Virology lab Flashcards
What is it called if a rash has some areas that are raised and some that are flat?
Maculopapular
Macular = flat lesions
What is a blotchy appearance rash typical of?
Measles
If the distribution of a rash is dermatomal, what is this typical of?
Shingles
What can virologists detect?
Infectious virus- virus isolation and EM
Protein components- antigens on the virus (p24 antigen in HIV, surface antigen in HBV, etc)
Genetic components of the virus (DNA or RNA)- quantitative/qualitative tests available
Host response- antibody or cell response
Why are cell culture and electron microscopy not used much anymore?
They have been replaced by PCR (genome detection)
What is sensitivity?
A test’s ability to correctly identify positive samples –> affects the rate of false negatives
What is specificity ?
A test’s ability to correctly identify negative samples –> affects the rat eof false-positives
What is a marker of recent infection?
IgM - IgG comes later on
What is essential for diagnosis and monitoring of HIV, HBV and HCV and also for CMV and EBV in immunocompromised?
Viral load- quantification of genomes
What typical sort of samples are used by virologists?
Throat swab, nasopharyngeal aspirate, bronchoaveolar lavage- detection of respiratory viruses by PCR
Stools- For rotavirus, adenovirus and norovirus antigen detection or PCR
Urine- for BK virus and 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 and or PCR
What is positive IgG with absent IgM consistent with?
Past infection or immunisation
What is antibody avidity testing used for?
Confirming a positive IgM result
A sample of serum is taken a few weeks apart and you detect which one binds more strongly to the sample.
What does antibody avidity mean?
The strength with which antibodies bind to a specific antigen
How does antibody avidity change throughout infection?
Early on in course of infection, avidity is low
Then you get maturation of antibody response so avidity gradually increases over a period of 3-6 months
If you have high antibody avidity then it makes it unlikely that infection occured in last 3 months
What is virus isolation in cell culture still useful for despite being slow and time consuming?
Phenotypic antiretroviral susceptibility testing (HSV)
What is immunofluorescence still useful for? What are its pros and cons?
DIRECT detection of viral antigen in clinical samples DIF (e.g. respiratory viruses),
Rapid and inexpensive but subjective and dependent on skill of technician and quality of sample
What are the recommended sample types used for investigation of respiratory tract infections?
Throat swab and nose swab Nasopharyngeal swab Nasopharyngeal aspirate Bronchoaveolar lavage Endotracheal tube secretion
SENT AWAY FOR PCR
What is multiplex PCR? How does it work?
Where you test for several viruses in one tube rather than single tube for each virus (makes it faster) - although you can only do 3/4 at a time
METHOD: markers of genes of different viruses are added. Bands are compared on PCR
What are the recommended sample types for CNS disease- meningitis and encephalitis?
- CSF for PCR (HSV, VZV, enterovirus etc)
- Stools and throat swab for enterovirus detection (PCR)
- Blood for serology and or PCR for west nile or japanese encephalitis virus infection and other arboviruses
What is PCR?
Polymerase chain reaction- Method for amplifying specific RNA (RT-PCR) or DNA sequences
30 cycles of denaturing, primer annealing and chain elongation
What is the starting block for PCR?
If you are looking for an RNA virus e.g. influenza then you first need to make a dsDNA copy of the RNA using REVERSE TRANSCRIPTASE
You then denature the dsDNA into two separate strands - achieved by heating
What is the most important enzyme in PCR?
Taq polymerase
What is phylogenetic analysis used for?
Investigating outbreaks
List some different diagnostic methods available for viruses.
Cell culture and EM Genome detection by PCR Genome sequencing - genotyping, antiviral resistance testing Antibody detection (serology - EIA) Antigen detection (Immunofluorescence, enzyme immunoassay EIA) Quantification of antibody/antigens Serotyping e.g. HIV Quantification of genomes - "viral load"
What is serology used for?
Testing for a specific antibody or viral antigen e.g. in serum (salive, CSF)
Compare the duration of IgM and IgG.
IgM ~3months (rapid increase, peak, levels offwithin 3 months)
IgG = lifelong (rises slowly on graph and remains high)
What can a 4th generation EIA of HIV detect?
Antibody and p24 antigen(at the core of the virus)
Describe serology tests for HIV.
- 4th gen EIA - detects both Ab and p24 antigen (if this is positive then a second assay is done for confirmatory testing to exclude false positives)
- Confirmed positives –> typing (HIV 1 or 2?)
- Blood sample released and EDTA blood taken to assess viral load, genotype and for baseline resistance testing.
What is electron microscopy useful for? What types of samples?
For viruses which are too small to be seen under microscope
Sample types: stool, vesicle fluids.
BUT rarely used now,
Describe the appearance of these viruses under EM:
Herpes
Rabies
Ebola
Herpes - core with 20 faces, enveloped virus.
Rabies - bullet shaped
Ebola - filamentous virus
What temperatures does PCR operate at?
Denature - 95oC
Annealing - ~50oC
Extension - 72oC (Taq polymerase and nucleotides)
What does Taq polymerase come from?
Thermophilus aquaticus
List 2 applications of sequencing viruses.
Antiviral resistance testing - vital for selecting the correct antiretroviral drugs
Phylogenetic analysis - tool for investigating outbreaks
