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
17
Q
What are the advantages and disadvantgaes of immunofluoresence?
A
-
Advantages
- Quick technique - only takes a couple hours to complete
-
Disadvantages
- Not very sensitive - need quite a lot of cells to be infected by virus in order for it to be detected
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
19
Q
What viral infections can immunochromatographic methods be used to diagnose?
A
- Dengue fever
- Test can detect presence of NS1 antigen in patient blood indicating presence of dengue virus
20
Q
What are the 3 different formats for ELISA (Enzyme-linked immunosorbent assay)?
A
- Indirect
- Direct (primarily antigen detection)
- Sandwich
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
22
Q
What happens to the class and quantity of antibodies produced over the course of a viral infection?
A
- When infected with a virus the immune response takes place resulting in production of antibodies
- IgM antibodies specific to the virus are produced first
- IgM antibodies present between 1-3 months
- After this. time period IgM declines and IgG is produced causing quantity of IgG to rise
23
Q
How can the detection of antibodies be used to diagnose a viral infection?
A
- Detection of IgM (can be non specific) - Presence of IgM indicates early stages of infection
-
Demonstration of seroconversion
- Negative IgG antibody at first
- Then presence of IgG antibody
24
Q
What is serology and what can it be used for?
A
- Serology, also known as reverse ELISA test, is the Indirect detection of the pathogen by detecting presence of antibodies within a sample
- Serology can be used to:
- Detect an antibody response in symptomatic patients
- Determine if vaccination has been successful
- Directly look for antigen produced by pathogens
25
What types of patient sample can be used for serology?
* **Blood/serum**
* **Semen**
* **Saliva** - useful for children as it's difficult to get a blood sample from them
26
How does serology work?
1. Get antigens of a specific virus and place it at the bottom of the well
2. Add patient sample to the well
3. If patient has virus-specific antibodies in their blood then they'll bind to the the viral antigens
4. 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
5. 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
27
How is serum obtained from the blood?
* Produced from processing blood
1. Blood is coagulated with micronized silica particles
2. Gel used to trap cellular components
3. Serum tubes are centrifuged for 10 min at 1000xg
4. Supernatant (serum) is removed and stored at 4ºC for short term or -20ºC for long term
28
What things does the serum contain?
* Proteins
* Antigens
* Antibodies
* Drugs (some)
* Electrolytes
29
Why is the detection of antigens and antibodies useful?
* 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
30
What is NAAT?
* NAAT = Nucleic acid amplification test
* Most common method used is PCR but tehre are others
* Can be used to detect DNA or RNA
* Has ability to multiplex using fluorescence probes - can look for several targets in one sample
* May be qualitative or quantitative
* Requires nucleic acid extraction prior to the amplification
31
What are the 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 treatement response - e.g. can measure HIV, HCV viral loads
32
What are the disadvantages of NAATs?
* May detect other viruses which are not causing the infection
* Highly sensitive and so may generate large numbers of amplicons - This may generate contamination
* Need to have an idea of what viruses you're looking for as you'll need primers and probes for a specific piece of viral DNA
33
What is multiplex PCR?
* The term used when more than one pair of primers is used in a PCR and so enables the amplification of multiple DNA targets in one tube
34
What is real-time PCR and what are its advantages?
* PCR where amplification and detection occur in real time - simultaneously by the release of fluorescence
* **Advantages**
* Avoids the use of gel electrophoresis or line hybridisation
* Allows the use of multiplexing
35
Describe the steps involved in real-time PCR using Taqman Probes
1. Taqman probe complimentary to region of interest, binds between primers
2. 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
3. Taqman probe hybridises to the region of interest
4. This occurs during the annealing phase of PCR
5. Fluorescence is prevented de to the proximity of quencher
6. Taq polymerase extends from the 3’ end of primer as normal.
7. The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe
8. 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
36
What things can inhibit PCR and how PCR inhibition be prevented?
* Substances such as haem and bile salts inhibit PCR
* Assays should always include an internal positive control as results could incorrectly be reported as negative
* **_NOTE:_** Include primers specific for the internal control
37
What is viral sequencing used for?
* Used to predict response of a specific virus to anti-virals
* Also used in investigations of particular viral outbreaks - can identify if someone has virus by showing that particular patient has identical sequences to suspected source of virus
38
What is the consensus sequence used for viral sequencing based on?
* Based on clinical observation of resistance or *in vitro* evidence
39
What combination of viral diagnosis methods would you use to diagnose and manage HIV?
* **Antibody and antigen detection for initial diagnosis**
* Screening test (EIA)
* Confirmatory test (EIA)
* **Quantify Viral load (using NAAT) at baseline and to monitor treatment response**
* Quantification of virus in blood
* **Resistance testing (sequencing)**
40
What is screening and when is it used?
* Screening - A specific confirmatory test used to test for specific infections in high risk groups
* Used because having virus may have an implication for others e.g. antenatal
* In these situations the patients are asymptomatic and so need a sensitive screening test to identify presence of particular virus
