Diagnosis Of Viral Infections Flashcards
What are possible tests to diagnose infection?
Electron microscopy - rarely used
Antigen detection
Virus isolation
Nuclei’s acid amplification tests
How can virus isolation in cell culture be used in viral diagnosis?
As viruses require host cells to replicate and may cause cytopathic effect (CPE) of cells when a patient sample containing a virus incubated within cell layer
Use different cell lines in test tubes or plates - selections of cell types important
Slow but occasionally useful
Old method not used now as much
What specimens can be used for antigen detection?
Nasopharyngeal aspirates - cell associated virus antigen
Blood - free antigen or whole virus
Vesicle fluid - whole virus
Faeces - whole virus
How can electron microscopy be used to diagnose viruses?
Viruses can be visualised with electron microscope
- Specimens are dried on a grid
- Can be stained with heavy metal
- Can be concentrated with application of antibody
- Beams of electrons are used to produce images
- Wavelength of electron beam is inch shorter than light, resulting in much higher resolution than light
Useful in charecterising emerging pathogens
Mostly replaces technique
What are the advantages of using electron microscopy to diagnose viruses?
Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses
What are the disadvantages of using electron microscopy to diagnose viruses?
Low sensitivity need 10^6 virgins/milileter - may be enough in vesicle secretion
Requires maintenance
Requires skilled operators
Cannot differentiate between viruses of the same virus family
What is the cytopathic effect?
Different viruses may give different appearances
Morphology of host cells are different
What are the most common methods used in antigen detection?
Direct immunofluorescence - cell associated antigens
Enzyme immunoassay - free soluble antigens or whole virus
Immunochromatograohic methods
What happens during direct immunoflorescnece detection of antigens?
Antigen bound to slide
Specific antibody to that antigen is tagged to a fluorochromes and mixed with sample
Viewed using a microscope equipped to provide ultraviolet illumination
What happens with immunochromatograohic methods?
Lateral flow tests
Used to test for pregnancy detection and virus testing
How is ELISA used for antigen detection?
Enzyme linked immunosorbent assay - a component of reaction is adhered to a solid surface
Three formats:
- indirect
- direct
- sandwich
Describe the stages that occur in a ELISA test
- Plate is coated with a capture
- Sample is added and any antigen present binds to capture antibody
- Enzyme conjugated primary antibody is added - binds to detection antibody
- Chromogenic substrate is added and converted by the enzyme to detectable form
How are antibodies detected using serology?
Indirect detection of pathogen
Used for organisms we cannot culture or not seen in electron microscopy
What is serology be used for?
Detect antibody response in symptomatic patients
Determine if vaccination has been successful
Not limited to blood and serum - can be performed on other bodily fluids
How is serum produced?
Routinely serum tubes are centrifuged for 10 minutes at 1000xg
Supernatant is removed and stored
Serum contains proteins, antigens, antibodies, drugs
How can viruses be diagnosed by antibody detection?
When infected with a virus the humoral immune response takes place resulting in production of immunoglobulins
- Detection of igM
- Or by demonstration of seroconversion
When can it be useful to detect antigen and antibody?
It allows us to establish weather acute or chronic infection
May have therapeutic implications
HIV, Hepatitis C and B
What is Nucleic acid amplification (NAAT)?
Can detect RNA or DNA of virus
Ability to multiplex using fluorescence probes
May be quantitative or qualitative
Eg. PCR
What are the stages of NAAT test?
- Specimen collection
- Extraction of nucleic acid
- DNA transcription of RNA viruses
- Cycles of amplification of DNA target
- Detection of amplicons
What are the advantages of using NAATS?
May be automated
Highly sensitive and specific
Generated hug e number of amplicons
Rapid - up to a few hours
Useful for steering viruses to make a diagnosis
Useful for monitoring treatments response
What are the limitations of using NAATS?
Generates large number of amplicons - may cause contamination
Need to have an idea of what viruses you are looking for as specific primers and probes required
Mutations in target sequence may lead to “dropout”
What is real time PCR?
Different chemistries but all similar
Amplification and detection occur in real time
Avoids the use of gel electrophoresis or line hybridisation
Allows the use of multiplexing
What is multiplex PCR?
Term used when more than one pair of primers is used in a PCR
Enables amplification of multiple DNA targets in one tube
Eg. Detection of multiple viruses in one CSF specimen
What is PCR inhibition?
Some substances inhibit PCR
- haem
- bile salts
Assays should contain a internal positive control as results could be false negative
Include primer specific for internal control material
Why can genome sequencing be beneficial for viral diagnosis?
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
New variants and vaccine efficacy
Can be used to predict response to anti virals
How can a combination of methods be used for diagnosis?
Antibody and antigen detection for initial diagnosis
NAAT at baseline and to monitor treatment response
Resistance testing - sequencing
How do you test for antiviral resistance?
Look for mutations known to cause resistance
Multiple viral enzyme targets
- reverse transcriptase
- integrase
- viral receptor binding
What is screening?
Testing for specific infections in at risk groups
Testing because it ,ay have a implication for others
Needs a sensitive screening test
May have false positives so a specific confirmatory test needed
