Diagnosis of Viral Infections Flashcards
What are 6 ways of testing for viruses?
β Electron microscopy β Virus isolation β Antigen detection β Antibody detection by serology β Nucleic acid amplification tests β Sequencing for genotype and detection of antiviral resistance
What magnification do viruses need?
β 20,000x
What magnification do bacteria, fungi and helminths need?
β 400-1000x
What has electron microscopy of viruses been replaced with?
β Molecular techniques
What can electron microscopy of viruses still be used for?
β Feces and vesicle specimens
How are virus specimens prepared for electron microscopy?
β specimens are dried on a grid
β they are stained with heavy metal (uranyl acetate)
β can be concentrated with application of antibody
β beams of electrons are used to produce images
Why does electron microscopy have a higher resolution than light microscopy?
β The wavelength of an electron beam is much shorter than light
β this results in much higher resolution than light microscopy
What are the 3 advantages of electron microscopy for viruses?
β Rapid
β detects viruses that cannot be grown in culture
β can visualise many different viruses
What are the 4 disadvantages of electron microscopy for viruses?
β Low sensitivity so you need 10^6 virions per ml
β requires maintenance
β requires skilled operators
β cannot differentiate between viruses of the same family
What does rotavirus cause?
β gastroenteritis
What does adenovirus cause?
β gastroenteritis
What does coronavirus affect?
β respiratory system
What does norovirus cause?
β Gastroenteritis
What does herpes simplex virus cause?
β vesicles
What does herpes (varicella zoster) virus cause?
β chickenpox
How can you differentiate between herpes viruses?
β You canβt with EM
β it depends on clinical context, site of vesicle and symptoms
What are the 4 types of poxviruses?
β Smallpox
β Monkeypox
β Cowpox
β Orf
What do viruses require to replicate?
β Host cells
How can you investigate cytopathic effect?
β Take a patient sample containing the virus sample
β incubate with a cell layer
β observe cytopathic effects
What two viruses have been discovered by the cytopathic effect technique?
β hMPV
β Nipha virus
How do you test antivirals?
β cell culture + antiviral
β look for inhibition of cytopathic effect
How do you identify viruses in cell cultures?
β using antigen detection
β neutralisation of growth
What viral components can be detected?
β Viral antigens
β they are usually proteins : either capsid or structural proteins
What do virus infected cells display?
β Viral antigens on their surfaces
What viruses do you take nasopharyngeal aspirates for?
β RSV
β Influenza
What viruses do you take blood samples for?
β Hepatitis B
β Dengue
What viruses do you take vesicle fluid samples for?
β Herpes simplex
β Varicella zoster
Which viruses do you take feces samples for?
β Rotavirus
β Adenovirus
What are the 3 most common virus detection methods?
β Direct immunofluorescence
β Enzyme immunoassay
β Immunochromatographic methods
How does immunofluorescence work?
β Antigen from infected host cells in sample bound to slide
β specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with sample
β viewed using a microscope that provides UV illumination
What virus is dengue caused by and how is it spread?
β Flavivirus
β arthropod vectors
What are the three types of ELISA test?
β direct
β indirect
β sandwich
How does antigen detection in the ELISA test work?
1) the plate is coated with a capture antibody (antibody that will capture an antigen you are interested in e.g one that the virus has)
2) The sample is added and any antigen present binds to capture antibody
3) Enzyme conjugated primary antibody is added and it binds to the detecting antibody
4) chromogenic substrate is added and is converted by the enzyme to a detectable form e.g color change
What is a negative result in an ELISA?
β no color change
What does the humoral system do when infected with a virus?
β Produce antibodies
How does diagnosis by antibody detection work?
β Detection of IgM
β demonstration of seroconversion
β negative IgG at first then positive
How do antibody classes vary during a viral infection?
β IgM antibodies specific to the virus are produced first
β IgM is present for a variable period 1-3m months
β IgM declines and IgG is produced
What 3 things can serology be used for?
β Detect an antibody response in symptomatic patients
β Determine if vaccination has been successful
β Directly look for antigen produced by pathogens
What fluids can serological tests be done on?
β blood
β serum
β semen
β saliva
What is serum produced from?
