Diagnosis of viral infections Flashcards
What are some possible test types to identify viruses?
Electron Microscopy
Virus isolation
Antigen detection
Antibody detection by serology
Nucleic Acid amplification tests( NAATs -PCR)
Sequencing for genotype and detection of antiviral resistance
How can we visualise microbes ?
By using electron microscopy
Outline Electron microscopy
Specimens will be dried on a grid
Then stained with heavy metal -uranyl acetate
Concentrated with application of antibody -Immunoelectron microscopy to concentrate the virus
Beams of electrons are used to produce an image
Wavelength of electron beam is much shorter than light and this results in a much higher resolution than light microscopy
What are the advantages of electron microscopy ?
Rapid
Detects viruses which cannot be grown in culture
Visualise many different viruses
What are the limitations of electron microscopy ?
Low sensitivity need 10^6 virions /ml
Maintenance
Skilled operators
Many viruses in same family look the same
What are some of the viruses which can be diagnosed using a electron microscope ?
Rotavirus (gastroenteritis) Adenovirus(gastroenteritis) Coronavirus Noravirus (calcivirus) Herpes virus that cause vesicles : -herpes simplex -Varicella zoster virus
EM cannot differentiate these different viruses depends on clinical context (in terms of HERPES)
Poxviruses
(Small pox,Monkey pox,cowpox
Describe the process of virus isolation in cell culture
Cells are intracellular obligates so require host cells to replicate and may cause a cytopathic effect of cells when patient sample containing a virus incubated with a cell layer.
Use cell lines in test tubes or plates
Example of discoveries :
hMPV and Nipha virus
Create a monolayer of cells and check for cytopathic effect
What is a cytopathic effect ?
structural changes in a host cell resulting from viral infection.
Different viruses may give different appearances
Identify virus using antigen detection techniques or neutralisation of growth -antiviral agents
Add antiviral and look for inhibition of cytopathic effect
What is Antigen detection ?
This is the direct detection of viral antigens
Viral antigens -proteins either capsid structural proteins or secreted proteins. Detected in cells or free in the blood/saliva /tissues/organs
Greater sensitivity -Nucleic acid detection instead
What are some possible specimens for viral antigen detection ?
Nasopharyngeal aspirates (NPA) cell associated virus antigens e.g.RSV,influenzxe
Blood(serum/plasma)
e.g.Hepatities B
Dengue (free antigen or whole virus )
Vesicle fluid
Herpes simplex , varicella zoster
(whole virus)
faces
-Rotavirus,adenovirus
What are the common methods of antigen detection?
Direct immunoflourescenece
-Cell associated antigens
Enzyme immunoassay
-Free soluble antigen s/whole virus
Immunochromatographic methods -lateral flow tests
Describe the process of immunofluorescence in antigen detection
Antigen from infected host cells is bound to a slide
Specifc antibody will bind and is tagged with a fluorochrome and mixed with sample
Viewed using a microscope equipped to provide ultraviolet illumination
What are immunochromatography tests?
These are also called lateral flow
combines separation of the sample molecules and reagents based on migration on a solid support by capillary flow. The identification and detection procedures are based on the antigen–antibody immune reaction.
What can be detected using immunochromatographic methods?
Dengue
Tropical infection caused by mosquito borne virus
Flavivirus
genus of positive-strand RNA viruses
Arthropod vector-Mosquitoes etc
Near patient test
Downside is they are not as specific as PCR
Outline the method of ELISA
This is enzyme-linked immunosorbent assay
A component of reaction is adhered to a solid surface
Three formats :
- Indirect
- Direct (primarily antigen detection)
- Sandwich -hepatitis surface antigen
What is the method of detection of antigens through ELISA
There is a micotiter tray with individual wells.
Plate is coated with a capture antibody
(Antibody will be absorbed to well)
Sample is added and any antigen present binds to capture antibody
(complementary antigen binds to antibody )
Enzyme-conjugated primary antibody is added ,binds to detecting antibody
(enzyme linked antibody specific for antigen )
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
A negative result will mean no colour change.
