L8 - Diagnosis of Viral Infections Flashcards
How can electron microscopy can be used in diagnosis?
Viruses can be visualised with electron microscope
Mostly replaced by molecular techniques
Possibly still useful for faeces and vesicle specimens
Useful in characterising emerging pathogens
What are the limitations of using electron microscopy for diagnosis?
low sensitivity need 106 virions/millilitre. May be enough in vesicle secretion/stool
Requires maintenance
Requires skilled operators
Cannot differentiate between viruses of the same virus family.
Describe how viral cells can be isolated and grown in cell culture?
A monolayer of cells is created on the bottom of a flat flask.
Clinical specimen is then added to it, and it is observed for a cytopathic effect.
Virus can then be identified using antigen detection techniques
State some specimens for Antigen detection?
Blood
Nasopharyngeal aspirates
vesicle fluid
feaces
What are the different types of antigen detection methods and what are they commonly used for?
Direct immunofluorescence
Cell associated antigens
Enzyme immunoassay
Free soluble antigens or whole virus
Immunochromatographic methods
Describe the Immunofluorescence technique of antigen detection?
Antigen (from infected host cells in sample) bound to slide
Specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with sample
Viewed using a microscope equipped to provide ultraviolet illumination
Describe the ELISA technique of antigen detection?
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
The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present. Negative result = NO colour change
What is Serology and what can it be used for?
Antibody detection
Detection of antibodies
Indirect detection of the pathogen
Serology can be used to:
Detect an antibody response in symptomatic patients
Determine if vaccination has been successful
(Directly look for antigen produced by pathogens)
Serological tests are not limited to blood & serum
can also be performed on other bodily fluids such as semen and saliva
What are the stages of a Nucleic Acid Amplification Test (NAAT)?
Specimen collection 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 After amplification Or real time
What are the advantages of using NAATs?
May be automated. POCT possible
Usually highly sensitive and specific, generates huge numbers of amplicons
Rapid – can be as quick as 15 minutes – usually a few hours
Useful for detecting viruses to make a diagnosis
- At first time of infection e.g. measles, influenza
- During reactivation e.g. cytomegalovirus
Useful for monitoring treatment response
- Quantitative e.g. HIV, HBV, HCV, CMV viral loads
What are the limitations of using NAATs?
Generates large numbers of amplicons. This may cause contamination.
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” e.g. S gene dropout seen with SARS-CoV-2 variants
What is Multiplex PCR?
Multiplex PCR is the term used when more than one pair of primers is used in a PCR. It enables the 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
