Rizalinda Sjahril

Basics of Diagnosis and Therapy

2009

D IA G N O S IS A N D
TH ER A P Y O F V IR A L
A N D FU N G A L
IN FEC TIO N

Isolation and Identifi

cation ofViruses
CELL and ORGAN CULTURE
Cells are derived from tissue source, allowed to grow in

nutrient media until a confluent one cell layer is

obtained.
Organ culture uses a tissue fragment with a specialized
function (e.g. Fetal trachea with ciliated epithelial cells)
Primary cell culture: cells derived from the initial growth of
cells from a tissue source
Secondary cell culture: redispersed and regrowth of
primary cell culture.
Cell lines: cells that have transformed spontaneously and
become immortal, or cells are obtained from cancerous
tissue

Isolation and Identifi

cation ofViruses

Detection of Viral growth

Observing CPE; alteration or cytopathic effect in

cells due to viral infection. Alteration may be

change in morphology or death of cells
Observing hemadsorption or interference on cells
that do not show CPE.
Interference: cells that do not show CPE after infected
with a virus, but the cells may show CPE if infected
with another virus (challenged). But the challenging
virus cannot infect the cell culture in the presence of
the first virus
Expressing infection on lymphocytes (EBV and HIV)

Isolation and Identifi

cation ofViruses
Quantitation of Viruses
Hemagglutination Assay:

Viruses have attachment proteins (=hemagglutinins) that can be

bound on red blood cells, thus causing hemagglutination.
Dilution of virus preparation reacted with a constant amount of red blood
cells will show the decreasing amount of hemagglutination. Agglutinated
RBCs settle dispersed on the bottom, but unagglutinated RBCs forms a
tight button shaped sedimentation on the bottom of the well/tube. The
titer is the reciprocal of the last dilution that still shows agglutination.
Plaque Assay
Virus at several different concentration is reacted into a one layer of
semisolid culture cells. The growth of any one virus is therefore limited in
its initial place, forming a round cleared area (plaque) due to the death of
infected cells. The number of plaques is then counted and calibrated
according to the dilution factor. Viral titer is the number of Plaque forming
units per millimeter (Pfu/ml)

Isolation and Identifi

cation ofViruses
In Vivo Isolation methods
Embryonated hens egg isolation and

propagation of influenza A virus

Animal inoculation suckling mouse
<48 hours of life are infected with virus,
observed for development of illness
(paralysis, convulsion, poor feeding,
death). Eg, arboviruses, rabies virus

 Viral Identification
Tentative identification based on CPE

characteristics may suggest a virus

Further identification are performed by;
Neutralization test
Serology
Cytology
Histology
Electron Microscopy

Im m unology for identifi

cation ofvirus

Antigen-antibody reaction :
Precipitation
Agglutination
Neutralization
Complement fixation
Immunofluorescence or radioisotope or
enzyme labelling
Antibody detection
Antigen detection

N ucleic Acid D etection

Analysis of Nucleic Acid:
Agarose gel electrophoresis
Restriction endonuclease Digestion
DNA hybridization
Polymerase Chain Reaction
Nucleic acid sequence analysis

AntiviralTherapy
General considerations
Viruses comprises of DNA or RNA, capsid and many

have lipid or lipoprotein envelope

Viruses uses the cellular structures for replication
unique for the virus
Target of inhibition includes each of the replication
steps:
Attachment
Penetration
Uncoating
RNA-directed DNA synthesis
Assembly
Release

AntiviralAgents
Summary of Antiviral Agents
Mechanism of
Action

Antiviral agent

Viral
spectrum

Inhib of viral
uncoating

Amantadine, rimantadine

Flu A

Neuraminidase
inhibition

Oseltamivir, Zanamivir

Flu A, Flu B

Inhib of viral DNA

polymerase

e.g. Acyclovir, Famciclovir, HSV,VZV

Valacyclovir
e.g. ganciclovir
CMV, HSV,
VZV

Inhib of viral reverse

transkriptase

e.g. Zidovudine,
dideoxyinosine,
dideoxycytidine
e.g. Lamivudine

HIV

Inhib of viral
protease

e.g. Saquinavir, Indinavir

HIV

Inhib of viral protein

synthesis

Interferon

HBV, HCV, HPV

HIV, HBV

FungiCellStructure
Typical eukaryotic features
Nucleus and nucleolus, linear chromosome
Sitoplasm contains organelles; mitochondria,

Golgi apparatus
Rigid cell wall distinguishes fungus from
mammalian cells, and a different composition of
cell wall that distinguishes them from bacteria
and plants

Identification of medically important molds

are based on the morphology and

development of reproductive elements
(conidia and spores)

Fungalterm s:
Conidia: asexual form of reproductive
elements
Spore: sexually
produced
elements
Conidiophore:
a
stalk structure where conidia buds
Macroconidia:
Large sized
conidia
Microconidia:
Small
sized
conidia
Chlamydoconidia=arthroconidia:

chlamydia that develops within the

hyphae
Ascospore: a sexual spore

D iagnosis offungalinfection
1. Direct Microscopic Examination: wet
2.
3.
4.
5.
6.
7.

mount
Antigen Detection: Fluorescent
antibody
Culture & Isolation
DNA Detection
Skin test
Serology
Histopathology (biopsy)

1. Microscopic Examination
Specimens : Skin scraping, sputum, Pus
Methods:
1.

Potassium hydroxide test: Skin scraping + KOH 10% and

place under a coverslip
* strong alkali digests the tissue elements such as epithelial cells,
leukocytes, debris

2.
3.

