diagnosis of viral infections Flashcards
electron microscopy
Viruses can be visualised with electron microscope
Mostly replaced by molecular techniques
Possibly still useful for faeces and vesicle specimens
Useful in characterising emerging pathogens
electron microscopy 2
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
advantages and disadvantages of electron microscopy
Limitations
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.
virus isolation in cell culture
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
Led to discovery of hMPV and Nipha virus in last 20 years and SARS-CoV-2 recently
Use different cell lines in test tubes or plates. Selection of cell types important.
Slow, but occasionally useful in anti-viral sensitivity testing
cytopathic effect
Different viruses may give different appearances
Different cell lines may support growth of different viruses
Identify virus using antigen detection techniques or neutralisation of growth
Cell culture plus antiviral – look for inhibition of cytopathic effect
describe 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. Possible specimens include:
Nasopharyngeal aspirates (NPA) (cell-associated virus antigens) e.g. 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 These techniques are being replaced by Nucleic acid detection methods due to improved test performance i.e. greater sensitivity
antigen detection
A variety of different methods can be used Commonest methods are Direct immunofluorescence Cell associated antigens Enzyme immunoassay Free soluble antigens or whole virus Immunochromatographic methods Often used at point of care for rapid diagnosis
immunofluorescene
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
immunochromatographic methods
Flavivirus
Arthropod vector
Common infection in returning travellers
Useful as a NPT (near patient test) – Point of care test (POCT)
ELISA for antigen detection
Enzyme-linked immunosorbent assay A component of reaction is adhered to a solid surface Three formats: Indirect Direct (primarily antigen detection) Sandwich
detection of antigen by 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,
binds to detecting antibody
Chromogenic substrate is added, and is converted by the enzyme to detectable form e.g. colour change
antibody detection by serology
Detection of antibodies
Indirect detection of the pathogen
Diagnostic mode of choice for organisms which are refractory to culture
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
serum
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
4ºC short term
-20ºC long term
Routinely serum tubes are centrifuged for 10 min at 1000xg
Serum contains proteins, antigens, antibodies, drugs (some)
and electrolytes
diagnosis by antibody detection
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
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
Modern Laboratory detection of antibodies and antigens in blood
Serology
Detection of antibody and or antigens
Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence
detection of antigen and antibody
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
This may have therapeutic implications
molecular diagnostic tests
Nucleic acid amplification (NAAT)
e.g. PCR although there are other examples*
Can detect 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 nucleic acid extraction prior to the amplification
stages of NAAT test
Specimen collection Extraction of nucleic acid DNA transcription for RNA viruses Cycles of Amplification of DNA target requires polymerase and dNTPs plus other reagents Detection of amplicons After amplification Or real time
advantages of using NAATs
May be automated. POCT 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
limitiations of using NAATs
Generates large numbers of amplicons. This may cause contamination.
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
real time PCR
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
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 e.g. detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2, VZV, enterovirus, mumps virus
PCR inhibition
Some substances inhibit PCR e.g. haem, bile salts. Assays should always include an internal positive control 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
genome sequencing
Partial or whole
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 naïve patients, or if clinical suggestion of resistance in drug experienced patients
