Diagnosis of viral infections Flashcards
What is an aid to diagnosis?
• Aid to diagnosis - history, examination & special investigations
What can rapid diagnosis of viral infections reduce?
• Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
What is the significance of test results dependent on?
• Significance of test results depend heavily on prevalence in population e.g. HIV
○ Very few tests that are right 100% of the time
§ Can get false positives/negatives
What are 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 type of microscope is used to see bacteria and fungi?
• Bacteria and fungi can be seen using a light microscope
What is protozoa and helminth seen by?
• Protozoa and helminths (worms) can be seen using the naked eye
What type of microscope is used to see viruses?
• Viruses are small so need electron microscopy
Steps involved in 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
○ Antibodies which bind the viruses and when mixed with the specimen, it clumps all the viruses together
§ Makes it so all the viruses are concentrated at one particular point – making it easier to see - 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
• EM cannot differentiate between Herpes Simplex Virus and Varicella zoster virus
§ Identification depends on clinical context, site of vesicle and symptoms
Advantages of electron microscope
Rapid – prep time is 15-30 mins
Detects viruses that cannot be grown in culture
Can visualize many different viruses
Disadvantages of electron microscope
Low sensitivity – 106 virions/ml may be enough in vesicle secretion
Sometimes there may not be enough viruses to be visualized
Requires maintenance
Required skilled operators
Cannot differentiate between viruses of the same virus family
What do viruses require to replicate and what effect may they cause?
○ 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
What is there in a cell culture and what do we do to it everyday?
• In a cell culture, there is a single layer of cells growing on a flat surface
○ Incubated everyday
○ Keep looking at culture until you see a cytopathic effect
Identifying virus using antigen detection techniques?
○ You may identify the virus because the culture used may only support the growth of one particular virus, so if a cytopathic effect is seen, the virus is present
○ May also do electron microscopy
Identifying virus using neutralisation of growth
Neutralisation – use the antibodies of the virus you think it is and if there is no cytopathic effect then the virus identity is confirmed
What can you see in cell culture plus antiviral?
○ Can see if you can inhibit the cytopathic effect of the virus using an anti-viral
What can you say if there still is a cytopathic effect in cell culture with anti-viral?
○ if there is still a cytopathic effect you can say that they have developed resistance to the anti-viral
What may infected cells display?
• Infected cells may display viral antigens on their surfaces
What are antigen detection techniques being replaced by?
• Antigen detection techniques are being replaced by Nucleic acid detection methods due to improved test performance
What are the most common methods for antigen detection?
○ Direct immunofluorescence
○ Enzyme immunoassay
○ Immunochromatographic methods
What is immunofluorescence to check for the presence of?
• This is to check for the presence of a virus in the cells of a patient
Steps involved to check for the presence of a virus in the cells of a patient
- i.e. take nasopharyngeal aspirate from patient
○ Put tube down nose and take fluid from nasopharynx - Take some cells from nasopharyngeal aspirate and layer it onto slide
○ Antigen (from infected host cells in sample) bound to slide - Let dry
- Specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with sample
- Antibody will bind to only the cells with the virus within them and will fluoresce
- Viewed using a microscope equipped to provide ultraviolet illumination
What is the advantage of immunofluorescence?
quick technique
What is the disadvantage of immunofluorescence?
○ May not be enough cells in the specimen with the virus to be able to see the fluorescence
What does the immunochromatographic method work similar to and how does it work?
• Works similar to pregnancy test whereby blood is put onto the stick and a heavy metal line is displayed depending on whether the virus is present or not
What happens to a component of the reaction in ELISA?
○ A component of reaction is adhered to a solid surface
What are the 3 formats of ELISA?
- Indirect
- Direct (primarily antigen detection)
- Sandwich
Steps involved in ELISA
- Plate is coated with a capture antibody
- Sample is added and any antigen present binds to capture antibody
- Enzyme-conjugated primary antibody is added and binds to detecting antibody
- Chromogenic substrate is added and is converted by the enzyme to detectable form e.g. colour change
How can we detect antibody instead of antigen and what is this known as?
• We can also detect the antibody instead of detecting the antigen (a reverse ELISA test) – known as serology
§ Look at antibody response after infection to determine which antigen was present
What response occurs when infected with a virus and what does this result in the production of?
• 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 over time
What can we look at to determine what stage of illness the patient is at?
Looking at concentration of Igs can determine what stage of illness the patient is at
What does a lot of IgM mean?
lots of IgM means early stages
What can diagnosis be made by of an illness involving antibodies?
• 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?
• Indirect detection of the pathogen
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 else can serological tests be performed on and why’s it useful?
○ Can also be performed on other bodily fluids other than blood and serum such as semen and saliva
§ Using saliva would be particularly useful for children as it is difficult to obtain a blood sample from a child
Steps involved in detecting measles antibodies
- Coat measles antigen to the bottom of the well
- Add patient sample
- If patient has measles antibody in their blood, it will bind to the antigen
- Can add another antibody (tagged with a fluorochrome) that will attach to the primary antibody
Look under microscope to see fluorescence
What is serum produced from?
Produced from processing blood
How often are serum tubes centrifuged?
• Routinely serum tubes are centrifuged for 10 min at 1000xg
What does serum contain?
• Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
What infections is detection of antigen and antibody 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
What is an example of a molecular diagnostic test?
NAAT
What can NAATS detect?
○ Can detect RNA or DNA
What does NAATS have the ability to do?
○ Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample
What does NAATS require prior to the amplification?
○ Requires nucleic acid extraction prior to the amplification
What are the advantages of NAATS?
- May be automated
- Highly sensitive and specific, generates huge numbers of amplicons
- Rapid
- Useful for detecting viruses to make a diagnosis
- Useful for monitoring treatment response
What are the limitations of using NAATS?
- Prone to getting contaminated
- Exquisitely sensitive and so may generate large numbers of amplicons
- Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target
When is the term multiplex PCR used and what can it detect?
• Multiplex PCR is the term used when more than one pair of primers is used in a PCR
§ Can detect multiple viruses at the same time
What does multiplex PCR enable the amplification of?
It enables the amplification of multiple DNA targets in one tube
What is real time PCR?
Real time as amplification AND detection occur in REAL TIME
What does real time PCR avoid the use of?
• Avoids the use of gel electrophoresis or line hybridisation
What does real time PCR allow the use of?
• Allows the use of multiplexing
What is cycle threshold?
number of times the amplification process needs to be done
What is specific taqman probes a type of?
• Type of real time PCR
Steps involved in specific taqman probes
- Taqman probe complimentary to region of interest, binds between primers
- 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 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 from 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 can inhibit PCR?
• Some substances inhibit PCR e.g. haem, bile salts
PCR can be inhibited, hence what should assays always include?
• Assays should always include an internal positive control as results could incorrectly be reported as negative
What type of viruses is organism sequencing for?
DNA or RNA viruses
What is organism sequencing used to predict?
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
What is organism sequencing useful for?
Useful for outbreak investigation by showing identical sequences in suspected source and recipient
Combination of methods in HIV diagnosis and management
- 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
What are the multiple viral enzyme targets in anti-viral resistance testing?
Multiple viral enzyme targets
- Reverse transcriptase, protease,
- Integrase,
- Viral receptor binding proteins)
What do we look for in anti viral resistance testing?
Look for mutations in the virus known to cause resistance
What are we testing in screening?
Testing for specific infections in at risk groups
We may get a false positive in screening hence what do we need?
May have some false positives, so need
-A specific confirmatory test
