Diagnosis of Viral Infections Flashcards
What are the possible test types for 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 can you see a virus?
Viruses need an electron microscope to be visualised
Nowadays it has been mostly replaced by molecular techniques
Possibly still useful for faeces and vesicle specimens
Useful in characterizing emerging pathogens
What is the electron microscopy procedure to see viruses?
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 and disadvantages of electron microscopy for virus visualisation?
Advantages:
- Rapid
- Detects viruses that cannot be grown in culture
- Can visualise many different viruses
Limitations:
- 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.
What is the cytopathic effect?
Different viruses may give different appearances
Different cell lines may support growth of different viruses
Identify virus using antigen detection techniques or neutralisation of growth
Cell culture plus antiviral – look for inhibition of cytopathic effect
How is antigen detection a method for viral diagnosis? What are the possible specimens?
Direct detection of the viral antigens.
Viral antigens, usually proteins – either capsid structural proteins or secreted proteins.
They can be detected in cells or free in blood, saliva or other tissues/organs.
Possible specimens include:
Nasopharyngeal aspirates (NPA) (cell-associated virus antigens)
- e.g. RSV, influenza
Blood (serum or plasma) (free antigen or whole virus)
- Hepatitis B
- Dengue
Vesicle fluid (whole virus)
- Herpes simplex, varicella zoster
Faeces (whole virus)
- Rotavirus, adenovirus
These techniques are being replaced by Nucleic acid detection methods due to improved test performance i.e. greater sensitivity
What are the most common methods of antigen detection?
A variety of different methods can be used
Commonest methods are:
- Direct immunofluorescence
· Used mostly for cell associated antigens
- Enzyme immunoassay
· Good for detecting free soluble antigens or whole virus
- Immunochromatographic methods e.g. lateral flow tests
Often used at point of care for rapid diagnosis
What is ELISA?
Enzyme-linked immunosorbent assay
- A component of reaction is adhered to a solid surface
Three formats:
- Indirect
- Direct (primarily antigen detection)
- Sandwich
Used for viruses, mainly hepatitis especially hepatitis B surface antigen
How is ELISA carried out?
- 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 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.
Negative result = NO colour change
How is serology used to detect viruses?
Detection of antibodies
Indirect detection of the pathogen
Diagnostic mode of choice for organisms which are refractory to culture
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
How is serum made?
Produced from processing blood
- Blood is coagulated with micronized silica particles
- Gel used to trap cellular components
Routinely serum tubes are centrifuged for 10 min at 1000xg
Supernatant (serum) is removed and stored
- 4ºC short term
- -20ºC long term
Routinely serum tubes are centrifuged for 10 min at 1000xg
Serum contains proteins, antigens, antibodies, drugs (some)and electrolytes
How does antibody detection detect viruses?
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 – usually 1 to 3 months
As IgM declines, IgG is produced
Quantity of IgG rises
Diagnosis can be made by
- detection of IgM (can be non specific)
- or by demonstration of seroconversion
· Negative IgG antibody at first
· Then presence of IgG antibody
How can hepatitis A be detected over time?
HAV infection followed by an incubation period
It will first be present in the serum
Then faeces
Then the person starts becoming symptomatic with things such as jaundice
ALT, an enzyme, will be released from the liver
IgM levels rise
IgG levels also rise after and as that peaks IgM levels start dropping
What is NAAT?
Nucleic acid amplification (NAAT)
- e.g. PCR although there are other examples* - 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 * LCR, SDA, LAMP
What are the stages of 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 and limitations of using NAAT?
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
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 real time PCR?
Different chemistries but all similar
Real time as amplification AND detection occur in REAL TIME i.e. simultaneously by the release of fluorescence
Avoids the use of gel electrophoresis or line hybridisation
Allows the use of multiplexing
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
What is meant by PCR inhibition?
Some substances inhibit PCR e.g. haem, bile salts.
Assays should always include an 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.
Include primers specific for the internal control material.
How could genome sequencing be used in viral diagnoses?
Partial or whole
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
New variants
- Diagnostic tests
- Vaccine efficacy
Can be Used to predict response to anti-virals e.g. for HIV in Rx naïve patients, or if clinical suggestion of resistance in drug experienced patients
Starting to think about using next generation sequencing to determine antiviral resistance, look at transmission of viruses.
How would you test for anti-viral resistance?
HIV as an example
Multiple viral enzyme targets
- Reverse transcriptase, protease,
- integrase,
- viral receptor binding proteins)
Look for mutations known to cause resistance.
Similar approach for hepatitis C, HSV, CMV (but different genes)