4.

Gram sputum or pus usually gram positive

Direct Immunofluorescence : to identify fungi in fixed tissue
section; using calcifluor that binds to polysaccharides in
cellulose and chitin, and the fluorescence is viewed under ultra
violet light.
Gomori Methenamine Silver (GMS) stain fungal cell black in
tissue section.

2. Detection of Fungal Antigen

Specimen: liqour
Method : latex agglutination test
Examples:

Cryptococcus
Histoplasma

3.Culture : Isolation & Identification

Specimens : Skin scraping, sputum, pus, liquor, blood
Medium :
Sabourauds dextrose agar (contains only glucose and peptones, pH 5.6).
Most bacteria fail to grow/grow poorly in this medium.
Temp. 25o-30oC, paired culture at 35oC and 25oC for dimorphic fungi
May be added with cycloheximide to inhibit the growth of some saprophytic
fungi /contaminants from the environment
Selective medium: Blood agar (or other enriched media) containing
chloramphenicol and gentamicin to obtain pure culture.
**But note that adding cycloheximide in Saboraud agar may inhibit the
growth of Cryptococcus neoformans, and chloramphenicol may inhibit the
yeast form of some dimorphic fungi.

Identification: to determine YEAST or MOLD

1.
2.
3.
4.

Colony: mycelium, septation, branching, pigmentation, spore or conidia

production.
Microscopic: morphology of conidia and conidiophore
Biochemical reactions.
DNA Probe

ect Methods Based on the Host Immune R

Skin test (dermal hypersensitivity testing): now

used most often to evaluate a patients immunity

Serology tests : Fungi is a poor antigen
1. Latex agglutination test : IgM
2. Immunodiffusion test: Ig G
3. Complement fixation test : IgG
Frequently false positive due to cross reactions.
Antibody detection: must be done 2-3 months
after the onset of disease

AntifungalChem otherapy
Most fungal infections are self-

limiting and require no

chemotherapy
For superficial mycoses topical
therapy is given
For deep mycoses that are
uncontrolled by the immune system
require prolonged use of relatively
toxic antifungals.

Antifungalaff
ecting the m em brane sterols
Polyenes: Nystatin and amphotericin B bind

to sterols in the cytoplasmic membrane of the

eukaryotic cells forming membrane channels
causing leak of small molecules from the
cytoplasm.
ACTIVE against most fungi

Azoles: imidazole, ketokonazole, triazoles,

fluconazole, itraconazole inhibits the enzyme

which is crucial for ergosterol synthesis
Allylamines: inhibits squalene epoxidase during
the ergosterol synthesis leading to accumulation
of squalene. Eg: terbinafine, naftifine

Antifungals that Aff

ect N ucleic Acid synthesis

5-Flucytosine: an antimetabolite

analog of cytosine, inhibits RNA, DNA

and protein synthesis.
ACTIVE on most YEASTS but not molds
Resistance develops so the use is

combined with amphotericin B

Antifungals that aff

ect cellw allsynthesis
Agents acting on glucan and chitin

synthesis are emerging

Echinocandins able to block glucan
synthesis; caspofungin may be
used against Candida and
Aspergillus. Nicomycins that disrupt
chitin synthesis are being developed

O ther antifungalagents
Griseofulvin: for superficial

mycoses (dermatophyte infection),

oral administration, concentrates in
the keratinized layers of the skin,
slow response
Potassium Iodide: effective only for
cutaneous sporotrichosis

Agent

Mech of
action

Mech of
resist

Route

Clinical
use

POLYENES
Nystatin
Amph. B

Disrupt
membrane

Strerol modifcn

AZOLES

Blocks ergost.
synthesis

Active efflux,
demethylase
alteration, or
overproduction

ALLYLAMINES
Terbinafine
Naftifine

Squalene
accumulation

? Active efflux

FLUCYTOSINE

RNA & DNA

Synthesis

Permease or
Oral
modifying enzymes
absent or decrsd

Candida,
Cryptococcus

ECHINOCANDI
NES

Block glucan
synthesis

unknown

IV

Aspergillus,
Candida

GRISEOFULVIN

Disrup
microtubules

unknown

Oral

Dermatophyte
s

POTASSIUM
IODIDE

Unknown

unknown

Oral

Sporothrix
Schenckii

TOLNAFTATE

Unknown

unknown

oral

Dermatophyt
es

Most fungi
Topical
intraveno
us
Varies;
Oral, IV,
topical

Oral
Topical

Candida

Dermatophyte
s

Further Readings
Sherris Medical Microbiology 4th ed,

an Introduction to Infectious
Diseases, Kenneth J Ryan & C.
George Ray (eds), 2004.
Bayley and Scotts Diagnostic
Microbiology, Baron, Peterson,
Finegold (eds).