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Fundamentals of P > Diagnosis of Viral Infections > Flashcards

Diagnosis of Viral Infections Flashcards

1
Q

What can aid a diagnosis

A

Aid to diagnosis - history, examination & special investigations

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2
Q

Rapid diagnosis of viral infections can reduce

A

Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics

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3
Q

Significance of test results depend heavily on

A

Significance of test results depend heavily on prevalence in population e.g. HIV

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4
Q

What affects test selection and interpretation

A

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

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5
Q

List possible test types

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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6
Q

Electron Microscopy = uses

A

Viruses can be visualised with electron microscope
Mostly replaced by molecular techniques
Possibly still useful for faeces and vesicle specimens
Useful in characterising emerging pathogens

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7
Q

Electron Microscopy - mechanism

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

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8
Q

Electron Microscopy - explain why resolution is higher than light microscopy

A

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

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9
Q

Electron Microscopy - advantages/disadvantages

A

Advantages:

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

Limitations:

low sensitivity need 106 virions/millilitre. May be enough in vesicle secretion/stool
Requires maintenance
Requires skilled operators
Cannot differentiate between viruses of the same virus family.

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10
Q

Herpes viruses that cause vesicles - examples

A

Herpes viruses that cause vesicles
Herpes simplex
Varicella zoster virus

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11
Q

Herpes viruses that cause vesicles - explain significance of site of vesicle

A

EM cannot differentiate these different viruses so depends on clinical context, site of vesicle and symptoms

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12
Q

Explain cytopathic Effect (CPE) of viruses

A 
Viruses require host cells to replicate and may cause a
Cytopathic Effect (CPE) of cells when a patient sample containing a virus incubated with a cell layer
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13
Q

Virus isolation in cell culture - characteristics

A

Old method, now replaced by molecular techniques, but still needed for research or for rare viruses

Led to discovery of hMPV and Nipha virus in last 20 years

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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14
Q

Cytopathic effect - explain how you would differentiate viruses

A

Different viruses may give different appearances

Different cell lines may support growth of different viruses

Identify virus using antigen detection techniques or neutralisation of growth

Cell culture plus antiviral – look for inhibition of cytopathic effect

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15
Q

Describe what allows detection of viruses

A

Viral antigens, usually proteins – either capsid structural proteins, secreted proteins can be detected. Infected cells may display viral antigens on their surfaces.

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16
Q

Nucleic acid detection methods - which techniques did this replace and why

A 
Nasopharyngeal aspirates (NPA)
e.g. RSV, influenza

Blood (serum or plasma)

  • Hepatitis B
  • Dengue

Vesicle fluid
- Herpes simplex, varicella zoster

Faeces
- Rotavirus, adenovirus

These techniques are being replaced by Nucleic acid detection methods due to improved test performance

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17
Q

Antigen detection - commonest methods are what and use

A

Commonest methods are:
Direct immunofluorescence
Enzyme immunoassay
Immunochromatographic methods

Often used at point of care for rapid diagnosis

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18
Q

Immunofluorescence - describe method

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

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19
Q

Immunochromatographic method - use example

A

e.g. diagnosis of dengue
Flavivirus
Arthropod vector
Common infection in returning travellers

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20
Q

Enzyme-linked immunosorbent assay - what is adhered to surface

A

Enzyme-linked immunosorbent assay

A component of reaction is adhered to a solid surface

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21
Q

Enzyme-linked immunosorbent assay - 3 formats

A

Three formats:
Indirect
Direct (primarily antigen detection)
Sandwich

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22
Q

Detection of Antigen by 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

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23
Q

ELISA test - when will the substrate change colour

A

The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present. Negative result = NO colour change

24
Q

Diagnosis by antibody detection - describe

A

When infected with a virus the humoral immune response takes place resulting in production of immunoglobulins i.e. antibodies
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

25
Q

Diagnosis by antibody detection - how can this be made

A

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
26
Q

Serology - define + when is it used

A

Indirect detection of the pathogen

Diagnostic mode of choice for organisms which are refractory to culture

27
Q

Serology can be used to:

A

Detect an antibody response in symptomatic patients
Determine if vaccination has been successful
Directly look for antigen produced by pathogens

28
Q

Serological tests are not limited to

A

Serological tests are not limited to blood & serum

  • can also be performed on other bodily fluids such as semen and saliva
29
Q

Serum - produced from processing blood

A

Produced from processing blood:

