Diagnosis of Viral Infections Flashcards
Why may it be important to do laboratory tests to diagnose diseases
- Not always possible to diagnose an infection clinically, Often requires a laboratory diagnostic test.
- Rapid diagnosis of viral infections can reduce need for unnecessary tests and inappropriate antibiotics
How are we able to run diagnostic tests
- Obtain consent from the patient
- 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
- Difference between diagnostic, monitoring
and screening tests
What are the possible test types we can run
- 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
Why may we use electron microscopes to visualise viruses
- Viruses are too small to be seen my normal microscopes
- useful in characterising emerging pathogens
- Possibly still useful for faeces and vesicle fluid specimens
Describe the steps in prepping a slide for electron microscopy
- 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
What are the advantages of using electron microscopy
- Rapid
- Detects viruses that cannot be grown in culture
- Can visualise many different viruses
What are the limitations of using electron microscopy
- low sensitivity
- Requires maintenance
- Requires skilled
operators - Cannot differentiate
between viruses of the same virus family.
How can we grow viruses in cell culture to help identify it
- Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE) when a patient sample containing a viruses incubated with a cell layer
- Old method, now replaced by molecular techniques, but still needed for research or rare viruses
- Led to discovery of hMPV and Nipah virus in last 20 years and SARS-CoV-2 recently
- 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 antigen detetction
Viral antigens, usually proteins – either capsid structural proteins or secreted proteins. They can be detected in cells or free in blood, saliva or other tissues/organs
What specimens can be used in antigen detection
- Nasopharyngeal aspirates (NPA) (cell-associated virus antigens) - e.g. RSV, influenza
- Blood (serum or plasma) (free antigen or whole virus) - Hepatitis B, Dengue
- Vesicle fluid
(whole virus), Herpes simplex, varicella zoster - Faeces (whole virus) - Rotavirus, adenovirus
What test types can we use for antigen detection
- Direct immunofluorescence
- Enzyme immunoassay
- Immunochromatographic methods
How does immunofluorescence work
- 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
- Long prep time and is not very sensitive
How does Immunochromatographic methods work
- Lateral flow tests for COVID-19
- Test for specific antigen
- Not as sensitive as PCR tests
What is the ELISA test of antigen detection
Enzyme-linked immunosorbent assay - A component of reaction is adhered to a solid surface
Three formats:
- Indirect
- Direct (primarily
antigen detection) - Sandwich
how does the ELISA test work
- 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
What is antibody detection by serology
- Detection of antibodies
- Indirect detection of the pathogen
- Diagnostic mode of choice for organisms which are
refractory to culture
What can serology be used for
- Detect an antibody response in symptomatic patients
- Determine if vaccination has been successful
- Directly look for antigen produced by pathogens
What can we diagnose with antibody detection
- When infected with a virus the humoral immune response takes place resulting in production of
immunoglobulins - 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
Why is detection of antigen AND antibody useful
This is useful for some infections such as: Hepatitis B, HIV, Hepatitis C
- This is because it allows therapeutic implications us to establish whether acute or chronic infection
- This may have therapeutic implications
What are molecular diagnostic tests
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
What are the stages in NAAT tests
- Specimen collection
- Extraction of nucleic acid
- DNA transcription for RNA viruses
- Cycles of Amplification of DNA target
– requires polymerase and dNTPs plus other
reagents - Detection of amplicons
– After amplification
– Or real time
What are the advantages of using NAATs
- May be automated. POCT possible
- Usually highly sensitive and specific, generates huge
numbers of amplicons - Rapid – can be as quick as 15 minutes – usually a
few hours - 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 loa
What are the limitations of using NAATs
- Generates 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.
- Mutations in target sequence may lead to “dropout” e.g. S gene dropout seen with SARS-CoV-2 variants
What is 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
What is multiplex PCR
- 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
What is 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
- Include primers specific for the internal
control material
-
how can we use genome sequencing in the diagnosis process
- Useful for outbreak
investigation by showing identical sequences in suspected source and recipient - New variants: Diagnostic tests, Vaccine efficacy
- Can be 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
How are a combination of methods are used in the diagnosis and management of HIV
Antibody and antigen detection for initial diagnosis:
- Screening test (EIA)
- Confirmatory test (EIA)
Viral load(NAAT) at baseline an dot monitor treatment response
- Quantification of virus in blood
Resistance testing (sequencing)
How can we test for ant-viral resistance susceptibility
Look for mutations known to cause resistance.
- Reverse transcriptase,
protease - integrase
- viral receptor binding
proteins
Why should we screen for these infections
- Testing for specific infections in
at risk groups - Testing because it may have
an implication for others e.g. antenatal - Needs a sensitive screening
test - May have some false
positives, so need - A specific confirmatory test
