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lolmyi > Diagnosis of viral infections > Flashcards

Diagnosis of viral infections Flashcards

1
Q

What are the types of tests to diagnose viral infections?

A

→Electron Microscopy

→Virus isolation (cell culture)

→Antigen detection

→Antibody detection by serology

→Nucleic acid amplification tests (NAATs e.g. PCR)

→Sequencing for genotype and detection of antiviral resistance

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2
Q

Which microbe scan be seen with light microscopy?

A

→fungi

→bacteria

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3
Q

What type of microscope do viruses need?

A

→EM

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4
Q

What are EM still useful for visualising?

A

→faeces and vesicle specimens

→characterizing emerging pathogens

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5
Q

Describe the process for EM visualisation

A

→Specimens are dried on a grid

→Can be stained with heavy metal e.g. 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

→Wavelength of electron beam is much shorter than light, resulting in much higher resolution than light microscopy

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6
Q

What are the advantages of EM?

A

→Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses

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7
Q

What are the limitations of EM?

A

→low sensitivity need 106 virions/millilitre.

→Requires maintenance

→Requires skilled operators

→Cannot differentiate between viruses of the same virus family.

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8
Q

How do rotavirus look like in EM?

A

→look like wheels

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9
Q

Which viruses cause vesicles?

A

→Herpes simplex

→Varicella zoster virus

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10
Q

How do you differentiate between herpes and VZV since they both cause vesicles?

A

→clinical context,

→site of vesicle
→symptoms

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11
Q

What is the vesicle derived from in herpes?

A

→Envelope membrane from cell that it has infected

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12
Q

Give examples of poxvirus

A

→Smallpox
→Monkeypox
→Orf
→Cowpox

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13
Q

How does virus isolation in cell culture help in diagnosis?

A

→Create a monolayer of cells and then add clinical specimen

→watch for cytopathic effect

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14
Q

How are viruses identified using cytopathic effects?

A

→antigen detection techniques or neutralisation of growth

→Cell culture plus antiviral – look for inhibition of cytopathic effect

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15
Q

What are viral antigens?

A

→usually proteins
→either capsid structural proteins or secreted proteins
→detected in cells or free in blood, saliva or other tissues/organs

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16
Q

What are possible specimens for antigen detection?

A

→Nasopharyngeal aspirates
→Blood (serum or plasma)
→Vesicle fluid
→Faeces

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17
Q

Why is antigen detection being replaced with nucleic acid detection?

A

→greater sensitivity

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18
Q

Which viruses are detected in nasopharyngeal aspirates?

A

→RSV, influenza

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19
Q

Which viruses are detected in blood?

A

→Hepatitis B
→Dengue

→free antigen or whole virus

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20
Q

Which viruses are detected in vesicle fluids?

A

→Herpes simplex,
→varicella zoster

→whole virus

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21
Q

Which viruses are detected in faeces?

A

→Rotavirus,
→adenovirus

→whole virus

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22
Q

What are three types of antigen detection?

A

→Direct immunofluorescence- Cell associated antigens

→Enzyme immunoassay- Free soluble antigens or whole virus

→Immunochromatographic methods

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23
Q

When is antigen detection mostly used?

A

→point of care

24
Q

Give an example of a virus detected using immunochromatographic

A

→dengue

→Non structural proteins circulating in blood

25
Q

What is ELISA?

A

→Enzyme-linked immunosorbent assay

→used to detect antigen and antibodies

26
Q

What are three formats of ELISA?

A

→Indirect
Direct (primarily antigen detection)
Sandwich

27
Q

Describe the process of ELISA

A

→Plate is coated with a capture antibody

→Sample is added and any antigen present binds to capture antibody

→Enzyme-conjugated primary antibody is added, binds to detecting antibody

→Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change

28
Q

When is there a colour change in ELISA?

A

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

29
Q

What can serology be used to detect?

A

→Detect an antibody response in symptomatic patients

→Determine if vaccination has been successful

→Directly look for antigen produced by pathogens

30
Q

What can serology tests be performed on?

A

→blood
→serum
→saliva
→semen

31
Q

How is serum processed from blood?

A

→Blood is coagulated with micronized silica particles

→Gel used to trap cellular components

32
Q

What does serum contain?

A

→antigens,
→antibodies,
→drugs (some)
→electrolytes

33
Q

Describe the Igs changes in response to infection

A

→IgM antibodies specific to the virus are produced first

→IgM present for a variable period – usually 1 to 3 months

→As IgM declines, IgG is produce

34
Q

How can diagnosis be made using Ig antibodies?

A

→detection of IgM (can be non specific)

→demonstration of seroconversion

35
Q

What are the levels of IgM and IgG in acute or recent Hep A infections?

A

→IgM positive

→IgG can be positive or negative

36
Q

What are the levels of IgM and IgG in Hep A in resolves infection/

A

→IgM negative

→IgG positive

37
Q

Why is antigen and antibody detection used for Hep B, C and HIV infections?

A

→establish whether acute or chronic infection

→therapeutic implications

38
Q

Describe NAAT detection

A

→PCR
→Can detect RNA or DNA

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

→May be qualitative or quantitative

→Requires nucleic acid extraction prior to the amplification

39
Q

Describe the stages of NAAT test

A

→Specimen collection- blood, saliva, csf

→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

40
Q

What can detection of amplicons be?

A

→After amplification

→Or real time

41
Q

What are the advantages of NAATs?

A

→be automated
→highly sensitive and specific
→rapid
→Useful for detecting viruses to make a diagnosis
→Useful for monitoring treatment response
- Quantitative e.g. HIV

42
Q

What are the limitations of using NAATs?

A

→Generates large numbers of amplicons
→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”

43
Q

What is RT-PCR?

A

→amplification AND detection occur in REAL TIME

44
Q

What are the benefits of RT-PCR?

A

→Avoids the use of gel electrophoresis or line hybridisation

→Allows the use of multiplexing

45
Q

What is multiplex PCR?

A

→when more than one pair of primers is used in a PCR

46
Q

What does multiplex PCR allow?

A

→amplification of multiple DNA targets in one tube

→e.g. detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2, VZV, enterovirus, mumps virus

47
Q

What is PCR Inhibition?

A

→Some substances inhibit PCR

48
Q

What are examples of PCR inhibitors?

A

→haem, bile salts

49
Q

Why should internal controls be used in PCR assays?

A

→so results are not read as false negatives

50
Q

What can internal controls be?

A

→RNA

→DNA

51
Q

What is genome sequencing useful for?

A

→outbreak investigation by showing identical sequences in suspected source and recipient
→vaccine efficacy

52
Q

What tests are involved in HIV diagnosis?

A

→Antibody and antigen detection for initial diagnosis
→Screening test (EIA)

→Confirmatory test (EIA)

53
Q

What tests are involved in monitoring treatment for HIV?

A

→Quantification of virus in blood

→NAAT

54
Q

What test is used for resistance testing in HIV?

A

→sequencing

55
Q

What does multiple viral enzyme target for anti-viral resistance?

A

→Reverse transcriptase, protease,

→integrase,

→viral receptor binding proteins)

56
Q

Why does screening ned confirmatory testing?

A

→some false positives

lolmyi (31 decks)

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