Blood is coagulated with micronized silica particles
Gel used to trap cellular components

30
Q

Serum - describe method

A

Routinely serum tubes are centrifuged for 10 min at 1000xg

Supernatant (serum) is removed and stored
4ºC short term
-20ºC long term

Routinely serum tubes are centrifuged for 10 min at 1000xg

31
Q

Serum contain

A

Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes

32
Q

Serological diagnosis of hepatitis A infection - Hepatitis A IgM: (list results for following)

No past or current infection or immunisation
Acute/recent infection
Resolved infection or immunisation

A

No past or current infection or immunisation = Negative

Acute/recent infection =
Positive

Resolved infection or immunisation = Negative

33
Q

Serological diagnosis of hepatitis A infection - Hepatitis A IgG: (list results for following)

No past or current infection or immunisation
Acute/recent infection
Resolved infection or immunisation

A

No past or current infection or immunisation = Negative

Acute/recent infection =
Negative/Positive

Resolved infection or immunisation = Positive

34
Q

The level and type of antibody varies

A

The level and type of antibody varies

with both the pathogen, exposure and time

35
Q

Serology - done by

A

Serology:

Detection of antibody and or antigens
Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence

36
Q

Detection of antigen and antibody - why is it useful = examples

A

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

37
Q

Detection of antigen and antibody - effect on sensitivity and window period

A

Combined antigen and antibody 4th Generation HIV tests detect both antibody and antigen in as assay. Results in increased sensitivity and a reduced window period (time between infection and being able to get a positive result).

38
Q

Nucleic acid amplification (NAAT) - characteristics

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

39
Q

Advantages of using NAATS

A

May be automated

Highly sensitive and specific, generates huge numbers of amplicons
Rapid
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 loads

40
Q

Limitations of using NAATs

A

May detect other viruses which are not causing the infection
Exquisitely sensitive and so may generate 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.

41
Q

Real time PCR - define and describe +ves

A

Different chemistries but all similar
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

42
Q

Multiplex PCR - define

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

43
Q

Specific Taqman Probes - describe suppresssion of quencher

A

Taqman probe complementary to region of interest, binds between primers.

Oligonucleotide probe with a fluorescent reporter at the 5’ and a quencher at the 3’.

The quencher prevents the reporter fluorescing when excited if in close proximity

44
Q

Specific Taqman Probes - fluorescence prevention

A

Taqman probe hybridizes to the region of interest

This occurs during the annealing phase of PCR

Fluorescence is prevented due to the proximity of quencher

45
Q

Specific Taqman Probes - describe removal of reporter

A

Taq polymerase extends from the 3’ end of primer as normal.
The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe.
The reporter is removed from the quencher and fluorescence can be detected.

46
Q

Specific Taqman Probes - amount of reaction components during exponential phase

A

For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number

47
Q

Real Time PCR - describe making it quantitative

A

Relative fluorescence can be plotted against the number of cycles
This can be used to determine relative concentrations of DNA present by construction of standard curve using standards of known concentration.

48
Q

Explain importance of having an internal positive control

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. The IC can be anything as long as RNA/DNA respectively depending on nature of target.

Include primers specific for the internal control material.

49
Q

Organism Sequencing - use

A

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

Useful for outbreak investigation by showing identical sequences in suspected source and recipient

50
Q

Organism Sequencing - consensus based on

A

Consensus sequence based on clinical observation of resistance or in vitro evidence

51
Q

Organism Sequencing - selection of minority species

A

Minority species sequencing

May be selected by treatment

52
Q

Describe sequence of virus testing with examples

A 
Antibody and antigen detection for initial diagnosis
Screening test (EIA)
Confirmatory test (EIA)
Viral load(NAAT) at baseline and to monitor treatment response
- Quantification of virus in blood

Resistance testing (sequencing)

53
Q

Multiple viral enzyme targets - examples

A

Multiple viral enzyme targets
Reverse transcriptase, protease,
integrase,
viral receptor binding proteins)

54
Q

Describe need for a screening test

A

Testing for specific infections in at risk groups
e.g. HIV, HBV and HCV

Testing because it may have an implication for others e.g. antenatal
HIV, HBV, rubella

In these situations the patients are asymptomatic
Needs a sensitive screening test

55
Q

Screening test - describe next steps if you get a false positive

A

May have some false positives, so need

A specific confirmatory test

Fundamentals of P (62 decks)

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