Week 9 Flashcards
Virus Diagnostics
Accurate virus diagnosis based on clinical examination alone of a case is not always reliable, except
during a known epidemic with large numbers of cases presenting with a similar clinical picture.
Rationale for Use of Laboratory Virus Diagnostics:
- Appropriate management of a patient with viral disease is dependent on the diagnosis. Rapid and
accurate diagnosis can avoid the use of unnecessary tests or treatments, and assist with prognosis.
- Selection of antiviral agents in treatment normally requires accurate identification of the infecting virus.
- Detection and surveillance of new or emerging viral pathogens for public health control.
Accurate diagnosis of viral infection and disease normally requires laboratory testing.
Diagnostic methods should satisfy a number of criteria including:
- Rapidity
- Simplicity
- Sensitivity
- Specificity
- Cost
Specimen Collection (virus)
Successful diagnosis of a viral infection is dependent on appropriate specimen collection:
- Right time means collection as soon as possible after the patient first presents with symptoms.
- Right place means collection from body location that constitutes a major site of viral replication
Viral Diagnostic Methods
Virus Isolation with In Vitro Culture
Direct Observation with Microscopy
Detection of Viral Antigen with Immunoassay
Detection of Viral Genetic Material with PCR
Detection of anti-Virus Antibodies in Acute and/or Convalescent Sera
Effect of Viral Replication of Cultured Cell
Cytopathic Effect (CPE)
Syncytia (cell fusion)
Haemadsorption
4 examples of Gastrointestinal Viruses in faecal samples
a) Calicivirus
b) Poliovirus
c) Rotavirus
d) Adenovirus
Direct Observation with Light Microscopy (Viruses)
Search for inclusion bodies (virus materials) in histological samples.
It is a low-specificity and low-sensitivity technique. However, it can beuseful for diagnosis of a few selected viral infections.
Steps for RT-PCR for COVID diagnosis
- nasopharyngeal swab
- Collected specimen
- RNA extraction
- RT-qPCR
- Test results
qPCR Fluorescent Probes
SYBR Green binds nonspecifically to dsDNA, but does not bind to ssDNA orRNA. SYBR Green added to PCR mixbecomes fluorescent only when bound,thus indicating that dsDNA is present, inthis case due to the amplification process.
Gene-specific fluorescent probes aremade by attaching a fluorescent dye to ashort DNA probe that matchesthe targetsequence being amplified. The dyefluoresces only when dsDNA of the correctsequence accumulates.
Cycle threshold
The Ct value is inversely proportional to the number of
initial copies of target viral nucleic acid sequence
Detection of anti-Virus Antibodies in Acute and/or Convalescent Sera
Criteria for primary infection diagnosis
- At least a 4-fold increase of IgG titer in acute serum compared to convalescent serum
- IgM detection
- Seroconversion
Criteria for diagnosis of reinfection/reactivation
- A strong increase of IgG titer
- Absence of or low IgM titer
techniques for Detection of anti-Virus Antibodies in Acute and/or Convalescent Sera
Techniques
- Enzyme immunoassays (ELISA)
- Radioimmunoassays(RIA)
- Complement fixation
- Western Blot
ELISA
A very sensitive immunological assay that is widely
used in diagnostic virology
An ELISA can detect as little as 0.01 nanograms of
antigen or antibody
- Fast (typically few hours)
- Low cost
- Lack of hazardous waste
- Long shelf life
- High specificity
- High sensitivity
Summary (Viruses)
Molecular diagnostic techniques based on the detection of viral antigen or genetic material offer greater
sensitivity and rapidity compared to traditional methods such as in vitro culture and direct microscopy.
Detection of anti-virus antibodies in serum can allow the detection of past viral infections even after the
virus has been eliminated from an individual.
