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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
Why is virus isolation in cell culture now an old method?
➝ it has been replaced by molecular techniques
26
Why is virus isolation in cell culture still used?
➝ needed for research | ➝ or for rare viruses
27
What three viruses have been discovered by the cytopathic effect technique?
➝ hMPV ➝ Nipha virus ➝ SARS-CoV-2
28
What is an advantage and disadvantage of virus isolation in cell culture technique?
➝ slow | ➝ occasionally useful in anti-viral sensitivity testing
29
How can you investigate cytopathic effect?
➝ Take a patient sample containing the virus sample ➝ incubate with a cell layer ➝ observe cytopathic effects
30
Why can you get different appearances of cytopathic effect?
➝ Different viruses may give different appearances | ➝ Different cell lines may support growth of different viruses
31
How do you test antivirals?
➝ cell culture + antiviral | ➝ look for inhibition of cytopathic effect
32
How do you identify viruses in cell cultures?
➝ using antigen detection | ➝ neutralisation of growth
33
What viral component can be detected and where?
➝ 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
34
What do virus infected cells display?
➝ Viral antigens on their surfaces
35
What viruses do you take nasopharyngeal aspirates for?
➝ RSV | ➝ Influenza
36
What type of antigens are we looking for when we take nasopharyngeal aspirates?
➝ cell-associated virus antigens
37
What viruses do you take blood samples for?
➝ Hepatitis B | ➝ Dengue
38
What type of antigens are we looking for when we take blood samples?
➝ free antigen | ➝ or whole virus
39
What viruses do you take vesicle fluid samples for?
➝ Herpes simplex | ➝ Varicella zoster
40
What are we looking for when we take vesicle fluid samples?
➝ whole virus
41
Which viruses do you take faeces samples for?
➝ Rotavirus | ➝ Adenovirus
42
What are we looking for when we take faeces sample?
➝ whole virus
43
What is replacing EM, cell culture and viral antigen detection techniques? Why?
➝ Nucleic acid detection methods | ➝ due to improved test performance i.e. greater sensitivity
44
What are the 3 most common virus antigen detection methods? What type of antigens can we detect?
➝ Direct immunofluorescence- cell associated antigens ➝ Enzyme immunoassay- free soluble antigens or whole virus ➝ Immunochromatographic methods
45
How does immunofluorescence 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 UV illumination
46
What virus is dengue caused by and how is it spread and who is is common in?
➝ Flavivirus ➝ arthropod vectors ➝ common infection in returning travellers
47
What can we diagnose using immunochromatographic methods?
➝ dengue
48
What is the advantage of immunochromatographic methods?
➝ are useful as a near patient test- NPT ➝ a point of care test - PCOT ➝ often used at point of care for rapid diagnosis
49
What is the disadvantage of immunochromatographic methods aka lateral flow test for diagnosing COVID-19?
➝ not as sensitive or specific as PCR test, NAATs
50
What is the full form of ELISA?
➝ Enzyme-linked immunosorbent assay
51
What are the three types of ELISA test?
➝ direct ➝ indirect (primarily antigen detection) ➝ sandwich
52
How can we detect antigen by ELISA?
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
When will the substrate change colour in ELISA?
➝ The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present.
54
What is a negative result in an ELISA?
➝ no colour change
55
What does the humoral system do when infected with a virus?
➝ Produce antibodies
56
For what organisms is antibody detection by serology a diagnostic mode of choice?
➝ for organisms which are refractory to culture
57
What are the signs of no past/ current infection or immunisation?
➝ patient negative for IgM | ➝ and negative for IgG
58
What are the signs of acute or recent Hepatitis A?
➝ patient positive for IgM | ➝ and can be negative/positive for IgG
59
What are the signs of immunisation or a resolved Hepatitis A infection?
➝ patient negative for IgM | ➝ and positive for IgG
60
What happens during second exposure to Hepatitis A?