β processing blood
How is serum produced?
β Blood is coagulated with micronized silica particles
β Gel is used to trap cellular components
How are serum tubes stored and at what temperature?
β Centrifuged for 10 minutes at 1000xg
β supernatant is removed and stored
β 4 C short term and -20C long term
What does serum contain?
β proteins β antigens β antibodies β Drugs β electrolytes
Describe how you would detect the measles antibody using an antigen down ELISA?
1) Attach measles antigen to the bottom of a well
2) Add the patients sample to the well
3) If the patient has measles antibodies in their blood they will bind to the antigen in the well
4) you add an animal antibody attached to an enzyme which will bind to the human antibody
5) at each stage you wash out the wells so you donβt have floating antibodies
How can you see what stage of Hepatitis A a patient is at?
β you measure their serum antibody levels
β IgM is produced first
β IgG persists
What are the signs of acute or recent Hepatitis A?
β Patient having IgM
What are the signs of immunisation or a resolved Hepatitis A infection?
β patient having IgG
What happens during second exposure to Hepatitis A?
β you will have IgG antibodies from the 1st exposure
β on second exposure there is a really high IgG response
β hardly any IgM
How do you detect antibodies and antigens in the blood?
β Enzyme immunoassays
in what 3 diseases is it useful to detect antigens and antibodies?
β Hepatitis B
β HIV
β hepatitis C
Why is it useful to detect antibodies and antigens?
β It allows us to establish whether it is an acute or chronic infection
What do molecular diagnostic tests require prior to amplification?
β Nucleic acid extraction
What can molecular diagnostic tests detect?
β RNA or DNA
What are the 5 advantages of molecular diagnostic tests?
β May be automated
β Highly sensitive and specific, generates huge numbers of amplicons
β rapid
β useful for detecting viruses to make a diagnosis
β useful for monitoring treatment response
Why do you need quantitative diagnostic tests?
β to measure viral load
What is NAAT?
β nucleic acid amplification
What are the 4 limitations of NAAT?
β May detect other viruses which are not causing the infection
β Exquisitely sensitive and can generate large numbers of amplicons
β May cause contamination
β you have to have an idea of what virus you are looking for as you need primers and probes specific to it
What does real time PCR avoid the use of?
β gel electrophoresis or line hybridisation
What is multiplex PCR?
β when more than one pair of primers is used in PCR
What does multiplex PCR enable?
β Amplification of multiple DNA targets in one tube
β detection of multiple viruses in one specimen of CSF
Describe how Specific Taqman probes work?
1) Taqman probe complementary to region of interest, binds between primers
2) Oligonucleotide probe with fluorescent reporter at the 5β end and a quencher at the 3β
3) The quencher prevents the reporter fluorescing when excited if in close proximity
4) Taqman probe hybridises to the region of interest
5) This occurs during the annealing phase of PCR
6) Fluorescence is prevented due to the proximity of the quencher
7) Taq polymerase extends from the 3β end of the primer as normal
8) The Taq possesses 5β-3β nuclease activity and hydrolyses the probe
9) The reporter is removed from the quencher and fluorescence can be detected
10) For any given cycle within the exponential phase, the amount of product and hence fluorescent signal is directly proportional to the initial copy number
What is the cycle threshold?
β The amount of cycles required to cross the threshold
What substances inhibit PCR?
β haem
β bile
What should assays include so false negatives donβt happen?
β An internal positive control
What is organism sequencing used for?
β to predict the response to anti-virals
β if there is resistance in drug experienced patients
What is the consensus sequence based on?
β clinical observation of resistance or in vitro evidence
How do you make an initial diagnosis of HIV?
β Antibody and antigen detection
How do you monitor the response to HIV?
β check the viral load with NAAT
β quantify the virus in the blood
What are the viral enzyme targets for antiviral resistance testing?
β Reverse transcriptase
β integrase
β Viral receptor binding proteins
Why do you screen for viruses?
β It may have an implication for others e.g antenatal
What are the three viruses you screen for?
β HIV
β HBV
β HCV
What is needed in addition to screening and why?
β may have some false positives so you need a specific confirmatory test
