Diagnosis of Viral Infections Flashcards
How can we tests for viruses?
- Electron Microscopy
- Virus isolation (cell culture)
- Antigen detection
- Antibody detection by serology
- Nucleic acid amplification tests (NAATs e.g. PCR)
- Sequencing for genotype and detection of antiviral resistance
What is the size of viruses?
- Viruses are tiny compared to other organisms
* Viruses need electron microscopy x20,000
How to we prepare a virus to visualise using an electron microscope?
- Specimens are dried on a grid
- Can be stained with heavy metal e.g. uranyl acetate
- Can be concentrated with application of antibody i.e. immuno- electron microscopy to concentrate the virus
- Beams of electrons are used to produce images
- Wavelength of electron beam is much shorter than light, resulting in much higher
What are the advantages and disadvantages of using electron microscopy?
Advantages
• Rapid
• Detects viruses that cannot be grown in culture
• Can visualise many different viruses
Disadvantages
• Low sensitivity need 106 virions/millilitre.
• Maybe enough in vesicle secretion/stool
• Requires maintenance
• Requires skilled operators
• Cannot differentiate between viruses of the same virus family.
• An expensive bit of an equipment
How do we isolate a virus in cell culture?
- Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE) of cells when a patient sample containing a virus incubated with a cell layer
- Old method, now replaced by molecular techniques, but still needed for research or for rare viruses
- Led to discovery of hMPV and Nipha virus in last 20 years
- Use different cell lines in test tubes or plates. Selection of cell types important.
- Slow, but occasionally useful in anti-viral sensitivity testing
- We can’t grow viruses very easily
- You can grow some viruses in a cell culture.
- If you can provide a virus with cells in a test tube, some viruses will be able to grow inside those cells and we can actually then grow those viruses from a clinical specimen from a patient. When a virus grow from those cells in a cell culture, it produces a CPE. It produces morphological changes in the cell. We can recognise the CPEs and hence work out the identity of viruses.
- hMPV a meta pneumo virus
- Niphaa discovered in Malaysia discovered in cell culture.
- It is found all over the world now.
- Different viruses have different affinities for cells and hence you need different cell lines.
- It is a slow process which can take several days
What is the cytopathic effect?
How can we identify viruses?
- Cells grow in monolayer in flask and have distinct morphology. Virus added and incubated and changes appearance (CPE).
- Different viruses may give different appearances.
- Different cell lines support growth of different viruses.
- Identify virus using antigen detection techniques or neutralisation of growth.
- Cell culture plus antiviral- look for inhibition of cytopathic effect.
- Test antiviral drugs and for resistance using cell culture techniques. E.g. Aciclovir for herpes.
What is antigen detection?
- • Direct detection of virus.
- • Viral antigen- usually capsid structural proteins (made up of capsomere) or secreted
- proteins that can be detected.
- • Infected cells may display viral antigens on their surfaces.
- • Acquire sample from patient depending on disease and virus.
- • Nasopharyngeal aspirates (NPA)- RSV (respiratory syncytial virus) and influenza.
- • Blood (serum or plasma)- hepatitis B and dengue.
- • Vesicle fluid- Herpes simplex or varicella zoster.
- • Faeces- rotavirus and adenovirus.
- • Nucleic acid detection methods now replaced this.
- • Commonest methods are direct immunofluorescence, enzyme immunoassay or
- immunochromatographic methods. Often used at point of care for rapid diagnosis. Faeces
- Rotavirus, adenovirus
What is immunofluorescence?
- Antigen (from infected host cells in sample) bound to slide
- Specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with sample
- Viewed using a microscope equipped to provide ultraviolet illumination
- NPA from a child
- Some cells are layered on a slide
- You get an AB with a fluorochrome. You mix the antibody with cells on the surface, and if a cell has the antibody, they will fluoresce
- Takes a few hours or so but is not that sensitive a test
What is ELISA?
Enzyme-linked immunosorbent assay A component of reaction is adhered to a solid surface Three formats: • Indirect • Direct (primarily antigen detection) • Sandwich
Direct ELISA- only one antibody is used which is directly conjugated to enzyme.
Indirect ELISA- Enzyme-conjugated secondary antibody added and binds to detecting
antibody (indirect ELISA) instead of the antigen.
In both direct and indirect, the antigen is immobilised of the plate.
Describe how ELISA works
- Plate is coated with a capture antibody
- Sample is added and any antigen present binds to capture antibody
- Enzyme-conjugated primary antibody is added, binds to detecting antibody
- Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change
The substrate only will change colour only if the enzyme- conjugated antibody and therefore also the antigen are present. Negative result = NO colour change
The substrate will change colour if the enzyme with the conjugated antibody is there.
How do we diagnose by antibody detection?
• IgM is what we produce first
• Then IgG will be produced.
• When infected with a virus the humoral immune response takes place resulting in production of immunoglobulins i.e. antibodies
• IgM antibodies specific to the virus are produced first
• IgM present for a variable period – usually 1 to 3 months
• As IgM declines, IgG is produced
• Quantity of IgG rises
• Diagnosis can be made by
o detection of IgM (can be non specific) – or
o demonstration of seroconversion
Negative IgG antibody at first
Then presence of IgG antibody
What is Serology and what can it be used for?
Indirect detection of the pathogen
Diagnostic mode of choice for organisms which are refractory to culture
Serology can be used to:
• Detect an antibody response in symptomatic patients
• Determine if vaccination has been successful
• Directly look for antigen produced by pathogens
Serological tests are not limited to blood & serum
can also be performed on other bodily fluids such as semen and saliva
What is serum?
• Produced from processing blood
o Blood is coagulated with micronized silica particles
o Gel used to trap cellular components
• Routinely serum tubes are centrifuged for 10 min at 1000xg
• Supernatant (serum) is removed and stored
• 4oC short term -20oC long term
• Routinely serum tubes are centrifuged for 10 min at 1000xg
• Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
• Only interested in the clear fluid
• This is a reversal, the antigen is now bound to the bottom of the well
• This is a kind of elisa test
How can we detect Hep A?
Can interpret stage of illness depending on the antibodies detected using reference
chart.
If no antibodies detected- this is somebody hat has never been infected with
hepatitis A.
If positive for IgM and positive or negative for IgG, this is an acute or recent
infection.
If negative for IgM and positive for IgG, this is a resolved or immunised infection.
The level and type of antibody varies with pathogen, exposure and time.
E.g. much higher IgG in second exposure than first and more immediate. Often no
IgM at all or just a little.
How is the combination of an antibody and antigen useful in detection of a viral infection?
This is useful for some infections such as
1. Hepatitis B
2. HIV
3. Hepatitis C
• This is because it allows us to establish whether acute or chronic infection
• This may have therapeutic implications
• Combined antigen and antibody 4th Generation HIV tests detect both antibody and antigen in as assay. Results in increased sensitivity and a reduced window period (time between infection and being able to get a positive result).
• Sometimes we try to look for both
• It can tell use what stage of the illness we are at
