Laboratory Diagnosis of Viral Infections
Laboratory Diagnosis of Viral Infections
DIAGNOSIS OF
VIRAL
INFECTIONS
DIAGNOSTIC METHODS IN VIROLOGY
1. Direct Examination
3. Serology
1. DIRECT EXAMINATION
•Most viruses enter the body through the respiratory or GI tract, so the
most obvious & appropriate specimen is respiratory tract secretions or
the GI tract secretions.
•The optimal specimens for viral culture are aspirates of fluids, exudates
or secretion, tissues, washings of upper air ways or stool specimens,
swab specimen are acceptable in most situations. Nasopharyngeal
washings are generally sufficient for respiratory viruses.
•Blood may be useful for entero viral infections in young children
& infants.
•To culture vesicular skin lesions the skin should be cleaned with
an alcohol swab and allowed to dry for at least one minute.
•The vesicle should then be unrolled with a sterile scalpel.
TRANSPORTATION &
STORAGE OF SPECIMEN
Transport medium
Inclusion bodies
1.2. ELECTRON MICROSCOPY
•Viruses are very small and most of them can be seen only by TEM
(transmission electron microscopy).
•TEM has therefore made a major contribution to virology, including
the discovery of many viruses, the diagnosis of various viral infections
and fundamental investigations of virus-host cell interactions.
• However, TEM has gradually been replaced by more sensitive
methods, such as the PCR.
•In research, new imaging techniques for fluorescence light microscopy
have supplanted TEM, making it possible to study live cells and
dynamic interactions between viruses and the cellular machinery.
• Nevertheless, TEM remains essential for certain aspects of virology.
• Expensive equipment
• Expensive maintenance
Fluorescence microscope
The working model of florescence microscope
Positive immunofluorescence
test for rabies virus antigen.
(Source: CDC)
CMV pp65 antigens
detected in nuclei of
peripheral blood
neutrophils
•It is the earliest method for the cultivation of viruses causing human
diseases.
•Reed & colleagues F in 1900 used human volunteers for their work
on yellow fever. In 1909, Landsteiner & Popper used monkeys to
isolate Polio virus.
•Rabbit, Guinea pig, mouse, rat are now commonly used in virus
cultivation.
•It needs to be noted that different animals are used for different
viruses & also there exists different routes to inoculation.
•The growth of virus in inoculated animals may be indicated by death,
disease or visible lesions.
•Disadvantages of animal inoculation are that they may interfere with
viral growth and that animals often harbour latent viruses.
Tissue culture
Tissue culture is often a generic term. Three types of tissue cultures
are available:
a. Organ culture
Small bits of organs are maintained in vitro for days & weeks. They
are used for cultivation of viruses which can be grown only in
specific organ.
b. Explants culture
Fragments of minced tissues is grown as explants embedded in
plasma clot. Eg. Adeno tissue explants for adenovirus.
c. Cell culture
It is routinely used based on their origin. Chromosomal characters &
the number of generation through which they can be maintained,
cell cultures are classified into 3 types
1. Primary cell culture
It consists of normal cells freshly taken from body & cultured. They are
capable of limited growth in culture & cannot be maintained in serial
culture. Eg. Monkey, kidney, human embryonic kidney, human amnion
& chick embryo cell culture. They are useful for isolation of viruses &
for vaccine production.
Sandwich ELISA
INSTRUMENTS REQUIRED
ELISA READER
DIRECT ELISA
INDIRECT ELISA
SANDWICH ELISA
Hemagglutination Inhibition test
The HI test involves three main components: antibodies, influenza
virus, and red blood cells that are mixed together in the wells (i.e.,
cups) of a microtiter plate