Download Diagnosis of viral infections

Diagnosis of viral infections
The laboratory diagnosis of viral infections
based on three general approaches
1-Direct detection of viral components either in cells derived
from infected tissues or free in fluid specimens.
2-Isolation of viruses, usually in cell cultures, followed by
identification.
3-Demonstration of a significant increase in serum levels of
antibodies to the etiological virus during the course of the
illness or the presence of antibodies of class [IgM] specific
to the etiological virus.
Specimens in viral infection
Timing
Specimens for virus isolation & direct detection as well as blood samples must be
collected within the first few days of the illness .
Choice of the specimens
This done by knowing the pathogenesis of the virus .
e.g. Polio virus infection, the neurological samples is not important in the diagnosis,
but throat wash is important, because the virus start replication in the oropharynx.
Clinical data
Because each type of virus need special type of tissue culture for replication, so
clinical data is important.
Specimen transport & storage
Specimens should delivered without delay, kept at neutral pH to avoid affection of
viral infectivity.
Keep the specimens at 4 °C if delay is expected for four days. (refrigeration).
If the delay is > four days the specimens should be kept at -70 °C (freezing).
If a virus suspected to be present is labile to freezing, the sample should be diluted
with an equal volume of 2SP(sucrose in phosphate buffer pH 7.2).
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Culture & isolation
Is the gold standard in viral diagnosis.
Viral Culture
Viruses don't grow on artificial media, they must be cultured on cell
cultures or other type of living system.
1- Emboryonated eggs
Inoculation of embryonated eggs used for growing of some viruses,(
mumps & influenza viruses).
Fertilized egg is incubated for (5-14) days after that inoculated.
The site of inoculation & the period of incubation is depend on the
type of virus.
Then embryonated examined for the evidence of viral replication
Death of the yolk sac.
Pocks formation as cytopathic effect on the chorion.
Examination of the embryonated egg fluid for the viral antigens.
2- Laboratory animals
In the past, mice & chimpanzees were inoculated with viruses, but now
this has been largely replaced by the use of cell cultures.
3- Cell cultures
 Cell culture originated from viable host cells & grow into a monolayer
on the sides of glass or plastic test tubes.
 Cells are kept moist & supplied with nutrients by keeping them
immersed in cell culture medium & maintenance medium.
 Cell culture incubated 1-4 weeks, depending on the type of the virus.
 Periodically the cell are inspected for the presence of virus, indicated
by areas of dead or dying cells called cytopathic effect (CPE).
Cell culture help in
1-Isolation & growth of a variety of viruses.
2-Studying viral replication.
3-Development of attenuated mutant strains, & vaccine
production.
A cell culture becomes a cell line once it has been passed (sub-cultured)
in vitro.
Cell lines are classified as
Primary cell lines.
Low passage cell lines.
Continuous cell lines.
Primary cell lines
Those cell lines that have been passed only once or twice since
harvesting. Further passage of primary cells results in decreased the
receptivity of the cell lines to viral infection.
Examples of the primary cell lines
1-Human embryonic kidney.
2-Guinea pig embryo.
3-Chick embryo.
4-Primary monkey kidney.
Low passage cell lines
Those cell lines that remain virus sensitive through 20 to 50 passages.
Human dipliod fibroblast cells such as lung fibroblast, are a commonly
used low passage cell line.
Examples
1-Human diploid fibroblast cell line.
2-MRC5 institution of the medical research.
3-WI 38.
Continuous cell lines
Those cell lines that can be passed & remain sensitive to virus
infections indefinitely.
Examples
1-HEp-2 human epidermiod cancer of the larynx.
2-HELLA cervical carcinoma.
Identification of viruses detected in cell culture
 Infected cell in cell culture change their morphology, & eventually lyse or detach
from the glass surface while dying.
 These changes in the infected cell called cytopathic effects of the virus (CPE).
 Viruses are detected in cell culture by the recognition of these (CPE).
 Viruses have distinct CPEs, just as colonies of bacteria on agar plates have unique
morphologies.
CPE may be quantitated as in the following
QUANTITATION
INTERPRETATION
Negative
uninfected monolayer
Equivocal(+/-)
atypical alteration of monolayer
involving few cells.
1+
1-25% of monolayer exhibit CPE
2+
25-50%of monolayer exhibit CPE
3+
50-75% of monolayer exhibit CPE
4+
75-100% of monolayer exhibit CPE
Viruses detected in cell culture identified by
1-Cell type that support viral replication.
