DIAGNOSTIC
MICROBIOLOGY
VIRUSES & PARASITES
Dr Nazia khan
OBJECTIVES

Discuss various techniques utilized in
diagnosis of viral infection with clinical
examples

Explain
principle
of
molecular
techniques in diagnostic microbiology.

Briefly discuss diagnostic modalities
available for parasitic infections with
clinical examples.
DIAGNOSTIC MICROBIOLOGY
Viruses & parasites not routinely
cultured on conventional agar
Diagnosis of viruses and parasites
require special procedures
Indirect methods are more suitable
then direct methods
Viruses are too small to be seen by
light microscopy
Conventional stains are ineffective
against them
DIAGNOSTIC MICROBIOLOGYDIRECT METHODS
MICROSCOPY
 Light microscopy useful for some
protozoa and parasites
 Scotch tape test-Enterobius ova
 Not fit for viruses
WET FILM EXAM
 Useful for motile Giardia,
Entameoba histolytica etc.
 Also for ovas, cysts of parasites
 Usually urine, stool examination
 Direct visualization of worm
DIAGNOSTIC MICROBIOLOGYDIRECT METHODS
STAINED SPECIMEN
 Negri bodies of Rabies virus
 Giemsa stain for Plasmodium in
blood
 ZN stain for Cryptosporidium
hominis
 Iodine stain for Giardia cyst,
Entamoeba cyst
 Viruses are not stained
Negri body
DIAGNOSTIC MICROBIOLOGYDIRECT METHODS
ELECTRON MICROSCOPY
 Not routinely used
 For certain viruses not grown in
cell cultures
 Example: Rotavirus in stool
Rota virus
 Zaire ebola virus was diagnosed by
EM in recent outbreak
 Monkeypox outbreak in USA
 Immune electron microscopy
improve virus detection by
reacting with specific antibodies
Ebola virus
DIAGNOSTIC MICROBIOLOGY-DIRECT
METHODS
IMMUNOFLUORESCENCE
Direct exam of viral particle
Monoclonal antibodies
against viral or parasitic
antigens
Fix on slide
Immuno-fluorescent
staining
Rapid and sensitive test
Cryptosporidium in stool
RSV in respiratory secretion
DIAGNOSTIC MICROBIOLOGYDIRECT METHODS
VIRUS CULTURE
 On special cell lines e.g. vero
cells, monkey kidney cells
 Specimens are inoculated on cell
lines
 Bactericidal chemicals added
 Incubated
 Observe effects on cell lines
 Cytolysis, syncytia formation
etc.
 Laborious, time consuming
 RSV, Measles, Influenza virus
DIAGNOSTIC MICROBIOLOGYDIRECT METHODS
VIRUS CULTURE NEWER
TECHNIQUES
 Immuno-fluorescent staining of
intracellular antigens on cultured
cells
 Use monoclonal antibodies against
virus antigen
 Label the antibodies
 Put these antibodies on cell lines
 Observe the fluorescence
 Advantage-diagnosis in 24 hours
possible
DIAGNOSTIC MICROBIOLOGYINDIRECT METHODS
ANTIGEN DETECTION
Rapid tests like swabs for
Influenza virus
ELISA TEST for HBsAg
detection, p24 antigens
Latex agglutination tests
Complement fixation
tests
MOVIE ELISA
DIAGNOSTIC MICROBIOLOGYINDIRECT METHODS
ANTIBODY DETECTION
Western Blot Immunoassays
Viral antigens are separated
on a filter paper
Serum is flooded on the filter
paper
Ag-Ab react
Dark bands of Ag-Ab visible
Can identify specific
antibodies
MOVIE WESTERN BLOT
DIAGNOSTIC MICROBIOLOGY
MOLECULAR DIAGNOSIS
A. PCR TEST
 PCR for HSV in CSF-sensitive
test for HSV encephalitis
 PCR for toxoplasma gondii in
amniotic fluid
B. NUCLEIC ACID PROBES
 FLUORESCENCE IN SITU
HYBRIDIZATION

EBV in cell cultures

Dengue virus
 DNA HYBRIDIZATION
 Leishmania tropica in skin smears
MOVIE PCR
MOVIE DNA PROBES