Diagnosis of Viral Infections Flashcards
Transmission Electron Microscopy
Based on transmitted electrons to see what is inside or beyond the surface
Pathogen that can caus human or animal disease but unlikely to be a serious hazard to laboratory workers, the community , livestock or the environment.
Laboratory exposure may cause serious infection, but effective treatment and preventive measures are available and risk of spread of infection is limited
Risk Group 2
Mechanism of Action of Immunochromatography
- Ab is immobilized on chromatographic paper, other is labeled with colloidal gold and infiltrated into sample pad
- Liquid sample is dropped on sample pad, Ag in sample forms an immunocomplex with Ab labled with colloidal gold
- Complex moves along with the liquid sample makes conact with the Ab immobilized followed by forming an immunocomplex with immobilized Ab generating color
Neutralization
Loss of infectivity through reaction of the virus with specific antibody
Competitive ELISA
Antigen of interest from the sample and purifed immobilized antigen compete for binding to teh capture antibody. Decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample
Fluorescence Antibody Test (FAT)
Antibodies are labelled with a fluorescent dye. Visible fluorescence appears following antigen-antibody reaction
Tissue Homogenization
Finely minced and homogenized tissue in glass or mechanical homogenizer
(Transmission/Scanning) electron microscopy produces images with a higher magnification and greater resolution
Transmission Electron Microscopy
Electron Microscopy
Used to demonstate viruses in sample and detect viruses that cannot be grown in-vitro
Direct FAT
Labelled Ab are added onto the samples Ag. Visible fluorescence appears at the binding sites of the specific Ab (Ab-Ag binding)
Complement Fixation Test
- Serum with Ab
- Ag binds with Ab
- Complement binds with Ag/Ab complexes
- Hemolysin sensitized RBCs serve as indicator
- RBC settle into a pellet
- No lysis occurs
In negative stain electron microscopy, the virus structure is (white/grey)
White
A microorganism that is unlikely to cause human or animal disease
Risk Group 1
Typical ELISA
- Antigen coated in a well
- Antibody tagged with enzyme
- Antgen binds to enzyme tagged antibody
- Wash the excess unbound antibodies
- Add substrate
- Enzyme tagged to antibody which is bound to antigen will change color
Point of Care (POC)
Diagnostic testing performed at or near the patients site of care
Assay
Qualittive or quantitative measurement of a target entity/analyte
Sandwich ELISA
Antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. Two antibodies must be very critically chosen to prevent cross reactivity or competition of binding sites
Immunochromatography
Form of POC test that is simple to perform, easy to carry and does not require specialized equipment
Direct ELISA
Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. Specificity of the primary antibody is very important
Indirect Immunohistochemistry Assy
Enzyme tagged to a secondary antibody that is specific against primary antibody
Pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures not available
Risk Group 4
(Transmission/Scanning) electron microscopy produces three-dimensional images
Scanning Electron Microscopy
Immunohistochemistry
Ab is tagged with an enzyme. Enzyme reacts with a substance to produce a colored product that can be visualized in the infected cells with a standard light microscope
Mechanism of Competitive Elisa
- Unlabeled Ab incubated in presence of its Ag
- Bound Ab/Ag complex added to Ag coated well
- Plate washed, unbound Ab removed
- Secondary Ab specific to primary Ab is added, second Ab is coupled with an enzyme
- Substrate is added - enzymes elicit chromogenic or fluorescent signal
- Reaction is stopped to prevent eventual saturation of signal
- Weak signal indicates presence of Ag in sample
To prevent spillage it is recommended to follow what system
Basic Triple Packaging System
Immunoblotting
- Ag separated on gel
- Blotting tank - proteins transferred to nitrocellulose sheet
- Specific antigen bind to corresponding labelled antibody
- Autoradiography
- Develop and fix autoradiograph
- Antigen bands visualized
Pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive meaures available.
