Diagnostic Virology Flashcards
When diagnosing a viral illness, taking a good history and performing a clinical examination will determine what virological tests should be requested, and can often give you the diagnosis. When talking a history, what is it important to include, especially for suspected viral infections?
Vaccination history, travel (especially in the previous 3 weeks), contact with animals/pets, contact with infected persons and occupation.
Some viral infections are easy to diagnose such as…?
shingles (dermatomal distribution)
chicken pox
What are the qualities of an ideal virological test?
- High specificity i.e. have a low level of cross reactivity (=> low rate of false positives)
- High Sensitivity: detect the virus or the antibody at very low levels (=> low rate of false negatives)
- Rapid: results should be available in a timely fashion.
- Non-invasive: reduces the risks of the procedure and easier to repeat if necessary.
- Cost effective
What are the 5 types of diagnostic lab tests for viral infections?
- Cell culture
- Electron microscopy
- Antibody detection (EIA)
- Antigen detection (Immunofluorescence, IF; enzyme immunoassay, EIA)
- Genome detection (polymerase chain reaction to detect viral DNA or RNA)
What is the main method of virus detection? What methods are very rarely used?
PCR
Virus isolation in cell culture and EM
What does quantification of the genomes allow assessment of? What is this essential for?
Viral load
Diagnosis and monitoring of many blood borne viruses such as HIV, HBV and HCV, and also for CMV and EBV in the immunocompromised
The type of samples to take will depend on the disease being investigated. What are the typical samples used and for what viruses?
- Throat swab, Nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), Endotracheal tube 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 (e.g. measles)
What is the difference between IgM and IgG in terms of when their levels rise following infection? State each of their durations
IgM is a marker of RECENT infection; approx. 3 months
IgG rises later on; lifelong
What does positive IgG and absent IgM indicate?
Past infection or immunisation (sensitive but not specific)
What test is used to confirm a positive IgM result?
Antibody avidity testing
avidity is defined as the ratio:
OD(8M urea)/OD(0.85% NaCl)
Urea is a denaturation agent which has greatest effect of preventing binding for low avidity antibodies
Describe how antibody avidity changes in infection.
Early on in the infection, avidity is LOW
Then you get maturation of the antibody response so the avidity will increase over a period of 3-6 months
If you have HIGH antibody avidity, then it is unlikely that the infection occurred in the last 3 months
What is targeted in the detection of HIV, and how?
Antibody and p24 antigen
4th generation EIA
What further tests are done following detection of the HIV antibody and p24 antigen?
- All reactive samples undergo confirmatory testing in a second assay to exclude false positives
- Confirmed positives undergo serotyping via recombinant immunoblot assays (HIV 1 vs 2 which differ in the antigenic determinants expressed on the cell surface)
- Repeat blood sample + EDTA blood for HIV viral load
What is immunofluorescence useful for? Pros and cons?
Useful for the direct detection of viral antigens in clinical samples (e.g. respiratory viruses); Relatively quick and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample
How does an ELISA (or EIA) work?
The antigen or antibody is bound onto a solid surface usually onto a micro-well plate and thereafter it is interacted with a counterpart antigen/ antibody. The counterpart antigen/ antibody is bound to an enzyme (direct ELISA) or alternatively can be detected by a secondary antibody that is in turn linked via conjugation to an enzyme (indirect ELISA). The detection of the assay is carried out by evaluating the enzyme activity of the conjugate which is implemented by incubation with a suitable substrate for the enzyme resulting in a generation of a quantifiable product. Generally, this results in a coloured end product which absorbs at a particular wavelength and can be correlated to the quantity of analyte in question present in the sample. Note that washing is carried out several times repeatedly after each step in order to ensure removal of unbound entities.
