Diagnosis Of Viral Infections Flashcards
1
Q
State the key concepts as to why we diagnose patients with viral infections (4)?
A
- Not always possible to diagnose a infection clinically (from symptoms alone). Often require a laboratory diagnostic test.
- Aid to diagnosis - history, examination & special investigations
- Rapid diagnosis of viral infections can reduce need for unnecessary tests, inappropriate antibiotics
- Important public health and infection control implications
2
Q
State the key concepts as to the importance of the test selection for diagnosing viral infections (3)?
A
- It helps to know the natural history of the pathogen in the type of patient you are testing as this will affect test selection and interpretation
- Consent - must be obtained for certain infections
- Difference between diagnostic, monitoring and screening tests
3
Q
State possible test types used in diagnosing viral infections (6)?
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
What technique can be used to visualise viruses and specifically what is the technique useful for?
A
- Viruses can be visualised with electron microscope
- Mostly replaced by molecular techniques
- Useful for faces and vesicle specimens and characterising emerging pathogens
5
Q
Describe the process of electron microscopy explaining the why electron is used over light for viruses?
A
- 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
- Allows for visibility of very small microbes like virus. Typically requires 20,000x mag. compared to other microbes (fungi, parasites) which can be seen at ×400-x1000, so these are much larger
6
Q
State advantages of electron microscopy (3)?
A
- Rapid
- Detects viruses that cannot be grown in culture
- Can visualise many different viruses
7
Q
State disadvantages of electron microscopy (4)?
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
List which viral infections may be diagnosed by electron microscopy and a key symptom they cause (6)?
A
- Rotavirus (causes gastoenteritis)
- Adenovirus (causes gastoenteritis)
- Coronavirus (resp. tract infection)
- Norovirus (calicivirus) (gastroenteritis)
- Herpes virus (group of virus) - Made up of herpes simplex, varicella zoster virus, cytomegalovirus (CMV), Epstein-barr virus (EBV): Causes vesicles
- Poxvirus e.g. smallpox, monkeypox, orf and cowpox: Causes skin lesions
9
Q
Why can EM not be the sole diagnostic tool in herpes virus and poxvirus?
A
- For herpes virus, EM cannot differentiate these different viruses so depends on clinical context, site of vesicle and symptoms
- For poxvirus, depends on the clinical context i.e. exposure history, geographic location, clinical features.
10
Q
Describe how viruses are isolated using cell culture and how it could be useful?
A
- 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
- Old method, now replaced by molecular techniques, but still needed for research or for rare viruses
- Use different cell lines in test tubes or plates. Selection of cell types important.
- Slow, but occasionally useful in v. rare infections where there are no other diagnostic techniques available
- Viral culture is a laboratory technique in which samples of a virus are placed to different cell lines which the virus being tested for its ability to infect. If the cells show changes, known as cytopathic effects, then the culture is positive.
11
Q
Describe the importance of the cytopathic effect from viruses in cell culture and what can this be used for?
A
- Different viruses may give different appearances/morphology to cell line via CE
- Different cell lines may support growth of different viruses
- What results can be used for
- Identify virus using antigen detection techniques or neutralisation of growth
- Cell culture plus antiviral - look for inhibition of cytopathic effect
12
Q
Describe antigen detection and what technique is being replaced by and why?
A
- Antigen detection: Direct detection of the viral antigens.
- Viral antigens, usually proteins - either capsid structural proteins or secreted proteins. They can be detected in cells or free in blood, saliva or other tissues/organs.
- These techniques are being replaced by Nucleic acid detection methods due to improved test performance i.e. greater sensitivity
13
Q
List the possible specimens used in antigen detection and link them with the relevant viruses e.g. RSV, influenza and Hepatitis B, Dengue and Herpes simplex, varicella zoster and Rotavirus, adenovirus?
A
- Nasopharyngeal aspirates (NPA) (cell-associated virus antigens): RSV, influenza
- Blood (serum or plasma) (free antigen or whole virus): Hepatitis B, Dengue
- Vesicle fluid (whole virus): Herpes simplex, varicella zoster
- Faeces (whole virus): Rotavirus, adenovirus
14
Q
State the commen methods of antigen detection, what they specfically
detect and when is it used? (3)
A
- Direct immunofluorescence: Cell associated antigens
- Enzyme immunoassay e.g. ELISA: Free soluble antigens or whole virus
- Immunochromatographic methods: Non-structural antigens e.g. circulating blood or saliva
- Often used at point of care for rapid diagnosis
15
Q
Describe the process of immunofluoresence?
A
- 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
16
Q
Describe what immunochromatographic methods is used for?
A
- e.g. diagnosis of dengue, COVID-19
- Flavivirus - any of a group of RNA viruses, mostly having arthropod vectors, which cause a number of serious human diseases including yellow fever, dengue, various types of encephalitis, and hepatitis C
- Arthropod vector
- Common infection in returning travellers
- Useful as a NPT (near patient test) - Point of care test (POCT)
17
Q
What is ELISA and state the 3 formats?
