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π“œπ‘’π’Ήπ’Ύπ’Έπ’Άπ“ π“œπ’Ύπ’Έπ“‡π‘œπ’·π’Ύπ‘œπ“π‘œπ‘”π“Ž > Diagnosis of Viral Infections > Flashcards

Diagnosis of Viral Infections Flashcards

1
Q

What are 6 ways of testing for viruses?

A 
➝ Electron microscopy
➝ Virus isolation
➝ Antigen detection
➝ Antibody detection by serology
➝ Nucleic acid amplification tests 
➝ Sequencing for genotype and detection of antiviral resistance
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2
Q

What magnification do viruses need?

A

➝ 20,000x

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3
Q

What magnification do bacteria, fungi and helminths need?

A

➝ 400-1000x

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4
Q

What has electron microscopy of viruses been replaced with?

A

➝ Molecular techniques

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5
Q

What can electron microscopy of viruses still be used for?

A

➝ Feces and vesicle specimens

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6
Q

How are virus specimens prepared for electron microscopy?

A

➝ specimens are dried on a grid
➝ they are stained with heavy metal (uranyl acetate)
➝ can be concentrated with application of antibody
➝ beams of electrons are used to produce images

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7
Q

Why does electron microscopy have a higher resolution than light microscopy?

A

➝ The wavelength of an electron beam is much shorter than light
➝ this results in much higher resolution than light microscopy

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8
Q

What are the 3 advantages of electron microscopy for viruses?

A

➝ Rapid
➝ detects viruses that cannot be grown in culture
➝ can visualise many different viruses

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9
Q

What are the 4 disadvantages of electron microscopy for viruses?

A

➝ Low sensitivity so you need 10^6 virions per ml
➝ requires maintenance
➝ requires skilled operators
➝ cannot differentiate between viruses of the same family

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10
Q

What does rotavirus cause?

A

➝ gastroenteritis

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11
Q

What does adenovirus cause?

A

➝ gastroenteritis

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12
Q

What does coronavirus affect?

A

➝ respiratory system

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13
Q

What does norovirus cause?

A

➝ Gastroenteritis

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14
Q

What does herpes simplex virus cause?

A

➝ vesicles

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15
Q

What does herpes (varicella zoster) virus cause?

A

➝ chickenpox

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16
Q

How can you differentiate between herpes viruses?

A

➝ You can’t with EM

➝ it depends on clinical context, site of vesicle and symptoms

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17
Q

What are the 4 types of poxviruses?

A

➝ Smallpox
➝ Monkeypox
➝ Cowpox
➝ Orf

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18
Q

What do viruses require to replicate?

A

➝ Host cells

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19
Q

How can you investigate cytopathic effect?

A

➝ Take a patient sample containing the virus sample
➝ incubate with a cell layer
➝ observe cytopathic effects

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20
Q

What two viruses have been discovered by the cytopathic effect technique?

A

➝ hMPV

➝ Nipha virus

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21
Q

How do you test antivirals?

A

➝ cell culture + antiviral

➝ look for inhibition of cytopathic effect

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22
Q

How do you identify viruses in cell cultures?

A

➝ using antigen detection

➝ neutralisation of growth

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23
Q

What viral components can be detected?

A

➝ Viral antigens

➝ they are usually proteins : either capsid or structural proteins

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24
Q

What do virus infected cells display?

A

➝ Viral antigens on their surfaces

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25
Q

What viruses do you take nasopharyngeal aspirates for?

A

➝ RSV

➝ Influenza

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26
Q

What viruses do you take blood samples for?

A

➝ Hepatitis B

➝ Dengue

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27
Q

What viruses do you take vesicle fluid samples for?

A

➝ Herpes simplex

➝ Varicella zoster

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28
Q

Which viruses do you take feces samples for?

A

➝ Rotavirus

➝ Adenovirus

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29
Q

What are the 3 most common virus detection methods?

A

➝ Direct immunofluorescence
➝ Enzyme immunoassay
➝ Immunochromatographic methods

30
Q

How does immunofluorescence work?

A

➝ 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 that provides UV illumination

31
Q

What virus is dengue caused by and how is it spread?

A

➝ Flavivirus

➝ arthropod vectors

32
Q

What are the three types of ELISA test?

A

➝ direct
➝ indirect
➝ sandwich

33
Q

How does antigen detection in the ELISA test work?

A

1) the plate is coated with a capture antibody (antibody that will capture an antigen you are interested in e.g one that the virus has)
2) The sample is added and any antigen present binds to capture antibody
3) Enzyme conjugated primary antibody is added and it binds to the detecting antibody
4) chromogenic substrate is added and is converted by the enzyme to a detectable form e.g color change

34
Q

What is a negative result in an ELISA?

A

➝ no color change

35
Q

What does the humoral system do when infected with a virus?

A

➝ Produce antibodies

36
Q

How does diagnosis by antibody detection work?

A

➝ Detection of IgM
➝ demonstration of seroconversion
➝ negative IgG at first then positive

37
Q

How do antibody classes vary during a viral infection?

A

➝ IgM antibodies specific to the virus are produced first
➝ IgM is present for a variable period 1-3m months
➝ IgM declines and IgG is produced

38
Q

What 3 things can serology be used for?

A

➝ Detect an antibody response in symptomatic patients
➝ Determine if vaccination has been successful
➝ Directly look for antigen produced by pathogens

39
Q

What fluids can serological tests be done on?

