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WEEK 12 SEM 4 > Diagnosis of viral infections > Flashcards

Diagnosis of viral infections Flashcards

1
Q

What are the key concepts?

A
  • cant always clinically diagnose a viral infection
  • need history, examination, investigation and imaging to diagnose
  • rapid investigations of viral infections reduce need for unnecessary tests and innappropriate antibiotics
  • test results depends on prevalence of disease in population
  • certain infections tests need consent e.g. HIV
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2
Q

What are the tests for virology?

A
  • electron microscopy (not used anymore)
  • virus isolation and 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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3
Q

What can be seen by light microscopy?

A
  • bacteria
  • fungi
  • helminths
  • protozoa
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4
Q

Why cant a virus be seen on light microscopy?

A

too small

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5
Q

What is the magnification of an electron microscope?

A

(x20,000

magnification)

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6
Q

How does an electron microscope work?

A
  1. electron beams
  2. electron beam wavelength much shorter than light wavelength and so it has high resolution
  3. electrons scatter when they pass thin sections of specimen
  4. electrons that dont scatter, pass through the specimen and make an image
  5. denser regions scatter less electrons and appear darker
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7
Q

What are the advantages of electron microscopy?

A
  • fast
  • dont need to culture anything on the virus
  • virus can be looked at on the same day
  • can detect viruses that you cant grow in culture
  • can visualise many different viruses
  • dont need to know what you are looking for before hand
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8
Q

What are the disadvantages of electron microscopy?

A
  • very low sensitivity = need sample of
    10^6 viral particles/ml to visulise them
  • require lot of maintenance
  • require lot of skilled operators
  • cant differentiate between viruses of same family e.g. HSV1, HSV2 and varicella zoster
  • need to do further tests like immunofluorescence or PCR to diagnose which herpes virus it is
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9
Q

What is a cytopathic effect?

A

effect virus has on host cell

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10
Q

How is viral isolation in cell culture conducted?

A
  1. incubate patients viral sample on a cell layer
  2. take cell culture every 2 days
  3. USE light microscopy to see CPE
  4. once visualised the cytopathic effect, use immunofluorescence to CONFIRM the virus
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11
Q

To culture herpes, what cell line is used?

A

human embryonic lung cell lines

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12
Q

What was viral isolation in cell culture used to discover?

A
  • hMPV and Nipha virus in the last 20

years

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13
Q

What can viral isolation in cell culture be used for?

A
  • sometimes used in anti viral sensitivity
    testing

in some viruses like herpesvirus, it can cause a
cytopathic effect in 24 to 48 hours

in other viruses such as cytomegalovirus, it can take up to 21 days

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14
Q

Which viruses are vieed by antigen detection?

A
  • respiratory syncytial virus and influenza from nasopharyngeal aspirates
  • hepatitis B or dengue from blood serum or plasma samples
  • herpes simplex and varicella zoster from fluid taken from skin vesicular swabs (from vesicular rashes)
  • rotavirus and adenovirus from faeces (cause gastroenteritis)
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15
Q

What is good about antigen detection technique?

A
  • detects actual virus not the immune response to it
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16
Q

What happens in direct immunofluorescence?

A
  1. nasopharyngeal aspirate is taken from a child
  2. aspirate has antigens of the virus
  3. sample is incubated and bound on a slide
  4. a specific antibody (with a fluorochrome) is added to the sample
  5. the sample is incubated
  6. microscope looks at the UV light
  7. bright apple green fluorescence is seen if the AB binds to the antigen on the cell
17
Q

What is enzyme immuno assay?

A

METHOD: ELISA

  1. microtiter plate has capture antibody (specific to the antigen you think you are detecting)
  2. add sample
  3. any antigen present binds to the antibody
  4. enzyme linked specific antibody is added
  5. forms a sandwish
  6. substrate is added
  7. enzyme converts the substrate
  8. this causes a colour change
  9. put the microtiter plate in a spectrophotmeter= detects optical density
18
Q

What are examples of immunochromatographic methods?

A
  • lateral flow devices (like pregnancy tests)

= used to detect dengue antigens

19
Q

How long is IgM persistent for?

A
  • persistent depending on infection - some 2 weeks, some 1 month or sipheles is there for years
20
Q

What is seroconversion?

A
  • check initially and dont see IgG, but then check later and see IgG
  • they have gone from IgG negative to IgG positive
21
Q

What is serology used to identify?

A
  • detect antibody response
  • see if vaccination is successful
  • look for antigens produced by pathogens
22
Q

What is serum?

A
  1. serum produced from processing blood
  2. blood coagulated with silica particles
  3. gel traps cellular components
  4. centrifuge blood tubes
  5. top fraction is serum (SUPERNATANT)
    serum is produced from processing blood
  6. REMOVE it from blood clot and investigate it
23
Q

Where is serum stored?

A
  • at 4C for short term storage

- at 20C for long term storage

24
Q

What does serum contain?

A

proteins, antigens, antibodies, drugs and electrolytes

25
Q

What does NAAT do?

A
  • detect RNA or DNA
  • Can amplify multiple DNA targets in 1 tube
  • qualitative or quantitative
  • requires nucleic acid extraction
26
Q

What are the advantages of NAAT?

A
  • automated
  • highly sensitive and specific
  • rapid- done on same day
  • useful to detect virus directly (not the immune response)
27
Q

What are the limitations of PCR?

A
  • can detect other viruses which are not causing the infection
  • can contaminate its own PCR environment
  • need to know virus before bc need to make primers and probes
28
Q

What happens in realtime PCR?

A
  1. there are reverse and forward primers
  2. taqman probe binds somewhere on the target region of interest between the forward primer and the probe)
  3. probe has fluorophore and a quencher (prevents dye fluorescing when fluorophore and quencher are close together)
  4. PCR starts
  5. taqman hybridizes to region of interest
  6. now fluorescence is still inhibited
  7. TAQ polymerase hydrolyses the probe
  8. quencher and fluorophore separate
  9. fluorescence is released
29
Q

What is a cycle threshold?

A

number of cycles it takes for the PCR to become positive

30
Q

Which substances inhibit PCR and what happens as a result of it?

A

HAEM AND BILE SALTS

31
Q

What do you need to have in a PCR?

A
  • internal control (DNA or RNA)
  • if no amplification of internal control it means that there is a false negative
  • test is only valid if both control and sample have amplified
32
Q

What are the combination methods used to test HIV?

A

antibody and antigen detection for initial
diagnosis
−
a screening test, and a confirmation test is done

WEEK 12 SEM 4 (5 decks)

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