➝ you will have IgG antibodies from the 1st exposure ➝ on second exposure there is a really high IgG response ➝ hardly any IgM
61
How do you detect antibodies and antigens in the blood? Give example
➝ Enzyme immunoassays | e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence
62
What is used in modern lab to detect antibodies and antigens in blood?
➝ automated analysers eg. Abbott Architect | ➝ rarely use Michael teeter trays
63
in what 3 diseases is it useful to detect antigens and antibodies?
➝ Hepatitis B ➝ HIV ➝ hepatitis C
64
Why is it useful to detect antibodies and antigens?
➝ It allows us to establish whether it is an acute or chronic infection ➝ this may have therapeutic implications
65
Why is looking at diff antigens and antibodies useful for Hepatitis B?
➝ we can better understand what type of infection the patient has i.e. none, acute, resolving, etc
66
What is NAAT?
➝ Nucleic acid amplification test ➝ they are molecular diagnostic tests ➝ such as PCR, LCR, LAMP, SDA
67
What do molecular diagnostic tests require prior to amplification?
➝ Nucleic acid extraction
68
What can molecular diagnostic tests detect?
➝ RNA or DNA
69
How can we look for several targets in one sample in molecular diagnostic tests?
➝ Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample
70
What are the stages of NAAT test?
➝ 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
What are the 5 advantages of molecular diagnostic tests?
➝ 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
Why do you need quantitative diagnostic tests?
➝ to measure viral load
73
What are the 4 limitations of NAAT?
➝ 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
When do we call it real time PCR?
➝ real time as amplification AND detection occur in real time ➝ i.e. simultaneously by the release of fluorescence
75
What does real time PCR avoid the use of? What does it allow the use of?
➝ avoids use of gel electrophoresis or line hybridisation | ➝ allows use of multiplexing
76
What is multiplex PCR?
➝ when more than one pair of primers is used in PCR
77
What does multiplex PCR enable?
➝ Amplification of multiple DNA targets in one tube | ➝ e.g. detection of multiple viruses in one specimen of CSF
78
Describe how Specific Taqman probes work?
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
What is the cycle threshold?
➝ The amount of cycles required to cross the threshold
80
What substances inhibit PCR?
➝ haem | ➝ bile salts
81
What should assays include so false negatives don't happen?
➝ An internal positive control
82
What can we use as an internal control?
➝ anything as long as DNA/RNA respectively depending on nature of target ➝ and primers specific for the internal control material
83
What is genome sequencing used for?
➝ to predict the response to anti-virals | ➝ if there is resistance in drug experienced patients
84
What is genome sequencing useful for?
➝ Useful for outbreak investigation by showing identical sequences in suspected source and recipient ➝ New variants Diagnostic tests Vaccine efficacy
85
What is the consensus sequence based on?
➝ clinical observation of resistance or in vitro evidence
86
How do you make an initial diagnosis of HIV?
➝ Antibody and antigen detection ➝ screening test (EIA) ➝ confirmatory test (EIA)
87
How do you monitor the response to HIV?
➝ check the viral load with NAAT at baseline | ➝ quantify the virus in the blood
88
How can we test for resistance in HIV?
➝ using sequencing ➝ look for mutations known to cause resistance ➝ similar approach for hepatitis C, HSV, CMV
89
What are the viral enzyme targets for antiviral resistance testing?
➝ Reverse transcriptase, protease ➝ integrase ➝ Viral receptor binding proteins
90
Who do we screen?
➝ testing for specific infections in risk groups
91
What are the three viruses you screen for?
➝ HIV ➝ HBV ➝ HCV
92
Why do you screen for viruses?
➝ It may have an implication for others e.g. antenatal ➝ HIV and HBV ➝ patients are asymptomatic ➝ needs a sensitive screening test
93
What is needed in addition to screening and why?
➝ may have some false positives so you need a specific confirmatory test

Microbiology (11 decks)

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