Flashcards in Virology Lab Deck (12):
What can we detect?
Infectious virus – virus isolation and EM
Protein components – antigens of the virus
Genetic components – DNA or RNA
Host response – antibody or cell responses
What are methods of diagnosing?
* Cell culture and electron microscopy – not used as commonly now, as replaced by PCR.
- PCR – Polymerase chain reaction – PCR detects specific sequences of DNA.
- Antibody detection – serology (EIA = Enzyme Immunoassay).
- Antigen detection – serology (EIA, IF = Immunofluorescence).
- Quantification of antibody or antigen.
- Serotyping – i.e. in HIV.
- Quantification of genomes – viral load.
> This is essential for diagnosis and monitoring of HIV, HBV, HCV and CMV/EBV in the immunocompromised.
- Genome sequencing – genotyping and antiviral resistance testing
What are the limitations of the tests?
False negatives results – SENSITIVITY – the tests ability to correctly identify positive samples
False positive results – SPECIFICITY – the tests ability to correctly identify negative samples
What samples can be tested?
Throat swab, nasopharyngeal aspirate (NPA), Bronchoalveolar lavage (BAL), ET (Endotracheal tube) secretions – PCR
Stools – rotavirus, adenovirus, norovirus, antigen detection (EIA) or PCR
Urine – BK virus and adenovirus – PCR
CSF – herpes viruses and enteroviruses – PCR
Blood (clotted) – serology (antibody detection)
Blood (EDTA) – PCR / viral load testing.
Saliva – serology and/or PCR (e.g. measles)
When is IgM produced and when is IgG produced?
IgM is a marker of RECENT INFECTION whereas IgG is created later in the host response and lasts longer
Both IgG and IgM are created in the acute phase of disease but the IgG levels only get higher and plateau whilst the IgM levels peak early and drop after a few weeks.
E.G. Positive IgG with absent IgM is consistent with past infection or immunisation
What is antibody avidity testing?
This confirms a positive IgM result
Avidity = strength with which antibodies bind to a specific antigen
Early on in infection avidity is low but it gets better over a period of months as the antibodies mature
Virus isolation in cell culture is rarely used now as its slow and time-consuming (expensive) but it is still useful for phenotypic antiretroviral susceptibility testing
Electron microscopy is also rarely used
What happens during HIV serology testing?
* We are on the 4th generation EIA for HIV – antibody and p24 antigen detection.
A positive result goes onto confirmatory testing in a second assay to reduce false positives.
Confirmed positives then undergo typing (HIV 1 vs. 2).
Blood samples are repeated and EDTA blood for the HIV viral load – for genotyping and baseline resistance testing.
Point-of-care testing also exists outside the lab (small equipment)
When is immunofluorescence used?
Useful for direct detection of viral antigens in clinical samples
Can also be used for typing and cell culture conformation
It is rapid and inexpensive but produces very subjective results as is very dependent upon skill of technician and quality of sample
What samples would you look at for a respiratory disease?
throat swab +/- nose swab
nasopharyngeal aspirate (NPA)
bronchoalveolar lavage (BAL)
endotracheal tube (ET) secretions
*Multiplex PCR – rather than use a single test tube for each virus, use several viruses in one tube – only do 3 or 4 at once
What samples would you take for a CNS disease (meninges/ encephalitis)?
CSF for PCR (HSV,VZV, enterovirus)
stool and throat swab (enterovirus detection)
blood for serology and/or PCR for west nile an dother arboviruses
How does a clinical history help identify viruses?
Meningitis or encephalitis – HSV, VZV and enterovirus
Young child with febrile fits – Add HHV-6 + parechovirus
Immunocompromised (HIV) – Add CMV, EBV, JC virus
Recent exotic travel – Japanese encephalitis, West Nile virus, equine encephalitides, tick borne encephalitis
Outbreaks – mumps