Diagnosis of Viral Infections Flashcards
What can aid a diagnosis
Aid to diagnosis - history, examination & special investigations
Rapid diagnosis of viral infections can reduce
Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
Significance of test results depend heavily on
Significance of test results depend heavily on prevalence in population e.g. HIV
What affects test selection and interpretation
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
List 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
Electron Microscopy = uses
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
Electron Microscopy - mechanism
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
Electron Microscopy - explain why resolution is higher than light microscopy
Wavelength of electron beam is much shorter than light, resulting in much higher resolution than light microscopy
Electron Microscopy - advantages/disadvantages
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.
Herpes viruses that cause vesicles - examples
Herpes viruses that cause vesicles
Herpes simplex
Varicella zoster virus
Herpes viruses that cause vesicles - explain significance of site of vesicle
EM cannot differentiate these different viruses so depends on clinical context, site of vesicle and symptoms
Explain cytopathic Effect (CPE) of viruses
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
Virus isolation in cell culture - characteristics
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
Cytopathic effect - explain how you would differentiate viruses
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
Describe what allows detection of viruses
Viral antigens, usually proteins – either capsid structural proteins, secreted proteins can be detected. Infected cells may display viral antigens on their surfaces.
Nucleic acid detection methods - which techniques did this replace and why
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
Antigen detection - commonest methods are what and use
Commonest methods are:
Direct immunofluorescence
Enzyme immunoassay
Immunochromatographic methods
Often used at point of care for rapid diagnosis
Immunofluorescence - describe method
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
Immunochromatographic method - use example
e.g. diagnosis of dengue
Flavivirus
Arthropod vector
Common infection in returning travellers
Enzyme-linked immunosorbent assay - what is adhered to surface
Enzyme-linked immunosorbent assay
A component of reaction is adhered to a solid surface
Enzyme-linked immunosorbent assay - 3 formats
Three formats:
Indirect
Direct (primarily antigen detection)
Sandwich
Detection of Antigen by ELISA
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
ELISA test - when will the substrate change colour
The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present. Negative result = NO colour change
Diagnosis by antibody detection - describe
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 by antibody detection - how can this be made
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 - define + when is it used
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
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
Produced from processing blood:
Blood is coagulated with micronized silica particles
Gel used to trap cellular components
Serum - describe method
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 contain
Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
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
No past or current infection or immunisation = Negative
Acute/recent infection =
Positive
Resolved infection or immunisation = Negative
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
No past or current infection or immunisation = Negative
Acute/recent infection =
Negative/Positive
Resolved infection or immunisation = Positive
The level and type of antibody varies
The level and type of antibody varies
with both the pathogen, exposure and time
Serology - done by
Serology:
Detection of antibody and or antigens
Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence
Detection of antigen and antibody - why is it useful = examples
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
Detection of antigen and antibody - effect on sensitivity and window period
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).
Nucleic acid amplification (NAAT) - characteristics
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
Advantages of using NAATS
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
Limitations of using NAATs
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 - define and describe +ves
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
Multiplex PCR - define
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
Specific Taqman Probes - describe suppresssion of quencher
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
Specific Taqman Probes - fluorescence prevention
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
Specific Taqman Probes - describe removal of reporter
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.
Specific Taqman Probes - amount of reaction components during exponential phase
For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number
Real Time PCR - describe making it quantitative
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.
Explain importance of having an internal positive control
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.
Organism Sequencing - use
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
Organism Sequencing - consensus based on
Consensus sequence based on clinical observation of resistance or in vitro evidence
Organism Sequencing - selection of minority species
Minority species sequencing
May be selected by treatment
Describe sequence of virus testing with examples
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)
Multiple viral enzyme targets - examples
Multiple viral enzyme targets
Reverse transcriptase, protease,
integrase,
viral receptor binding proteins)
Describe need for a screening test
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
Screening test - describe next steps if you get a false positive
May have some false positives, so need
A specific confirmatory test
