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Bien Ag Nina Ian John ͞G͟ Rachel Mark Jocelle Edo Gienah Jho Kath Aynz Je Glad Nickie

Ricobear Teacher Dadang Niňa Arlene Vivs Paul fie Rico F. Ren Mai Revs Mavis Jepay Yana Mayi Serge Hung Tope

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most easy to handle but the range of viruses supported is often
DIAGNOSTIC METHODS IN VIROLOGY limited.
ñ Direct Examination
p Antigen Detection
- Immunofluorescence CELL CULTURES
- ELISA ñ Growing virus may produce
p Electron Microscopy 1. CYTOPATHIC EFFECT (CPE) - such as the ballooning
- Morphology Of Virus Particles of cells or syncytia formation, may be specific or non-
- Immune Electron Microscopy specific.
p Light Microscopy
- Histological Appearance
- Inclusion Bodies
p Viral Genome Detection
- Hybridization With Specific Nucleic Acid
Probes
- Polymerase Chain Reaction (PCR)

ñ Indirect Examination (Virus Isolation)


p Cell Culture
- Cell Culture
- Eggs r
- Animals
p Eggs
- Pocks on Cam
- Haemagglutination
- Inclusion Bodies
p Animals
Cytopathic effect of enterovirus 71 and HSV in cell culture.
- Disease Or Death Note the BALLOONING OF CELLS (B)
(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)
ñ Serology
p Detection of rising titres of antibody between acute
Π
and convalescent stages of infection, or the detection
of IgM in primary infection.
Classical Techniques Newer Techniques
1. Complement fixation tests 1. Radioimmunoassay (RIA)
(CFT)
2. Haemagglutination inhibition 2. Enzyme linked
tests immunosorbent assay (EIA)
3. Immunofluorescence 3. Particle agglutination
techniques (IF)
4. Neutralization tests 4. Western Blot (WB)
5. Counter- 5. RIBA, Line immunoassay
immunoelectrophoresis

VIRUS ISOLATION
ñ Cell Cultures are most widely used for virus isolation

ñ 3 types of cell cultures:
1. Primary cells - Monkey Kidney
2. Semi-continuous cells - Human embryonic kidney and
skin fibroblasts
3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK 
ñ Primary cell culture are widely acknowledged as the best cell
culture systems available since they support the widest range of Measles 
viruses. However, they are very expensive and it is often
difficult to obtain a reliable supply. Continuous cells are the

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SYNCYTIUM FORMATION(S) in cell culture caused by RSV (top), and
measles virus (bottom).
(courtesy of Linda Stannard, University of Cape Town, S.A.)

2. HAEMADSORPTION- cells acquire the ability to stick


to mammalian red blood cells.
VIRUSES ISOLATED BY CELL CULTURE
Viruses readily isolated Viruses less frequently isolated
A Herpes Simplex A Varicella-Zoster
  A Cytomegalovirus A Measles
A Adenoviruses A Rubella
A Polioviruses A Rhinoviruses
A Coxsackie B viruses A Coxsackie A viruses
 A Echoviruses
A Influenza
A Parainfluenza
A Mumps
A Respiratory Syncytial Virus


106 virus particles per ml required for visualization, 50,000 - 60,000
Syncytial Formation(S) caused by mumps virus and Haemadsorption magnification normally used. Viruses may be detected in the following
(H)of erythrocytes onto the surface of the cell sheet.
(courtesy of Linda Stannard, University of Cape Town, S.A.) specimens.
ñ Faeces
p Rotavirus
ñ Confirmation of the identity of the virus may be carried out
p Adenovirus
using neutralization, haemadsorption-inhibition or
p Norwalk like viruses
immunofluorescence tests.
p Astrovirus
p Calicivirus
PROBLEMS WITH CELL CULTURE:
ñ Vesicle Fluid
ñ Long period (up to 4 weeks) required for result.
p HSV
ñ Often very poor sensitivity, sensitivity depends on a large extent p VZV
on the condition of the specimen.
ñ Skin scrapings
ñ Susceptible to bacterial contamination.
p Papillomavirus
ñ Susceptible to toxic substances which may be present in the p Orf
specimen. p Molluscum contagiosum
ñ Many viruses will not grow in cell culture e.g. Hepatitis B,
Diarrheal viruses, parvovirus, papillomavirus.
Adenov
RAPID CULTURE TECHNIQUES

