Diagnosis Of Viral Infections Flashcards
- Is it always possible to diagnose an infection clinically?
Not always possible to diagnose a infection clinically. Often require a laboratory diagnostic test
- What kind of information would the doctor need to diagnose a patient?
Aid to diagnosis - history, examination & special investigations
- Why is it important to diagnose viral infections quickly ?
Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
- Give 6 possible test mechanisms 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
- Which microbes can be seen using light microscopy (x400-x1000) ?
Bacteria
Fungi
Protozoa
Helminths (might be seen by naked eye)
- Can we see viruses with light microscopy? if no, what kind of microscope can we see them with?
No we cant
Viruses can be seen with electron microscopy x20,000
- What is electron microscopy useful for ?
- > Faeces and Vesicle Specimens
- >Characterising emerging pahogens
- How does electron microscopy work?
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 three advantages of electron microscopy?
Rapid
Detects viruses that cannot be grown in culture
Can visualise many different viruses
- What are four limitations of electron microscopy?
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.
- Explain what
Rotavirus (gastroenteritis),
Adenovirus (gastroenteritis),
Coronavirus (respiratory tract infection), Nonovirus (gastroenteritis)
Astrovirus (gastroenteritis)
all appear like on an Electron Microscope?
Rotavirus (gastroenteritis) = circular shaped , has a distinct lining
Adenovirus (gastroenteritis), = Circular, all white
Coronavirus (respiratory tract infection), =looks like a dog bone, two circles joined by a line in the middle
Nonovirus (gastroenteritis) = lots of small circles
Astrovirus (gastroenteritis) =small white circles
- Which viruses shows vesicles in an EM?
How do we differentiate between these two
Herpes Viruses:
- Herpes Simplex
- Varicella Zoster Virus
EM cant differentiate between these two viruses so we need to use clinical context, site of vesicle and symptoms to differentiate?
- Can EM differentiate between the different types of Poxvirus ( eg Smallpox, Monkeypox , Orf, Cowpox) ?
No it cant
Depends on clinical context to diagnose
- How does isolating a virus in a cell culture work?
Use different cell lines in test tubes or plates. Selection of cell types important.
Slow, but occasionally useful in anti-viral sensitivity testing
- What has viral isolation in cell culture led to the discovery of in the past 20 yrs?
Led to discovery of hMPV and Nipha virus in last 20 years
- Viral infection in cell culture is an old method and has been replaced for diagnosis but in what situations could we use this technique?
Old method, now replaced by molecular techniques, but still needed for research or for rare viruses
- Explain the process of viral isolation in cell culture?
Use different cell lines in test tubes or plates. Selection of cell types important
Different viruses viruses may give different appearances
Different cell lines may support growth of different viruses
Identify virus using antigen detection techniques or neutralisation of growth
- What is one advantage and disadvantage if using viral isolation in cell culture?
Slow, but occasionally useful in anti-viral sensitivity testing
- What would you look for if your cell culturing a virus PLUS an antiviral is present?
look for inhibition of cytopathic effect
- Can we detect the viruses by their antigens?
Viral antigens, usually proteins – either capsid structural proteins, secreted proteins can be detected. Infected cells may display viral antigens on their surfaces.
- For the following patient samples explain which viruses you would expect to find there:
- Nasopharyngeal Aspirates (NPA)
- Blood (Serum or plasma)
- Vesicle Fluid
- Faeces
Nasopharyngeal aspirates (NPA): e.g. RSV, influenza
Blood (serum or plasma): Hepatitis B Dengue Vesicle fluid: Herpes simplex, varicella zoster Faeces Rotavirus, adenovirus
- What is direct detection of the antigen being replaced by and why?
These techniques are being replaced by Nucleic acid detection methods due to improved test performance
- What are the 3 most common techniques for antigen detection?
Direct immunofluorescence
Enzyme immunoassay
Immunochromatographic methods
- Explain the process of how we can detect an antigen using immunofluorescence?
