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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)
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
c Y
SYNCYTIUM FORMATION(S) in cell culture caused by RSV (top), and
measles virus (bottom).
(courtesy of Linda Stannard, University of Cape Town, S.A.)
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
c
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
WE ERN BLO
COMPLEMENT FIXATION TE
c ~
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.
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
c 6
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.
c M
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.
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
c