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Life Protection > Diagnosis of Viral Infections > Flashcards

Diagnosis of Viral Infections Flashcards

1
Q

What are the advantages of testing for viral infections?

A

Not always possible to diagnose an infection clinically - often need diagnostic test
Aid to diagnosis - history, examination and special investigations
Rapid diagnosis reduces need for unnecessary tests and antibiotics
NOTE- consent needed for some tests, eg for HIV

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2
Q

Give the possible test types

A
  • Ag detection
  • Ab detection by serology (indirect test)
  • Nucleic acid amplification test (NAATs e.g. PCR)
  • Sequencing for genotype and detection of antiviral resistance
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3
Q

Describe Ag detection tests and what virus each type of sample can detect

A

Infected cells may display viral Ags on surface
Nasopharyngeal aspirates (NPA) can detect: RSV, influenza
Blood (serum or plasma) can detect: hep B, dengue
Vesicle fluid can detect: herpes, VZV
Faeces can detect: rotavirus, adenovirus. Also viral gastroenteritis diagnosis by antigen detection

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4
Q

Give the benefits and limitations of Ag detection tests

A

Benefits of using Ag detection: easy set up, can be done out of hours or at patient bedside
Being replaced by nucleic acid detection due to improved test performance:
- direct detection of the virus
- sensitivity PCR to detect a pathogen= much higher than Ag detection

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5
Q

There are different methods for antigen detection kits, including direct immunofluorescence. Describe this and its limitations

A

Antigen bound to a slide
Specific Ab to that Ag is tagged to a fluorochrome and is mixed w the sample
Slide viewed using microscope which gives off UV illumination
Apple green fluoresence seen when virus is present in cell
- very time consuming - 4 hours
- not particularly sensitive

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6
Q

There are different methods for antigen detection kits, incl immunochromatographic. Describe this

A

e.g. diagnosis of dengue
- flavivirus
- arthropod vector
- common infection in returning travellers
Here hay development of a control line and a test line in the positve sample - like covid LFT

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7
Q

There are different methods for antigen detection kits, incl ELISA. Describe this and its limitations

A
  • a serological technique
  • enzyme linked immunoabsorbent assay
  • three different formats and variations:
    • indirect
    • direct (primarily antigen detection)
    • sandwich
      Limitations: labour intensive, difficult to quantify
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8
Q

Describe the method by which antigens are detected by ELISA

A
  • Coat plate w a capture antibody
  • Add sample, any antigen present will bind to the capture antibody
  • Add enzyme-conjugated primary Ab; this binds to detecting antibody - forming a sandwich
  • Add substrate, which is converted by the enzyme to detectable form
    -Sera colour change
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9
Q

Describe diagnosis by antibody detection

A

Viral infection diagnosis can be made by:
- detection of IgM (can be non-specific)
- or by demonstration of seroconversion: negative antibody at first (no IgM/IgG, then presence of antibody when infected)

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10
Q

Serology is indirect pathogen detection- what can it be used for?

A

Can be used to:
Detect Ab response in symptomatic patients
Determine if vaccination has been successful
Directly look for Ag produced by pathogens

Serological tests are not limited to blood and serum- works on semen and saliva

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11
Q

What is Serum- what does it contain, how is it made?

A

Serum contains proteins, Ags, Abs, drugs and electrolytes
Produced from processing blood: blood is coagulated with micronized silica particles
Gel is used to trap cellular components
Serum tubes usually centrifuged for 10 mins at 1000xg
Supernatant (serum) is removed and stored at 4ºC short term, 20 long term

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12
Q

Describe the serological diagnosis of Hep A

A
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13
Q

What specific infectons is antigen and Ab detection useful for?

A
  • hep B
  • HIV
  • hep C
    This is bc it allows us to establish whether it is an acute or chronic infection
    May have therapeutic implications
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14
Q

What is Ab avidity?

A
  • avidity is a measure of the overall binding strength
  • IgG avidity will increase w time after infection
  • used to approximately time when an infection occurred
  • useful when timing some infections that can cause congenital infection e.g. CMV and toxoplasma → did the infection occur prior to conception → so unlikely baby will be affected?
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15
Q

Describe the molecular diagnostic tests

A
  • nucleic acid amplification - NAAT, eg 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
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16
Q

What are the limitations of NAATs?

