Diagnosis of viral infections Flashcards by BLACK H

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

How well did you know this?

Not at all

Perfectly

Which microbe scan be seen with light microscopy?

→fungi

→bacteria

How well did you know this?

Not at all

Perfectly

What type of microscope do viruses need?

→EM

How well did you know this?

Not at all

Perfectly

What are EM still useful for visualising?

→faeces and vesicle specimens

→characterizing emerging pathogens

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

What are the advantages of EM?

→Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses

How well did you know this?

Not at all

Perfectly

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 well did you know this?

Not at all

Perfectly

How do rotavirus look like in EM?

→look like wheels

How well did you know this?

Not at all

Perfectly

Which viruses cause vesicles?

→Herpes simplex

→Varicella zoster virus

How well did you know this?

Not at all

Perfectly

How do you differentiate between herpes and VZV since they both cause vesicles?

→clinical context,

→site of vesicle
→symptoms

How well did you know this?

Not at all

Perfectly

What is the vesicle derived from in herpes?

→Envelope membrane from cell that it has infected

How well did you know this?

Not at all

Perfectly

Give examples of poxvirus

→Smallpox
→Monkeypox
→Orf
→Cowpox

How well did you know this?

Not at all

Perfectly

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 well did you know this?

Not at all

Perfectly

How are viruses identified using cytopathic effects?

→antigen detection techniques or neutralisation of growth

→Cell culture plus antiviral – look for inhibition of cytopathic effect

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

What are possible specimens for antigen detection?

→Nasopharyngeal aspirates
→Blood (serum or plasma)
→Vesicle fluid
→Faeces

How well did you know this?

Not at all

Perfectly

Why is antigen detection being replaced with nucleic acid detection?

→greater sensitivity

How well did you know this?

Not at all

Perfectly

Which viruses are detected in nasopharyngeal aspirates?

→RSV, influenza

How well did you know this?

Not at all

Perfectly

Which viruses are detected in blood?

→Hepatitis B
→Dengue

→free antigen or whole virus

How well did you know this?

Not at all

Perfectly

Which viruses are detected in vesicle fluids?

→Herpes simplex,
→varicella zoster

→whole virus

How well did you know this?

Not at all

Perfectly

Which viruses are detected in faeces?

→Rotavirus,
→adenovirus

→whole virus

How well did you know this?

Not at all

Perfectly

What are three types of antigen detection?

→Direct immunofluorescence- Cell associated antigens

→Enzyme immunoassay- Free soluble antigens or whole virus

→Immunochromatographic methods

How well did you know this?

Not at all

Perfectly

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