Laboratory Diagnosis

• Why are definitive laboratory tests for viral diseases developed?

  • Patient management
  • Availability of some antivirals
  • Rapid advancements in drug therapies require proper diagnosis
  • Screening blood supply from donors
  • Tracking novel viral strains
  • Initiating disease-specific control measures
  • Surveillance
• PROVING CAUSATION OF VIRAL DISEASES 

  • Based on Koch’s postulates for proving causation in bacterial diseases
  • Challenges with adapting for viruses:
  • Asymptomatic carrier state
  • Subclinical infections
  • Rivers 6 criteria for viruses 
  • Fredericks and Relman
  • Used biotechnology and PCR to offer updated causation guidelines
• Rivers’s Causation Criteria

  1. Isolate virus from diseased hosts
  2. Cultivation of virus in host cells
  3. Proof of filterability
  4. Production of a comparable disease when the cultivated virus is used to infect experimental animals
  5. Reisolation of the same virus from the infected experimental animal
  6. Detection of a specific immune response to the virus.
• VIRAL DIAGNOSTICS IN THE CLINICAL LABORATORY

  • >60% of infectious diseases seen by physicians are caused by viruses
  • Accurate and rapid detection and diagnosis key to successful treatment 
  • Factors influencing laboratory outcome:
  • Type and quality of specimen
  • Transport condition and time
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  1. MICROSCOPY
  2. VIRAL ANTIGEN DETECTION
  3. CULTURE
  4. NUCLEIC ACID DETECTION
  5. ANTIBODY DETECTION
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  
  

  Microscopy

  • Light Microscopy:
  • Used to observe intracellular inclusions
  • Immunohistochemistry (HIC)
  • Electron Microscopy:
  • Used to observe individual virus particles
  • Immunoelectron microscopy
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  CULTURE

  • Cell cultures used for virus isolation and identification
  • Monitored for cytopathic effects (CPEs)
  • Centrifugation culture (Shell vial technique)
  • Rapid diagnosis
  • Detects viral antigens before CPEs are present
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  Nucleic Acid Detection

  • Nucleic acid-amplification tests (NAATs):
  • Detect viral nucleic acids

  1. PCR and RT-PCR technology
  2. NASBA and TMA are non-PCR methods

  • Diagnosis
  • Management of patient: 

  1. Ex: HIV, hepatitis C patients
  2. Monitor viral load
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  ANTIBODY DETECTION

  • Presence of antibodies are an indirect measure of viral infection
  • Patient serum contains antibodies

  1. Recent viral infection: lgM
  2. Re-infection with the same virus: lgG

  • Methods

  1. Indirect immunofluorescent assays (IFA)
  2. ELISA
  3. Western Blot
• Microarrays and Protein Arrays: New Options in Viral Diagnostics

  • DNA Microarrays
  • Diagnostic
  • Detect agents of bioterror
  • Detect presence or absence of viral pathogenicity genes
  • Patient management
  • Vaccine quality control
  • Study of host gene responses to viral infections
• Protein Arrays

  • A spin-off of the DNA chip using arrays of antibodies immobilized on chips
• PCR-Based strategies

  • Used to measure and monitor viral load
  • PCR
  • RT-PCR
  • Compare viral load measurement after treatment to baseline measurement

  Plaque reduction assays

  • Gold standard for measuring effects of antiviral drugs
  • Toxicity assays performed
• WORKING WITH VIRUSES IN THE RESEARCH LABORATORY 

  • Viruses need a “host system”
  • Viruses can be grown in:
  • Animals
  • Embryonated eggs
  • Tissue (cell) cultures (preferred method
  • Optimal growth conditions vary greatly
  • Monolayers in flasks
  • Suspension cell growth
• Vertical flow laminar hood - Air filtered through high-efficiency particulate air (HEPA) filter. Removes 99.97% of particles ≥0.3 µm
• Cytopathic Effects (CPEs)

  • Visual changes in host from viral infections:
  • Formation of inclusion bodies
  • Rounding of the cells
  • Shrinkage
  • Increased retractability
  • Fusion/syncytia formation
  • Aggregation
  • Loss of adherence
  • Cell lysis/death
• • CPEs occur as a result of 
  • Entry into host cell
  • Inhibition of cellular transcription or stimulation of cellular RNA polymerase activity
  • Virus interactions with RNA processing pathways
  • Virus interactions with ribosomes
  • Host responses to viral infection
  • Inclusion Bodies
  • Subtle intracellular abnormalities
  • Can be indicative of specific viruses
• Hemadsorption (or hemagglutination).

  • Adherence of red blood cells to other cells, including virally infected cells
  • Envelope protein called hemagglutinin
  • Influenza A virus
  • Parainfluenza virus
  • Togavirus
  • Hemadsorption assays
• COMMON METHODS USED TO STUDY VIRUSES IN THE RESEARCH LABORATORY

  • Plaque assays
  • Tissue culture infectious dose (TCID)50
  • Neutralization, hemagglutination, and hemagglutination-inhibition assays
  • Transformation (focus) assays
  • Interference assays
  • PCR-based methods
  • Detection of viral enzymes
• Plaque Assays - Quantitative assay measuring number of viruses in a prepared virus stock
• TCID50 - Endpoint dilution assay. Developed before plaque assay and still used for viruses that do not form plaques but do produce CPEs
• Transformation (Focus) Assays

  • Determine Immortalization of cells in culture 
  • Ex: Tumor viruses
  • Transformed cell characteristics
  • Immortalization
  • Loss of contact inhibition
  • Anchorage Independence
  • Tumorigenicity
• Neutralization and Hemagglutination Inhibition Assays

  • Detect and quantify virus and strain-specific neutralizing antibodies
  • Without neutralizing antibodies. CPEs and hemagglutination occur
  • Hemagglutination assays are also used to quantitate or titer virus stocks in research labs.
• Interference Assays

  • Detect viruses that do not cause visual CPEs
  • Interference phenomenon
  • Non-visual CPEs detected by their ability to interfere with replication of second virus added to same culture
• PCR-Based Methods

  • Uses:
  • Discover emerging or reemerging viruses
  • Nucleic acid sequencing 
  • Study virus replication in vitro
  • Restriction fragment length polymorphism (RFLP)
  • Real-time PCR
  • Combined with fluorescent resonance energy transfer (FRET) in biomedical research
• LABORATORY SAFETY 

  • Labs classified by biosafety level (BSL)
  • BSL-1 (minimum containment)
  • BSL-2
  • BSL-3
  • BSL-4 (maximum containment)
  • Protective equipment varies by BSL
• FIVE APPROACHES FOR LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

  VIRAL ANTIGEN DETECTION

  • Enzyme-linked immunosorbent assay (ELISA) 
  • Inexpensive
  • Technical easy to perform 
  • Rapid turnaround