What is serology ?
This is the indirect way of detecting antibodies which the body has produced in response to the virus.
In symptomatic patients
Determines if vaccination has been successful
Not limited to blood /serum
can be performed on semen /saiva
What is serum ?
This is produced by processing blood
Blood is coagulated with micronized silica particles
Gel is used to trap cellular components
Routinely serum tubes are centrifuged for 10 min at 1000xg
Supernatant (serum) is removed and stored
Serum tubes are centrifuged for 10 mins at 1000xg
Serum contains: proteins ,antigens ,antibodies ,drugs and electrolytes
How can we diagnose a patient by using antibody detection ?
When infected with a virus the humoral immune response takes place resulting in production of immunoglobulins i.e. antibodies
IgM antibodies specific to the virus are produced first
IgM present for a variable period -1-3 months
As IgM declines , IgG is produced
The diagnosis can therefore be made by detection of IgM -Non specific -can lead to false positive
or demonstration of seroconversion
Negative IgG antibody at first
Then presence of IgG antibody
What is IgM?
Immunoglobulin M -one of the several antibody types
-Largest
What is IgG?
This is the main antibody which is found in blood and extracellular fluid
Allowing the control of infection
How do laboratories carry out serology ?
Detection of antibody and or antigens
Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immunochemiluminescence
Outline Nucleic acid amplification (NAAT)
An example is PCR but others do exist
Can detect RNA/DNA (of the virus)
Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample
Qualitative /quantitative
Requires nucleic acid extraction prior to the amplification
What are the different stages of NAAT test ?
1.Specimen collection (Blood,salvia,CSF,Faeces,respiratory) 2.Extraction of the nucleic acid 3.DNA transcription for RNA viruses 4.Cycles of amplification of DNA target -Requires polymerase and dNTPS plus other reagents (Threshold of detection)
- Detection of amplicons
- After amplification
- real time
What are the advantages of using NAATS?
They may be automated, POCT possible
Usually highly sensitive and specific ,generates huge numbers of amplicons
Rapid -quick as 15mins -a few hours
Useful for detecting viruses in nucleic acid to make a diagnosis
First time infections + reactivations (latent viruses)
Useful for monitoring treatment responses
-Quantitative
What are some limitations of using NAATS?
They will generate large numbers of amplicons and this may lead to contamination.
Need to have an idea of what viruses you are looking for as will require primers and probes that are specific to the target
Mutations in target sequence may lead to “dropout” e.g. S gene dropout seen with SARS-CoV-2 variants
(The target will no longer allow primers to bind to it )
There were false negative results
What is Real time PCR
Real time because amplification and detection occur in real time -simultaneously by the release of fluorescence
Avoids the use of gel electropheresis /line hybridisation
Allows the use of multiplexing
(way of sending multiple signals or streams of information over a communications link at the same time )
Define Multiplex PCR
Term used when more than one pair of primers is used in a PCR.
(Looking for more than one virus)
Enables amplification of multiple DNA targets in one tube
Detection of multiple viruses in one CSF specimen e.g. HSV1,HSV2,VZV ,enterovirus ,mumps virus
How can we ensure there are no false negative ?
There are substances which can inhibit PCR for e.g. haem ,bile salts
Assays should always include internal positive control as results could incorrectly be reported as negative
The IC can be anything as long as RNA/DNA respectively depending on nature of target .
Primers which are always amplified
Why is Genome sequencing used and why is it important ?
-Partial /whole
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
New variants:
Diagnostic tests
Vaccine efficacy
Used to predict response to antivirals e.g. HIV in Rx naïve patients or clinical suggestion of resistance in durg experienced patients
-Was it mutation ? what was the reason
Outline how we can combine methods .For e.g in HIV diagnosis and management
SLIDE
What is anti-viral resistance testing ?
HIV as example :
Multiple viral enzyme targets :
Three main viral enzymes
Look for mutations known to cause resistance
Define screening
This is the testing for specific infections in a risk group
SLIDE
