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M&I: Pathogen Detection & Diagnosis of Infection > Diagnosis Of Viral Infections > Flashcards

Diagnosis Of Viral Infections Flashcards

1
Q

What is the purpose of a laboratory diagnostic test?

A
  • Reduce need for unnecessary tests and inappropriate antibiotics
  • Public health implications
  • Natural history of the pathogen to treat
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2
Q

Name some possible test types for viral infections

A
  • Electron microscopy
  • Virus isolation
  • Antigen detection
  • Antibody detection by serology
  • Nucleic acid amplification tests (NAATs)
  • Sequencing for genotype and detection of antiviral resistance
  • Immunochomatographic methods
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3
Q

Why might we still use electron microscopy in a viral lab?

A
  • Characterising emerging pathogens (so it was used to characterise COVID)
  • Possibly still useful for feaces and vescile specimens
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4
Q

What are the advantages of using electron microscopy?

A
  • Rapid
  • Detects viruses that cannot culture
  • Visualisation of viruses
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5
Q

What are the limitations of electron microscopy?

A
  • Low sensitivity - need 106 virons/ml
  • Requires maintenance
  • Requires skilled operators
  • Cannot differentiate between viruses of the same family
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6
Q

Name some viral infections that may be diagnosed with electron microscopy

A

Viruses that have a distinct shape but we can not tell the difference between members of the same family so like which herpes virus it is

  • Rotavirus
  • Norovirus
  • Herpes
  • Adenovirus
  • Coronavirus
  • Poxviruses such as smallpox and chickenpox
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7
Q

Describe the apperance of herpes virus under an electron microscope

A

Seems a bit like a fried egg - it is an enveloped virus and so the central ‘yolk’ is the virus and the rest is the envelope

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8
Q

How can we grow a virus in a lab?

A

We can use different cell lines in tubes ior plates and infect them with a virus - the right cell line is needed for each virus
- is slow but can be useful for research or rare viruses even though it has been replaced by molecular techniques

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9
Q

In the detection of viral antigens for diagnosis, where are the antigens usually found from the virus?

A

Antigens would usually be

  • structural proteins
  • secreted proteins
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10
Q

What techniques are replacing viral antigen detection?

A

Replaced by Nucleic acid detection methods due to improved test performance I.e greater sensitivity

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11
Q

Name the 3 most common types of viral antigen detection

A
  • Immunochromatographic methods = lateral flows
  • Direct immunofluorescence
    • Cell associated antigens
  • Enzyme immunoassay
    • Free soluble antigens or whole virus
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12
Q

Describe the principles of direct immunofluorescence in the detection of viral antigens

A
  • Take a swab to collect cells then deposit them on a microscope slide, the idea is that we want to see if there is viral antigen inside the cells.
  • We then add an antibody that we know will bind to the viral antigens and the antibody has a fluorochrome attached - then wash the cells and view the slide with UV light
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13
Q

How could we use ELISA for viral diagnosis?

A
  • We can use it to detect viral antigens (or even body antibodies)
  • Commonly use it for detection of hepatitis
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14
Q

What are the 3 types of ELISA?

A
  • indirect
  • direct (primarily for antigen detection)
  • sandwhich
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15
Q

Fully explain how ELISA works

A
  • Antibodies that we know will bind the antigen are fixed to the bottom of the well - we then add the specimen and if antigen there it will stick to the antibody.
  • We then add a second antibody that is also complimentary to the measles antigen this second antibody is conjugated to an enzyme that produces a product that produces a colour
  • the enzyme is only present if the second antibody conjugated to the antigen
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16
Q

Why do we call it serology?

A

Bc the specimen that we normally use is serum - we can store this for long periods of time

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17
Q

Explain how we can use ELISA for detection of antibodies

A
  • The red dots are antigen and they are fixed to the bottom of the well (this could be HepA antigen)
  • So antibodies stick to antigen, wash out any remaining
  • Then we stick another antibody (made in mice) on to the first antibody this second antibody has an enzyme …
  • makes purple colour
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18
Q

Roughly when after infection with hepatitis A do symptoms start?

A

Around 3 weeks

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19
Q

Describe some of the symptoms of hepatitis A

A
  • Generally unwell
  • jaundice due to liver damage
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20
Q

What is ALT and why is it clinically significant?

A

Is an enzyme of the liver that we can detect when there is liver damage from hepatitis A

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21
Q

Describe when we can detect serum and faecal antigens and IgM and IgG for Hep A

A
  • first antibody is IgM then IgG after around 4 weeks
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22
Q

What is NAAT?

A
  • Nucleic acid amplification
  • Tests that detect RNA or DNA of the virus you are looking for
  • PCR e.g.
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23
Q

For retroviruses, what extra step do we need to do to prepare them in a sample when looking for the virus with NAATs

A

Convert the RNA → DNA with reverse transcriptase before NAAT

24
Q

Name some advantages of NAATS

A
  • May be automated. POCT possible
  • Usually highly sensitive and specific, generates huge numbers of amplicons
  • Rapid - can be as quick as 15 minutes - usually a few hours
  • 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
Q

Which type of viral infection is NAATS especially useful for detecting?

