2: Diagnostic Virology Flashcards
What are the 4 things that you can detect in a virology lab?
Infectious virus
Protein components (antigens) on virus
Genetic components of virus
Host response to virus
What tubes do you use for PCR and serology?
PCR = 6ml EDTA (Pink) Serology = 5ml SST
What is PCR
Polymerase Chain Reaction
Method for AMPLIFYING specific DNA or RNA sequences
What is serology
Enzyme immunoassay to detect antibodies/antigens
What are other diagnostic tests for viruses?
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
What are the different types of samples used?
• 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
What is serology used for?
Diagnostic exam of blood serum (no coag factors) - can detect up the ANTIBODY produced by the patient in response to a virus
What would you detect in serology of different viruses?
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
Explain HIV serology testing
- Enzyme Immunoassay (EIA) - detect antibody and p24 antigen
- If positive result -> confirmatory testing to exclude FALSE POSITIVES
- If confirmed positive -> undergo TYPING (is it HIV 1 or 2?)
- Repeat blood sample and EDTA blood for HIV VIRAL LOAD (for genotyping + baseline antiviral resistance testing)
What is antibody avidity testing?
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
Why is virus isolation in cell culture rarely used?
Time-consuming and slow
BUT it is still useful for phenotypic antiretroviral susceptibility testing
What is immunofluorescence useful for?
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
Name 5 tests you can do for resp. virus PCR
Throat/nose swab
Nasopharyngeal swab/aspirate
Bronchoalveolar Lavage (BAL)
Endotracheal tube (ET) secretions
Name 7 resp. viruses. How do you confirm which one it is?
Influenza Parainfluenza Rhino/Adeno/Coronavirus RSV HPMV
Use MULTPLEX PCR to confirm which one
What diagnostic tests would you do for meningitis/encephalitis (CNS disease)?
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
What diagnostic tests would you do for diarrhoea and vomiting? Name viruses that you might expect to see
Stool - preferred
Vomit - lower yield
PCR or antigen detection assay (EIA)
Check for norovirus, rotavirus, adenovirus, sapovirus, astrovirus
Explain the steps of PCR
Need: DNA, DNA Taq polymerase, DNA primers, free nucleotides
- For RNA virus (e.g. influenza), make dsDNA copy of viral RNA using reverse transcriptase
- Denature dsDNA into 2 strands - heat at 95c
- Primer annealing - cool to 55c to allow primers to bind
- 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
How is PCR used?
Sequencing viral genomes (genotyping)
Antiviral resistance testing
Phylogenetic analysis (for investigating outbreaks)
What are tests for viral hepatitis?
Hep A/E IgM
Hep B surface antigen
Hep C antibody
Abnormal LFTs commonly due to viral hep so should get checked
What tests do you do for returning travellers?
Syphylis EIA test + toxoplasma serology
Toxoplasmosis = caused by Toxoplasma gondii (protozoan parasite)
Transmitted through uncooked meat + food/drink contaminated with faeces of infected cats
Enlarged lymph nodes, fever, rash, small red patches. What should you test for?
EBV + CMV IgG
also ALWAYS check HIV antibodies if theres any possibility
