Diagnosis of viral infections Flashcards
Why do we use laboratory diagnostic tests?
- to confirm/diagnose an infection
- to monitor patient’s disease (e.g. monitor response to treatment)
- to screen patients who are perfectly well to see whether they have a particular infection (e.g. pregnant women)
Examples of laboratory diagnostic tests 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 we visualise a virus?
Electron microscopy
-useful in characterising emerging unknown pathogens never seen before
Method of electron microscopy to visualise virus
1) Specimens dried on a grid
2) Can stain with either heavy metal (e.g. uranyl acetate) or antibody (i.e. immuno-electron microscopy)
3) Beams of electrons produce images
Resolution of electron microscopy compared to light microscopy
The wavelength of electron beams is much shorter than light, resulting in electron microscopy having a much higher resolution than light microscopy
Advantages of electron microscopy
- Rapid
- Detects viruses that cannot be grown in culture
- Can visualise many different viruses
- High Resolution
Limitations of electron microscopy
- Low sensitivity; need 10^6 virions/millilitre present. May be enough in vesicle secretion/stool
- Expensive equipment
- Requires maintenance
- Requires skilled operators
- Cannot differentiate between viruses of the same virus family
What does a rotavirus look like under EM?
“Wheel” Appearance
What does an adenovirus look like under EM?
Icosahedral capsids
What does a coronavirus look like under EM?
“Crown-like” structure around a “dumbbell” shaped virus
What does an astrovirus look like under EM?
“Star-like” structure
What do herpesviruses HSV and VZV look like under EM?
Herpes viruses such as Herpes simplex and Varicella zoster virus cause vesicles (fried egg appearance)
- disadvantage is that EM cannot differentiate between the two
- diagnosis would depend on clinical context
Clinical presentation of HSV vs VZV
If patient presented with fever and itchy widespread vesicular rash all over the body → chances are it is a VZV infection → chicken pox
If patient was older and presents with vesicles in one dermatome/one nerve distribution of the body → chances are it is shingles (reactivated VZV infection).
If patient presented with vesicular lesions just around the lip → herpes simplex
What does a poxovirus look like under EM?
Ball of wool
Examples:
- smallpox
- monkeypox
- cowpox
- Orf
EM can’t differentiate these (like HSV and VZV) and so diagnosis depends on clinical context
How can we grow viruses in vitro?
Virus Isolation in Cell Culture
-require host cells to replicate
What effect does the virus have on cells within a cell culture?
Cytopathic Effect (CPE), which can suggest what the identity of the virus :
- different viruses give different appearances
- different cells lines support the growth of different viruses
What can virus isolation in cell culture be used for?
Anti-viral sensitivity testing
-cell culture + anti-viral= look for inhibition of cytopathic effect
How can we identify a virus from its cytopathic effect?
Using antigen detection techniques
-direct detection of the virus
How do we use antigen detection techniques to detect a virus?
Virally infected cells may display viral antigens on their surface, therefore we acquire a specimen from a patient and do a test to detect those viral antigens
What are some common specimens we acquire to detect viral antigens?
· Nasopharyngeal aspirates (NPA) → for RSV, influenza
· Blood (serum or plasma) → for Hepatitis B or Dengue
· Vesicle fluid → for Herpes simplex, varicella zoster
· Faeces → for Rotavirus, adenovirus
Methods used for antigen detection
- Direct Immunofluorescence
- Enzyme Immunoassay (ELISA)
- Immunochromatographic methods
*so rapid that they are often used at point of care (e.g. bed-side) for rapid diagnosis
How is direct immunofluorescence used for antigen detection?
1) Infected host cells are extracted from an obtained specimen and are layered onto a microscopic slide, meaning the antigens from those infected host cells are bound to the slide.
2) Add specific antibody (polyclonal or monoclonal) to that antigen. That antibody is tagged to a fluorochrome (labelled antibody) and mixed with sample.
3) If there are any cells which have the virus in/on them, the antibody will bind to those cells and cells will fluoresce, and this can be viewed under an ultraviolet microscope.
What is the disadvantage of using direct immunofluorescence for antigen detection?
Not sensitive
-need many infected cells in the specimen to be able to see the virus
How is immunochromatography used for antigen detection?
1) Patient’s blood is obtained and loaded onto apparatus containing antigen-specific antibody
2) Visible line appears on the apparatus where there is binding of the antigen in the patient’s blood with the antibody
What is immunochromatography used in the diagnosis of?
Used in the diagnosis of dengue:
- flavivirus
- arthropod vector
*common infection in returning travellers
How is ELISA used for antigen detection?
1) Well is coated with a capture antibody. This is antibody we know will capture the particular antigen we are interested in.
2) Patient sample is added and any complementary antigen present binds to capture antibody.
3) Enzyme-conjugated primary antibody specific for test antigen is added, binds to the captured antigen, forming a sandwich
4) Chromogenic substrate for the enzyme is added, and the reaction between the two produces a product that causes a visible colour change
What is required for the chromogenic substrate in ELISA to change colour?
The substrate will only 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 viral infections?