ñ Available whereby viral antigens are detected 2 to 4 days after


inoculation.
ñ CMV DEAFF test : the best example, whereby
p The cell sheet is grown on individual cover slips in a
plastic bottle.
p Following inoculation, the bottle then is spun at a low Œotav
speed for one hour (to speed up the adsorption of the
virus) and then incubated for 2 to 4 days.
(courtesy of Linda Stannard, University of Cape Town, S.A.)
p The cover slip is then taken out and examined for the
presence of CMV early antigens by
immunofluorescence. IMMUNE ELECTRON MICROSCOPY
ñ The sensitivity and specificity of EM may be enhanced by
immune electron microscopy. There are two variants:
p 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.
p Solid phase immune electron microscopy (SPIEM) - the
DEAFF test for CMV
(Virology Laboratory, Yale- grid is coated with specific anti-sera. Virus particles
New Haven Hospital)

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present in the sample will be absorbed onto the grid by Complement Fixation est in Microtiter Plate.
the antibody. Rows 1 and 2 exhibit complement fixation obtained with
acte and convalescent phase serm specimens,
PROBLEMS WITH EM respectively. (2-fold serm diltions were sed) he
ñ Expensive equipment observed 4-fold increase is significant and indicates
recent infection.
ñ Expensive maintenance
ñ Require experienced observer
ñ Sensitivity often low
EROLOGY
ñ Criteria for diagnosing Primary Infection ELI A FOR HIV ANIBODY
p 4 fold or more increase in titre of IgG or total antibody
between acute and convalescent sera
p Presence of IgM
p Seroconversion
p A single high titre of IgG (or total antibody) - very
unreliable
ñ Criteria for diagnosing Reinfection
p fold or more increase in titre of IgG or total antibody
between acute and convalescent sera 
p Absence or slight increase in IgM

Typical erological Profile After Acte Infection

Microplate ELI A for HIV antibody:


Colored wells(C) indicate REACIVIY

WE ERN BLO

Note that dring reinfection, IgM may be absent or


present at a low level transiently

COMPLEMENT FIXATION TE 

HIV-1 Western Blot


Lane1: Positive Control
Lane 2: Negative Control
ample A: Negative
ample B: Indeterminate
ample C: Positive

U EFULNE OF EROLOGICAL RE UL

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ñ How useful a serological result is depends on the individual p VZV
virus. ñ Blood
ñ For example, for viruses such as rubella and hepatitis A, the p CMV (pp65 antigenaemia test)
onset of clinical symptoms coincide with the development of
antibodies. The detection of IgM or rising titres of IgG in the
serum of the patient would indicate active disease.
ñ However, many viruses often produce clinical disease before
the appearance of antibodies such as respiratory and diarrhoeal
viruses. So in this case, any serological diagnosis would be
retrospective and therefore will not be that useful.
ñ There are also viruses which produce clinical disease months or
years after seroconversion e.g. HIV and rabies. In the case of IMMUNOFLUORESCENSE
these viruses, the mere presence of antibody is sufficient to
make a definitive diagnosis.

PROBLEMS WITH SEROLOGY


ñ Long period of time required for diagnosis for paired acute and
convalescent sera.
ñ Mild local infections such as HSV genitalis may not produce a
detectable humoral immune response.
ñ Extensive antigenic cross-reactivity between related viruses e.g.
HSV and VZV, Japanese B encephalitis and Dengue, may lead to
false positive results.
ñ immunocompromised patients often give a reduced or absent
humoral immune response.
ñ Patients with infectious mononucleosis and those with Positive immunofluorescence test for
connective tissue diseases such as SLE may react non- rabies virus antigen. (Source: CDC)
specifically giving a false positive result.
ñ Patients given blood or blood products may give a false positive
result due to the transfer of antibody.

CSF ANTIBODIES
ñ Used mainly for the diagnosis of herpes simplex and VZV
encephalitis
ñ CSF normally contain little or no antibodies
ñ presence of antibodies suggest meningitis or
meningoencephalitis

CSF antibody titre >_1_is indicative of Meningitis


Serum antibody titre100

ñ Diagnosis depends on the presence of an intact blood-brain


barrier (Virology Laboratory, Yale-New Haven
Hospital)

CMV pp65 Antigenaemia Test

RAPID DIAGNOSIS BASED ON THE DETECTION OF VIRAL ANTIGENS


ñ Nasopharyngeal Aspirate
p RSV
p Influenza A and B
p Parainfluenza
p Adenovirus
ñ Faeces
p Rotaviruses
p Adenoviruses
p Astrovirus
ñ Skin
p HSV

