DIAGNOSTIC VIROLOGY
- No. of virions shed from the lesion
First clues to the diagnosis of a viral
infection
- Patient’s history
- Symptoms
PURPOSE OF VIRAL LABORATORY
STUDIES
1) To confirm the diagnosis by identifying
the viral agent of infection
2) To define the disease process
3) To monitor the disease
epidemiologically
4) To educate physicians and patients
CARDINAL RULES IN SPECIMEN
COLLECTION
1) Appropriate time of collection
SPECIMENS FOR VIRAL DIAGNOSIS
- Early in disease process
decreases dramatically each day
after the lesion manifests
2) Go “where the action is”
- Aspirated secretions – maximum
recovery
- Swabs – use cotton, Dacron or
nylon
- Ca alginate: toxic to many viruses
3) Transport
- Medium: buffered isotonic
solution + some type of protein
e.g., albumin
- Plus antibiotics
- Refrigerated or on ice (not frozen)
- Delay of 4-7 days: -70oC (not -
20oC: ice crystals)
PSALM 56:3 When I am afraid, I put my trust in You. 1
THE CHOICE OF LAB METHOD OF ADVANTAGE
DIAGNOSIS DEPENDS ON
- Result available quickly, usually within
- Type of viral disease a few hours.
- Capability the lab
DISADVANTAGE
- Availability of the method
DIAGNOSTIC METHODS IN VIROLOGY - Often very much reduced sensitivity
compared to cell culture, can be as low
1) Direct Examination as 20%. Specificity often poor as well.
2) Indirect Examination (Virus Isolation) - Requires good specimens.
3) Serology - The procedures involved are often
tedious and time-consuming and thus
DIRECT EXAMINATION
expensive in terms of laboratory time.
1) Antigen Detection
RAPID DIAGNOSIS: VIRAL ANTIGEN
- immunofluorescence, ELISA etc.
DETECTION
2) Electron Microscopy
- morphology of virus particles 1) Nasopharyngeal Aspirate
- immune electron microscopy - RSV
3) Light Microscopy - Influenza A and B
- histological appearance - Parainfluenza
- inclusion bodies - Adenovirus
4) Viral Genome Detection 2) Faeces
- hybridization with specific nucleic acid - Rotaviruses
probes - Adenoviruses
- polymerase chain reaction (PCR) - Astrovirus

PSALM 56:3 When I am afraid, I put my trust in You. 2

 3) Skin
- HSV
- VZV
4) Blood
- CMV (pp65 antigenaemia test)
ELECTRON MICROSCOPY
- 106 virus particles per ml required
for visualization, 50,000 - 60,000
magnification normally used.
Viruses may be detected in the
following specimens.
1) Faeces
- Rotavirus, Adenovirus
- Norwalk like viruses
- Astrovirus, Calicivirus
2) Vesicle fluid
- HSV
- VZV
3) Skin scrapings
- papillomavirus, orf
- molluscum contagiosum
IMMUNE ELECTRON MICROSCOPY
- The sensitivity and specificity of EM
may be enhanced by immune electron
microscopy. There are two variants:-
1) Classical Immune electron
microscopy (IEM) - the sample is
treated with specific anti-sera
before being put up for EM. Viral
particles present will be
agglutinated and thus congregate
together by the antibody.
2) Solid phase immune electron
microscopy (SPIEM) - the grid is
coated with specific anti-sera. Virus
particles present in the sample will
be absorbed onto the grid by the
antibody.
PSALM 56:3 When I am afraid, I put my trust in You.
DISADVANTAGE
- Expensive equipment
- Expensive maintenance
- Require experienced observer
- Sensitivity often low
CYTOLOGY (LIGHT MICROSCOPY)
- Rapid diagnosis of viral infections that
produce cytopathic effects (CPE)
◼ Changes in cell morphology
◼ Cell lysis
◼ Vacuolation
◼ Syncytia
◼ Inclusion bodies
MOLECULAR METHODS
- Based on the detection of viral
genome
- in practice, the role played in a
routine diagnostic virus laboratory is
still small compared to conventional
methods.
- It is certain though that the role of
molecular methods will increase
rapidly in the near future.
CLASSICAL MOLECULAR TECHNIQUES
They depend on the use of specific
DNA/RNA probes for hybridization.
- Dot-blot,
- Southern blot,
- in-situ hydridization
- the sensitivity of these techniques is
not better than conventional viral
diagnostic methods.
- usually more tedious and expensive
than conventional techniques, never
found widespread acceptance
3
DNA PROBE 1) PRIMARY CELL CULTURE
- derived from normal adult tissues,
- known DNA fragment used to identify ◼ e.g. Monkey Kidney
closely related DNA or RNA - can be passed (subcultured) once or
sequences 2X
- the best cell culture systems available
since they support the widest range of
viruses.
OTHER NEWER MOLECULAR - very expensive
TECHNIQUE - often difficult to obtain a reliable
supply.

