Diagnosis of Viral Infections Flashcards Preview

CLINICAL PATHOLOGY > Diagnosis of Viral Infections > Flashcards

Flashcards in Diagnosis of Viral Infections Deck (34)
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1

It is not always possible to diagnose a infection clinically. Often we require a laboratory diagnostic test. 

Other factors that aid diagnosis are history, examination and special investigations 

2

Why is it important to get a rapid diagnosis? 

A rapid diagnosis of viral infecrions can reduce the need for uneccesary tests and inappropriate antibiotics. 

It is also an important public health tool, as it has infection control implications

3

What are the three types of laboratory tests with regards to viral infections? 

  • Diagnostic tests 
  • Monitoring tests 
  • Screening tests 

4

List some possible test types of diagnosis of viral infections 

 

  • Electron microscopy 
  • Virus isolation 
  • Antigen detection 
  • Antibody detection by serology 
  • Nucleic acid amplification tests (NAATs e.g PCR) 
  • Sequencing by genotype and detection of antiviral resistance 

5

How do we visuallise viruses? 

By an electron microscope

  • However they have mostly been replaced my molecular techniques 
  • However still used for faeces and vesicles 
  • And characterising emerging pathogens 

6

How does electron microscopy work? 

  1. Specimens are dried on a grid
  2. Can be stained with heavy metal e.g. uranyl acetate
  3. Can be concentrated with application of antibody e.g. immunoelectron microscopy to concentrate the virus
  4. Beams of electrons are used to produce images
  5. Wavelength of electron beam is much shorter than light resulting in much higher resolution than light microscopy = sharper resolution of image

7

What are some advantages of EM? 

  • Rapid 
  • Detects virus that cannot be grown in culture 
  • Allows for many different viruses to be visuallised 

8

Disadvantages of EM

  • Low sensitivity need 106 virions/millilitre. May be enough in vesicle secretion/stool
  • Requires maintenance
  • Requires skilled operators
  • Cannot differentiate between viruses of the same family

9

Which two herpes viruses will cause vesicles, and would an EM be able to distinguish between them? 

 

  • Herpes simplex and varicella zoster virus will cause vesicles
  • EM cannot differentiate between these different viruses so depends on clinical context, site of vesicle and symptoms
  • Herpes contain a virion inside an envelope,’ fried egg appearance’ but as mentioned mainly depends on clinical context.

10

Describe virus isolation 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 replicated by molecular techniques) however is still needed for research of rare viruses 

  • Use different cell lines in test tubes or plates. Selection of cell types is important as different viruses may have different affinities for different cell types 

    • Slow, but occasionally useful in anti-viral sensitivity testing 

11

Once what happens once you have a cytopathic effect, how can you further analyse viruses?

Once you add a specimen to the cell culture and asses to see if there was a cytopathic effect

  • Different viruses will give different appearances
  • Different cells lines will only support growth of specific viruses
  • Identify virus using antigen detection techniques or neutralisation of growth
  • Cell culture plus antiviral à Look for inhibition of cytopathic effect

12

What are some common methods for antigen detection 

  • Direct immunofluorescence 
  • Enzyme immunoassay 
  • Immunochromatographic methods 

13

Give a brief description of 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
  • Any cells which have the virus will fluoresce 

14

Briefly describe the immunochromatographic method

e.g diagnosis of dengue (Flavivirus, arthropod vector, common infection in returning travellers)

Patients’ blood is added to the well

A line will appear if there is binding of the antigen in the patient’s blood to the antibody in the specific kit causing precipitation

15

Describe how elisa works for antigen detection

Enzyme linked immunosorbent assay = ELISA 

A component of reaction is adhered to solid surface

Three formats

  • Indirect
  • Direct (primary antigen detection)
  • sandwich

16

Describe how ELISA works

  1. Plate is coated with capture antibody 
  2. The sample is added and any antigen present binds to the capture antibody 
  3. An enzyme conjugated primary antibody is added, which binds to the detecting antibody 
  4. After washing away the unconjugated sample, a chromogenic substance is added and is converted by the enzyme to a detectable form e.g colour change 
  • The substrate will only change colour only if the enzyme conjugated antibody and therefore also the antigen are present 
  • A negative result = NO COLOUR CHANGE 

17

How can we carry out diagnosis using antibodies? 

