Diagnostic Virology

Question 1

What diagnostic tools are available in virology?

Answer 1

• history (e.g. travel)
• physical examination (rashes?)
• lab tests (non-specific; virological;)

Question 2

Shingles rash

Answer 2

single dermatome infected

Question 3

What can be detected in virology?

Answer 3

• Infectious virus (virus isolation & EM) -> done very rarely
• Protein components (antigens) of the virus (p24 antigen in HIV, surface antigen in HBV, etc) -> done quite commonly
• Genetic components of the virus (DNA or RNA) - quantitative or qualitative tests are available
• The host response (e.g. antibody or cell responses) -> done most commonly

Question 4

What diagnostic methods are used?

Answer 4

• cell culture (VERY rarely, not really done anymore)
• electron microscopy (very rarely)
• antibody detection (serology - EIA)
• antigen detection: (Immunofluoroescence -IF; enzyme immunoassay - EIA)
• Genome detection - PCR (done very commonly!)
• quantification of AB or antigens
• Serotyping (e.g. HIV)
• quantification of genomes (viral load - essential for diagnosis and monitoring of HIV, HBV, HCV and also CMV and EBV in immunocompromised)
• genome sequencing (genotyping, antiviral resistance testing)

Question 5

Sensitivity vs. Specificity

Answer 5

All assays give rise to false negative and false positive results

• Sensitivity: the test’s ability to correctly identify positive samples (sensitive = few false negative results)
• Specificity: the test’s ability to correctly identify negative samples (specific = few false positive results)

Question 6

What are the typical samples used in virology?

Answer 6

• Throat swab, Nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET secretions – for detection of respiratory viruses by (IF or) PCR
• Stools - for rotavirus, adenovirus & norovirus antigen detection (EIA) or PCR
• Urine – for BK virus & adenovirus PCR
• CSF - for herpes viruses and enteroviruses PCR
• Blood (clotted) - for serology (antibody detection)
• Blood (EDTA) - for PCR / viral load testing
• Saliva – for serology &/or PCR (eg measles)

Question 7

Serology in virology

Answer 7

• HIV serology (antibody + p24 antigen)
• Hepatitis A IgM and IgG
• HBV surface Ag/Ab, eAg/eAb, core Ab, core IgM
• HCV serology (antibody +/- core antigen)
• CMV and EBV IgM & IgG
• VZV IgG
• Measles, mumps, rubella IgM & IgG
• Parvovirus B19 IgM & IgG

Question 8

IgM and IgG testing

Answer 8

• Both present in acute phase
• IgM stays for ~ 3 months
• IgG stays lifelong
• IgM tests are very non-specific - quite many false positives

Question 9

What is AB avidity testing? When and Why is it used?

Answer 9

• IgM tests are quite non-specific
• antibodies are bound, then washed with urea
• if high avidity they stay attached, if they have low avidity they are washed away
• higher avidity in chronic than in acute sample

Question 10

What does HIV serology involve?

Answer 10

• 4th generation EIA: Ab + p24 Ag detection
• All reactive samples undergo confirmatory testing in a second assay to exclude non-specific reactivity (false positives)
• Confirmed positives undergo typing (HIV 1 vs 2)
• Repeat blood sample + EDTA blood for HIV viral load required from all new +ves (also genotyping and baseline resistance testing)

Question 11

Virus isolation in cell cultures

Answer 11

• Now rarely used - Reference lab only
• Slow, time consuming (hence expensive)
• Still useful for phenotypic antiviral susceptibility testing (HSV)
• poor sensitivity and specificity in general

Question 12

Electron Microscopy

Answer 12

• Viruses are too small to be seen by light microscopy
• They can be visualised using an electron microscope
• Sample types: stool and vesicle fluids
• Now rarely used

Question 13

Immunofluorescence

Answer 13

• IF
• Still occasionally used for the direct detection of viral antigens in clinical samples (DIF) (e.g. respiratory viruses)
• Rapid and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample
• patient sample -> put it on a glass -> incubate with ABs that are flour marked

Question 14

Respiratory Tract infections

Answer 14

• Throat swab +/- nose swab
• Nasopharyngeal swab
• Nasopharyngeal aspirate (NPA)
• Bronchoalveolar lavage (BAL)
• Endotracheal tube (ET) secretions
  
  • > for respiratory virus PCR!

(Influenza, parainfluenza, RSV, rhinovirus, human metapneumovirus [HMPV], adenovirus, bocavirus, +/- coronavirus…)
“multiplex PCR”

Question 15

Multiplex PCR

Answer 15

• look for multiple gene targets

- look for end product via electrophoresis

Question 16

CNS disease - meningitis / encephalitis

Answer 16

• CSF for PCR (HSV, VZV, enterovirus…)
• Stools and throat swab for enterovirus detection (ie by PCR)
• Blood for serology and/or PCR for West Nile and/or Japanese Encephalitis virus infection and other arboviruses

Question 17

D&V

Answer 17

• Stool (preferred)
• Vomit – lower yield …
• PCR or antigen detection assays (EIA)
  (norovirus, rotavirus, adenovirus, sapovirus, astrovirus)

Enteric viruses vs enteroviruses!

Question 18

PCR

Answer 18

• Polymerase Chain Reaction
• Method for amplifying specific RNA (RT-PCR) or DNA sequences
  Cycle ( x 30):

RNA -> dsDNA via reverse trasncriptase
1. Denaturation
2. Primer annealing
3. Chain elongation (Tam polymerase)
REPEAT

Question 19

Sequencing applications

Answer 19

• genotyping
• antiviral resistance tetinf
• phylogenetic analysis

Question 20

What is being increasingly used?

Answer 20

point of care testing (but limited spec and sens)