Risk Group 3
Diagnostic laborator requires what data
Epidemiological Data
Case History
Clinical Signs
Gold Standard Test
Diagnostic test that is considered to be the most accurate and best available under a particular condition or set of conditions
Characteristics of a BSL-4 Lab
Maximum containment
Workers wear one piece, positively air pressurized suit
Negative air pressure mus be maintained in lab rooms
Incoming and outgoing air is HEPA-filtered
Sterilization through double door autoclave
Suit decontamination shower after leaving lab
Potential hazards associated wtih transportation of pathogens
Breakage of container resulting in spilling
resulting in exposure
Delay in package delivery
Plasma
Produced when whole blood is collected in tues that are treated with an anticoagulent. Blood does not clot in plasma tube. Cells are then removed by centrifugation
Agar Gel Immunodiffusion Test
- Antigen and Antibody placed in separate wells of an agar gell
- Antigen and Antibody diffuse toward each other
- Thin white line is formed due to precipitation of antigen/anitbody complex
Hemadsorption Inhibition Assay Mechanism
- Infected monolayer cells are incubated with known specific Ab
- Attempts made to wash away Ab
- Pretreated monolayer cells are incubated with RBCs - binding is inhibited
Biosecurity
Laboratory biosecurity describes the protection, control and accountability for valuable biological materials within laboratories in order to prevent their unauthorized access, loss, theft, misuse, diversion or intentional release
Positive Predictive Value (PPV)
Probability of a positive result accurately indicating the presence of infection
Negative Staining Electron Microscopy
- Virus sample is mixed with solution of heavy metal salt that is highly opaque to electrons
- Mixture is then spread on thin layer on carbon-coated copper grid and dried
- After bombardment with electron beam, stain absorbs electrons in much higher amounts
Indirect FAT
IFAT employs secondary antibody labeled with fluorescent marker that recognizes the primary Ab bound to Ag
Negative Predictive Value (NPV)
Probability that a negative test result accurately indicates the absence of infection
Hemadsorption
Glycoproteins inserted into host cell membrane at sites of budding of enveloped viruses, allows monolayer cells to adsorb erythrocytes on their cell membranes
Plasma - Clotting Factors =
Serum
Biosafety
Laboratory biosafety describes the contaiment principles, technologies and practices that are implemented to prevent the unintentional exposure to pathogens and toxins or their accidental release
Specificity
Probability that cases without infection willl have a negative result using the test under evaluation
Detection of viruses can be through
Culture
Inoculation in eggs
Direct Immunohistochemistry Assay
Enzyme tagged with primary Ab that binds to Ag, upon successful Ag-Ab binding, tagged enzyme catalyzes substrate to produce color product
Biosafety Cabinets (BSC)
Enclosed, ventilated laboratory workspace for safely working with material contaminated with pathogens requiring a defined biosafety level
Site from which the specimen is collected will be influenced by
Clinical signs and knowledge of the pathogenesis of suspected virus
Diagnosis of viral infections by gross evaluation involves
Clinical Sings
Necropsy
Histopathology
Aerosol
Very small droplets of fluid that can spread via air
Indirect ELISA
Primary antibodies are not labeled, but detected instead with enzyme conjugated secondary antibodies that recognize the primary antibodies
Agglutination
Method using the property of specific antibodies to bind many antigens into single clumps thereby forming large complexes, whihc are easily precipitated. Precipitation can be macroscopically or microscopically visible
Viral Transport Media (VTM)
Stabilize the infectivity of specimens, especially swabs. Prevents specimen from drying, helps maintain viral viability and retards growth of microbial contaminants
Scanning Electron Microscopy
Based on scattered electrons, focuses on the samples surface and composition
For virus isolation when is the best time to collect a sample?
Soon after the onset of symptoms
________________________
First three days after onset, greatly reduced beyond 5 days
Sensitivity
Probability that cases with the infection will have a positive result using the test under evaluation
For serological tests when is the best time to collect samples?
Two blood specimen
One during the acute phase
Second during the convalescence period
Biohazard
Biological substance that pose a threat to the health of living organisms, primarily humans
Successful detection of viruses from a sample depends on:
Collection of sample from correct site
Correct time
Most appropriate animal
Proper transport and storage
Correct diagnostic test
Proper interpretation of results
IgM Class-Specific Antibody Assay
IgM appear early in infection but drop to low levels within 1-2 months and generally altogether within 3 months, they are usually indicative of recent infection
Neutralization Assay
- Presence of unneutralized virus detected
- Virus and serum mixed under apprpriate conditions and incoculated into cell culture
- Ag/Ab reaction
- Ab bound virus becomes non infectious
Serum
Clear yellowish fluid obtained upon separating whole blood into its solid and liquid components after it has been allowed to clot
Hemagglutination and Hemagglutination inhibition method relies on the property of some pathogens to
Nonspecifically agglutinate erythrocytes
