L9 Lab diagnosis Flashcards
What is the direct approach to viral diagnosis
Demonstration of virus in material from patient approach
What is the indirect approach to viral diagnosis
Demonstration of an immune response to virus (serology)
How are viruses isolated
In tissue culture as need to grow in cells
How is tissue culture grown
In flask lying flat down
Cells stick to plastic until confluence growth and scrap cells off surface
Red liquid - cells
What do uninflected human embryonic lung fibroblasts look like under light microscope
Long thin shape of cells
What do rhinovirus infected lung fibroblasts look like
Round and shrivelled typical of rhinovirus
Cytopathic effect CPE
What do cytomegalovirus infected fibroblasts look like
Causes rounded and swollen cells for CPE effect
What is important when trying to isolate many viruses
Not all cells have a receptive for every virus - need to provide a range of different cell types if trying to isolate many viruses
What are Hep2 cells infected with respiratory syncytial virus like
Huge gaps in cell sheet
Large multi nucleated giant cells
Virus caused these cells to fuse together - syncytia
How is a genital ulceration tested
Scrap cells off swab
Break swab into transport medium as need cells alive not dried
What is in transport medium
Antibiotics to stop overgrowth
Where are viral antigens expressed
In cells as viruses replicate in cells
What is early antigen fluorescent foci
Antigen expressed early in cell cycle
Make antibody for this and tag with fluorescent marker
What is virus isolation like
Can be slow
Why is detection of early antigen fluorescent foci good
Can do within 24 hrs rather than waiting for CPEs which take long
What are the pros of virus isolation in tissue culture
Positive result unequivocal
What are the cons of virus isolation in tissue culture
May be slow
Can’t be universal (can be hard to grow in cell culture)
How is virus isolation sped up
Haemaosdrption
Detection of early antigen fluorescent foci
What is DEAFF
Detection of Early Antigen Fluorescent Foci
What happens if the virus can’t be grown to isolate
Try use electron microscopes
Virus isolation in tissue culture
What is the con with electron microscopy
Some viruses look the same e.g. all herpes viruses look the same so doesn’t know which one it is
What are the pros of electron microscopy
Rapid technique when positive
Independent of variability
What are the cons of electron microscopy
Insensitive- need a lot of virus to see it
Semi specific- can’t tell which herpesvirus it is
If you can’t grow or see the virus what can be done to workout the virus
Need to find the viral proteins (antigens) and viral genetic material (genome)
What is the principle of immunofluorescence
Antigen within of on virus
Cells from patient onto slide add monoclonal AB that are fluorescent
Wash unbound AB
Dry slide
What is the immunofluorescence technique like
Very adaptable technique
What happens if the cells are infected in principle of immunofluorescence
Cell fluorescent under UV
What happens if the cells are uninfected in the principle of immunofluorescence
No binding of AB and no fluorescence
What are the pros of antigen detection
Rapid technique
Widely applicable
What are the cons of antigen detection
Some subjectivity needs a lot of training
Cell debris fluorescent
Where are cells taken from for antigen detection
Throat swab
Nasopharynx swab
What detection is used for respiratory virus diagnosis
Antigen detection
What does genome detection involve
Genome amplification e.g. PCR assay
How can genome detection be quantitative
E.g. monitoring of HIV viral load in response to therapy - how much virus there is, can monitor the response to the treatment
What is the problem with antibody testing
Incubation period there is no AB to detect
How can diagnosis of recent infection be tested
Demons of antigen specific IgM
Demonstration of rise in AB titre
What antibodies appear and when in infection
IgM appear first
Followed quickly by IgG
What antibody remains for potentially life
IgG
What does it mean if IgM detected
Patient recently infected
What is important to watch about antibody titre
Rise in AB
Goes from undetectable to high
Only in response to infection
What is an ELISA
Enzyme linked immunisorbent assay
What is good about ELISA
Optical density reading
Straightforward interpretation
What is adaptable with enzyme immunoassay
Adapt to detect virus specific IgM response
Pros of ELISA
Quick diagnosis
Very flexible technique
Other than infection why is the window period important
Blood donation - check no HIV infection
What are the ways to demonstrate virus in patient material
Virus isolation in tissue culture
Electron microscopy
Antigen detection
Genome detection