2) SEMI-CONTINUOUS CELL
CULTURE
- derived from embryonic tissues;
◼ e.g., Human embryonic kidney
and skin fibroblasts
PCR AND RT-PCR

- DNA or RNA is extracted 3) CONTINUOUS CELL CULTURE

- PCR - derived from malignant tissues
- Gel electrophoresis ◼ e.g., HeLa, Vero, Hep2, LLC-MK2,
MDCK
INDIRECT METHOD (VIRUS ISOLATION - can be subcultured indefinitely
AND IDENTIFICATION) - most easy to handle
- Disadvantage: the range of viruses
VIRUS ISOLATION
supported is often limited
1) Cell culture
VIRAL DETECTION
- cytopathic effect (CPE)
- haemabsorption 1) Cytopathic Effect (CPE) – may be
- immunofluorescence specific or non-specific
2) Eggs ◼ ballooning of cells
- pocks on CAM ◼ syncytia formation
- haemagglutination 2) Hemadsorption – cells infected with
- inclusion bodies some viruses express hemagglutinin
3) Animals that bind erythrocytes
- Disease or death
CONFIRMATION OF IDENTITY
CELL CULTURE
1) Neutralization test
- are most widely used for virus - (Virus + specific Ab) + cell culture
isolation. → no CPE
2) Hemagglutination-inhibition (HAI)
test

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 - (Virus + specific Ab) + rbc → no HA - Many viruses will not grow in cell
3) Immunofluorescence test culture
- Virus + specific Ab-fluorescein ◼ e.g. Hepatitis B, diarrheal viruses,
→ fluorescence parvovirus, papillomavirus.
PROBLEMS SEROLOGY

- Long period (up to 4 weeks) required - Detection of rising titres of antibody

for result. between acute and convalescent
- Often very poor sensitivity stages of infection, or the detection of
- Susceptible to bacterial contamination. IgM in primary infection.
- Susceptible to toxic substances which
may be present in the specimen.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)

2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay

CRITERIA: DIAGNOSING PRIMARY

INFECTION
- 4 fold or more increase in titer of IgG
or total antibody between acute and
convalescent sera
- Presence of IgM
- Seroconversion
- A single high titre of IgG (or total
antibody) - very unreliable
CRITERIA: DIAGNOSING REINFECTION
- 4 fold or more increase in titer of IgG
or total antibody between acute and WESTERN BLOT
convalescent sera
- HIV-1 Western Blot
- Absence or slight increase in IgM
- Lane1: Positive Control
TYPICAL SEROLOGICAL PROFILE AFTER - Lane 2: Negative Control
ACUTE INFECTION - Sample A: Negative
- Sample B: Indeterminate
- Note that during reinfection, IgM may
- Sample C: Positive
be absent or present at a low level
transiently

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 - Extensive antigenic cross-reactivity
between related viruses e.g. HSV and
VZV, Japanese B encephalitis and
Dengue, may lead to false positive
results.
- Immunocompromised patients often
give a reduced or absent humoral
immune response.
- Patients with infectious mononucleosis
and those with connective tissue
USEFULNESS OF SEROLOGICAL TEST diseases such as SLE may react non-
specifically giving a false positive
DEPENDS ON THE INDIVIDUAL VIRUS
result.
- For example, rubella and hepatitis A - Patients given blood or blood products
viruses may give a false positive result due to
◼ the onset of clinical symptoms the transfer of antibody.
coincide with the development of
CSF ANTIBODIES
antibodies.
◼ The detection of IgM or rising titres - Used mainly for the diagnosis of
of IgG in the serum of the patient herpes simplex and VZV encephalitis
would indicate active disease - CSF normally contain little or no
- However, many viruses (respiratory antibodies
and diarrheal viruses) often produce - presence of antibodies suggest
clinical disease before the appearance meningitis or meningoencephalitis
of antibodies - Diagnosis depends on the presence of
◼ So in this case, any serological an intact blood-brain barrier
diagnosis would be retrospective
and therefore will not be that
useful.
- Viruses like HIV and rabies produce
clinical disease months or years after
seroconversion
◼ the mere presence of antibody is
sufficient to make a definitive
diagnosis
PROBLEMS WITH SEROLOGY
- Long period of time required for
diagnosis using paired acute and
convalescent sera.
- Mild local infections such as HSV
genitalis may not produce a detectable
humoral immune response.

PSALM 56:3 When I am afraid, I put my trust in You. 6