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
- Give an example of a virus we can diagnose using immunochromatographic methods?
Diagnosis of Dengue
- What do you know about Dengue?
Flavivirus
Arthropod vector
Common infection in returning travellers
27, What are the three formats of the ELISA test?
Indirect
Direct (primarily antigen detection)
Sandwich
- Explain the process of antigen detection by ELISA test?
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 antibody
Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change
- Explain how the production of the types of antibodies change whilst infected with a virus
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
- What type of antibodies can we detect in the ELISA test?
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
- What is serology?
Examining patient samples
- Is serology using indirect or direct detection of the pathogen?
Indirect
- What can serology be used to do?
Detect an antibody response in symptomatic patients
Determine if vaccination has been successful
Directly look for antigen produced by pathogens
- What samples of a patient can we take to perform a serological test?
Serological tests are not limited to blood & serum
can also be performed on other bodily fluids such as semen and saliva
- Explain the process of serology specifically using patients serum?
- Serum is produced from processing blood :
- Blood is coagulated with micronized silica particles
- Gel used to trap cellular components
2.Routinely serum tubes are centrifuged for 10min at 1000xg
- Supernatant (serum) is removed and stored:
- 4 degrees short term
- -20 degrees long term - Serum contains proteins,antigens , antibodies, drugs and some electrolytes
- Explain the serological diagnosis of Hep A infection?
If then patient has no past or present infection or immunisation = Negative for Hep A IgM and IgG antibodies
If the patient has an acute/recent infection = Positive for Hep A IgM and depending on how long they’ve had the infection a positive (long term) or negative (recent) Hep A IgG antibodies
If the patient has a resolved infection/Immunity = Negative for IgM antibodies and positive for IgG
- What are some modern laboratory detections of antibodies and antigens in blood?
ELISA or related technology e.g. microparticle immuno-chemiluminescence
- Sometimes we can have detection of antigen AND antibody, which infections would this be useful for and why?
This is useful for some infections such as
Hepatitis B
HIV
Hepatitis C
This is because it allows us to establish whether acute or chronic infection
- Give a name of a molecular diagnostic test we can use and an example ?
Nucleic acid amplification (NAAT)
for eg PCR
- How does NAAT work?
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
- What are 5 advantages of using NAATs?
May be automated
Highly sensitive and specific, generates huge numbers of amplicons
Rapid
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
- What are three limitations of NAATs?
May detect other viruses which are not causing the infection
Exquisitely sensitive and so may generate 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
- 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 multiplexing?
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 are specific Taqman Probes?. How do they work?
Oligonucleotide probe with a fluorescent reporter at the 5’ and a quencher at the 3’(The quencher prevents the reporter fluorescing when excited if in close proximity)
- Taqman probe complimentary to region of interest, binds between primers
- Taqman probe hybridises to the region of interest
This occurs during the annealing phase of PCR
Fluorescence is prevented due to the proximity of quencher - 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 form the quencher and fluorescence can be detected.
For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number
- What is the cycle threshold/CT?
In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level).
- How can we make real time PCR quantitative?
Relative fluorescence can be plotted against the number of cycles
This can be used to determine relative concentrations of DNA present by construction of standard curve using standards of know concentration.
- Give an example of some substances which inhibit PCR?
haem, bile salts
- How can we counteract PCR inhibition?
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
- What can sequencing the organism tell us?
DNA or RNA viruses
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
Consensus sequence based on clinical observation of resistance or in vitro evidence
Minority species sequencing
May be selected by treatment
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
- Explain how you would combine methods for HIV diagnosis and managment?
Antibody and antigen detection for initial diagnosis: Screening test (EIA) Confirmatory test (EIA)
Viral load(NAAT) at baseline and to monitor treatment response Quantification of virus in blood
Resistance testing (sequencing) to confer resistance before and during treatment
- Explain how you would do anti-viral resistance testing using 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)
- What should be screened for regularly in patients?
Testing for specific infections 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 positives, so need
A specific confirmatory test
