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
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
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 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