A
  • May detect other viruses not causing the infection
  • V 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
17
Q

What are the advantages of 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
18
Q

What is nucleic acid extraction??

A
  • RNA or DNA
  • manual or automated extraction
    • throughput
    • human error
  • required to provide template for PCR reaction
  • different methods
    • cost
    • hands on time
    • sensitivity/efficacy
19
Q

What are the components of PCR mix and what are they used for?

A

Primer - short fragment, sticks to one end of the PCR strand
Nucleotides - elongate DNA during amplification
Taq polymerase - heat stable enzyme, incorporates nucleotides into growing DNA strand
MgCl2 - needed by taq polymerase to have activity
Buffer - creates optimal enzyme conditions

20
Q

What is the difference between realtime and multiplex PCR?

A

real time PCR: amplification and detection occur in real time
Avoids the use of gel electrophoresis or line hybridisation
Allows the use of multiplexing

Multiplex PCR: >1 pair of primers is used in a PCR
Enables amplification of multiple DNA targets in 1 tube
e.g. multiple virus detection in 1 resp sample

21
Q

Describe PCR

A
  • taqman probe binds between primers
  • oligonucleotide probe w a fluorescent reporter at the 5’ and a quencher at the 3’
  • the quencher prevents the reporter fluorescing when excited if in close proximity

Annealing phase: taqman probe hybridises to region of interest
Fluorescence prevented due to the proximity of quencher

taq possesses 5’ to 3’ nuclease activity and hydrolyses the probe
- the reporter is removed from the quencher and fluorescence can be detected
- for any given cycle within the exponential phase, the amount of produce, and hence fluorescence signal, is directly proportional to the initial copy number

22
Q

What is the cycle threshold/CT?

A
  • with PCR, CT value can quantify how much viral target is there using an arbitrary threshold
    If CT value above threshold, PCR is positive
  • so the CT value= cycle at which the PCR has crossed the arbitrary level of fluorescence
23
Q

Which substances inhibit PCR and what are the implications of this?

A
  • haem, bile salts inhibit PCR
  • assays should always inc an internal positive control as results could incorrectly be reported as negative
  • the control can be anything you’re not going to be expecting it in that sample type
24
Q

What are the common sample types and what are they used to diagnose by PCR?

A
  • clotted blood for serology
  • EDTA blood for PCR reactions
  • CSF: PCR for causes of viral meningitis
  • tissues: CMV, enteroviruses by PCR
  • urines: for PCR e.g. CMV
25
Q

Describe common bacterial/fungal/parasitic and viral pathogens found in CSF

A
26
Q

Describe common bacterial/fungal/parasitic and viral pathogens found in skin and urine

A
27
Q

Describe common bacterial/fungal/parasitic and viral pathogens found in the genital tract

A
28
Q

Compare blood results for bacterial vs viral infections

A

Bacterial usually cause a higher CRP than viral
Raised WBC count in both
Raised neutrophil count in bacterial infections
Raised lymphocyte count in viral infections. Some viruses cause neutropenia e.g. HIV, EBV
Culture, microscopy, serology to diagnose bacteria infection
PCR and serology to diagnose viral infection

29
Q

Compare EBV pharyngitis with streptoccoal phanryngitis

A
30
Q

Explain how a combination of methods is used to diagnose HIV

A

Ab and Ag detection for initial diagnosis: screening and confirmatory test (EIA)
Viral load (NAAT) at baseline and to monitor treatment response
Resistance testing (sequencing): look for mutations, appropriate antiviral regimens selected based on results

31
Q

Describe Mumps- its route of spread and infectious periods

A

Route of spread – aerosol, hand/fomite
contact with salivary or resp secretions
Average incubation period
Infectious period – 1 week before to after
the parotitis.

32
Q

What is the clinical presentation and diagnosis for mumps?

A

Presentations – parotitis, aseptic meningitis, deafness and pancreatitis (rare).
May need lab confirmation – clotted blood for IgM and IgG, CSF (PCR), saliva (IgM and PCR).

Life Protection (35 decks)

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