe Ab
 Chronic hepatitis B infection
 The persistence of HBs Ag beyond 6 months after infection
 Transmission
 Humans are reservoir and vector
 Spread by direct contact including exchange of body secretions, receiving
contaminated blood products, and percutaneous injection of virus
 Site of latency: Liver
 Disease: Acute infection with resolution (90%); fulminant hepatitis (1%); chronic
hepatitis (9%); chronic infection
 IP is 12 weeks
 Chronic infection and carriers (10-16% in Phil)
 Consequence: liver cirrhosis and/or hepatocellular carcinoma
 Diagnosis: Serology and viral antigen detection
 Oncogenic: Liver cancer
 Prevention: HBV vaccine; hepatitis B immune globulin

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 RNA VIRUSES

Picornaviridae
 Single-stranded genome; icosahedral, non-enveloped; 25-30 nm
 Human Enteroviruses
 Poliovirus: 3 serotypes
 Coxsackie A virus: 24 serotypes
 Coxsackie B virus: 6 serotypes
 Echoviruses: 34 serotypes
 Human Hepatitis A virus (Human Enterovirus 72)
 IP is 2-6 weeks
 Anorexia, malaise, nausea, diarrhea, abdominal discomfort, fever,
chills and in some cases jaundice
 No chronic form
 Serology
 Acute hepatitis A infection is diagnosed by detection of IgM
anti-HAV
 IgM anti-HAV appear 4 weeks after the infection and
disappear about 3-4 months after infection
 Presence of IgG indicates immunity
 Human Rhinoviruses: 113 serotypes (Common colds virus)
* Foot-and-Mouth Disease (Aphthovirus) in lower animals
 Transmission of Enteroviruses
 Fecal-oral route
 Diseases caused by Enteroiviruses
 Poliomyelitis (Polio), Herpangina (Coxsackie A), Pleurodynia (Coxsackie
B), Aseptic meningitis (many types), Hand-foot-mouth disease (Coxsackie
A), Pericarditis and myocarditis (Coxsackie B), Acute hemorrhagic cystitis
(Enterovirus 70), Fever, Myalgia, “Flu”
 Detection: Cell culture (PMK and HDF), serology
 Treatment: Supportive
 Prevention: Avoid contact with virus; vaccination for polio
Caliciviridae
 Single-stranded genome; icosahedral capsid; non-enveloped; 35-40 nm

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  Human pathogens
 Norwalk agent
 Transmission: Fecal-oral route
 Disease: Most frequent cause of acute viral gastroenteritis in
adults
 Detection: Immunoelectron microscopy, Serology, difficult to
cultivate
 Treatment: Supportive
 Prevention: Food and water precautions
 Hepatitis E virus (HEV)
 Now classified as separate family HEPEVIRIDAE
 Cause hepatitis similar clinically to Hepatitis A
 Mortality rate is 10-20% in pregnant women
 Diagnosis is mainly by serology; difficult to cultivate

Astroviridae
• Single-stranded, (+) sense; 28-30 nm, nonenveloped
• Star-shaped on E/M
• Human Astrovirus: 8 serotypes
– Cause childhood diarrheas
• Detected by electron microscopy

Togaviridae
 Single-stranded genome, positive-sense; enveloped, icosahedral capsid;
 60-70 nm
 Genus Alphavirus: Mosquito-borne viruses
 Eastern, Western, Venezuelan equine encephalitis viruses
 Chikungunya virus (transmitted by Aedes aegypti)

 Genus Rubivirus
 Rubella virus (German measles)
 Transmission: Respiratory, transplacental
 Disease: Rubella (German measles), Congenital rubella (first
trimester)
 Detection: Serology
 Treatment: Supportive
 Prevention: Rubella vaccine

Filoviridae
 Single-stranded genome; long, filamentous (helical) capsid; enveloped
 80x1000 nm
 Human Pathogens
 Marburg virus
 Ebola virus
 Zoonotic: From infected African green monkeys
 Transmission: Direct contact with infected secretions especially
blood
 Disease: African hemorrhagic fever
 Severe hemorrhagic fever and liver necrosis
 Mortality rate of 90%

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  Detection: Electron microscopy, ELISA for detection of viral Ag
and Ab, cell culture (monkey kidney cells)
 Treatment is supportive
 Prevention: avoid contact with virus and export prohibition on wild-
caught monkeys