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ñ PCR allows the in vitro amplification of specific target DNA
ADVANTAGES sequences by a factor of 106 and is thus an extremely sensitive
ñ Result available quickly, usually within a few hours. technique.
ñ It is based on an enzymatic reaction involving the use of
DISADVANTAGES synthetic oligonucleotides flanking the target nucleic sequence
ñ Often very much reduced sensitivity compared to cell culture, of interest.
can be as low as 20%. Specificity often poor as well. ñ These oligonucleotides act as primers for the thermostable
ñ Requires good specimens. Taq polymerase. Repeated cycles (usually 25 to 40) of
ñ The procedures involved are often tedious and time-consuming denaturation of the template DNA (at 94oC), annealing of
and thus expensive in terms of laboratory time primers to their complementary sequences (50oC), and primer
ECIMEN FOR ROUTINE TE T extension (72oC) result in the exponential production of the
Throat specific target fragment.
Clinical Category Blood Faeces CSF Other
swab ñ Further sensitivity and specificity may be obtained by the
1. Meningitis + + + +
nested PCR.
2. Encephalitis + + + + Brain biopsy
3. Paralytic ñ Detection and identification of the PCR product is usually
+ + + +
disease carried out by agarose gel electrophoresis, hybridization with a
4. Respiratory Nasopharyngeal specific oligonucleotide probe, restriction enzyme analysis, or
+ +
illness aspirate
5. Hepatitis +
DNA sequencing.
6. Gastroenteritis + ñ Advantages of PCR:
7. Congenital
+ Urine, saliva
p Extremely high sensitivity, may detect down to one
diseases viral genome per sample volume
Lesion sample e.g.
8. Skin lesions + + vesicle fluid, skin p Easy to set up
scrapping p Fast turnaround time
9. Eye lesions Eye swab ñ Disadvantages of PCR
10. Myocarditis + Pericardial fluid
p Extremely liable to contamination
11. Myositis + +
12. Glandular fever + p High degree of operator skill required
Autopsy p Not easy to set up a quantitative assay.
13. Post Mortem +
p A positive result may be difficult to interpret,
especially with latent viruses such as CMV, where any
After use, swabs should be broken into a small bottle containing 2 ml seropositive person will have virus present in their
of virus transport medium. Swabs should be sent to the laboratory as blood irrespective whether they have disease or not.
soon as possible without freezing. Faeces, CSF, biopsy or autopsy ñ These problems are being addressed by the arrival of
specimens should be put into a dry sterile container. commercial closed systems such as the Roche Cobas Amplicor
which requires minimum handling. The use of synthetic internal
MOLECULAR METHOD competitive targets in these commercial assays has facilitated
ñ Methods based on the detection of viral genome are also the accurate quantification of results. However, these assays
commonly known as molecular methods. It is often said that are very expensive.
molecular methods is the future direction of viral diagnosis.
ñ However in practice, although the use of these methods is chematic of PCR
indeed increasing, the role played by molecular methods in a
routine diagnostic virus laboratory is still small compared to
conventional methods.
ñ It is certain though that the role of molecular methods will
increase rapidly in the near future.

CLASSICAL MOLECULAR TECHNIQUES


ñ Dot-blot, Southern blot, in-situ hydridization are examples of
classical techniques. They depend on the use of specific
DNA/RNA probes for hybridization.
ñ The specificity of the reaction depends on the conditions used
for hybridization. However, the sensitivity of these techniques is
not better than conventional viral diagnostic methods.
ñ However, since they are usually more tedious and expensive
than conventional techniques, they never found widespread
acceptance.

POLYMERASE CHAIN REACTION

Each cycle doubles the copy number of the target

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OTHER NEWER MOLECULAR TECHNIQUES
ñ Branched DNA is essentially a sensitive hydridization technique
which involves linear amplification. Whereas exponential
amplification occurs in PCR.
ñ Therefore, the sensitivity of bDNA lies between classical
amplification techniques and PCR. Other Newer molecular
techniques depend on some form of amplification.
ñ Commercial proprietary techniques such as LCR, NASBA, TMA
depend on exponential amplification of the signal or the target.
ñ Therefore, these techniques are as susceptible to contamination
as PCR and share the same advantages and disadvantages.
ñ PCR and related techniques are bound to play an increasingly
important role in the diagnosis of viral infections.
ñ DNA chip is another promising technology where it would be
possible to detect a large number of viruses, their pathogenic
potential, and their drug sensitivity at the same time.

COMPARISON BETWEEN PCR AND OTHER NUCLEIC ACID


AMPLIFICATION TECHNIQUES

Target Signal Commercial


Method Amplification Amplification
Thermocycling Sensitivity
Examples

Exponentia Roche
PCR No Yes High
l Amplicor
Exponentia Abbot
LCR No Yes High
l LCX
Exponentia Organon
NASBA No No High
l Teknika
Exponentia
TMA No No High Genprobe
l
Qß- Exponentia
No No High None
Replicase l
Branched Chiron
DNA No Linear No Medium Quantiple
x


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