2: Diagnostic Virology

Question 1

What are the 4 things that you can detect in a virology lab?

Answer 1

Infectious virus
Protein components (antigens) on virus
Genetic components of virus
Host response to virus

Question 2

What tubes do you use for PCR and serology?

Answer 2

PCR = 6ml EDTA (Pink)
Serology = 5ml SST

Question 3

What is PCR

Answer 3

Polymerase Chain Reaction

Method for AMPLIFYING specific DNA or RNA sequences

Question 4

What is serology

Answer 4

Enzyme immunoassay to detect antibodies/antigens

Question 5

What are other diagnostic tests for viruses?

Answer 5

Quantification of antibody/antigen (i.e. how strong is AB response)

Serotyping (e.g. HIV)

Quantification of GENOME = viral load - can be used to determine if patient is immune or not. Essential for diagnosis and monitoring of HIV/HBV/HCV, and also for CMV/EBV in immunocompromised

Genome sequencing - includes genotyping + antiviral resistance test

Question 6

What are the different types of samples used?

Answer 6

• For respiratory viruses (PCR) --> fever, cough, require oxygen 
	○ Throat swab,
	○ Nasopharyngeal aspirate 
	○ Bronchoalveolar lavage (BAL)
	○ Endotracheal tube (ET) secretions 

• For rotavirus, norovirus and adenovirus
○ Stools
○ Antigen detection

• For BK and adenovirus
○ Urine

• For herpes and enteroviruses
○ CSF

• For serology (clotted antibodies)
○ Blood clotted

• For PCR/viral load
○ Blood EDTA

• For serology e.g. measles
○Saliva

Question 7

What is serology used for?

Answer 7

Diagnostic exam of blood serum (no coag factors) - can detect up the ANTIBODY produced by the patient in response to a virus

Question 8

What would you detect in serology of different viruses?

Answer 8

HIV = antibody + p24 antigen
HBV = surface Ag/Ab, eAg/eAb, core Ab, core IgM
HCV = antibody +/- core antigen
CMV/EBV = IgM + IgG
Hep A = IgM + IgG
MMR = IgM + IgG
Parvovirus B19 = IgM + IgG
VZV = IgG

IgM = Recent infection (IMmediate)
IgG = G for Gone- Past infection/immunisation

Question 9

Explain HIV serology testing

Answer 9

1. Enzyme Immunoassay (EIA) - detect antibody and p24 antigen
2. If positive result -> confirmatory testing to exclude FALSE POSITIVES
3. If confirmed positive -> undergo TYPING (is it HIV 1 or 2?)
4. Repeat blood sample and EDTA blood for HIV VIRAL LOAD (for genotyping + baseline antiviral resistance testing)

Question 10

What is antibody avidity testing?

Answer 10

A way of confirming a positive IgM result
Avidity = strength with which antibodies bind to specific antigen
Early infection -> LOW avidity
Antibody then matures so you get gradual increase of avidity over 3-6 months

HIGH antibody avidity = unlikely that infection has occured within last 3 months

Question 11

Why is virus isolation in cell culture rarely used?

Answer 11

Time-consuming and slow

BUT it is still useful for phenotypic antiretroviral susceptibility testing

Question 12

What is immunofluorescence useful for?

Answer 12

Direct detection of viral antigens in clinical samples (e.g. resp viruses)
Can be used for typing and cell culture confirmation

Rapid/inexpensive BUT subjective, depends on skill of technician and quality of sample

Question 13

Name 5 tests you can do for resp. virus PCR

Answer 13

Throat/nose swab
Nasopharyngeal swab/aspirate
Bronchoalveolar Lavage (BAL)
Endotracheal tube (ET) secretions

Question 14

Name 7 resp. viruses. How do you confirm which one it is?

Answer 14

Influenza 
Parainfluenza
Rhino/Adeno/Coronavirus 
RSV
HPMV

Use MULTPLEX PCR to confirm which one

Question 15

What diagnostic tests would you do for meningitis/encephalitis (CNS disease)?

Answer 15

CSF - for PCR (HSV, VZV, enterovirus)
Stools + Throat swab - Detect enterovirus by PCR
Blood - for serology and/or PCR for West Nile/Japanese Encephalitis (+other arboviruses)

If patient is immunocompromised test for CMV, EBV and JC viruses as well

Question 16

What diagnostic tests would you do for diarrhoea and vomiting? Name viruses that you might expect to see

Answer 16

Stool - preferred
Vomit - lower yield
PCR or antigen detection assay (EIA)

Check for norovirus, rotavirus, adenovirus, sapovirus, astrovirus

Question 17

Explain the steps of PCR

Answer 17

Need: DNA, DNA Taq polymerase, DNA primers, free nucleotides

1. For RNA virus (e.g. influenza), make dsDNA copy of viral RNA using reverse transcriptase
2. Denature dsDNA into 2 strands - heat at 95c
3. Primer annealing - cool to 55c to allow primers to bind
4. Chain elongation - heat to 75c (Taq polymerase synthesises new DNA)

Cycle through this 30 times. Each time you DOUBLE the amount of target sequence. EXPONENTIAL expansion

Question 18

How is PCR used?

Answer 18

Sequencing viral genomes (genotyping)
Antiviral resistance testing
Phylogenetic analysis (for investigating outbreaks)

Question 19

What are tests for viral hepatitis?

Answer 19

Hep A/E IgM
Hep B surface antigen
Hep C antibody

Abnormal LFTs commonly due to viral hep so should get checked

Question 20

What tests do you do for returning travellers?

Answer 20

Syphylis EIA test + toxoplasma serology

Toxoplasmosis = caused by Toxoplasma gondii (protozoan parasite)
Transmitted through uncooked meat + food/drink contaminated with faeces of infected cats

Question 21

Enlarged lymph nodes, fever, rash, small red patches. What should you test for?

Answer 21

EBV + CMV IgG

also ALWAYS check HIV antibodies if theres any possibility