A
- ELISA: Enzyme-linked immunsorbent assay
- A component of reaction is adhered to a solid surface
- Three formats: Indirect, Direct (primarily antigen detection), Sandwich
18
Q
Describe the detection of antigens by ELISA (sandwich format)?
A
- 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
- Chromogenic = colourless substance. If in presence of enzyme, becomes coloured
- The substrate only will change colour only if the enzyme-conjugated antibody and therefore also the antigen are present.
- Negative result = NO colour change
19
Q
What is serology used for in diagnosing viral infection?
A
- Serology method: Detection of antibodies
- Indirect detection of the pathogen
- Diagnostic mode of choice for organisms which are refractory to culture - Can’t be detected by most other techniques
20
Q
What can serotology be used for?
A
- 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
- Serological tests are not limited to blood & serum: Can also be performed on other bodily fluids such as semen and saliva
21
Q
Describe the process of extracting serum for serology?
A
- Produced from processing blood: Blood is coagulated with micronized silica particles, Gel used to trap cellular components
- Routinely serum tubes are centrifuged for 10 min at 1000xg
- Supernatant (serum) is removed and stored: 4°C short term, 20°C long term
- Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
22
Q
How is the diagnosis made via antibody detection and describe how this occurs?
A
- 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
- IqM present for a variable period - usually 1 to 3 months
- As IgM declines, IgG is produced -> Quantity of IgG rises
23
Q
How is the diagnosis made via antibody detection?
A
- Diagnosis can be made by
1. Detection of IgM (can be non specific)
2. Demonstration of seroconversion - Negative IgG antibody at first
- Then presence of IgG antibody
24
Q
Describe Hep IgM and IgG presence (either positive or negative) in no past infection, acute infection and resolved infection of Hep A?
A
VD
25
State the methods involved in serology?
- Usually by
- Enzyme immunoassays e.g. ELISA
- Related technology e.g. microparticle immuno chemiluminescence
- These will detect antibody and or antigens
26
What is the advantage behind detecting both antigen and antibody together for viral diagnostic?
- This is useful for some infections as it allows for investigation of different markers such as: Hepatitis B, HIV, Hepatitis C
- This is because it allows us to establish whether acute or chronic infection
- This may have therapeutic implications
27
State and describe the molecular diagnostic test used for viral diagnostic?
- Nucleic acid amplification test (NAAT)
e.g. PC although there are other examples* *LCR, SDA, LAMP
- Can detect viral 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 nuclei acid extraction prior to the amplification
28
Describe the stages of NAAT test? (5)
1. Specimen collection
2. Extraction of nucleic acid
3. DNA transcription for RNA viruses
4. Cycles of Amplification of DNA target: Requires polymerase and dNTPs plus other reagents
5. Detection of amplicons (amplified DNA): After amplification, Real time
29
State the advantages of using NAATs? (5)
- May be automated. POCT (point of care test) possible
- Usually highly sensitive and specific, generates huge numbers of amplicons
- Rapid: can be as quick as 15 minutes - usually a few hours
- 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
30
Describe the limitations behind using NAATS? (3)
- Generates large numbers of amplicons. This may cause contamination - false + ve reactions
- Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target.
- Mutations in target sequence may lead to "dropout" e.g. S gene dropout seen with SARS-CoV-2 variants: Primers are unable to recognise
31
Describe real time PC and what allows for the use of?
- 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
32
What is multiplex PCR?
- 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 and thus the detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2, VZV, enterovirus, mumps virus
33
Describe how PC inhibition can occur and how this is overcome?
- Some substances inhibit PC e.g. haem, bile salts
- Assays should always include an internal positive control (IC) 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
34
What is the use of genome sequencing in viral diagnostics?
- Detects partial or whole viral sequences
- Useful for outbreak investigation by showing identical sequences in suspected source and recipient
- New variants: Diagnostic tests, Vaccine efficacy
- Can be used to predict response to anti-virals e.g. for HIV in Rx naive patients, or if clinical suggestion of resistance in drug experienced patients
35
Describe the combination of methods used for HIV diagnosis and management?
1. Antibody and antigen detection for initial diagnosis
• Screening test (EIA)
• Confirmatory test (EIA)
2. Viral load (NAAT) at baseline and to monitor treatment response
a. Quantification of virus in blood
3. Resistance testing (sequencing)
EM of HIV in cell culture BUT EM not used for routine diagnosis
36
Describe anti-viral resistance testing in HIV and what it looks for?
- Antiviral resistance testing involves identification of the mutations using sequencing methods (genotyping) and determination of their effect on viral drug susceptibility using in-vitro cell-based assays (phenotyping)
- 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)
37
Describe the purposes of screening
- Screening: 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 and HBV,
- In these situations the patients are asymptomatic
38
Describe the process behind screening
- Needs a sensitive screen test
- May have some false positive screen tests so then perform
- A confirmatory test
