Diagnosis of viral infections

Question 1

Describe how electron microscopy can diagnose viral infections

Answer 1

• Replaced now by modern techniques
• Useful in characterising emerging pathogens
• Patient sample is stained and concentrate with antibody. Beams of electrons are used to produce images.
• Much higher resolution than light microscopy - but low sensitivity
• Can visualise many viruses, quick
• Requires maintenance and skilled operators
• However EM cannot differentiate herpes viruses so depends on clinical context, symptoms and site of vesicle

Question 2

Describe how virus isolation can diagnose viral infections

Answer 2

• Virus require host cells to replicate and may cause a cytopathic effect of cells when a patient sample containing a virus is incubated within a cell layer
• Different viruses may give different appearances –> identify virus using antigen detection techniques or neutralisation of growth
• Old method and slow but can be useful

Question 3

Describe how antigen detection can diagnose viral infections

Answer 3

Viral antigens can be detected in cells or free in blood, saliva or other tissues/organs

They are being replaced by nucleic acid detection methods due to improved test performance

Commonest methods: direct immunofluorescence or enzyme immunoassay or immunochromatographic methods

• Immunofluorescence - swab taken onto microscope slide. Antibody which will bind to antigens which is attached to fluorochrome which can be viewed and provide ultraviolet illumination (green = viral antigens)
• Immunochromatographic - lateral flow tests
• ELISA - indirect, direct and sandwich - Hep B detection

Question 4

Describe how antigen detection by serology can diagnose viral infections

Answer 4

• Detection of antibodies = indirect detection of the pathogen
• Used in symptomatic patients - to determine if vaccination has been successful
• Can be: blood, serum, semen and saliva
• Serology = Serum is the supernatant of blood once it has clotted and been centrifuged. Serum contains proteins, antigens, antibodies, some drugs and electrolytes.
• Diagnosis - IgM antibodies specific to the virus are produced first, present for 1 to 3 months. As IgM declines, IgG is produced and rises over time. So diagnosis is made by detection of IgM. Negative IgG at first, then presence of IgG antibody
• Detect both antigen and antibody - you get a good overview of the infection and to make decisions about the stage of illness and what treatments

Question 5

Describe how molecular dianostic tests can diagnose viral infections

Answer 5

NAATs - nucleic acid amplification tests

• Test is designed to detect RNA or DNA of the virus your looking for
• Can be qualitative or quantitative
• Requires nucleic acid extraction prior to the amplification
• Specimen collection, extraction of nucleic acid, DNA transcription for RNA viruses, cycles of amplification of DNA target, detection of amplicons
• Advantages: automated, highly sensitive and specific, rapid. Useful for monitoring treatment response
• Limitations - generates large numbers of amplicons which can cause contamination, need to have an idea of viruses as you need primers

Multiplex PCR - more than one primer is used to enable amplification of multiple DNA targets in one tube

Sequencing for genotype and detection of antiviral resistance

• Partial or whole - useful for outbreak investigation by showing identical sequences in suspected source
• Useful for new variants - diagnostic tests and vaccine efficacy
• Used to predict response to anti-virals