Paramyxoviridae
 Single-stranded genome; enveloped helical capsid
 150 nm
 Human Pathogens
 Measles virus or Rubeola
 Transmission: Contact with respiratory secretions; extremely
contagious
 Disease: Measles (Rubeola); Subacute sclerosing panencephalitis
(SSPE)
 Detection: Cell culture (PMK) and serology; Warthin Finkeldey
giant cells
 Treatment: Supportive
 Prevention: Measles vaccine
 Mumps virus
 Transmission: Person-to-person contact, presumably respiratory
droplets
 Disease: Mumps (Epidemic Parotitis)
 Detection: Cell culture (PMK) and serology
 Treatment: Supportive
 Prevention: Mumps vaccine
 Parainfluenza viruses types 1-4
 Transmission: Contact with respiratory secretions
 Disease:
 Adults: Upper respiratory infection, rarely Pneumonia
 Children: Croup (Acute laryngotracheobronchitis), Bronchiolitis
and Pneumonia
 Detection: Cell culture (PMK), Shell vial culture, and FA
 Epidemiology: 4 serotypes; disease occurs year-round
 Treatment: Supportive
 Prevention: Avoid contact with virus
 Respiratory syncytial virus (RSV)
 Transmission: Person-to-person by hand and respiratory contact
 Disease: Primarily in infants and children
 Infants: Bronchiolitis, Pneumonia, and Croup
 Children: Upper respiratory infection
 Detection: Cell culture (HEp-2), EIA, FA stain
 Epidemiology: Nosocomial transmission occur readily
 Treatment: Supportive
 Prevention: Avoid contact with virus, Prevent nosocomial infection
Orthomyxoviridae
 Single-stranded genome; enveloped helical capsid
 80-120 nm
 Influenza viruses types A, B, C
 Avian flu: H5N1
 Swine flu: H1N1

49
  2 important envelope glycoproteins
 Hemagglutinin (HA)
 Neuramidase (NA)
 Transmission: Contact with respiratory secretions
 Disease: Influenza (malaise, headache, myalgia, cough); Primary influenza
pneumonia
 Detection: Cell culture (PMK), EIA, FA stain
 Epidemiology
 Viral subtypes based on hemagglutinin (H) and neuraminidase (N)
 Can infect humans and animals
 Antigenic shift (major variation) and antigenic drift (minor) results to local
or worldwide outbreaks
 Treatment: Supportive
 Prevention: Influenza vaccine
Bunyaviridae
 Enveloped helical nucleocapsids
 100 nm
 Human Pathogens
 Mosquito-borne
 Sandfly fever virus
 Rift Valley fever virus
 Hantaan virus (Korean hemorrhagic fever)
 Zoonotic (rodents)
 Sin nombre
-Hemorrhagic fever with pulmonary syndrome
Flaviviridae
 Enveloped icosahedral nucleocapsids
 45-55 nm
 Mosquito-borne
 Dengue virus
 4 serotypes: 1, 2, 3, 4
 Transmission: bite of Aedes aegypti mosquito
 Disease: Classical Dengue fever
 Dengue hemorrhagic fever/ Dengue shock syndrome
(immune-
mediated reactions)
 Danger signs: decreasing platelet count and increasing hematocrit
 Detection: Serology
 Prevention: Vector control
 Japanese encephalitis virus
 Transmission: Bite of infected Culex mosquito
 Disease: Encephalitis
 Detection: Serology
 Prevention: Vector control
 Yellow fever virus
 St Louis encephalitis virus
 Zika virus (microcephaly virus; Aedes aegypti)

 Hepatitis C virus (HCV)

 Hepatitis C infection previously referred as non-A, non-B hepatitis
 Responsible for 90% of post-transfusion hepatitis

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  Chronic infection
 Consequence of liver cirrhosis and/or hepatocellular carcinoma
 Serology: detection of anti-HCV

Coronaviridae
 Enveloped helical nucleocapsids
 120 nm
 Petal-shaped spikes producing a crown-like structure
 Human Coronavirus
 SARS virus
 Transmission: Close contact with infected persons
(“superspreaders”)
 Disease
 Originated from China, first outbreak in Nov. 2002
 I.P. average of 6 days; early symptoms of fever, malaise,
chills, headache, dizziness, cough and shortness of
breath; progress rapidly to severe acute respiratory
disease; high mortality rate especially among the elderly
 Detection: Culture (Vero monkey kidney cells); Serology by ELISA
or FAT
 Epidemiology: Originated from pigs and domestic fowls
 Treatment: Supportive
 Prevention: Isolation of patients, quarantine of those exposed,
travel restrictions, wearing of protective gears

 MERS-CoV (Middle East Respiratory Syndrome Coronavirus; from

camels and bats)

