44. Diagnosis of Viral Infections Flashcards
What are key concepts?
- Not always possible to diagnose a infection clinically. Often require a laboratory diagnostic test.
- Aid to diagnosis - history, examination & special investigations
- Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
- Important public health and infection control implications
- Significance of test results depend heavily on prevalence in population e.g. HIV
- 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
- Consent - must be obtained for certain infections
- Difference between diagnostic, monitoring and screening tests
What are possible test types?
- 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
Expand on the use of electron microscopes.
- Mostly replaced by molecular techniques - possibly still useful for faeces and vesicle specimens
- 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
+ Rapid
+ Detects viruses that cannot be grown in culture
+ Can visualise many different viruses
- 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.
Virus isolation in cell culture
- 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
- 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
What is a cytopathic effect?
- 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
Antigen detection
•A variety of different methods can be used
•Commonest methods are
- Direct immunofluorescence
- Enzyme immunoassay
- Immunochromatographic methods
•Often used at point of care for rapid diagnosis
ELISA for antigen detection
•Enzyme-linked immunosorbent assay •Three formats: - Indirect - Direct (primarily antigen detection) - Sandwich
- 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
Diagnosis by antibody detection
•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
•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
Serology
•Indirect detection of the pathogen
•Diagnostic mode of choice for organisms which are refractory to culture
•Serology can be used to:
- Detect an antibody response in symptomatic patients
- Determine if vaccination has been successful
- Directly look for antigen produced by pathogens
•Serological tests are not limited to blood & serum
- can also be performed on other bodily fluids such as semen and saliva
Serum
Produced from processing blood
- Blood is coagulated with micronized silica particles
- Gel used to trap cellular components
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
Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
- Detection of antibody and or antigens
- Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence
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
+ 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
- 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.
Real time PCR
- 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
Describe how specific Taqman probes work.
Taqman probe complimentary 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
Taqman probe hybridises to the region of interest
This occurs during the annealing phase of PCR
Fluorescence is prevented de to the proximity of quencher
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 form the quencher and fluorescence can be detected.
For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number.
PCR Inhibition
- 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.
Combinations of methods e.g. HIV diagnosis and managment
•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)
