Diagnosis of Viral Infections

Question 1

What is a key concept about diagnosing viral infection?

Answer 1

It is not always possible to diagnose a infection clinically. Often, we require a laboratory diagnostic test.

Other factors that aid diagnosis:

• history
• examination
• special investigations

Question 2

Why is it good to get rapid diagnosis?

Answer 2

A rapid diagnosis of viral infections can reduce the need for unnecessary tests and inappropriate antibiotics.

It is also an important public health tool, as it has infection control implications.

Question 3

What are the three types of laboratory tests with regards to viral infections?

Answer 3

• diagnostic tests
• monitoring tests
• screening tests

Question 4

List some possible tests.

Answer 4

• 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

Question 5

How do we visualise viruses?

Answer 5

Viruses can be visualised with an electron microscope.

They have mostly been replaced by molecular techniques. However, they are possibly still useful for faeces and vesicle specimens.
They’re also useful in characterising emerging pathogens.

Question 6

How does an electron microscope work?

Answer 6

The specimens are dried on a grid.

They can be stained with heavy metal e.g. uranyl acetate.
They can also be concentrated with application of antibody i.e. immuno-electron microscopy to concentrate the virus.

Beams of electrons are then used to produce images.

It uses a wavelength of electron beam that is much shorter than light, resulting in a much higher resolution than light microscopy.

Question 7

List some advantages of using an electron microscope.

Answer 7

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

Question 8

List some disadvantages of using an electron microscope.

Answer 8

• Low sensitivity - need 106 virions/millilitre to be able to visualise (may be enough in vesicle secretion/stool)
• Requires maintenance
• Requires skilled operators
• Cannot differentiate between viruses of the same virus family

Question 9

Which two herpes viruses cause vesicles, and would an electron microscope be able to distinguish between them?

Answer 9

Herpes viruses that cause vesicles are:

• Herpes simplex
• Varicella zoster virus

An electron microscope cannot differentiate between these different viruses so, it depends on the clinical context, site of vesicle and symptoms present.

Question 10

Describe virus isolation in cell culture.

Answer 10

Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE) of cells when a patient sample containing a virus is incubated with a cell layer.
Thus is an old method, now replaced by molecular techniques, but still needed for research or for rare viruses. It led to discovery of hMPV and Nipha virus in last 20 years.

We use different cell lines in test tubes or plates. The selection of cell types is important as different viruses may have different affinities for different cells.

It’s slow, but occasionally is useful in anti-viral sensitivity testing.

Question 11

What are some common methods used for antigen detection?

Answer 11

A variety of different methods can be used. The commonest methods are:

• Direct immunofluorescence
• Enzyme immunoassay
• Immunochromatographic methods

It is often used at point of care for rapid diagnosis.

Question 12

Briefly, describe immunofluorescence.

Answer 12

The antigen (from infected host cells in sample) is bound to the slide.

A specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with the sample.

It is then viewed using a microscope equipped to provide ultraviolet illumination.
Any cells that have the virus in them will fluoresce.

Question 13

Briefly, describe the immunochromatographic method.

Answer 13

You get a bit of the patients blood, and you add it to the machine.

You’ll see a line appear on it where you get the binding on an antigen in the patient’s blood with an antibody in the particular kit, and it causes precipitation of heavy metals and you see a visible line.

This is called the immunochromatographic method.

Question 14

Briefly, describe ELISA for antigen detection.

Answer 14

ELISA is the enzyme-linked immunosorbent assay.

There are three formats:

• Indirect
• Direct (primarily antigen detection)
• Sandwich

Question 15

Describe how the ELISA test works.

Answer 15

1. The plate is coated with a capture antibody.
2. The sample is added and any antigen present binds to the capture antibody.
3. An enzyme-conjugated primary antibody is added, which binds to the detecting antibody.
4. After washing away the unconjugated sample, a chromogenic substrate is added, and is converted by the enzyme to a detectable form e.g. colour change.

The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present.
A negative result = NO colour change.

Question 16

Describe how we can diagnose someone’s stage of illness using antibody detection.

Answer 16

When infected with a virus, the humoral immune response takes place, resulting in the production of immunoglobulins i.e. antibodies.

IgM antibodies specific to the virus are produced first; they are present for a variable period – usually 1 to 3 months.
As IgM declines, IgG is produced, and the quantity of IgG rises.

A 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)

Question 17

Describe serology.

Answer 17

It is the indirect detection of the pathogen.

It is the 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 blood & serum - they can also be performed on other bodily fluids such as semen and saliva.

Question 18

Describe serum’s acquirement, storage and use.

Answer 18

Serum is produced from processing blood:

• the blood is coagulated with micronized silica particles
• a gel is used to trap cellular components

Routinely, serum tubes are centrifuged for 10 min at 1000xg.

Supernatant (serum) is removed and stored at:
- 4ºC for the short term
- -20ºC for the long term
At the right temperature, serum can be stored for years.

Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes.

Question 19

Describe the method by which antibodies are detected in the blood.

Answer 19

We get the viral antigen we want to detect, and we stick it to the bottom of the well. We then add the patient sample to it. If the patient has got the antibody to the viral antigen in their blood, they will bind to the antigen stuck to the bottom of the well.

We then add another antibody that is conjugated to a colour-producing enzyme, which will bind to the original antibody. In this situation, we could use a goat or a mouse antibody that will bind to a human antibody.
After each addition of antibody, you wash away the remaining unbound antibodies, so that we are only measuring the ones that we have.

