Diagnosis Of Viral Infections Flashcards
(55 cards)
What is the purpose of a laboratory diagnostic test?
- Reduce need for unnecessary tests and inappropriate antibiotics
- Public health implications
- Natural history of the pathogen to treat
Name some possible test types for viral infections
- Electron microscopy
- Virus isolation
- Antigen detection
- Antibody detection by serology
- Nucleic acid amplification tests (NAATs)
- Sequencing for genotype and detection of antiviral resistance
- Immunochomatographic methods
Why might we still use electron microscopy in a viral lab?
- Characterising emerging pathogens (so it was used to characterise COVID)
- Possibly still useful for feaces and vescile specimens
What are the advantages of using electron microscopy?
- Rapid
- Detects viruses that cannot culture
- Visualisation of viruses
What are the limitations of electron microscopy?
- Low sensitivity - need 106 virons/ml
- Requires maintenance
- Requires skilled operators
- Cannot differentiate between viruses of the same family
Name some viral infections that may be diagnosed with electron microscopy
Viruses that have a distinct shape but we can not tell the difference between members of the same family so like which herpes virus it is
- Rotavirus
- Norovirus
- Herpes
- Adenovirus
- Coronavirus
- Poxviruses such as smallpox and chickenpox
Describe the apperance of herpes virus under an electron microscope
Seems a bit like a fried egg - it is an enveloped virus and so the central ‘yolk’ is the virus and the rest is the envelope
How can we grow a virus in a lab?
We can use different cell lines in tubes ior plates and infect them with a virus - the right cell line is needed for each virus
- is slow but can be useful for research or rare viruses even though it has been replaced by molecular techniques
In the detection of viral antigens for diagnosis, where are the antigens usually found from the virus?
Antigens would usually be
- structural proteins
- secreted proteins
What techniques are replacing viral antigen detection?
Replaced by Nucleic acid detection methods due to improved test performance I.e greater sensitivity
Name the 3 most common types of viral antigen detection
- Immunochromatographic methods = lateral flows
- Direct immunofluorescence
- Cell associated antigens
- Enzyme immunoassay
- Free soluble antigens or whole virus
Describe the principles of direct immunofluorescence in the detection of viral antigens
- Take a swab to collect cells then deposit them on a microscope slide, the idea is that we want to see if there is viral antigen inside the cells.
- We then add an antibody that we know will bind to the viral antigens and the antibody has a fluorochrome attached - then wash the cells and view the slide with UV light
How could we use ELISA for viral diagnosis?
- We can use it to detect viral antigens (or even body antibodies)
- Commonly use it for detection of hepatitis
What are the 3 types of ELISA?
- indirect
- direct (primarily for antigen detection)
- sandwhich
Fully explain how ELISA works
- Antibodies that we know will bind the antigen are fixed to the bottom of the well - we then add the specimen and if antigen there it will stick to the antibody.
- We then add a second antibody that is also complimentary to the measles antigen this second antibody is conjugated to an enzyme that produces a product that produces a colour
- the enzyme is only present if the second antibody conjugated to the antigen
Why do we call it serology?
Bc the specimen that we normally use is serum - we can store this for long periods of time
Explain how we can use ELISA for detection of antibodies
- The red dots are antigen and they are fixed to the bottom of the well (this could be HepA antigen)
- So antibodies stick to antigen, wash out any remaining
- Then we stick another antibody (made in mice) on to the first antibody this second antibody has an enzyme …
- makes purple colour
Roughly when after infection with hepatitis A do symptoms start?
Around 3 weeks
Describe some of the symptoms of hepatitis A
- Generally unwell
- jaundice due to liver damage
What is ALT and why is it clinically significant?
Is an enzyme of the liver that we can detect when there is liver damage from hepatitis A
Describe when we can detect serum and faecal antigens and IgM and IgG for Hep A
- first antibody is IgM then IgG after around 4 weeks
What is NAAT?
- Nucleic acid amplification
- Tests that detect RNA or DNA of the virus you are looking for
- PCR e.g.
For retroviruses, what extra step do we need to do to prepare them in a sample when looking for the virus with NAATs
Convert the RNA → DNA with reverse transcriptase before NAAT
Name some advantages of NAATS
- May be automated. POCT possible
- Usually highly sensitive and specific, generates huge numbers of amplicons
- Rapid - can be as quick as 15 minutes - usually a few hours
- Useful for detecting viruses to make a diagnosis
- At first time of infection e.g. measles, influenza
- During reactivation e.g. cytomegalovirus
- Useful for monitoring treatment response
- Quantitative e.g. HIV, HBV, HCV, CMV viral loads
