Diagnosis of Viral Infections Flashcards by Maaria M

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

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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

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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

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electron micropyle is useful in what?

characterising emerging pathogens

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advantages of electron microscopy?

Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses

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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.

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which virus has a wheel shape?

rotavirus

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shape of adenovirus (gastroenteritis)?

glycosohedral shape

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coronavirus shape?

respiratory tract infection

crown like structure around a dumbell shape

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Astrovirus shape?

star

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Poxvirus - different types and what is the shape?

ball of wool

Smallpox
Monkeypox
Orf
Cowpox

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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

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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

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different cell lines may…

support growth of different viruses

-this is because different viruses have affinities for different cells

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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

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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