  • When infected with a virus the humoral immune response takes place = production of immunoglobulins (antibodies) 
  • IgM antibodies specific to virus produced first = 1-3 months 
  • As IgM declines, IgG is produced 

Diagnosis can be made by: 

  • Detection of IgM (can be non-specific) 
  • Or by demonstration of seroconservation (negative IgG antibody at first, then presence of IgG antibody) 

18

What is serology? 

Scientific study of serum and other bodily fluids, usually for diagnostic identification of antibodies in the serum = indirect detection of pathogen 

19

What can serology be used for? 

  • 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 and amp = they can be performed on other bodily fluid such as semen + saliva 

20

Describe the serological results that can help diagnose the stage of infection of Hepatitis A 

  • We can use antibodies to assess whether a patient has been infected or not
  • Negative for hepatitis IgM and IgG means that they will have never been infected by hepatitis A or immunised
  • Acute or recent infection you will have positive IgM and negative IgG
  • Resolved infection or immunisation you will have negative IgM and positive IgG
  • You can only be infected by hepatitis A once however there are other infections where you can be infected again

21

Why are we sometimes unable to detect viruses based on IgM levels? 

  • With second exposure, since we've made memory cells to the infection often we get a very IgG response 
    • You get a slight raise in IgM but often IgM doesnt raise at all 
  • So some viruses you can't detect by looking at IgM. You would have to look for them  by detecting a rise in IgG thats there 
    • Its rare to use this method but can still be used if someone has pre-existing antibodies 

22

Detection of antigen and antibody 

  • This is useful for some infections such as: 
    • Hepatitis B, HIV and Hepatitis C
    • This is because it allows us to establish whether acute or chronic infection
    • This may have therapeutic implications

23

Describe molecular diagnostic tests for viral detection 

  • An example of nuclear acid amplification test (NAAT is PCR but there are others) 
  • It can detect RNA/DNA (depends on viral genome) 
    • Design primers which are complementary to the DNA from virus ,these will only anneal if the virus is present 
  • Abillity to multiplex using fluorescent probes = look for several targets in one sample 
  • May be quantitative or qualitative 
  • Requires NA extraction prior to amplification 

24

List some advantages of 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

25

What are some limitations of 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, require highly stringent conditions
  • Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target

26

Describe multiplex PCR 

  • Term used when one pair of primers is used in PCR 
  • It enables amplification of multiple DNA targets in one tube e.g detection of multiple viruses in one CSF specimen 
    • HSV1, HSV2, VZV, enterovirus, mumps virus

27

Describe real time PCR

  • Measures the accumulation of DNA product in real time 
    • Amplification and detection occur at the same time by the release of fluorescence 
  • It avoids the use of gel electrophoresis or line hybridisaiton 
  • Can allow the use of multiplexing 

28

Describe how the specific Taqman probes work 

 

  • A primer will be annealed to the DNA strand and a taqman probe will hybridise to the region of interest (occurs during annealing phase) 
  • The probe will contain a fluorescent reporter and a quencher which will stop it glowing when it is in close proximity to the reporter 
  • When the probe binds DNA an enzyme called Taq polymerase posses 5'-3' nuclease activity and hydroyluse the probe
  • Now seperated from the quencher, the reporter will glow 
  • Every time you get this process of amplification, you get fluorescence 
    • Hence, the fluorescence signal directly proportional to initial copy number 

29

What is the CT? 

Cycle Threshold = the number of times we actually need to do the amplification process 

 

30

How can PCR can be inhibited? 

  • Substances can inhibit PCR (e.g Haem, bile salts) 
  • Assays should always include an internal posotive control as the results could be incorrectly reported as negative 
    • We can include primers specific for the internal control of material 

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