Finally, we add the substrate that produces the colour that tells us if the antibody for the antigen is present or not.

Question 20

Describe the different serological results that can help diagnose the stage of infection of Hepatitis A.

Answer 20

With no past or current infection or immunisation:
HEP A IGM: negative
HEP A IGG: negative

With an acute/recent infection:
HEP A IGM: POSITIVE
HEP A IGG: negative/POSITIVE

With resolved infection or immunisation:
HEP A IGM: negative
HEP A IGG: POSITIVE

Question 21

Why are we sometimes unable to detect viruses based on the IgM levels?

Answer 21

With a second exposure, since we’ve made memory cells to the infection, often we get a very IgG response. You can get a slight raise in IgM, but often IgM doesn’t raise at all.

So, some viruses you can’t detect by looking at IgM. You would have to look for them by detecting a rise in the IgG that’s there.

It’s quite rare to use this method, but it can still be used to diagnose if someone has had pre-existing antibodies.

Question 22

Describe molecular diagnostic tests for viral detection.

Answer 22

An example of a nucleic acid amplification test (NAAT is PCR (although there are others).

It can detect RNA or DNA, depending on the virus.

It has the ability to multiplex using fluorescence probes i.e. we can look for several targets in one sample.

It may be qualitative or quantitative (to allow you to figure out how much DNA was there to begin with).

It requires nucleic acid extraction prior to the amplification.

Question 23

List some advantages of using NAAT.

Answer 23

• they may be automated, making it less toilsome
• they are highly sensitive and specific, and generate huge numbers of amplicons
• they’re rapid (which is useful especially in diagonosis)
• useful for detecting viruses to make a diagnosis (both at the first time of infection [e.g. measles, influenza], or during reactivation [e.g. cytomegalovirus])
• useful for monitoring treatment response: we use it quantitatively e.g. HIV, HBV, HCV, CMV viral loads (detect amount in blood, use treatment, then look to see if amount falls)

Question 24

List some disadvantages of using NAAT.

Answer 24

• we may detect other viruses which are not causing the infection
• it is exquisitely sensitive and so may generate large numbers of am plicons; this may cause contamination
• we need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target

Question 25

Briefly, describe multiplex PCR.

Answer 25

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.

Question 26

Briefly, describe real time PCR.

Answer 26

It has a different chemistry to multiplex, but they’re all similar.

It’s considered real time as amplification AND detection occur in REAL TIME i.e. simultaneously by the release of fluorescence.

It avoids the use of gel electrophoresis or line hybridisation.
It can allow the use of multiplexing.

Question 27

Describe how specific Taqman probes work.

Answer 27

Along with the primer for our sample of DNA, we also have a probe that will bind to the bit of DNA we’re trying to amplify. That probe has a fluorescent reporter on it which will shine when we shine light on it. It’s got a quencher nearby that actually stops it glowing when it is in close proximity to the reporter.

When the probe binds to the DNA, an enzyme called Taq polymerase actually cleaves the reporter bit from the probe. Now separated from the quencher, the reporter will glow.

Every-time you get this process of amplification, you will get fluorescence.
Hence, the fluorescence signal is directly proportional to the initial copy number.

Question 28

What is the cycle threshold?

Answer 28

The cycle threshold is the number of times we actually need to do the amplification process.

Question 29

How can PCR be inhibited?

Answer 29

Some substances inhibit PCR (e.g. haem, bile salts).

Assays should always include an internal positive control as the results could incorrectly be reported as negative.

We can include primers specific for the internal control of material.

Question 30

Describe organism sequencing.

Answer 30

This can be used for DNA or RNA viruses.

It is used to predict responses to anti-virals e.g. for HIV in Rx naïve patients, or if there is a clinical suggestion of resistance in drug-experienced patients.

A consensus sequence is made based on clinical observation of resistance or in vitro evidence.

It’s useful for outbreak investigations by showing identical sequences in the suspected source and recipient.

Question 31

Describe how you would use a combination of methods in HIV diagnoses and management.

Answer 31

For the antibody and antigen detection for initial diagnosis:

• screening test (EIA)
• confirmatory test (EIA)

To detect the viral load at baseline and to monitor treatment response:

• (NAAT)
• quantification of virus in blood

We would also do resistance testing (sequencing) to confer resistance before and during treatment.

Question 32

Describe anti-viral resistance testing.

Answer 32

Using HIV as an example, there are multiple viral enzyme targets:

• reverse transcriptase, protease
• integrase
• viral receptor binding proteins

We can look for mutations in various genes in the virus which are known to be associated with resistance.

We know they are associated by doing cell culture techniques, to see if the antivirus actually works on the virus. If it doesn’t and it has a particular mutation, you know the virus is resistant due to that mutation.

Thus, when you sequence the virus and notice the mutations, you can predict that they will be resistant to certain antivirals.

We use a similar approach for hepatitis C, HSV, CMV (but different genes).

Question 33

Briefly, describe screening.

Answer 33

We use screening to test for specific infections in at risk groups (drug addicts, etc.).
- e.g. HIV, HBV and HCV

We also do this testing because it may have an implication for others e.g. antenatal.
- e.g. HIV, HBV, rubella

In these situations the patients are asymptomatic, we need a sensitive screening test.
We may also get some false positives, so we need a specific confirmatory test.