Antibody detection
-indirect detection of pathogen which involves looking for ANTIBODIES in patient’s sample
What is serology?
study of blood serum to identify whether people have been exposed to a particular pathogen
-not limited to blood serum, can also be performed on other bodily fluids e.g. semen and saliva
Antibody response to viral infection
When infected with a virus, the humoral immune response takes place resulting in production of antibodies:
· IgM antibodies specific to the virus are produced early on (first) against a virus
· IgM present for a variable period- usually 1 to 3 months
· As IgM declines, IgG is produced and 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)
Uses of serology
- Detect an antibody response in symptomatic patients
- Determine if vaccination has been successful
- Directly look for antigen produced by pathogens
How is blood serum obtained?
Blood is coagulated with micronized silica particles
Gel is used to trap cellular components
Serum tubes are centrifuged for 10 minutes at 1000xg. The supernatant (serum) is removed and stored at appropriate temp.
Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes.
Method of Detecting Antibodies in Blood Serum
REVERSE ELISA:
1) Antigen for a specific virus is coated to the bottom of the well
2) Patient sample is added to it
3) If the patient has antibodies against the antigen, they will bind to the antigen adsorbed to the bottom of the well
4) Mixture is washed to rid of any antibodies not bound to antigens at the bottom of the well
5) Enzyme-conjugated antibody is added. This antibody will bind to the antibodies bound to the antigens at the bottom of the well.
6) Mixture is washed to rid of any antibodies not bound to antibodies at the bottom of the well
7) Chromogenic substrate for the enzyme is added, and the reaction between the two produces a product that causes a visible colour change
How does the presence of IgM or IgG aid in the diagnosis?
Can interpret what stage the illness is at:
If you took a test and couldn’t find any antibodies in that patient:
-patient has never previously been infected with Hepatitis A & never been immunised
If patient has IgM antibodies
-infection must be acute/recent
If patient only has IgG antibodies
-infection has resolved or patient has had immunisation
Detection of antibodies AND antigens in serology
Via ELISA or related technology such as microparticle immune-chemiluminescence
Useful for:
- HepB
- HepC
- HIV
Helps establish whether infection is acute or chronic
How do we use molecular diagnostic tests to diagnose viral infections?
Nucleic Acid Amplification Tests (NAATs) →PCR
1) Extract piece of genomic DNA from virus and sequence it to make a primer complementary to the viral DNA
2) Patient DNA is denatured to form single strands
3) Primer added
4) If sample contains viral DNA, primer will bind to template by DNA polymerase elongation, forming a dsDNA molecule
5) Cyclical process, resulting in large amount of DNA which can be detected
What are the advantages of using NAATs?
- Automated
- Highly sensitive and specific
- Generates huge numbers of amplicons
- Useful for detecting viruses to make a diagnosis
- Useful for monitoring treatment response
What are the limitations of using NAATs?
- May detect other viruses not causing the infection
- TOO sensitive and generates large numbers of amplicons which could cause contamination, meaning high stringent conditions necessary
- Need to have an idea of what virus you are looking for as will need primers and probes that are specific for that target
What is multiplex PCR?
more than one pair of primers is used in a PCR, enabling the amplification of multiple DNA targets in one tube
e.g. detection of multiple viruses in one CSF specimen such as HSV1, HSV2, VZV, enterovirus, mumps virus to test for meningitis as they can all cause the disease.
What is real-time PCR?
Along with PCR reagents an organism specific fluorescent DNA probe is also added which can be detected in real time PCR machine simultaneously during the amplification process
What is used to monitor real-time PCR cycle?
Specific Taqman Probes:
· Taqman fluorescent probe complementary to the region of interest will bind between primers
· This oligonucleotide probe has a fluorescent reporter at the 5’ end and a quencher at the 3’ end.
· The quencher prevents the reporter fluorescing when excited if in close proximity
· Taqman probe hybridises to region of interest during annealing phase of PCR
· Fluorescence is still prevented due to the proximity of the quencher to the reporter
· Taq polymerase extends from the 3’ end of primer as normal
· The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe
· The reporter is removed from the quencher and fluorescence can be detected
What is the CT value in the real time PCR?
number of cycles required for the fluorescent signal to cross the threshold
-allows for quantitation and detects presence/absence of amplicon
How can we quantitate real time PCR?
· Relative fluorescence can be plotted against the number of cycles (Ct value)
· This can be used to determine relative concentrations of standard curve using standards of known concentration
PCR Inhibitors
The PCR process can be affected by compounds that interfere with the interaction between DNA and Taq polymerase, and thus inhibit the reaction.
Many body fluids contain substances that can inhibit PCR:
- haem
- bile salts
What is the effect of PCR inhibition?
Can incorrectly report results as negative
Therefore, we add an internal positive control in PCR assay
-if we get a negative test, we need to know it is negative because there is no virus DNA there, rather than the fact that there is an inhibitor there (hence use a control)
Virus Sequencing
Looking at the virus genome
- used to predict response to anti-virals
- useful for outbreak investigation by showing identical sequences in suspected source and recipient
Anti-viral resistance testing
If anti-viral doesn’t work on virus, then virus has a particular mutation and is resistant.
Sequence viral genes known to be associated with resistance to anti-virals (e.g. integrase, reverse transcriptase in HIV)
Allows us to predict whether a patient would be resistant to a particular anti-viral treatment they are on
Screening purposes of Laboratory Diagnostic Tests
Testing for specific infections in those that are well/healthy but in at risk groups:
- E.g. HIV, HBV and HCV
Testing because it may have an implication for others e.g. antenatal:
- HIV, HBV, rubella
· In these situations, the patients are asymptomatic
· Needs a sensitive screening test
· May have some false positive, so need a specific confirmatory test
