Diagnosis of Viral Infections Flashcards
What are 6 different test types that can diagnose a viral infection?
1) Electron microscopy
2) Virus isolation (cell culture)
3) Ag detection
4) Ab detection by serology (indirect)
5) Nucleic acid amplification (NAATS - PCR)
6) Sequencing for genotype and detection of antiviral resistance
Describe Ag detection tests and what virus each type of sample can detect
Which method is it being replaced by?
DIRECT detection of virus
Infected cells may display viral antigens on their surfaces
- VESICLE FLUID (VZV, HSV)
- NASOPHARYNGEAL ASPIRATES (RSV, influenza)
- BLOOD (hep B, dengue)
- FAECES (rotavirus, adenovirus)
(being replaced by NUCLEIC ACID detection due to improved test performance)
What are the 3 most commonest methods used for antigen detection? (immuno…)
1) Direct immunofluorescence (UV microscope) - ag bound to slide w specific tagged Ab added
2) Immunochromatographic methods (lateral flow for covid)
3) Immunoassay (ELISA, ELFA,CLIA)
- indirect, sandwich, direct
Describe the method by which antigens are detected by ELISA
- Coat plate w a capture antibody
- Add sample, any antigen present will bind to the capture antibody
- Add enzyme-conjugated primary Ab; this binds to detecting antibody - forming a sandwich
- Add substrate, which is converted by the enzyme to detectable form = colour change
Describe diagnosis by antibody detection
Diagnosis can be made by:
- detection of IgM (can be non-specific)
- demonstration of seroconversion (no IgM/IgG, then presence of antibody when infected)
What is the difference between antibody affinity and avidity?
Give its effect on IgG and when else it can be used
Affinity = strength of interaction between single antigenic determinant and single antigen binding site
Avidity = overall binding strength
- IgG avidity will increase w time after infection
- Used to approx time when infection occurred (useful in congenital infections)
Serology is indirect pathogen detection- what are 3 things it can be used for?
What type of samples can be used?
1) Detect Ab response in symptomatic patients
2) Determine if vaccination has been successful
3) Directly look for Ag produced by pathogens
Serological tests are not limited to blood and serum- works on CSF, semen and saliva
Describe NAATs and give 3 pros and cons each
e.g. PCR, LAMP
- Can detect RNA/DNA
- Multiplex (fluorescent probes) - can look at several targets in one sample
- May be qualitative/ quantitative
- May require nucleic acid extraction prior to amp and signal detection
Pros:
- Automated, Rapid
- Highly sensitive and specific
- Diagnosis and treatment monitoring
Cons:
- Sensitive to contamination
- May detect other viruses not causing infection
- 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 are the 6 components of PCR mix and what are they used for?
1) Primer - short fragment, sticks to one end of the PCR strand
2) Nucleotides - elongate DNA during amplification
3) Taq polymerase - heat stable enzyme, incorporates nucleotides into growing
4) DNA strand
5) MgCl2 - needed by taq polymerase to have activity
6) Buffer - creates optimal enzyme conditions
What is the difference between realtime and multiplex PCR?
Real time PCR:
- Amplification and detection occur in real time
- Avoids the use of gel electrophoresis or line hybridisation
- Allows the use of multiplexing
Multiplex PCR:
- >1 pair of primers is used in a PCR
- Enables amplification of multiple DNA targets in 1 tube
- e.g. multiple virus detection in 1 resp sample
What is the cycle threshold/CT?
- With PCR, CT value can quantify how much viral target is there using an arbitrary threshold
- If CT value above threshold, PCR is positive
so the CT value= cycle at which the PCR has crossed the arbitrary level of fluorescence
Explain how a combination of methods is used to diagnose HIV
- Ab and Ag detection for initial diagnosis: screening and confirmatory test (EIA)
- Viral load (NAAT) at baseline and to monitor treatment response
- Resistance testing (sequencing): look for mutations, appropriate antiviral regimens selected based on results
Compare blood results and test types for bacterial vs viral infections
- Bacterial usually cause a higher CRP and neutrophil count than viral
- Raised WBC count in both
- Raised lymphocyte count in viral infections. Some viruses cause neutropenia e.g. HIV, EBV
- Culture, microscopy, serology to diagnose bacteria infection
- PCR and serology to diagnose viral infection
