DiagnosIs and Therapy of

Viral and Fungal Infection

BAGIAN MIKROBIOLOGI FKUH

BAGIAN MIKROBIOLOGI FKUH

Isolation and Identifi

cation
ofViruses (1)
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)

Isolation and Identifi

cation
ofViruses(2)
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 of
Viruses(3)
Detection of Viral growth
1. Observing CPE; alteration or

cytopathic effect in cells due to viral

infection. Alteration may be change
in morphology or death of cells

Tissue culture of epitheloid cells

Tissue culture of fibroblasts cells.

Enteroviru
s

Paramyxovir
us

Herpetoviru
s

Isolation and Identifi

cation
ofViruses(4)
2. 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(5)
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.

Plate H AI

Isolation and Identifi

cation
ofViruses(6)
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)

Plaque form ed in an agar plate

http://www.virology.ws/2009/07/06/d

etecting-viruses-the-plaque-assay/

Isolation and Identifi

cation
ofViruses(7)
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

Immunology for identification of virus

Antigen-antibody reaction :

Precipitation
Agglutination
Neutralization
Complement fixation
Immunofluorescence or radioisotope
or enzyme labelling
Antibody detection
Antigen detection

Nucleic Acid Detection

Analysis of Nucleic Acid:
Agarose gel electrophoresis
Restriction endonuclease

Digestion
DNA hybridization
Polymerase Chain Reaction
Nucleic acid sequence analysis

Antiviral Therapy
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

Antiviral Agents
Summary of Antiviral Agents
Mechanism of
Action

Inhib of viral
uncoating
Neuraminidase
inhibition
Inhib of viral
DNA polymerase

Inhib of viral
reverse
transkriptase

Antiviral agent

Viral spectrum

Amantadine,
rimantadine
Oseltamivir,
Zanamivir
e.g. Acyclovir,
Famciclovir,
Valacyclovir
e.g. ganciclovir

Flu A

e.g. Zidovudine,
dideoxyinosine,
dideoxycytidine

HIV

Flu A, Flu B
HSV,VZV
CMV, HSV,
VZV

HIV, HBV

Antiviral Agents
Summary of Antiviral Agents
Mechanism of
Action

Antiviral agent

Viral spectrum

Inhib of viral
e.g. Saquinavir,
protease
Indinavir
Inhib of viral
Interferon
protein synthesis

HIV

Inhib of viral RNA Ribavirin

polymerase

RSV, HCV,
Lassa fever

Antisense
Fomivirsen
inhibition of viral
mRNA synthesis

CMV

HBV, HCV,
HPV

Fungi Cell Structure

Identification of medically

important molds are based on

the morphology and
development of reproductive
elements (conidia and spores)

P endahuluan

(1)

Eukariota, beda dari tumbuhan & hewan

Dinding sel rigid dan bukan selulosa
tetapi kitin dan glukan.
Heterotropik, tidak memiliki klorofil,
tidak dapat mengolah makanan sendiri
Dalam kaitan ini mampu menyebabkan
kerusakan antara lain menyebabkan
penyakit pada manusia/makhluk hidup lain

P endahuluan

(2)

Strukturnya sederhana, tidak ada

pembagian organ atau jaringan

Terdiri atas bangunan seperti filamen
yang disebut HIFA atau sel independen
yaitu SPORA elemen jamur
Struktur yang sederhana tersebut dapat
berubah- rubah sesuai keperluan, hal itu
penting dalam perannya sebagai
penyebab penyakit

SU M B ER

(1)

Jamur tumbuh bebas di alam

Pada umumnya tidak patogen
Jamur patogen juga ditemukan tumbuh

bebas di alam dan alam bertindak

sebagai sumber penularan

SU M B ER

(2)

Jamur juga dapat tumbuh di dalam tubuh

manusia tanpa menyebabkan kelainan

Hidup sebagai bagian flora normal di
dalam tubuh manusia
Hidup dalam keseimbangan ekologis
dengan mikroorganisme lain, hal itu
berperan dalam mencegah timbulnya
penyakit.

Fungal terms :
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:

Conidia that develops within the

hyphae
Ascospore: a sexual spore

Diagnosis of fungal infection

1. Direct Microscopic Examination:
2.
3.
4.
5.
6.
7.

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

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. Gram sputum or pus usually gram
positive

Microscopic Examination
Specimens : Skin scraping, sputum, Pus
Methods:
3.

4.

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.

Detection of Fungal Antigen

Specimen: liqour
Method : latex agglutination test
Examples:

Cryptococcus
Histoplasma

Detection of Fungal Antgen)

1. 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.
2. Gomori Methenamine Silver (GMS)
stain fungal cell black in tissue
section.

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

Culture : Isolation & Identification

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. Colony: mycelium, septation,

branching, pigmentation, spore or

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

ect Methods Based on the Host Immune Resp

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

Antifungal Chemotherapy (1)

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.

Antifungal affecting
the membrane sterols (2)
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

Antifungal affecting
the membrane sterols (3)
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 Affect

Nucleic Acid synthesis (4)
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 affect

cell wall synthesis (5)
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

Other antifungal agents (6)

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

TABLE ANTI FUNGAL (1)

Agent

Mech of
action

Mech of
resist

POLYENES
Nystatin
Amph. B

Disrupt
membrane

Strerol modifcn Topical Most fungi

Intra
venous

AZOLES

Blocks
ergost.
synthesis

Active efflux,
demethylase
alteration, or
overproduction

ALLYLAMINES

Squalene
? Active efflux
accumulation

Terbinafine
Naftifine
FLUCYTOSINE

RNA & DNA

Synthesis

Permease or
modifying
enzymes
absent or
decrsd

Route Clinical use

Varies;
Oral,
IV,
topical

Candida

Dermatophytes
Oral
Topical
Oral

Candida,
Cryptococcus

TABLE ANTI FUNGAL (2)

Agent

Mech of
action

Mech of
resist

Route Clinical use

POLYENES
Nystatin
Amph. B

Disrupt
membrane

Strerol modifcn

ECHINOCANDINES

Block glucan
synthesis

unknown

IV

Aspergillus,
Candida

GRISEOFULVIN

Disrup
microtubules

unknown

Oral

Dermatophytes

POTASSIUM IODIDE

Unknown

unknown

Oral

Sporothrix
Schenckii

TOLNAFTATE

Unknown

unknown

oral

Dermatophytes

Most fungi
Topical
Intra
venous

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).

TERIMA KASIH

Terima Kasih

KOPKUM MAA KAB