DVIP Flashcards
What are the types of tests to diagnose viral infections?
→Electron Microscopy
→Virus isolation (cell culture)
→Antigen detection
→Antibody detection by serology
→Nucleic acid amplification tests (NAATs e.g. PCR)
→Sequencing for genotype and detection of antiviral resistance
Which microbe scan be seen with light microscopy?
→fungi
→bacteria
What type of microscope do viruses need?
→EM
What are EM still useful for visualising?
→faeces and vesicle specimens
→characterizing emerging pathogens
Describe the process for EM visualisation
→Specimens are dried on a grid
→Can be stained with heavy metal e.g. uranyl acetate
→Can be concentrated with application of antibody i.e. immuno-electron microscopy to concentrate the virus
→Beams of electrons are used to produce images
→Wavelength of electron beam is much shorter than light, resulting in much higher resolution than light microscopy
What are the advantages of EM?
→Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses
What are the limitations of EM?
→low sensitivity need 106 virions/millilitre.
→Requires maintenance
→Requires skilled operators
→Cannot differentiate between viruses of the same virus family.
How do rotavirus look like in EM?
→look like wheels
Which viruses cause vesicles?
→Herpes simplex
→Varicella zoster virus
How do you differentiate between herpes and VZV since they both cause vesicles?
→clinical context,
→site of vesicle
→symptoms
What is the vesicle derived from in herpes?
→Envelope membrane from cell that it has infected
Give examples of poxvirus
→Smallpox
→Monkeypox
→Orf
→Cowpox
How does virus isolation in cell culture help in diagnosis?
→Create a monolayer of cells and then add clinical specimen
→watch for cytopathic effect
How are viruses identified using cytopathic effects?
→antigen detection techniques or neutralisation of growth
→Cell culture plus antiviral – look for inhibition of cytopathic effect
What are viral antigens?
→usually proteins
→either capsid structural proteins or secreted proteins
→detected in cells or free in blood, saliva or other tissues/organs
What are possible specimens for antigen detection?
→Nasopharyngeal aspirates
→Blood (serum or plasma)
→Vesicle fluid
→Faeces
Why is antigen detection being replaced with nucleic acid detection?
→greater sensitivity
Which viruses are detected in nasopharyngeal aspirates?
→RSV, influenza
Which viruses are detected in blood?
→Hepatitis B
→Dengue
→free antigen or whole virus
Which viruses are detected in vesicle fluids?
→Herpes simplex,
→varicella zoster
→whole virus
Which viruses are detected in faeces?
→Rotavirus,
→adenovirus
→whole virus
What are three types of antigen detection?
→Direct immunofluorescence- Cell associated antigens
→Enzyme immunoassay- Free soluble antigens or whole virus
→Immunochromatographic methods
When is antigen detection mostly used?
→point of care
Give an example of a virus detected using immunochromatographic
→dengue
→Non structural proteins circulating in blood
What is ELISA?
→Enzyme-linked immunosorbent assay
→used to detect antigen and antibodies
What are three formats of ELISA?
→Indirect
Direct (primarily antigen detection)
Sandwich
Describe the process of ELISA
→Plate is coated with a capture antibody
→Sample is added and any antigen present binds to capture antibody
→Enzyme-conjugated primary antibody is added, binds to detecting antibody
→Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change
When is there a colour change in ELISA?
→substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present
What can serology be used to detect?
→Detect an antibody response in symptomatic patients
→Determine if vaccination has been successful
→Directly look for antigen produced by pathogens
What can serology tests be performed on?
→blood
→serum
→saliva
→semen
How is serum processed from blood?
→Blood is coagulated with micronized silica particles
→Gel used to trap cellular components
What does serum contain?
→antigens,
→antibodies,
→drugs (some)
→electrolytes
Describe the Igs changes in response to infection
→IgM antibodies specific to the virus are produced first
→IgM present for a variable period – usually 1 to 3 months
→As IgM declines, IgG is produce
How can diagnosis be made using Ig antibodies?
→detection of IgM (can be non specific)
→demonstration of seroconversion
What are the levels of IgM and IgG in acute or recent Hep A infections?
→IgM positive
→IgG can be positive or negative
What are the levels of IgM and IgG in Hep A in resolves infection/
→IgM negative
→IgG positive
Why is antigen and antibody detection used for Hep B, C and HIV infections?
→establish whether acute or chronic infection
→therapeutic implications
Describe NAAT detection
→PCR
→Can detect RNA or DNA
→Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample
→May be qualitative or quantitative
→Requires nucleic acid extraction prior to the amplification
Describe the stages of NAAT test
→Specimen collection- blood, saliva, csf
→Extraction of nucleic acid
→DNA transcription for RNA viruses
Cycles of
→Amplification of DNA target
requires polymerase and dNTPs plus other reagents
→Detection of amplicons
What can detection of amplicons be?
→After amplification
→Or real time
What are the advantages of NAATs?
→be automated
→highly sensitive and specific
→rapid
→Useful for detecting viruses to make a diagnosis
→Useful for monitoring treatment response
- Quantitative e.g. HIV
What are the limitations of using NAATs?
→Generates large numbers of amplicons
→Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target
→Mutations in target sequence may lead to “dropout”
What is RT-PCR?
→amplification AND detection occur in REAL TIME
What are the benefits of RT-PCR?
→Avoids the use of gel electrophoresis or line hybridisation
→Allows the use of multiplexing
What is multiplex PCR?
→when more than one pair of primers is used in a PCR
What does multiplex PCR allow?
→amplification of multiple DNA targets in one tube
→e.g. detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2, VZV, enterovirus, mumps virus
What is PCR Inhibition?
→Some substances inhibit PCR
What are examples of PCR inhibitors?
→haem, bile salts
Why should internal controls be used in PCR assays?
→so results are not read as false negatives
What can internal controls be?
→RNA
→DNA
What is genome sequencing useful for?
→outbreak investigation by showing identical sequences in suspected source and recipient
→vaccine efficacy
What tests are involved in HIV diagnosis?
→Antibody and antigen detection for initial diagnosis
→Screening test (EIA)
→Confirmatory test (EIA)
What tests are involved in monitoring treatment for HIV?
→Quantification of virus in blood
→NAAT
What test is used for resistance testing in HIV?
→sequencing
What does multiple viral enzyme target for anti-viral resistance?
→Reverse transcriptase, protease,
→integrase,
→viral receptor binding proteins)
Why does screening ned confirmatory testing?
→some false positives
