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Microbiology > 8- Diagnosis of viral infections > Flashcards

8- Diagnosis of viral infections Flashcards

1
Q

What do we often require to diagnose an infection?

A

➝ laboratory diagnostic test

➝ as it is not always possible to diagnose clinically

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2
Q

What 3 factors act to aid diagnosis?

A

➝ history
➝ examination
➝ special investigations

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3
Q

Why is rapid diagnosis of viral infections necessary?

A

➝ reduce need for unnecessary tests
➝ and inappropriate antibiotics
➝ important public health and infection control implications

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4
Q

Why is it helpful to know natural history of pathogen in the patient you are testing?

A

➝ will affect test selection and interpretation

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5
Q

What are 6 ways of testing for viruses?

A

➝ Electron microscopy
➝ Virus isolation (cell culture)
➝ Antigen detection
➝ Antibody detection by serology
➝ Nucleic acid amplification tests (NAATs eg. PCR)
➝ Sequencing for genotype and detection of antiviral resistance

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6
Q

What magnification do viruses need?

A

➝ x20,000

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7
Q

What magnification do bacteria, fungi and protozoa, helminths need?

A

➝ 400-1000x

➝ naked eye for protozoa, helminths

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8
Q

What has electron microscopy of viruses been replaced with?

A

➝ Molecular techniques

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9
Q

What can electron microscopy of viruses still be used for?

A

➝ Faeces and vesicle specimens

➝ characterising emerging pathogens

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10
Q

How are virus specimens prepared for electron microscopy?

A

➝ specimens are dried on a grid
➝ can be stained with heavy metal (uranyl acetate)
➝ can be concentrated with application of antibody i.e. immuno-electron microscopy to concentrate the virus
➝ beams of electrons are used to produce images

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11
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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12
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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13
Q

What are the 4 disadvantages of electron microscopy for viruses?

A

➝ Low sensitivity need 10^6 virions per ml. May be enough in vesicle secretion/stool.
➝ requires maintenance
➝ requires skilled operators
➝ cannot differentiate between viruses of the same family

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14
Q

What does rotavirus cause?

A

➝ gastroenteritis

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15
Q

What does adenovirus cause?

A

➝ gastroenteritis

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16
Q

What is OC43 and what does it cause?

A

➝ one of the four ‘seasonal coronaviruses’

➝ causes mild respiratory tract infections

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17
Q

What does norovirus (calicivirus) cause?

A

➝ Gastroenteritis

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18
Q

What 2 herpes viruses cause vesicles?

A

➝ Herpes simplex

➝ Varicella zoster virus

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19
Q

How can we differentiate between different herpes viruses?

A

➝ EM cannot differentiate these diff viruses
➝ depends on clinical context
➝ site of vesicle
➝ symptoms

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20
Q

What does herpes (varicella zoster) virus cause?

A

➝ chickenpox

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21
Q

What are the 4 types of poxviruses?

A

➝ Smallpox
➝ Monkeypox
➝ Cowpox
➝ Orf

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22
Q

How can we differentiate between different poxvirus?

A

➝ depends on clinical context i.e. exposure history, geographic location, clinical features

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23
Q

What do viruses require to replicate?

A

➝ Host cells

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24
Q

What can virus replication cause in vitro/ cell culture?

A

➝ may cause cytopathic effect

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25
Q

Why is virus isolation in cell culture now an old method?

A

➝ it has been replaced by molecular techniques

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26
Q

Why is virus isolation in cell culture still used?

A

➝ needed for research

➝ or for rare viruses

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27
Q

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

A

➝ hMPV
➝ Nipha virus
➝ SARS-CoV-2

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28
Q

What is an advantage and disadvantage of virus isolation in cell culture technique?

A

➝ slow

➝ occasionally useful in anti-viral sensitivity testing

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29
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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30
Q

Why can you get different appearances of cytopathic effect?

A

➝ Different viruses may give different appearances

➝ Different cell lines may support growth of different viruses

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31
Q

How do you test antivirals?

A

➝ cell culture + antiviral

➝ look for inhibition of cytopathic effect

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32
Q

How do you identify viruses in cell cultures?

