Lecture 7 - Diagnosis of Infection Flashcards
The first part of the Diagnosis Infection is Exposure History. What aspects of Exposure history include?
- Time of illness onset and/or appearance of symptoms
- Time and place of exposure
- Past infectious diseases or vaccinations
- Immunological status
- Exposure sources (people, animals, food, environment)
A Physical Examination is required to assess the patient. What types of things are examined in a physical examination?
Overt physical signs (rash, animal bites, etc)
Blood pressure, pulse, body temperature
Coughing, chest pain
Local infection
Systemic infection
What are Diagnostic Tests used for? What types of things are used for Diagnostic Testing?
Used to narrow down list of possible infections
Dependent on exposure history and physical exam
Epidemiological information critical
Invasive and non-invasive
Tests can take time and can be expensive
Invaluable for treatment, surveillance, and research
Definitive laboratory Tests are used for viral diseases. Why?
Patient management
Symptom vs. specific treatments
Availability of some antivirals
Rapid advancements in drug therapies
Screen blood supply for viruses
Track novel viral strains
Surveillance
What are Koch’s Postualtes and how they relate to viruses?
- Microbe must be associated with every case of the disease but not in healthy hosts.
- Microbe must be isolated from the diseased host and grown in pure culture.
- The cultured microbe must cause the same disease when innoculated into a healthy host.
- Microbe must be re-isolated from the intentionally infected host.
Viruses can be difficult to isolate and culture
What are the steps to proving caustation of viral diseases? There are six.
- Isolate virus from diseased hosts.
- Cultivation of virus in host cells.
- Proof of filterability.
- Production of a comparable disease when the cultivated virus is used to infect experimental animals (same or related species).
- Re-isolation of the same virus from the infected experimental animal.
- Detection of a specific immune response to the virus.
What are other ways to prove causation of viral diseases? (Slide 2)
- Nucleic acid sequence of the pathogen should be present in all cases of infectious disease
- No pathogen-associated nucleic acid sequences should be present in healthy hosts or tissue.
- Nucleic acid sequences of pathogen should no longer be detected upon resolution of disease.
- The nucleic acid sequence copy number that correlates with disease severity is more likely to be the cause of the disease.
- Clinical features and pathologies observed are consistent with the biological properties of the suspected pathogen.
- The pathogen or its antigens can be detected in disease tissue containing the nucleic acid sequences of the suspected pathogen.
- Sequence-based evidence of the pathogen should be reproducible.
Viral Diagnosis in Lab
What types of specimens are collected to diagnose?
- Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum (50% of all specimens)
- Eye infections: throat and conjunctival swab/scraping
- Gastrointestinal tract infections: stool and rectal swabs
- Vesicular rash: vesicle fluid, skin scrapings
- Maculopapular rash: throat, stool, and rectal swabs
- Central nervous system (CNS; encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid
- Genital infections: vesicle fluid or swab
- Urinary tract infections: urine
- Bloodborne infections: blood
What are the general approaches for the diagnosis of viral infections?
- Microscopy
- Detection of viral antigens
- Culture
- Nucleic Acid detection
- Antibody detection
What is an antigen?
A foreign substance that stimulates an immune response in the form of antibody formation or a cell-mediated response. Beacteria and viruses are common sources of antigens.
What is an Antibody?
A three-lobed globulin molecule found in the blood or other body fluids that can be produced by th epresenced of an antigen (invading microorganism). It has a destructive influence on the anitgen.
What is involved in the Microscopy approach?
Observation using immunofluorescence microscopy
Fluorescent antibodies detect viral antigens
Immunocytochemistry
Electron microscopy
Visualize virus particles in a sample
Useful to visual viruses that can’t be grown in cell cultures
Immunoelectron microscopy
Not sensitive, difficult, and expensive
- Fluorescent Microscopy
- Immunohistrochemistry
- Electron Microscopy
What is involved in the detection of Viral antigens?
Enzyme Linked Immunosorbent Assay (ELISA)
Rapid
Inexpensive
Technically easy to perform
Very sensitive
What is involved in the culture approach?
Viruses need living host cell for replication
Use of cell cultures for routine virus isolation
No single cell line can support all viruses
Cell cultures monitored for cytopathic effects (CPE)
What is involved in Nucleic ACid Detection Approach?
Nucleic Acid Amplification Tests (NAATs)
Detect of viral nucleic acids
PCR (DNA viruses) and RT-PCR (RNA viruses)
Diagnosis
Management of patient
e.g., HIV, hepatitis C patients
What is PCR methodology?
PCR allows for viral detection
What is Antibody Detection?
- Presence of antibodies are an indirect measure of viral infection
- Patient serum contains antibodies
- Recent viral infection: IgM
- Re-infection with same virus: IgG
- ELISAs (already discussed)
- Western blotting
- Separation of viral proteins by size(SDS-PAGE)
What are microarrays and protein arrays?
DNA microarrays (“DNA chip”)
Diagnostic, including novel viruses
Detect agents of bioterror
Patient management
Vaccine quality control
Host gene expression responses to viral infection
What is involved in microarrays?
- Nucleic acid extracted from sample
- RNA or DNA is amplified, incorporating fluorescence probes
- Amplified, labeled nucleic acids added to “chip” spotted with complementary probes representing known viruses
- High-throughput microaraay reader used to interpret signals to identify presence/absence of virusl infection
What is involved in Protein Arrays?
Spin-off of the DNA chip
Chips probed with blood sample from a patient.
5.3 Viral Load Testing and Drug Susceptibility Testing
PCR-Based Strategies
Used to monitor viral loads
PCR
RT-PCR
HIV and hepatitis C
Plaque reduction assays
Measure effects of antiviral drugs
Experimentally test vaccine efficacy
What is a Plaque Assay?
- Quantitative assay
- Monolayers infected with serial dilutions of virus
- Agarose overlay to limit spread
- Plaques visualized by staining cells with dyes (crystal violet)
- Only measures the # of infectious particles in a sample
5.4 Working with Viruses in the Research Laboratory
What should you think about when growing viruses in a lab?
- Viruses need a “host” system.
- Viruses can be grown in:
- Animals (some only grow in animals!)
- Embryonated eggs
- Tissue (cell) cultures (preferred method)
- Virologist’s facility
Laminar flow hoods
Contains HEPA filter
What are Cytopathic Effects (CPE)?
- CPE occur as a result of viral replication cycle
- CPE of infected cells can be observed with inverted light microscopes
- Rounding/detachment from plastic flask
- Syncytia/fusion
- Shrinkage
- Increased refractility
- Aggregation
- Loss of adherence
- Cell lysis/death
- Inclusion bodies
What is Hemadsorption?
Adherence of red blood cells to other cells, including virally infected cells.
Influenza virus
Parainfluenza virus
Measles virus
Envelope protein hemagglutinin (HA)
What are some common methods used to study viruses in a research lab?
Plaque assays
Endpoint dilution method or tissue culture infectious dose (TCID)50
Neutralization, hemagglutination, and hemagglutination inhibition assays
Transformation (focus) assays
Interference assays
PCR-based methods
Detection of viral enzymes
What is TCID50 assays?
Quantitative assay
Monolayers infected with serial dilutions of virus
Infected cultures scored for CPE
Tissue culture infectious dose 50
≥50% of cultures display CPE
What is viral Transformation?
Transformation/focus assays
Immortalization of cells in culture
Loss of contact inhibition/foci formation
Ability to form tumors in animals
Describe the Lab safety Levels
