Diagnosis of viral infections Flashcards
Key concepts of diagnosing of Viral Infections…
→ Not always possible to diagnose a infection clinically.
→ Often require a laboratory diagnostic test
→ Aid to diagnosis - history, examination & special investigations
→ Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
→ Important public health and infection control implications
→ It helps to know the natural history of the pathogen in the type of patient you are testing as this will affect test selection and interpretation
→ Consent - must be obtained for certain infections
→ Difference between diagnostic, monitoring and screening tests
What are some examples of possible test types?
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
What can viruses be visualised with? What has this been replaced with mostly?
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
How are specimens prepared/viewed with electron microscopy?
→ 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 some advantages and limitations of electron microscopy?
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 do we know about virus isolation in cell culture as a diagnostic technique?
What is a cytopathic effect?
Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE)of cells when a patient sample containing a virus incubated with a cell layer
→ Old method, now replaced by molecular techniques, but still needed for research or for rare viruses
→ Led to discovery of hMPV and Nipha virus in last 20 years and SARS-CoV-2 recently
→ Use different cell lines in test tubes or plates. Selection of cell types important
→ Slow, but occasionally useful
→ Different viruses may give different appearances (cells give off different cytopathic effects- once you see cytopathic effect, still need to identify and confirm what type of virus)
→ 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 does antigen detection, as a type of diagnostic technique, work?
What variety of methods can be used with antigen detection?
→ Antigen detection is the 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
A variety of different methods can be used
Commonest methods are:
–Direct immunofluorescence:
*Cell associated antigens
–Enzyme immunoassay:
*Free soluble antigens or whole virus–
Immunochromatographic methods
*Often used at point of care for rapid diagnosis
How does the diagnostic tool of immunofluorescence work?
→ 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
→ If fluorochrome is there, you will see green fluorescence (red means antibodies haven’t bound to antigen)
How can an ELISA be used for antigen detection?
*Enzyme-linked immunosorbent assay
→ A component of reaction is adhered to a solid surface
*Three formats:
→ Indirect
→ Direct (primarily antigen detection)
→ Sandwich
*Detection of Antigen by ELISA:
1. Plate is coated with a capture antibody
2. Sample is added and any antigen present binds to capture antibody
3. Enzyme-conjugated primary antibody is added, binds to detecting antibody
4. 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 does serology work for detecting antibodies?
Why do we call it serology?
→ Detection of antibodies
→ Indirect detection of the pathogen(not detecting the actual virus but antibodies produced in response to the virus)
→ 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 - we mostly look at antibodies but occasionally also antigens
→ Serological tests are not limited to blood & serum
– can also be performed on other bodily fluids such as semen and saliva
We call it serology because we usually use serum.
SERUM:
*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 can a diagnosis be made by 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 – 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 do Nucleic acid amplification tests work in summary as molecular diagnostic tests?
What stages does a nucleic acid amplification test go through? (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
Stages of a 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 some advantages and limitations of using NAATs?
Advantages:
*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:
*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(looking at more than 1 virus when testing)
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
Also need a control within the assay to prevent negative results due to PCR inhibition.
How can we use Genome Sequencing?
*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
