Diagnosis of Viral Infections Flashcards
possible test types used nowadays?
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
Visualisation of Microbes - how can they be seen?
bacteria, protozoa, fungi and helminths can all be seen with light microscopy
viruses are much smaller, can be viewed with an electron microscope
how does electron microscopy work?
Specimens dried on a grid and stained with heavy metal e.g. uranyl acetate
Can be concentrated with application of antibody
Beams of electrons are used to produce images
Wavelength of electron beam is much shorter than light, resulting in much higher resolution than light microscopy
electron micropyle is useful in what?
characterising emerging pathogens
advantages of electron microscopy?
Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses
limitations of electron microscopy?
low sensitivity - may be enough in vesicle secretion/stool (needs high conc of virus)
Requires maintenance and skilled operators
Expensive
Cannot differentiate between viruses of the same virus family.
which virus has a wheel shape?
rotavirus
shape of adenovirus (gastroenteritis)?
glycosohedral shape
coronavirus shape?
respiratory tract infection
crown like structure around a dumbell shape
Astrovirus shape?
star
Poxvirus - different types and what is the shape?
ball of wool
Smallpox
Monkeypox
Orf
Cowpox
what are 2 herpes viruses that cause vesicles?
Herpes simplex (cold sores or genital herpes which can cause little blisters) Varicella zoster virus (chickenpox or shingles)
they both look like a virion inside an envelope – the envelope has come from a human cell which the virus infected.
given they both look the same, distinguishing which viral infection the patient has is based on clinical context.
- if the patient has a fever and an itchy white rash all over the body, it will probably be varicella zoster virus
- if there are vesicles in just one area of the body, would be zoster/shingles
- cold sore would be Herpes Simplex
Virus isolation in cell culture
Viruses require cells to grow so we can’t grow them on an agar plate like we can with bacteria
- if you can provide a virus with cells in a test tube, viruses will be able to grow inside those cells (patient sample containing a virus is incubated with a cell layer)
- when viruses grow inside these cells, it produces a Cytopathic effect (CPE), and we see morphological changes in those cells
slow process (several days to get a results) but occasionally useful in anti-viral sensitivity testing - we now have other methods which are used more regularly
different cell lines may…
support growth of different viruses
-this is because different viruses have affinities for different cells
uses of viral neutralisation cultures?
set up a neutralisation culture, add antibodies against the suspected virus in the culture - cytopathic effect will be inhibited, then you can work out what virus it is
Important uses:
- test out anti-viral drugs by adding them and seeing if the cytopathic effect is inhibited - if it is inhibited, you know the drug will work
- you can see if a virus has developed anti-viral resistance
Antigen (viral) Detection
-what are antigens and how are they detected?
proteins displayed on the surface of cells (structural proteins), or floating in the blood of someone with an infection. Infected cells may display viral antigens on their surfaces.
eg. Hep B antigen in the blood, herpes simplex antigen in vesicle fluid, rotavirus in faeces
detected by
Direct immunofluorescence
Enzyme immunoassay
Immunochromatographic methods
Immunofluoresence - how does it work?
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
Immunochromatographic methods
get some of the patients blood and add it to the well and see a line appear where you get antigen-antibody binding
-causes precipitation of heavy metals and a line forming
ELISA (Enzyme-linked immunosorbent assay)
A component of reaction is adhered to a solid surface
3 formats:
Indirect
Direct (primarily antigen detection)
Sandwich
- have a well, antibody attached to bottom and add patient sample
- if the patient sample includes the antigen, it will be bound by the antibody
- you then add another antibody which is conjugated with an enzyme
- this conjugated antibody will bind to the antigen
- sandwich formed
- the enzyme changes the colour of a chomogenic substrate which is added
The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present. Negative result = NO colour change
Diagnosis by antibody detection
IgM antibodies specific to the virus are produced first
As IgM declines, IgG is produced
Quantity of IgG rises
Diagnosis can be made by detection of IgM or by demonstration of seroconversion
Negative IgG antibody at first
Then presence of IgG antibody
Serology - what can it be used for?
Indirect detection of the pathogen
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
Serum
Produced from processing blood
Routinely serum tubes are centrifuged
Supernatant (serum) is removed and stored
Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
why might you have antibodies in your body?
because of infection or immunisation
secondary exposure to an antibody leads to a massive increase in what?
IgG
Modern Laboratory detection of antibodies and antigens in blood
Serology
Detection of antibody/antigens via enzyme immunoassays or related technology e.g. microparticle immuno-chemiluminescence
- useful for some infections such as: Hepatitis B, HIV, Hepatitis C
- this is because it allows us to establish whether acute or chronic infection
- may have therapeutic implications
Molecular diagnostic tests
Nucleic acid amplification (NAAT), e.g. PCR
For example: is there any adenovirus DNA in the patient sample – looking for this
- Extract DNA from adenovirus and sequence the whole virus so you know the genetic makeup
- get primers, short bit of complementary DNA, which will bind to the DNA from the adenovirus
- denature the patient DNA so it comes apart, and add adenovirus primers
- the primers will bind to the adenovirus DNA parts if its present in the human sample
- add DNA polymerase so double stranded DNA is formed, then denature again and again until you can detect large amounts of DNA in the sample
Advantages of using NAATS
May be automated
Highly sensitive and specific, generates huge numbers of amplicons
Rapid
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
Limitations of using NAATs
May detect other viruses which are not causing the infection
Exquisitely sensitive and so may generate large numbers of amplicons. This may cause contamination.
Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target.
Real time PCR
Real time as amplification AND detection occur in REAL TIME i.e. simultaneously by the release of fluorescence
Avoids the use of gel electrophoresis or line hybridisation
Multiplex PCR
term used when more than one pair of primers is used in a PCR
enables the amplification of multiple DNA targets in one tube e.g. detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2
Specific Taqman Probes
Probe will bind to bit of DNA you want to amplify, will glow IF the reporter is cut off by Taq polymerase
check slide 40
Organism Sequencing
DNA or RNA viruses
Used to predict response to anti-virals
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
Combinations of methods e.g. HIV diagnosis and management
Antibody and antigen detection for initial diagnosis Screening test (EIA) Confirmatory test (EIA)
Viral load(NAAT) at baseline and to monitor treatment response Quantification of virus in blood
Resistance testing (sequencing) to confer resistance before and during treatment
Screening
Testing for specific infections in at risk groups
Testing because it may have an implication for others e.g. antenatal
Needs a sensitive screening test
May have some false positives, so need a specific confirmatory test
