Diagnosis of viral infection

Question 1

List which viral infections may be diagnosed by electron microscopy

Answer 1

• Hepatitis Viruses: Liver biopsy specimens (B, C) or fecal samples (for A)
• Rotavirus: stool samples
• Herpesviruses: tissue samples, such as skin scrapings or biopsy specimens
• Adenoviruses: respiratory secretions, conjunctival scrapings, or stool samples
• Norovirus: stool samples
• Coronaviruses: respiratory secretions
• Influenza Viruses: respiratory secretions
• Poxviruses: skin scrapings or biopsy specimens
• Rabies virus: brain tissue samples taken post-mortem

Question 2

Describe the principles of serologic diagnosis of viral infections

Answer 2

Serologic diagnosis refers to the identification of viral infections through the detection of antibodies in a patient’s blood (serum) that have been produced in response to a viral infection

1) Principle of Immune Response:

• When a foreign substance like a virus enters the body, the immune system recognises this antigen and produces specific antibodies against it

2) Detection of Antibodies or Antigens:

• serologic assays can either detect the presence of antibodies produced in response to a viral infection (e.g., ELISA, Western Blot) or they can detect the viral antigen itself (e.g., Rapid Influenza Diagnostic Tests, RIDTs)

3) Type of Antibodies:

• The type of antibody detected can give clues about the stage of infection
• For instance, the presence of IgM antibodies typically indicates a recent or acute infection, while the presence of IgG antibodies suggests a past infection or immunity due to vaccination
• Some tests can also detect IgA antibodies, which are often found at mucosal surfaces and can indicate a localised infection

4) Antibody Titer:

• The quantity or concentration of antibodies, known as the antibody titer, can also be measured
• A significant rise in antibody titer between acute and convalescent serum samples usually confirms a current or recent infection

5) Seronegative/seropositive:

• Individuals who have no detectable antibodies to a particular virus in their blood are referred to as seronegative, vice versa

6) Cross-reactivity:

• Some viruses may share certain antigenic determinants, leading to the production of cross-reactive antibodies
• result in false-positive results
• Advanced techniques such as Western blot or neutralisation tests can often overcome this issue by being able to differentiate between closely related viruses

Question 3

Describe the principles of antigen detection and PCR for diagnosis of viral infections

Answer 3

Antigen Detection:

immunoassays that detect the presence of a specific viral antigen, implying current viral infection, not as sensitive as molecular tests; higher chance of false negatives

1) Sample Collection:

• blood, urine, or nasal swabs
• then prepared for testing, which might involve dilution, addition of buffers, or other treatments

2) Antigen-Antibody Reaction:

• Specific antibodies labelled with a signal-generating molecule (like a fluorescent dye or enzyme) are added to the sample
• If the viral antigen is present in the sample, it will bind to the antibodies

3) Signal Generation:

• The signal-generating molecule attached to the antibody will create a measurable signal if antigen-antibody binding has occurred

4) Measurement:

• The generated signal is then measured, typically by a machine. If the signal exceeds a certain threshold, the test is considered positive

The method, while less sensitive than PCR, has the advantage of being faster and often easier to perform, allowing for rapid, point-of-care testing

PCR (Polymerase Chain Reaction):

PCR is a technique used to amplify small segments of DNA; very sensitive

1) Sample Collection and Preparation:

• swab from the respiratory tract, a blood sample, stool, or other bodily tissues or fluids
• The sample is then processed to extract the viral genetic material

2) Reverse Transcription (for RNA Viruses):

• If the virus has an RNA genome (like SARS-CoV-2 or Influenza), the RNA is first reverse-transcribed into complementary DNA (cDNA) using an enzyme called reverse transcriptase

3) PCR Amplification:

• The PCR process involves a series of temperature changes that are designed to denature the DNA (separate the DNA strands), anneal specific short DNA primers that are complementary to the sequences of interest, and extend (synthesise new DNA) from these primers using a heat-stable DNA polymerase

4) Detection:

• The amplified DNA can then be detected
• often done in real-time (during the PCR process) using fluorescent dyes that intercalate into the amplified DNA, with the amount of fluorescence being proportional to the amount of DNA
• The test is considered positive if the fluorescence exceeds a certain threshold

PCR tests are more complex and time-consuming than antigen tests and require specialised laboratory equipment and trained personnel

They are often used in a confirmatory role when the results of less sensitive tests are unclear, or for initial diagnosis during the early stages of an outbreak when rapid tests may not yet be available

Question 4

Describe the methods employed for the detection of antiviral resistance

Answer 4

1) Genotypic Testing:

• involves sequencing the gene or genes of a virus known to harbour mutations that can confer resistance
• Polymerase chain reaction (PCR) is the most commonly used method for amplification. Once the regions of interest have been amplified, they are sequenced to determine the specific nucleotide sequence
• This sequence is then compared to the reference sequence of a susceptible virus strain to identify any mutations
• The presence of certain mutations can infer resistance to specific antiviral drugs
• faster and less expensive than phenotypic testing but requires knowledge of which mutations confer resistance

2) Phenotypic Testing:

• the virus is cultured in the presence of varying concentrations of an antiviral drug
• The virus’s ability to replicate in the presence of the drug is then measured, typically using an assay that detects viral enzymes or viral-induced cytopathic effects
• Phenotypic assays can provide a more direct measure of resistance than genotypic assays and can detect resistance even if the responsible mutation is unknown
• The concentration of drug required to inhibit viral replication by 50% (IC50) is often compared to a reference strain to quantify resistance
• Phenotypic testing is more labour-intensive and expensive than genotypic testing but can detect resistance even if the responsible mutation(s) are unknown

3) Next-Generation Sequencing (NGS):

• NGS allows for the sequencing of large portions of the viral genome, or even the entire genome, to identify mutations
• As such, it can provide a more comprehensive picture of potential resistance than traditional genotypic testing

4) Digital PCR:

• This is a highly sensitive method that can quantify the number of viral particles with a specific mutation in a given sample
• This can be helpful for detecting minor viral populations with resistance-associated mutations that might be missed by other methods