Reoviridae
 Naked icosahedral nucleocapsid
 75 nm
 Wheel-like appearance
 Human Rotavirus: 1-5
 Transmission: Fecal-oral, survives well on inanimate objects
 Disease: Gastroenteritis in infants and children 6 months to 2 years
 Detection: EIA, Latex agglutination
 Epidemiology: 4 serotypes; nosocomial infections can occur easily
 Treatment: Supportive especially fluid replacement
 Prevention: Avoid contact with the virus
Rhabdoviridae
 Bullet-shaped enveloped helical nucleocapsid
 180x75 nm
 Rabies virus
 Transmission: Bite of a rabid animal (most common); Inhalation; Corneal
transplant
 Reservoir: vampire bats
 Disease: Rabies
 Detection: FA staining; Serology; Negri bodies
 Treatment: Supportive
 Prevention: avoid contact with saliva of infected animal or person;
vaccinate animals; Rabies vaccine and hyperimmune antirabies globulin

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Arenaviridae
 Enveloped helical nucleocapsid
 110-130 nm
 Human Pathogens
 Lymphocytic choriomeningitis virus (LCM)
 Argentinian (Junin) and Bolivian (Machupo) viruses
 Lassa virus

Retroviridae
 Enveloped particles containing a coiled nucleocapsid within a probably
icosadehdral core shell
 Reverse transcriptase
 80-100 nm
 Human Retroviruses
 Human T cell Lymphotropic virus type I (HTLV-I)
 associated with adult human T cell leukemia and lymphoma
 Human Immunodeficiency virus 1 and 2 (HIV)
 Lentivirus
 HIV-1 and HIV-2
 HIV-1 has 3 groups: M, N, O (predominant M type contains
10 subtypes or “clades”, A-J)
 HIV-2 has 5 subtypes A-E
 Enveloped RNA virus; 100-120 nm
 Virus encodes its genetic information in RNA and uses a unique
viral enzyme called reverse transcriptase to copy its genome into
DNA
 Latency: CD4+T cells
 HIV genome: genes
 gag, pol,pro,env: code for proteins directly involved in viral
replication (structural proteins)
 gag: viral core proteins (p24)
 pol: reverse transcriptase, protease, integrase
 pro: which encodes protease enzyme
 env: envelope glycoproteins (gp 120, gp 41)
 tat, rev, nef, vif
 regulatory or accessory proteins
 HIV infection
 Target cells are
 CD4+T cells
 Monocytes and macrophages
 Dendritic cells and Langerhans cells
 Transformed B cells
 Astrocytes, Oligodendrocytes, Microglial cells
 Pathogenesis
 CD4 receptors are needed for attachment to host
cell
 Co-receptor /second receptor needed for fusion
and entry into target cells
 CCR5 : predominant co-receptor for
macrophages

52
  CXCR4 : predominant co-receptor for
lymphocytes
 Transmission
 HIV can be isolated from the body fluids and
infected cells but virus infected cells appear to be
the major vehicle for transmission
 Highest concentration in the semen and
blood
 Sexual transmission
 Parenteral
 Vertical transmission
 Persons at high risk
 Homosexuals or heterosexuals with
multiple sexual partners
 Intravenous drug abusers
 Persons receiving multiple blood
transfusion
 Babies of infected mothers
 Medical and paramedical workers
 Clinical manifestations (CDC)
 IP 4-12 weeks
 Early or Acute
 High virus production
 Viremia and widespread seeding of
lymphoid tissues
 Nonspecific illness
 Spontaneously resolve in 2-4 weeks
 Chronic or Latent
 Latency and ARC (AIDS related complex)
 Relative containment of the virus
 HIV antibody concentration at its peak
 Patients either asymptomatic of develop
persistent generalized lymphadenopathy
 ARC: unexplained weight loss, fever, oral
lesions
 Final or Crisis or Full blown AIDS
 Breakdown of host defense
 Dramatic increase in viremia
 Disappearance of HIV antibodies
 CDC guideline: any HIV-infected person
with fewer than 200 CD4+T cells/uL
 Opportunistic infections
 Protozoa: Toxoplasma,
Cryptosporidium, Isospora
 Fungi: Candida albicans,
Cryptococcus neoformans,
Pneumocystis
 Viruses: Cytomegalovirus, Herpes
virus, Varicella-Zoster virus

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  Bacteria: Mycobacterium avium-
intracellulare, Mycobacterium
tuberculosis,
Listeria monocytogensis
 Secondary neoplasms: Kaposi sarcoma,
lymphomas, cervical cancer, anogenital
cancer
 Neurologic disease