A

➝ using antigen detection

➝ neutralisation of growth

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33
Q

What viral component can be detected and where?

A

➝ Viral antigens
➝ they are usually proteins : either capsid or structural proteins
➝ can be detected in cells or free in blood
➝ saliva
➝ or other tissues/organs

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34
Q

What do virus infected cells display?

A

➝ Viral antigens on their surfaces

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35
Q

What viruses do you take nasopharyngeal aspirates for?

A

➝ RSV

➝ Influenza

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36
Q

What type of antigens are we looking for when we take nasopharyngeal aspirates?

A

➝ cell-associated virus antigens

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37
Q

What viruses do you take blood samples for?

A

➝ Hepatitis B

➝ Dengue

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38
Q

What type of antigens are we looking for when we take blood samples?

A

➝ free antigen

➝ or whole virus

39
Q

What viruses do you take vesicle fluid samples for?

A

➝ Herpes simplex

➝ Varicella zoster

40
Q

What are we looking for when we take vesicle fluid samples?

A

➝ whole virus

41
Q

Which viruses do you take faeces samples for?

A

➝ Rotavirus

➝ Adenovirus

42
Q

What are we looking for when we take faeces sample?

A

➝ whole virus

43
Q

What is replacing EM, cell culture and viral antigen detection techniques? Why?

A

➝ Nucleic acid detection methods

➝ due to improved test performance i.e. greater sensitivity

44
Q

What are the 3 most common virus antigen detection methods? What type of antigens can we detect?

A

➝ Direct immunofluorescence- cell associated antigens
➝ Enzyme immunoassay- free soluble antigens or whole virus
➝ Immunochromatographic methods

45
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 equipped to provide UV illumination

46
Q

What virus is dengue caused by and how is it spread and who is is common in?

A

➝ Flavivirus
➝ arthropod vectors
➝ common infection in returning travellers

47
Q

What can we diagnose using immunochromatographic methods?

A

➝ dengue

48
Q

What is the advantage of immunochromatographic methods?

A

➝ are useful as a near patient test- NPT
➝ a point of care test - PCOT
➝ often used at point of care for rapid diagnosis

49
Q

What is the disadvantage of immunochromatographic methods aka lateral flow test for diagnosing COVID-19?

A

➝ not as sensitive or specific as PCR test, NAATs

50
Q

What is the full form of ELISA?

A

➝ Enzyme-linked immunosorbent assay

51
Q

What are the three types of ELISA test?

A

➝ direct
➝ indirect (primarily antigen detection)
➝ sandwich

52
Q

How can we detect antigen by ELISA?

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- complementary
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. colour change

53
Q

When will the substrate change colour in ELISA?

A

➝ The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present.

54
Q

What is a negative result in an ELISA?

A

➝ no colour change

55
Q

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

A

➝ Produce antibodies

56
Q

For what organisms is antibody detection by serology a diagnostic mode of choice?

A

➝ for organisms which are refractory to culture

57
Q

What are the signs of no past/ current infection or immunisation?

A

➝ patient negative for IgM

➝ and negative for IgG

58
Q

What are the signs of acute or recent Hepatitis A?

A

➝ patient positive for IgM

➝ and can be negative/positive for IgG

59
Q

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

A

➝ patient negative for IgM

➝ and positive for IgG

60
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

61
Q

How do you detect antibodies and antigens in the blood? Give example

A

➝ Enzyme immunoassays

e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence

62
Q

What is used in modern lab to detect antibodies and antigens in blood?

A

➝ automated analysers eg. Abbott Architect

➝ rarely use Michael teeter trays

63
Q

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

A

➝ Hepatitis B
➝ HIV
➝ hepatitis C

64
Q

Why is it useful to detect antibodies and antigens?

A

➝ It allows us to establish whether it is an acute or chronic infection
➝ this may have therapeutic implications

65
Q

Why is looking at diff antigens and antibodies useful for Hepatitis B?

A

➝ we can better understand what type of infection the patient has i.e. none, acute, resolving, etc

66
Q

What is NAAT?