2-Time need to detect CPE.
3-Morphology of the CPE.
Morphology of CPE
These CPE suggestive of infection by a family, but don't allow determination of the
species of the virus.
Examples of CPE
(a) Herpesviruses: loss of architecture & rounding of cells in 24-48 hours.
(b) Paramyxoviruses: (measles virus, respiratory syncytial virus) cells coalesce into
large multinucleated syncytia in 3-5 days.
Syncytia formed by membrane fusion of infected & non infected cells.
(c) Cytomegalovirus (CMV) appearance of foci of cell lysis in 2-3 weeks. (plaques)
(d) Viruses may cause the appearance of inclusion bodies in infected cells.
Viral inclusions
They are intracellular structures formed by:
a- aggregates of viruses within an infected cell.
b- abnormal accumulation of viral components from inefficient viral maturation.
 Inclusion bodies are visible by conventional microscopy.
 Location & appearance of inclusion bodies provide information about infecting
virus.
E.g. stained smear from based of skin vesicle used to detect VZV or HSV inclusions
is called a Tzank test.
Human embryo skin muscle cells were infected with human CMV &
stained at selected times to demonstrate:
(A) uninfected cells, (B) late CPE (nuclear inclusions, cell enlargement),
(C) cell degeneration (D) a focus of cell lysis in a cell monolayer (
plaque).
For definitive identification of the virus
1- Fluorescent-labeled antisera
available for most viruses, & used for culture conformation.
2 - Viral neutralization
used to identify viruses with many serotype for which
fluorescent labeled antisera are not available.
3 - Acid liability test
used to differentiate enteroviruses from rhinoviruses.
4 - Some viruses, such as influenza, parainfluenza, & mumps,
can be detected by hemadsorption, since infected cells
contain viral hem-adsorbing glycoprotein in their outer
membranes.
5 - Other methods
EM, or immune electron microscopy to detect the virus
inside the infected cells.
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Shell vial method
A rapid modification of a conventional cell culture.
Virus is detected more quickly, by staining host cell for viral antigens
before the development of CPE.
Here the monolayer of the cell culture is grow on cover slip (shell
vial) & specimens are inoculated onto the shell vial cell layer by low
speed centrifugation, which enhances viral infectivity.
So the shell vial method
(a) Shorten the time of virus to get in the cell.
(b) Enhance replication of the virus.
(c) Decrease the time of CPE of the virus to occurs.
Used for viruses required long incubation before producing CPE.
e.g. CMV need four weeks to shown CPE in conventional cell culture,
by shell vial method few days required for CPE to appear.
Rapid diagnostic tests
These tests in comparison to the cell culture (required days), few
hours required after receiving the specimens to detect the virus.
These tests includes
1-Direct EM examination of the specimens.
e.g. examination of the stool for Rotavirus & Hepatitis A.
2-Immune electron microscopy.
Allows visualization of virus particles present in numbers too small
for easy direct detection
3-Antigen detection method
The antigen of the virus is detected by immunofluorescent technique.
e.g. detection of the RSV antigen in the respiratory secretions.
4-Agglutination method
Here antibody is added to the specimen to detect the presence of the
viral antigen.
5-Molecular technique (Nucleic acid hybridization)
Use to detect viral genome & genome sequence in clinical specimens.
Examples of the molecular methods
A- Insitu hybirdization technique.
use single stranded, complementary nucleic acid probes.
It is highly sensitive & specific
This technique used to identify
a- Adenovirus in nasopharyngeal washings.
b- CMV in urine.
c- HIV in blood of seronegatine individuals
B-PCR.
Increases the sensitivity of nucleic acid hybridization by increases the
amount of viral genome obtained from the patient.
This approach is useful for rapid detection of the virus in patient
specimens when the amount of viral nucleic acid in patient sample is
small.
6-Serology
Serological test used to detect antibodies produced against viral
antigens.
From these serological tests we have
1-Radioimmunoassay RIA.
2-Enzyme immune assay.
3-Indirect immunofluorescent tests.
4-Hemagglutination inhibition test, measure antibodies directed
against hemagglutinating viruses, such as influenza viruses.
5-Complement fixation test.
The aim of the serological tests
1- Detect a significant increase in the titer of antibodies to the
etiological virus.
2- The presence of IgM specific antibodies to the etiological virus.