Diagnostic Virology

Question 1

Immunofluorescence

Answer 1

• Useful for the direct detection of viral antigens in clinical samples
  (e. g. respiratory viruses)
• Can be used for typing and culture confirmation
• Relatively quick and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample

Question 2

Enzyme Immunoassay (EIA’s) for HIV antibody detection

Answer 2

• Detection of specific antibody is an indirect method of detecting infection
• Non-specific reactions can be a problem
• Important to use multiple formats (generally use 3 different assays)
• Interpretation of results must take the clinical circumstances into account.

Question 3

HIV antibody tests

Answer 3

• In UK we use 3 tests
  
  • 3 formats
  • Different manufactures (different antigens)
  • Test from clot and separated serum
  • Ask for repeat tests from all new positives

Question 4

Polymerase chain reaction

Answer 4

• Target amplification to allow detection and quantification over very large dynamic ranges (> 5-8 logs)
• Can be very sensitive (as low as 1 genome copy)
• Can subtype viruses from PCR products
• Problems with contamination. This can be overcome using “Real Time” PCR.

Question 5

Understand what tests are available for diagnosing viral infections.

Answer 5

Virological tests- ideal tests should have the following qualities

• High specificity i.e. have a low level of cross reactivity.
• Sensitive- detect the virus or the antibody at very low levels
• Rapid- results should be available in a timely fashion.
• Non-invasive. This reduces the risks of the procedure and makes then easier to repeat if necessary.
• Cost effective. Most virology tests only cost a few pounds each but some of the molecular tests are significantly more expensive, so use them wisely.

Diagnostic Methods

• Cell culture
• Electron microscopy
• Antibody detection
• Antigen detection
• Genome detection
• Either look for the virus or look for the body’s response to it.

Question 6

Know what clinical samples to take to enable you to make the correct diagnosis.

Answer 6

Typical Samples Used

• Throat swab- for PCR (EDTA blood)
• Stools- for EM, rotavirus EIA and norovirus PCR
• CSF- PCR for herpes
• Nasopharyngeal aspirate (NPA) for respiratory viruses using IF or PCR
• Urine- for PCR
• Blood (clotted)- for antibody detection
• Blood (EDTA)- for PCR

What can we measure?

• Detect the infectious virus (virus isolation)
• Detect protein components of the virus (p24 antigen in HIV, surface antigen in HBV, etc)
• Detect genetic components of the virus (cDNA or RNA)- quantitative or qualitative tests are available
• Detect the host response (e.g. antibody or cell responses)

Question 7

Appreciate the range of viruses that can cause human disease.

Answer 7

Serology

• HIV antibody
• HCV antibody
• HBV sAg, eAG e-antibody
• Herpes antibodies (HSV, EBV, CMV, VZV etc)
• Hepatitis A IgG and IgM
• Rubella, parvovirus B19 (erythrovirus)
• Measles, mumps serology

Respiratory infection

• Nasopharyngeal aspirate (NPA)
• Throat swab
• Broncheo-alveolar lavage (BAL)
• Blood for PCR if disseminated infection suspected e.g. adenovirus in immunocompromised patient
• Acute and convalescent serology

CNS disease- meningitis/encephalitis

• CSF for PCR
• Stools and throat swab for enterovirus detection
• Serology for West Nile virus infection and other arboviruses. This is why a travel history is so important.
• Worrying about acute infections, anyone who has been in the country for more than 3 weeks will not have one of these.

Diarrhoea and vomiting

• Stool
• Vomiting can be used to detect viruses by electron microscopy, but…
• PCR or antigen detection assays for noroviruses best used for stool samples.

Cell culture

• Still a gold standard
• Relatively slow but still useful
• Main method of herpes simplex and respiratory virus detection.
• Isolates useful for sensitivity testing.
• Needed in “forensic” cases
• Expensive