A

➝ Nucleic acid amplification test
➝ they are molecular diagnostic tests
➝ such as PCR, LCR, LAMP, SDA

67
Q

What do molecular diagnostic tests require prior to amplification?

A

➝ Nucleic acid extraction

68
Q

What can molecular diagnostic tests detect?

A

➝ RNA or DNA

69
Q

How can we look for several targets in one sample in molecular diagnostic tests?

A

➝ Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample

70
Q

What are the stages of NAAT test?

A

➝ Specimen collection
➝ Extraction of nucleic acid
➝ DNA transcription for RNA viruses
➝ Cycles of Amplification of DNA target- requires polymerase and dNTPs plus other reagents
➝ Detection of amplicons- After amplification Or real time

71
Q

What are the 5 advantages of molecular diagnostic tests?

A

➝ May be automated. PCOT possible.
➝ Highly sensitive and specific, generates huge numbers of amplicons
➝ rapid
➝ useful for detecting viruses to make a diagnosis- at first time of infection or during reactivation
➝ useful for monitoring treatment response- can be quantitative or qualitative

72
Q

Why do you need quantitative diagnostic tests?

A

➝ to measure viral load

73
Q

What are the 4 limitations of NAAT?

A

➝ Exquisitely sensitive and can generate large numbers of amplicons- which may cause contamination
➝ need to have an idea of what viruses you are looking for as you need primers and probes specific for that target
➝ Mutations in target sequence may lead to ‘dropout’ e.g. D gene dropout seen with SARS-CoV-2 variants
➝ May detect other viruses which are not causing the infection

74
Q

When do we call it real time PCR?

A

➝ real time as amplification AND detection occur in real time
➝ i.e. simultaneously by the release of fluorescence

75
Q

What does real time PCR avoid the use of? What does it allow the use of?

A

➝ avoids use of gel electrophoresis or line hybridisation

➝ allows use of multiplexing

76
Q

What is multiplex PCR?

A

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

77
Q

What does multiplex PCR enable?

A

➝ Amplification of multiple DNA targets in one tube

➝ e.g. detection of multiple viruses in one specimen of CSF

78
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

79
Q

What is the cycle threshold?

A

➝ The amount of cycles required to cross the threshold

80
Q

What substances inhibit PCR?

A

➝ haem

➝ bile salts

81
Q

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

A

➝ An internal positive control

82
Q

What can we use as an internal control?

A

➝ anything as long as DNA/RNA respectively depending on nature of target
➝ and primers specific for the internal control material

83
Q

What is genome sequencing used for?

A

➝ to predict the response to anti-virals

➝ if there is resistance in drug experienced patients

84
Q

What is genome sequencing useful for?

A

➝ Useful for outbreak investigation by showing identical sequences in suspected source and recipient
➝ New variants
Diagnostic tests
Vaccine efficacy

85
Q

What is the consensus sequence based on?

A

➝ clinical observation of resistance or in vitro evidence

86
Q

How do you make an initial diagnosis of HIV?

A

➝ Antibody and antigen detection
➝ screening test (EIA)
➝ confirmatory test (EIA)

87
Q

How do you monitor the response to HIV?

A

➝ check the viral load with NAAT at baseline

➝ quantify the virus in the blood

88
Q

How can we test for resistance in HIV?

A

➝ using sequencing
➝ look for mutations known to cause resistance
➝ similar approach for hepatitis C, HSV, CMV

89
Q

What are the viral enzyme targets for antiviral resistance testing?

A

➝ Reverse transcriptase, protease
➝ integrase
➝ Viral receptor binding proteins

90
Q

Who do we screen?

A

➝ testing for specific infections in risk groups

91
Q

What are the three viruses you screen for?

A

➝ HIV
➝ HBV
➝ HCV

92
Q

Why do you screen for viruses?

A

➝ It may have an implication for others e.g. antenatal
➝ HIV and HBV
➝ patients are asymptomatic
➝ needs a sensitive screening test

93
Q

What is needed in addition to screening and why?

A

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

Microbiology (11 decks)

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