A

Latent reactivating or chronic infections

26
Q

Name some disadvantages of NAATS

A
  • Generates 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.
  • Mutations in target sequence may lead to “dropout” e.g. S gene dropout seen with SARS-CoV-2 variants
27
Q

What is real-time PCR?

A
  • Amplification AND detection occur in real time by the release of fluorescence and so no gel electrophoresis needs to be done - faster
  • allows the use of multiplexing
28
Q

What is multiplex PCR?

A
  • Multiplex PCR is when mre than one pair of primers os used in a PCR.
  • So we can do one PCR for multiple pathogens e.g. for the detection of the x known viruses that can cause meningitis
  • only one sample needed and one tube
29
Q

In the diagnosis and management of HIV how do we use NAATs and other viral tests?

A

Antibody and antigen detection for the intitial diagnosis (screening test and confirmatory test)

  • Viral load measured by qPCR (NAAT)
  • Resistance testing done by gene sequencing
30
Q

What can we use gene sequencing for in the management of viral disease?

A
  • Can test for resistace to drugs/treatment (looking at specific mutations in antibiotic/antiviral resistance genes).
  • For HIV:
    • Multiple viral enzyme targets
      • Reverse transcriptase protease.
      • integrase
      • viral receptor binding proteins
31
Q

(Consolidation session) Name 3 lab techniques used to diagnose HIV/monitor progression

A
  • PCR (qPCR or other NAATs) is the most sensitive
  • ELISA to detect HIV antibody (or antigen?)
  • Flow cytometry (FACS?) to monitor disease progression by monitoring the %of CD4+ T cells in serum over time
32
Q

When can we use serology?

A

We can use serology to detect if an antibody response is present in a symptomatic patient or determine if vaccination has been successful

33
Q

What is serology mostly used for?

A

Serology is mostly used for the detection of viral antibodies

34
Q

How does virus isolation work?

A
  • Put different cell lines in test tubes or plates and then grow them
35
Q

Why would you do a cell culture?

A
  • Check for cytopathic effects of an antiviral
  • Different cell lines supporting growth of a different virus
36
Q

Where can you detect antigens?

A

In cells or free in blood, saliva or other tissues

37
Q

What can you detect in nasopharyngeal aspirates?

A

Cell associated virus antigens of:
RSV
Influenza

38
Q

What can you detect in blood?

A

Free antigens or whole virus of:
Hep B
Dengue

39
Q

What can you detect in vesicle fluid?

A

Whole virus of:
Herpes simplex
Varicella zoster

40
Q

What can you detect in the faeces?

A
  • Whole virus of:
  • Rotavirus
  • Adenovirus
41
Q

When do you use antibody detection by serology?

A

If the organism doesn’t like culture

42
Q

What can serology be used to do?

A

Detect an antibody response in symptomatic patients
Check if vaccination has been successful

43
Q

What are the different methods of antibody detection by serology?

A

Direct immunofluorescence
ELISA

44
Q

How do you carry out direct immunofluorescence?

A

Antigen bound to slide
Specific antibody to antigen is tagged to fluorochrome and mixed with the sample
Viewed using a microscope equipped with a UV light

45
Q

What are the three formats of an ELISA?

A

Indirect
Direct
Sandwich

46
Q

When are immunochomatographic methods used?

A

At point of care for rapid diagnosis

47
Q

What is the method of NAATs?

A

Specimen collection
Extraction of nucleic acid
DNA transcription for RNA viruses
Cycles of amplification of DNA polymerase
Detection of amplicons

48
Q

What are the advantages of NAATs?

A

May be automated
Highly sensitive and specific
Generates huge number of amplicons
Rapid
Useful for detecting viruses to make a diagnosis and monitoring treatment response

49
Q

What are the limitations of using NAATs?

A

Generates large numbers of amplicons (possibly causing contamination)
Need to have an idea of what you’re looking for
Mutations in target sequence may lead to dropout

50
Q

What is multiplex PCR?

A

When more than one pair of primers is used in the PCR

51
Q

What are the viral enzyme targets in antiviral resistance testing?

A

Reverse transcriptase, protease
Integrase
Viral receptor binding proteins

52
Q

What is serum produced from?

A

Processing blood

53
Q

What does serum contain?

A

Proteins,
antigens,
antibodies,
drugs,
electrolytes

54
Q

What techniques are replacing viral antigen detection?

A

Replaced by Nucleic acid detection methods due to improved test performance I.e greater sensitivity

55
Q

When can we use serology?

A

We can use serology to detect if an antibody response is present in a symptomatic patient or determine if vaccination has been successful

M&I: Pathogen Detection & Diagnosis of Infection (2 decks)

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