Laboratory Diagnosis of Viral

Infections

Fourth Medical, 2007

Prof. Widad Al-Nakib, FRCPath.
 Overview of Diagnostic Methods

• In general, diagnostic tests can be

grouped into 3 categories:
(1) Direct examination
(2) Indirect examination
(3) Serology
 Contd..
• In direct examination, the clinical specimen is
examined directly for the presence of virus
particles, virus antigen or viral nucleic acids.
• In indirect examination, the specimen is
inoculated into cell culture, eggs or animals in an
attempt to grow the virus: this is called virus
isolation.
• In Serology, the diagnosis is based on the
detection of antibody and generally constitute by
far the bulk of the work of any virology
laboratory.
1. Direct Examination of Specimens
• Light microscopy, histological
appearance - e.g. inclusion bodies
• Electron Microscopy, virus particle are
directly seen in clinical specimens,
example, stools, vesicular fluids
• Antigen detection by
immunofluorescence, ELISA etc.
• Molecular techniques for the direct
detection of viral genomes
 Contd..
• The specimen used for direct detection and
virus isolation is very important.
• A positive result from the site of disease
would be of much greater diagnostic
significance than those from other sites.
• For example, in the case of herpes simplex
encephalitis, a positive result from the CSF or
the brain would be much greater significance
than a positive result from an oral ulcer, since
reactivation of oral herpes is common during
times of stress.
 2. Indirect Examination

• Cell culture - cytopathic effect (CPE),

haemadsorption, confirmation by
neutralization, interference,
immunofluorescence etc.
• Eggs pocks on CAM - haemagglutination,
inclusion bodies
• Animals disease or death confirmation by
neutralization
Virus Growth in Embryonated Eggs
Virus Cytopathic Effect (CPE) in Cell
Cultures
 3. Serology

• Detection of rising titres of

antibody between acute and
convalescent stages of infection
• Detection of IgM in primary
infection.
Secondary Immune Response
Immune Response After Hepatitis A
Virus Infection
 “Classical” Serological Techniques

1. Complement-Fixation (CFT)
2. Haemagglutination inhibition tests
(HAI)
3. Immunofluorescence techniques (IF)
4. Neutralization tests (NT)
5. Single Radial Haemolysis (SRH)
 “Newer” Serological Techniques

1. Radioimmunoassay (RIA)
2. Enzyme Immunoassay (EIA)
3. Particle agglutination
4. Western Blot (WB)
5. Recombinant immunoblot assay (RIBA)
 Immunofluorescence

•
 Light Microscopy
• Replicating virus often produce histological changes in
infected cells. These changes may be characteristic or
non-specific.
• Viral inclusion bodies are basically collections of
replicating virus particles either in the nucleus or
cytoplasm.
• Examples of inclusion bodies include the negri bodies
and cytomegalic inclusion bodies found in rabies and
CMV infections, respectively.
• Although not sensitive or specific, histology
nevertheless serves as a useful adjunct in the diagnosis
of certain viral infections.
Rabies Negri Bodies
 Electron Microscopy (EM)

• Virus particles are detected and identified

on the basis of morphology. You need
1,000,000 particles per ml. to see the virus
• A magnification of around 50,000 is
normally used.
• EM is now mainly used for the diagnosis
of viral gastroenteritis by detecting viruses
in faeces e.g. rotavirus, adenovirus,
astrovirus, calicivirus and Norwalk-like
viruses
 CONTD..
• Occasionally it may be used for the detection of
viruses in vesicles and other skin lesions, such
as herpesviruses, papillomaviruses and
poxviruses.
• The sensitivity and specificity of EM may be
enhanced by immune electron microscopy,
whereby virus specific antibody is used to
agglutinate virus particles together and thus
making them easier to recognize, or to capture
virus particles onto the EM grid.
Electron Microscopy (EM)
 New Molecular Techniques

• Polymerase chain reaction (PCR)

• Ligase chain reaction (LCR)
• Nucleic acid based amplification (NASBA)
• Branched DNA (bDNA)
• ALL depend on some form of amplification, either
the target nucleic acid, or the signal itself.
 Molecular Techniques

• PCR is being increasingly used for viral

diagnosis, especially as the cost of the assay
come down and the availability of closed
automated systems that could also perform
quantification for example, measurement of
Viral Load (Quantitative PCR).
Principle of PCR
 Amplification of different
enteroviruses
M 1 2 3 4 5 6 7 8 9 10 11 12 13

Lane M – 100bp DNA marker

Lane 1 – Cox A7 Lane 14 – Polio 1 (Sabin)

Lane 2 – Cox A9 Lane 16 – Echo 4
Lane 3 – Cox A16 Lane 17 – Echo 6
200 -
Lane 5 – Cox B1 Lane 18 – Echo 9
Lane 6 – Cox B2 Lane 19 – Echo 11
M 14 15 16 17 18 19 20 21 22 23 24 25
Lane 7 – Cox B3 Lane 20 – Echo 17
Lane 9 – Cox B4 Lane 21 – Echo 30
Lane 10 – Cox B5 Lane 22 – Echo 34

200 -
Lane 11 – Cox B6 Lane 23 – negative
Lane 12 – Entero 70 Lane 24 – Cox B5 (old)
Lane 13 – Entero 71 Lane 25 – Echo 9 (old)
 Rapid and Direct Virus Diagnosis

• With new techniques such as PCR we are now able

to detect viral genomes in very small quantities (by
amplifying the viral genome) in body tissues ( in
situ PCR), body fluids (CSF, pericardial and
vesicular fluids), excretions (urine) and blood.
Thus, allowing us to establish a direct relationship
between the causative virus and the disease, in a very
short period of time.
 Contd..
Detecting Viral Genome- a direct association.

• Blood - viraemia
• CSF - viral meningitis and encephalitis
• Pericardial fluids – viral carditis
• Vesicular fluids – herpetic lesions
• BAL – viral pneumonia
• Cells and tissues- chronic and persistent viral
infections.
Viral Load Predicting Disease and
Survival
CMV DNA Load
– Independent marker of CMV disease and
survival( Patients responding to preemptive therapy with
undetectable plasma CMV viral load)
– Lower risk of disease, higher rate of survival

HIV RNA Load

- Determine severity of infection
- Monitor the efficacy of antiviral chemotherapy
- Determine drug resistance if no change in viral
load
 HIV Resistance Testing

Genotype
• Sequence of nucleotide bases
• Specific base changes or mutations causing
resistance
Phenotype
• Traits or behavior resulting from genotype
• Antiviral susceptibility