Medical Microbiology: Diagnosis of Viral Infections Flashcards
1
Q
Why are laboratory tests often needed to diagnose an infection?
A
- Many viral infections present in similar ways so it’s not possible to diganose the infection clinically
2
Q
Why is rapid diagnosis of viral infections important?
A
- Can reduce need for unnecessary tests
- Can prevent inappropriate use of antibiotics
- Can prevent other people from getting the infection
3
Q
What are the possible test types for diagnosis of viral infections?
A
- 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
4
Q
How can you visualise microbes?
A
- Bacteria and fungi can be seen using a light microscope (x400-x1000)
- Protozoa and helminths can be seen using naked eye as well as light microscope
- Viruses can only be seen using electron microscope (x20,000)
5
Q
How does electron microscopy work?
A
- Specimens are dried onto a grid
- Specimens are then stained with a heavy metal e.g. uranyl acetate
- Specimens can be concentrated with application of antibody e.g. immuno-electron microscopy to concentrate the virus
- Involves mixing antibodies which bind the virus into the specimen which clumps all the viruses together.
- This means that all the viruses are concentrated at one particular point and this makes them easier to view
- Beams of electrons fired at specimen to produce images
- Wavelength of electron beam is much shorter than light, resulting in much higher resolution image than light microscopy
6
Q
What are the advantages of electron microscopy?
A
- Rapid
- Detects viruses that cannot be grown in culture
- Can visualise many different viruses
7
Q
What are the disadvantges of electron microscopy?
A
- 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
8
Q
Name some viruses that can be visualised using electron microscopy
A
- Rotavirus
- Adenovirus
- Coronavirus
- Norovirus (caicivirus)
- Astrovirus
9
Q
Why can’t viruses be grown in a cell-free medium?
A
- Viruses require host cells to replicate and may cause a Cytopathic Effect (CPE) of cells when a patient sample containing a virus is incubated with a cell layer
- NOTE: Viruses can however be grown in cell culture
10
Q
What is meant by the term “cytopathic effect?”
A
- Morphological changes in cells which can be recognised by skilled professional to work out the identity of the virus
11
Q
How can you check to see if a virus is growing within cell culture?
A
- Patient sample added to cell culture
- Cell culture then incubated everyday
- Look at cell culture everyday until you see a cytopathic effect
- Cytopathic effect indicated presence of virus within cells of cell culture
12
Q
How can you identify a particular virus growing in cell culture?
A
- Use cell culture that only supports growth of one type of virus - if cytopathic effect occurs then you know that particular virus is growing
- Electron microscopy
- Antigen detection techniques
- Neutralisation techniques - Culture antibodies for virus you think it is and then add it to cell culture containing virus if cytopathic effect is inhibited then you know that virus is present
13
Q
What is antigen detection and what specifc viral antigens can be detected?
A
- Antigen detection - Direct detection of the virus
- Viral antigens such as capsid structural proteins and secreted proteins can be detected
14
Q
Where can viral antigens be detected from within a patient?
A
- Nasopharyngeal aspirates (NPA) e.g. RSV, influenza
- Blood (serum or plasma) e.g. Hepatitis B, Dengue fever
- Vesicle fluid e.g. Herpes simplex, varicella zoster
- Faeces e.g. Rotavirus, adenovirus
15
Q
What are some common methods for viral antigen detection?
A
- Direct immunofluorescence
- Enzyme immunoassay
- Immunochromatographic methods
16
Q
How does antigen detection via immunofluorescence work?
A
- Take nasopharyngeal aspirate from patient
- Cells from nasopharyngeal aspirate are extracted and placed onto a microscope slide
- Cells are then left to dry
- Specific antibody (polyclonal or monoclonal) to the viral antigen is tagged to a fluorochrome and mixed with sample
- Sample is viewed using a microscope equipped to provide ultraviolet illumination
- If any cells within the sample have the virus within them they will be bound by the antibody causing that cell to fluoresece
18
Q
How do immunochromatographic methods work?
A
- Get a sample of patients blood and place it on the sample well in the test kit
- If viral antigen present in the patients blood you’ll see a line appear on test kit where antigen has bound to specific antibody for that viral antigen present within the test kit
21
Q
How does antigen detection via the direct ELISA format work?
A
- Plate is coated with a capture antibody
- Patient sample is added and any antigen present binds to capture antibody
- Enzyme-conjugated primary antibody is added and binds to the detecting antibody
- Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change
26
Q
How does serology work?
A
- Get antigens of a specific virus and place it at the bottom of the well
- Add patient sample to the well
- If patient has virus-specific antibodies in their blood then they’ll bind to the the viral antigens
- Primary antibody with a fluorochrome attached to it is then added to the well and is able to bind to the antibody already within the well
- Look for fluoresence under the microscope
- NOTE: After each stage a washing step is done to wash away all the antibodies that aren’t bound to either a viral antigen or another antibody
29
Q
Why is the detection of antigens and antibodies useful?
A
- Useful for some infections such as: Hepatitis B, HIV and Hepatitis C
- This is because it allows us to establish whether a person has an acute or chronic infection
- May also have therapeutic implications
35
Q
Describe the steps involved in real-time PCR using Taqman Probes
A
- 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 de 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
* NOTE: For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number
