diagnosis of viral infections

Question 1

electron microscopy

Answer 1

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

Question 2

electron microscopy 2

Answer 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

Question 3

advantages and disadvantages of electron microscopy

Answer 3

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.

Question 4

virus isolation in cell culture

Answer 4

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

Question 5

cytopathic effect

Answer 5

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

Question 6

describe antigen detection

Answer 6

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

Question 7

antigen detection

Answer 7

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

Question 8

immunofluorescene

Answer 8

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

Question 9

immunochromatographic methods

Answer 9

Flavivirus
Arthropod vector
Common infection in returning travellers
Useful as a NPT (near patient test) – Point of care test (POCT)

Question 10

ELISA for antigen detection

Answer 10

Enzyme-linked immunosorbent assay
A component of reaction is adhered to a solid surface
 Three formats:
Indirect
Direct (primarily antigen detection)
Sandwich

Question 11

detection of antigen by ELISA

Answer 11

Plate is coated with a capture antibody

Sample is added and any antigen present binds to capture antibody

3. 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

Question 12

antibody detection by serology

Answer 12

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

Question 13

serum

Answer 13

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

Question 14

diagnosis by antibody detection

Answer 14

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

Question 15

Modern Laboratory detection of antibodies and antigens in blood

Answer 15

Serology
Detection of antibody and or antigens
Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence

Question 16

detection of antigen and antibody

Answer 16

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

Question 17

molecular diagnostic tests

Answer 17

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

Question 18

stages of NAAT test

Answer 18

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

Question 19

advantages of using NAATs

Answer 19

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

Question 20

limitiations of using NAATs

Answer 20

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

Question 21

real time PCR

Answer 21

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

Question 22

multiplex PCR

Answer 22

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

Question 23

PCR inhibition

Answer 23

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

Question 24

genome sequencing

Answer 24

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

Question 25

combinations of methods e.g. HIV diagnosis and management

Answer 25

Antibody and antigen detection for initial diagnosis
Screening test (EIA)
Confirmatory test (EIA)

Viral load(NAAT) at baseline and to monitor treatment response
Quantification of virus in blood

Resistance testing (sequencing)
to confer resistance before

Question 26

anti viral resistance testing

Answer 26

HIV as an example
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)

Question 27

screening

Answer 27

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
Needs a sensitive screening test
May have some false positives, so need
A specific confirmatory test