10 Laboratory diagnosis of virus infections Flashcards
what are the approaches to viral diagnosis
- demonstration of virus in material derived from the patient
- demonstration of immune response to virus (serology) (only immune response if been infected)
how is virus grown to test
virus isolation in tissue culture (very difficult to isolate viruses as don’t grow in inanimate surface – has to grow in tissue culture)
Tissue culture flask incubate laying down, cells grow until surface completely covered, then can scrap the cells off and inoculate glass tubes, lay them on side and cells will settle around base of tube
what is the light microscope used for in virus growth
Observation for cytopathic effects
Inspect growth using light microscope
If can see effects of the virus growing
how is a sample of genital ulceration done
- swab ulcer, place in viral transport medium (isotonic solution with some AB to stop overgrowing – and so does not die)
- virus in cells
what is virus isolation like
may be slow
where do viruses replicate
in cells
what is made in viral replication
viral antigens will be expressed within the cells some viruses express a haemagglutinin molecules
what is haemadsorption
adherence of red blood cells to the surface of something (as a virus or cell)
how can viral antigens be detected
Detection of early antigen fluorescent foci
Look under UV light
Can see that there is a nucleus infected
what are the advantages of virus isolation
- sensitive
- ‘catch all’ (don’t need to know in advance which virus it is)
- generates isolates for further study
- detects viable virus
- adaptation for rapid result
what are the disadvantages of virus isolation
- slow
- labour-intensive
- multiple cell lines required
- not applicable to all viruses
what are the methods in demonstration of virus in patient-derived material
- Virus isolation in tissue culture
- Electron microscopy
- Genome detection
- Antigen detection
what are the pros of electron microscopy
> Rapid technique when positive
> Independent of viability
what are the cons of electron microscopy
> Insensitive
> Semi-specific (e.g. can tell you it is a herpesvirus but not which one)
what is electron microscopy used for
viral gastroenteritis, vesicle fluid
what happens in the principle of immunofluorescence
Principle of antigen detection
virus is replicating in the cell, viral proteins being expressed
Make monoclonal antibody against viral antigens tag with a fluorescent label
Need patient cells – put down on the slide
Spot the cells on slide then dry them
Add monoclonal AB (wait 30mins)
Wash unbound
Look under fluorescent microscope
(one spot should shine – where the virus is)
what are the pros of antigen detection by immunofluorescence
> Rapid technique
> Widely applicable
what are the cons of antigen detection by immunofluorescence
Some subjectivity (know what is the debri and what is the cell fluorescence)
what is antigen detection by immunofluorescence used for
respiratory virus diagnosis
what does genome detection usually involve
genome amplification e.g. polymerase chain reaction assay
- Highly sensitive assays
Genome detection
Advantages
- speed
- high sensitivity
- wide range of applications
- wide range of utility
- detection of un-cultivable viruses
Genome detection Disadvantages
- expensive
- high set-up costs
- rigorous QC systems required
- target sequence must be known and highly conserved
what are the approaches to viral diagnosis
- Demonstration of virus in material derived from the patient
- Demonstration of an immune response to virus (serology)
demonstration of immune response to virus (serology)
- Demonstration of antigen-specific IgM
2. Demonstration of rise in antibody titre
what are enzyme immunoassays adapted to detect
adapt to detect virus-specific IgM response
what is the benefit of ELISA
diagnosis is rapid ie on day of presentation – by the time patient is ill IgM should be present
what is the significance of window period
- Diagnosis of HIV infection is by detection of anti-HIV
- In window period, patient is anti-HIV negative, but HIV positive
- Blood donation would be infectious
what is the approach to viral diagnosis
nucleic acid detection (RNA or DNA) techniques usually used now
