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Microbiology and Immunology - SGUL (Sem 4) > Diagnosis of Viral Infections > Flashcards

Diagnosis of Viral Infections Flashcards

1
Q

Why may it be important to do laboratory tests to diagnose diseases

A
  • Not always possible to diagnose an infection clinically, Often requires a laboratory diagnostic test.
  • Rapid diagnosis of viral infections can reduce need for unnecessary tests and inappropriate antibiotics
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2
Q

How are we able to run diagnostic tests

A
  • Obtain consent from the patient
  • It helps to know the natural history of the pathogen in the type of patient you are testing as this will affect test selection and interpretation
  • Difference between diagnostic, monitoring
    and screening tests
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3
Q

What are the possible test types we can run

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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4
Q

Why may we use electron microscopes to visualise viruses

A
  • Viruses are too small to be seen my normal microscopes
  • useful in characterising emerging pathogens
  • Possibly still useful for faeces and vesicle fluid specimens
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5
Q

Describe the steps in prepping a slide for electron microscopy

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 using electron microscopy

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 using electron microscopy

A
  • low sensitivity
  • Requires maintenance
  • Requires skilled
    operators
  • Cannot differentiate
    between viruses of the same virus family.
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8
Q

How can we grow viruses in cell culture to help identify it

A
  • Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE) when a patient sample containing a viruses incubated with a cell layer
  • Old method, now replaced by molecular techniques, but still needed for research or rare viruses
  • Led to discovery of hMPV and Nipah virus in last 20 years and SARS-CoV-2 recently
  • Use different cell lines in test tubes or plates. Selection of cell types important
  • Slow, but occasionally useful in anti-viral sensitivity testing
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9
Q

What is antigen detetction

A

Viral antigens, usually proteins – either capsid structural proteins or secreted proteins. They can be detected in cells or free in blood, saliva or other tissues/organs

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10
Q

What specimens can be used in antigen detection

A
  • Nasopharyngeal aspirates (NPA) (cell-associated virus antigens) - e.g. RSV, influenza
  • Blood (serum or plasma) (free antigen or whole virus) - Hepatitis B, Dengue
  • Vesicle fluid
    (whole virus), Herpes simplex, varicella zoster
  • Faeces (whole virus) - Rotavirus, adenovirus
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11
Q

What test types can we use for antigen detection

A
  • Direct immunofluorescence
  • Enzyme immunoassay
  • Immunochromatographic methods
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12
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 ultraviolet illumination
  • Long prep time and is not very sensitive
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13
Q

How does Immunochromatographic methods work

A
  • Lateral flow tests for COVID-19
  • Test for specific antigen
  • Not as sensitive as PCR tests
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14
Q

What is the ELISA test of antigen detection

A

Enzyme-linked immunosorbent assay - A component of reaction is adhered to a solid surface

Three formats:

  • Indirect
  • Direct (primarily
    antigen detection)
  • Sandwich
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15
Q

how does the ELISA test work

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
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16
Q

What is antibody detection by serology

A
  • Detection of antibodies
  • Indirect detection of the pathogen
  • Diagnostic mode of choice for organisms which are
    refractory to culture
17
Q

What 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
18
Q

What can we diagnose with antibody detection

A
  • When infected with a virus the humoral immune response takes place resulting in production of
    immunoglobulins
  • 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 produced
  • Quantity of IgG rises
  • Diagnosis can be made by – detection of IgM (can be non specific) – or by demonstration of seroconversion
  • Negative IgG antibody at first
  • Then presence of IgG antibody
19
Q

Why is detection of antigen AND antibody useful

A

This is useful for some infections such as: Hepatitis B, HIV, Hepatitis C
- This is because it allows therapeutic implications us to establish whether acute or chronic infection
- This may have therapeutic implications

20
Q

What are molecular diagnostic tests

A

Nucleic acid amplification (NAAT)

  • e.g. PCR although there are
    other examples
  • 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
21
Q

What Arte the stages in NAAT tests

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
22
Q

What are the advantages of using 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 loa
23
Q

What are the limitations of using 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
24
Q

What is Real Time PCR

A
  • Real time as amplification AND detection occur in REAL TIME i.e. simultaneously by the release of fluorescence
  • Avoids the use of gel electrophoresis or line hybridisation
  • Allows the use of multiplexing
25
Q

What is multiplex PCR

A
  • Multiplex PCR is the term used when more than one pair of primers is used in a PCR
  • It enables the amplification of multiple DNA targets in one tube
26
Q

What is PCR inhibition

A
  • Some substances inhibit PCR e.g. haem, bile salts
  • Assays should always include an internal positive control as results could incorrectly be reported as negative
  • Include primers specific for the internal
    control material
    -
27
Q

how can we use genome sequencing in the diagnosis process

A
  • Useful for outbreak
    investigation by showing identical sequences in suspected source and recipient
  • New variants: Diagnostic tests, Vaccine efficacy
  • Can be Used to predict response to anti-virals e.g. for HIV in Rx naïve patients, or if clinical suggestion of resistance in drug experienced patients
28
Q

How are a combination of methods are used in the diagnosis and management of HIV

A

Antibody and antigen detection for initial diagnosis:

  • Screening test (EIA)
  • Confirmatory test (EIA)

Viral load(NAAT) at baseline an dot monitor treatment response

  • Quantification of virus in blood

Resistance testing (sequencing)

29
Q

How can we test for ant-viral resistance susceptibility

A

Look for mutations known to cause resistance.

  • Reverse transcriptase,
    protease
  • integrase
  • viral receptor binding
    proteins
30
Q

Why should we screen for these infections

A
  • Testing for specific infections in
    at risk groups
  • Testing because it may have
    an implication for others e.g. antenatal
  • Needs a sensitive screening
    test
  • May have some false
    positives, so need
  • A specific confirmatory test

Microbiology and Immunology - SGUL (Sem 4) (27 decks)

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