Diagnosis of viral infections

Question 1

How do we visualise microbes?

Answer 1

• Larger microbes such as bacteria, fungi and protozoa/ helminths can be seen using light microscopy
• Viruses are much smaller and so have to be observed using EM

Question 2

How does EM work?

Answer 2

• Beams of electrons are used to produce images
• Wavelength of electron beam is much shorter than light, resulting in much higher resolution
• They scatter when they pass through thin sections, but transmitted ones that dont scatter are used to produce an image
• Denser regions cause more scatter and so appear darker

Question 3

Pros and Cons of EM

Answer 3

Pro

• Rapid
• Detects viruses that cannot be grown in culture
• Can visualise may different viruses

Con

• Low sensitivity - need a lot
• Requires maintenance and skilled operators
• Cannot differentiate between viruses of the same family

Question 4

How do we look at viruses isolated in cell culture?

Answer 4

• Viruses require host cells to replicate
• May cause a cytopathic effect of cells when infected patient sample is incubated with a cell layer
• old method, replaced by molecular techniques
• Still needed for research and rare viruses

Question 5

What cytopathic effects can be caused?

Answer 5

• Different viruses may give different appearances
• Different cell lines may support growth of different viruses
• Identify virus using ag detection techniques or neutralisation of growth

Question 6

What are the commonest methods of ag detection?

Answer 6

• Direct immunofluorescence
• Enzyme immunoassay
• immunochromatographic methods

Question 7

How do we use immunofluorescence?

Answer 7

• Ag from infected host cells in sample bound to slide
• Specific ab (poly/monoclonal) to the ag that is tagged to a fluorochrome and mixed with a sample
• Viewed using a microscope equipped to provide UV illumination

Question 8

How do we used ELISA for ag detection?

Answer 8

• Plate is coated with capture antibody
• Patient sample is added; complementary ag binds to ab
• Enzyme-linked ab specific for test ag is added, binds to ag - forming sandwich
• Enzyme’s substrate is added, and reaction produces a product that causes a visible colour change

Question 9

What can serology be used for?

Answer 9

• Detect an ab reponse in symptomatic patients
• Determine if vaccination was successful
• Directly look for ag produced by pathogens

Question 10

How do we get serum?

Answer 10

• Coagulate blood with micronised silica particles
• Use gel to trap cellular components
• Centrifuged
• Serum contains proteins, ags, abs, drugs and electrolytes

Question 11

What will ab levels be like at different stages of infection?

Answer 11

• No previous/ current ifection or vaccine = no IgM or IgG
• First exposure - IgM will increase at first, then IgG after a while
• Resolved infection/ immunisation = no IgM, but IgG present (memory)
• Secondary infection = massive increase in IgG faster, same IgM

Question 12

Why would we test for detection of ag AND ab?

Answer 12

• Allows us to establish whether acute or chronic infection
• Combine tests give increased sensitivity and reduced window period (can get result quicker after infection)
• Used for Hep B, C and HIV

Question 13

What molecular diagnostic tests do we use?

Answer 13

• Nucleic acid amplification
• eg. PCR
• Can detect RNA or DNA
• Can use fluorescent probes to look a several targets
• Qualitative or quantitative

Question 14

Pros and Cons of Nucleic acid amplification

Answer 14

Pro

• automated
• Highly sensitive and specific
• Rapid
• Useful for detecting viruses for diagnosis + monitoring treatment response

Con

• may detect other viruses not causing infection
• Very sensitive, so generates large number of amplicons - may cause contamination
• Need to know what we are looking for as use specific probes and primers

Question 15

What is real time PCR?

Answer 15

• Amplification and detection occur in real time - simultaneously by the release of fluorescence
• avoids use of electrophoresis or line hybridisation
• Allows use of multiplexing

Question 16

What is multiple PCR?

Answer 16

• More than one pair of primers is used in a PCR

- Enables amplification of multiple DNA targets in one tube

Question 17

What are Taqman probes?

Answer 17

• Complimentary to region of interest, binds between primers
• Has a fluorescent reporter at 5’ and quencher at 3’
• Quencher prevents fluorescence when excited if in close proximity
• probe hybridises to region during annealing phase - fluorescence prevented due to proximity of quencher
• Taq polymerase moves from 5’-3’ and hydrolyses the probe
• This releases the reporter, stopping the quencher’s activity - detect fluorescence
• the amount of fluorescence is proportional to initial copy number

Question 18

What is the cycle threshold?

Answer 18

• Used to perform quantitation and presence/ absence of amplicon detection
• Cycle number at which the fluorescence cross the threshold line (proportional to number of copies of DNA)
• A low CT indicates lots of the viral target - not many cycles are required to reach the CT

Question 19

How can you make real time PCR quantitiave?

Answer 19

• Relative fluorescence can be plotted against number of cycles
• Can be used to determine relative conc of DNA present by construction of standard curve against standards of known concentration

Question 20

What substances nhibit PCR?

Answer 20

• Some substances such as haem and bile salts

- Need to include positive control as results could be incorrectly reported as negative

Question 21

When would we do organism sequencing?

Answer 21

• DNA or RNA viruses
• Used to predict response to antivirals
• Consensus sequence based on clinical observation of resistance or in vitro evidence
• Useful for outbreak investigation by showing identical sequences in suspected source and recipient