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