*** Only 10% of HIV-infected individuals will develop full

blown AIDS after a chronic phase lasting for 7-10 years

 Laboratory diagnosis
 Culture
 Antigen detection
 PCR
 Serology: HIV antibody detection
 ELISA: screening test
 Western blot or IF: confirmatory test

 Treatment
 AZT
 Treat infections resulting from immunosuppression
 Prevention
 Avoid contact with infected blood and blood
products and other secretions
 Blood for transfusion is screened for antibodies to
HIV 1 and 2

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 SEROLOGY OF HEPATITIS

HEPATITIS VIRUSES
• Hepatitis A, B, C, D, E, F, G
• Hepatitis F and G
– RNA viruses
• Target cells are liver cells or hepatocytes
• All are RNA viruses except Hepatitis B
• All are transmitted by parenteral route except Hepatitis A and E
– Hepatitis A and E transmitted through the fecal-oral route
Hepatitis A Virus (HAV)
• Picornaviridae
• IP is 2-6 weeks
• Anorexia, malaise, nausea, diarrhea, abdominal discomfort, fever, chills and in
some cases jaundice
– No chronic form
• Serology
– Acute hepatitis A infection is diagnosed by detection of IgM anti-HAV
– IgM anti-HAV appear 4 weeks after the infection and disappear about 3-4
months after infection
– Presence of IgG indicates immunity
Hepatitis B Virus (HBV)
• Hepadnaviridae
• IP is 12 weeks
• Chronic infection (10-16% in Phil)
• Chronic carriers
• Consequence: liver cirrhosis and/or hepatocellular carcinoma
• Found in all body fluids
• HBV Structure
• 42 nm Dane particle
• 27 nm core (nucleocapsid)
• Spherical or tubular particles are envelope components without nucleic
acids
• Serologic Markers of HBV
• Hepatitis B surface antigen (HBsAg): A protein on the surface of the
hepatitis B virus (HBV); it can be detected in high levels in serum during
acute or chronic HBV infection. The presence of HBsAg indicates that the
person is infectious. The body normally produces antibodies to HBsAg as
part of the normal immune response to infection. HBsAg is the antigen
used to make Hepatitis B vaccine.
• Hepatitis B surface antibody (anti-HBs): The presence of anti-HBs is
generally interpreted as indicating recovery and immunity from HBV
infection. Anti-HBs also develops in a person who has been successfully
vaccinated against hepatitis B.
• Total hepatitis B core antibody (anti-HBc): Appears at the onset of
symptoms in acute hepatitis B infection and persists for life. The presence
of anti-HBc indicates previous or ongoing infection with HBV in an
undefined time frame.

55
 • IgM antibody to hepatitis B core antigen (IgM anti-HBc): Positivity
indicates recent infection with HBV (≤6 months). Its presence usually
indicates acute infection.
• Hepatitis B e antigen (HBeAg): A secreted product of the nucleocapsid
gene of the hepatitis B virus that is found in serum during acute and
chronic hepatitis B infection. Its presence indicates that the virus is
replicating and the infected person has high levels of HBV.
• Hepatitis B e antibody (HBeAb or anti-HBe): Produced by the immune
system temporarily during acute HBV infection or consistently during or
after a burst in viral replication. Spontaneous conversion from e antigen to
e antibody (a change known as seroconversion) is a predictor of long-
term clearance of HBV in patients undergoing antiviral therapy and
indicates lower levels of HBV.
• HBV DNA: Indicates an active HBV infection
Hepatitis C Virus (HCV)
• Flaviviridae
• Hepatitis C infection previously referred as non-A, non-B hepatitis
• Responsible for 90% of post-transfusion hepatitis
• Chronic infection: >50%
• Consequence of liver cirrhosis and/or hepatocellular carcinoma
• Serology
• Anti-HCV by ELISA
• Confirmation by Western blotting
• PCR
• Also confirmatory
• Identify specific serotype
Hepatitis D Virus (HDV)
• Delta agent
– Defective RNA virus that requires an external HBV envelope (helper
virus) to become infectious
• Occurs as either
– Acute form or coinfection: <5% chronicity
– Chronic or superinfection: 80% chronicity

Hepatitis E Virus (HEV)

• Hepeviridae
• No chronic disease
• Similar to HAV in structure, transmission and clinical infection
• Responsible for 20% mortality rate in infected pregnant women

Summary of Hepatitis Serology

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 END OF VIROLOGY

GOOD L U C K !!!
jmb2016

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