Diagnosis Of Viral Infections

Question 1

What are possible tests to diagnose infection?

Answer 1

Electron microscopy - rarely used

Antigen detection

Virus isolation

Nuclei’s acid amplification tests

Question 2

How can virus isolation in cell culture be used in viral diagnosis?

Answer 2

As viruses require host cells to replicate and may cause cytopathic effect (CPE) of cells when a patient sample containing a virus incubated within cell layer

Use different cell lines in test tubes or plates - selections of cell types important

Slow but occasionally useful

Old method not used now as much

Question 3

What specimens can be used for antigen detection?

Answer 3

Nasopharyngeal aspirates - cell associated virus antigen

Blood - free antigen or whole virus

Vesicle fluid - whole virus

Faeces - whole virus

Question 4

How can electron microscopy be used to diagnose viruses?

Answer 4

Viruses can be visualised with electron microscope

1. Specimens are dried on a grid
2. Can be stained with heavy metal
3. Can be concentrated with application of antibody
4. Beams of electrons are used to produce images
5. Wavelength of electron beam is inch shorter than light, resulting in much higher resolution than light

Useful in charecterising emerging pathogens

Mostly replaces technique

Question 5

What are the advantages of using electron microscopy to diagnose viruses?

Answer 5

Rapid

Detects viruses that cannot be grown in culture

Can visualise many different viruses

Question 6

What are the disadvantages of using electron microscopy to diagnose viruses?

Answer 6

Low sensitivity need 10^6 virgins/milileter - may be enough in vesicle secretion

Requires maintenance

Requires skilled operators

Cannot differentiate between viruses of the same virus family

Question 7

What is the cytopathic effect?

Answer 7

Different viruses may give different appearances

Morphology of host cells are different

Question 8

What are the most common methods used in antigen detection?

Answer 8

Direct immunofluorescence - cell associated antigens

Enzyme immunoassay - free soluble antigens or whole virus

Immunochromatograohic methods

Question 9

What happens during direct immunoflorescnece detection of antigens?

Answer 9

Antigen bound to slide

Specific antibody to that antigen is tagged to a fluorochromes and mixed with sample

Viewed using a microscope equipped to provide ultraviolet illumination

Question 10

What happens with immunochromatograohic methods?

Answer 10

Lateral flow tests

Used to test for pregnancy detection and virus testing

Question 11

How is ELISA used for antigen detection?

Answer 11

Enzyme linked immunosorbent assay - a component of reaction is adhered to a solid surface

Three formats:
- indirect
- direct
- sandwich

Question 12

Describe the stages that occur in a ELISA test

Answer 12

1. Plate is coated with a capture
2. Sample is added and any antigen present binds to capture antibody
3. Enzyme conjugated primary antibody is added - binds to detection antibody
4. Chromogenic substrate is added and converted by the enzyme to detectable form

Question 13

How are antibodies detected using serology?

Answer 13

Indirect detection of pathogen

Used for organisms we cannot culture or not seen in electron microscopy

Question 14

What is serology be used for?

Answer 14

Detect antibody response in symptomatic patients

Determine if vaccination has been successful

Not limited to blood and serum - can be performed on other bodily fluids

Question 15

How is serum produced?

Answer 15

Routinely serum tubes are centrifuged for 10 minutes at 1000xg

Supernatant is removed and stored

Serum contains proteins, antigens, antibodies, drugs

Question 16

How can viruses be diagnosed by antibody detection?

Answer 16

When infected with a virus the humoral immune response takes place resulting in production of immunoglobulins

• Detection of igM
• Or by demonstration of seroconversion

Question 17

When can it be useful to detect antigen and antibody?

Answer 17

It allows us to establish weather acute or chronic infection

May have therapeutic implications

HIV, Hepatitis C and B

Question 18

What is Nucleic acid amplification (NAAT)?

Answer 18

Can detect RNA or DNA of virus

Ability to multiplex using fluorescence probes

May be quantitative or qualitative

Eg. PCR

Question 19

What are the stages of NAAT test?

Answer 19

1. Specimen collection
2. Extraction of nucleic acid
3. DNA transcription of RNA viruses
4. Cycles of amplification of DNA target
5. Detection of amplicons

Question 20

What are the advantages of using NAATS?

Answer 20

May be automated

Highly sensitive and specific

Generated hug e number of amplicons

Rapid - up to a few hours

Useful for steering viruses to make a diagnosis

Useful for monitoring treatments response

Question 21

What are the limitations of using NAATS?

Answer 21

Generates large number of amplicons - may cause contamination

Need to have an idea of what viruses you are looking for as specific primers and probes required

Mutations in target sequence may lead to “dropout”

Question 22

What is real time PCR?

Answer 22

Different chemistries but all similar

Amplification and detection occur in real time

Avoids the use of gel electrophoresis or line hybridisation

Allows the use of multiplexing

Question 23

What is multiplex PCR?

Answer 23

Term used when more than one pair of primers is used in a PCR

Enables amplification of multiple DNA targets in one tube

Eg. Detection of multiple viruses in one CSF specimen

Question 24

What is PCR inhibition?

Answer 24

Some substances inhibit PCR
- haem
- bile salts

Assays should contain a internal positive control as results could be false negative

Include primer specific for internal control material

Question 25

Why can genome sequencing be beneficial for viral diagnosis?

Answer 25

Useful for outbreak investigation by showing identical sequences in suspected source and recipient

New variants and vaccine efficacy

Can be used to predict response to anti virals

Question 26

How can a combination of methods be used for diagnosis?

Answer 26

Antibody and antigen detection for initial diagnosis

NAAT at baseline and to monitor treatment response

Resistance testing - sequencing

Question 27

How do you test for antiviral resistance?

Answer 27

Look for mutations known to cause resistance

Multiple viral enzyme targets
- reverse transcriptase
- integrase
- viral receptor binding

Question 28

What is screening?

Answer 28

Testing for specific infections in at risk groups

Testing because it ,ay have a implication for others

Needs a sensitive screening test

May have false positives so a specific confirmatory test needed