2. Virology lab Flashcards
Which disease is a widespread macularpapular (flat) rash typical of?
Measles
Which disease is a dermatomally distributed vesicular (raised) rash typical of?
Shingles (varicellar zoster)
What can we detect in the (virology) lab?
- Infectious viruses and their components
- Protein components of the virus (antigens)
- Genetic components of the virus (DNA or RNA)
- The host response e.g. antibodies and cell responses
What is the viral load?
Amount of viral nucleic acid present in the blood or other bodily fluids
What is sensitivity and specificity?
- Sensitivity - the test’s ability to correctly identify positive sample (less false negative results)
- Specificity - the test’s ability to correctly identify negative samples (less false positive results)
What samples can you use for detection of respiratory viruses?
- Throat swap
- Nasopharyngeal aspirate (NPA)
- Bronchoalveolar lavage (BAL)
IF / PCR
What samples can you use for a rotavirus, adenovirus and norovirus detection?
• Stools
Antigen detection (EIA) / PCR
What samples can you use for BK virus and adenovirus detection?
• Urine
PCR
What samples can you use for herpes viruses and enteroviruses?
• CSF
PCR
What type of blood can you use for serology (antibody detection)?
Clotted blood
What samples can you use for viral load testing/PCR?
Blood (EDTA)
What does serology consist of and give examples?
• Testing for specific antibodies or viral antigens in serum or other body fluids (saliva, CSF)
- HIV (antibody + p24 antigen)
- Hepatitis A - IgM + IgG
- CMV and EBV - IgM + IgG
- M, M, R - IgM + IgG
- VZV - IgG
What is the difference between the IgM and IgG results?
- IgM - marker for recent or acute infection (duration: 3 months)
- IgG - in absence of IgM - infection at some time in the past, or immunisation (duration: life long)
What is the specificity like for IgM tests and how can you determine the significance of a positive IgM result?
- Low specificity
- Therefore, more false positives
• Antibody avidity test for significance of positives
How does antibody avidity change over the duration of an infection?
- Low avidity at acute stages of infection
* Maturation of immune response - avidity increases
What does a low and high antibody avidity indicate?
- Low - recent/acute infection
* High - past infection
What generation of EIA are we on?
- 4th generation
* Ab + p34 Ag detection
What does confirmatory testing in HIV serology involve?
- Second assay
- Exclude non-specific reactivity (false positives)
- Confirmed positives undergo typing (HIV 1 vs. 2)
- Repeat blood samples and EDTA blood is required for HIV viral load from all new positives
- Genotyping and baseline resistance testing
How does serology allow early treatment?
- Identifies infection at slightly earlier stages
- Before antibodies reach detectable levels
- Highly automated - more samples tested, quickly and cheaply
- However, more false negatives
What is virus isolation in cell culture useful for?
Phenotypic antiviral susceptibility testing
Why is virus isolation in cell culture only performed in specialised laboratories?
Because it’s slow and time consuming
How can you visualise viruses and what samples would you used?
- Electron microscopy
- Limited - time consuming and costly
- Stool and vesicle fluid samples
How can you directly detect viral antigens in clinical samples and when is this method used?
- Immunofluorescence
- Out-of-hours respiratory virus testing
- Rapid and inexpensive
- Subjective and dependent on technician skill and sample quality
What disease usually causes hyper-inflated lungs in young children and how can you detect it?
- Bronchiolitis
- RSV
- Immunofluorescence and x-ray
What type of PCR do you use for respiratory virus testing?
- Multiplex PCR assay
- Can test for more than one virus per tube (3 or 4)
- Quicker and more cost effective
Which CNS diseases should you consider having obtained the following information: • meningitis/encephalitis • young child with febrile fits • immunocompromised • recent travel to endemic region • SSPE (progressive brain inflammation)
- Meningitis/encephalitis - HSV, VZV, enterovirus
- Young child with febrile fits - HHV-6, parechovirus
- Immunocompromised - CMV, EBV, JC virus
- Recent travel to endemic region - consider Japanese Encephalitis, West Nile virus, equine encephalitides, tick borne encephalitis
- SSPE - measles (antibody index)
Where is it better to take a sample from during diarrhoea and vomiting?
- Stool preferred
* Vomit - low yield
What is the difference between enteric and enteroviruses?
- Enteric viruses - causes diarrhoea and vomiting e.g. norovirus, rotavirus
- Enteroviruses - genus of RNA viruses (intestinal transmission)
How do you amplify RNA/DNA virus sequences?
• PCR
• Starting block is double-stranded DNA
- if you have RNA: make a ds-DNA copy of the viral RNA
- reverse transcription, using reverse transcriptase from retroviruses
• Strands denatured (95°C),
• Primers annealed (50°C)
• Chain elongation - taq polymerase (72°C)
• Sequence doubles at each completed cycle - exponential amplification
What type of PCR detects the amplification of DNA in real time, qualitatively detecting gene expression through the creation of complementary DNA (cDNA) transcripts from RNA?
RT-PCR (qPCR - quantitive)
What is phylogenetic analysis used for?
Investigation of outbreaks and transmission incidence