A

➝ blood
➝ serum
➝ semen
➝ saliva

40
Q

What is serum produced from?

A

➝ processing blood

41
Q

How is serum produced?

A

➝ Blood is coagulated with micronized silica particles

➝ Gel is used to trap cellular components

42
Q

How are serum tubes stored and at what temperature?

A

➝ Centrifuged for 10 minutes at 1000xg
➝ supernatant is removed and stored
➝ 4 C short term and -20C long term

43
Q

What does serum contain?

A 
➝ proteins
➝ antigens
➝ antibodies 
➝ Drugs
➝ electrolytes
44
Q

Describe how you would detect the measles antibody using an antigen down ELISA?

A

1) Attach measles antigen to the bottom of a well
2) Add the patients sample to the well
3) If the patient has measles antibodies in their blood they will bind to the antigen in the well
4) you add an animal antibody attached to an enzyme which will bind to the human antibody
5) at each stage you wash out the wells so you don’t have floating antibodies

45
Q

How can you see what stage of Hepatitis A a patient is at?

A

➝ you measure their serum antibody levels
➝ IgM is produced first
➝ IgG persists

46
Q

What are the signs of acute or recent Hepatitis A?

A

➝ Patient having IgM

47
Q

What are the signs of immunisation or a resolved Hepatitis A infection?

A

➝ patient having IgG

48
Q

What happens during second exposure to Hepatitis A?

A

➝ you will have IgG antibodies from the 1st exposure
➝ on second exposure there is a really high IgG response
➝ hardly any IgM

49
Q

How do you detect antibodies and antigens in the blood?

A

➝ Enzyme immunoassays

50
Q

in what 3 diseases is it useful to detect antigens and antibodies?

A

➝ Hepatitis B
➝ HIV
➝ hepatitis C

51
Q

Why is it useful to detect antibodies and antigens?

A

➝ It allows us to establish whether it is an acute or chronic infection

52
Q

What do molecular diagnostic tests require prior to amplification?

A

➝ Nucleic acid extraction

53
Q

What can molecular diagnostic tests detect?

A

➝ RNA or DNA

54
Q

What are the 5 advantages of molecular diagnostic tests?

A

➝ May be automated
➝ Highly sensitive and specific, generates huge numbers of amplicons
➝ rapid
➝ useful for detecting viruses to make a diagnosis
➝ useful for monitoring treatment response

55
Q

Why do you need quantitative diagnostic tests?

A

➝ to measure viral load

56
Q

What is NAAT?

A

➝ nucleic acid amplification

57
Q

What are the 4 limitations of NAAT?

A

➝ May detect other viruses which are not causing the infection
➝ Exquisitely sensitive and can generate large numbers of amplicons
➝ May cause contamination
➝ you have to have an idea of what virus you are looking for as you need primers and probes specific to it

58
Q

What does real time PCR avoid the use of?

A

➝ gel electrophoresis or line hybridisation

59
Q

What is multiplex PCR?

A

➝ when more than one pair of primers is used in PCR

60
Q

What does multiplex PCR enable?

A

➝ Amplification of multiple DNA targets in one tube

➝ detection of multiple viruses in one specimen of CSF

61
Q

Describe how Specific Taqman probes work?

A

1) Taqman probe complementary to region of interest, binds between primers
2) Oligonucleotide probe with fluorescent reporter at the 5’ end and a quencher at the 3’
3) The quencher prevents the reporter fluorescing when excited if in close proximity
4) Taqman probe hybridises to the region of interest
5) This occurs during the annealing phase of PCR
6) Fluorescence is prevented due to the proximity of the quencher
7) Taq polymerase extends from the 3’ end of the primer as normal
8) The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe
9) The reporter is removed from the quencher and fluorescence can be detected
10) For any given cycle within the exponential phase, the amount of product and hence fluorescent signal is directly proportional to the initial copy number

62
Q

What is the cycle threshold?

A

➝ The amount of cycles required to cross the threshold

63
Q

What substances inhibit PCR?

A

➝ haem

➝ bile

64
Q

What should assays include so false negatives don’t happen?

A

➝ An internal positive control

65
Q

What is organism sequencing used for?

A

➝ to predict the response to anti-virals

➝ if there is resistance in drug experienced patients

66
Q

What is the consensus sequence based on?

A

➝ clinical observation of resistance or in vitro evidence

67
Q

How do you make an initial diagnosis of HIV?

A

➝ Antibody and antigen detection

68
Q

How do you monitor the response to HIV?

A

➝ check the viral load with NAAT

➝ quantify the virus in the blood

69
Q

What are the viral enzyme targets for antiviral resistance testing?

A

➝ Reverse transcriptase
➝ integrase
➝ Viral receptor binding proteins

70
Q

Why do you screen for viruses?

A

➝ It may have an implication for others e.g antenatal

71
Q

What are the three viruses you screen for?

A

➝ HIV
➝ HBV
➝ HCV

72
Q

What is needed in addition to screening and why?

A

➝ may have some false positives so you need a specific confirmatory test

π“œπ‘’π’Ήπ’Ύπ’Έπ’Άπ“ π“œπ’Ύπ’Έπ“‡π‘œπ’·π’Ύπ‘œπ“π‘œπ‘”π“Ž (17 decks)

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