Diagnosis Of Viral Infections 1,2,3 Flashcards
Describe the classification of infective micro organisms by risk group.
-Risk group 1: no/low individual & community risk
-Risk group 2: moderate individual risk & low community risk
-Risk group 3: high individual risk, low community risk
>pathogen that causes serious human or animal disease but doesnt spread from one infected invidual to another
>effective T&P
-Risk group 4: high individual risk & community risk
>pathogen that causes serious human or animal disease that can be readily transmitted from one individual to another (directly/indirectly)
>NO effective T&P
Describe BSL4.
-max containment lab
-dangerous & exotic pathogens in risk group 4 like Ebola virus
-neg air pressure in lab room
-incoming & outgoing hair is HEPA filtered (high efficiency particulate air)
-sterilization thru double door auto claving system
-suit decontamination shower after leaving
Definitions!
- Biohazard
-biological substances that pose a threat to health of living organisms - Biosafety
-containment principles, technologies, practices that are implemented to prevent unintentional exposure to pathogens & toxins (or accidental release) - Aerosol
-sm droplets of fluid that spread via air (viruses can be spread this way) - Bio security
-protection, control, & accountability for valuable biological materials (VBM) in labs to prevent their unauthorized access, loss, theft, misuse, diversion, or intentional release - Assay
-qual/quantitative measurement of a target entity/analyte like drug or bio molecule - Gold standard test
-diagnostic test considered to be most accurate & best avail under particular condition or set of conditions
Describe how the timing of sample collection is important for 3 different types of tests.
- Virus isolation
-specimens collected soon after onset of symptoms bc max amount of titers of virus are present
>chance of viral recovery is best during 1st 3d after onset & is reduced beyond 5d - Serological test
-2 blood specimen collected
>1 during acute phase of illness
>2 during convalescence period - Molecular diagnostics
-PCR
-collected during early part of illness
Describe why transport & storage of samples are important.
-viral transport medium (VTM) = swabs
-to prevent spills
>triple packing system
Describe the different way to diagnose viral infections.
- CS
- Necropsy (gross eval)
- Histopathology
- Cultivation/isolation of virus in cells/tissue culture
- Inoculation in eggs
- Electron microscopy
-virus in sample & virus that cant be grown in vitro
Describe the different types of electron microscopy.
- Negative stain
-electron beam = stain absorbs electrons in higher amounts than the sample
-parts of the viral particles that aren’t penetrated by the stain appear as electron lucent (low affinity, less electron density) VS opaque (high affinity, electron dense) background
-fluid matrix must have approx 10^6-10^7 virions per ml to detect virus particles - Scanning electron microscope
- Transmission electron microscope
Describe the differences between TEM & SEM.
- TEM
-transmitted electrons
-sees what’s inside or beyond the surface
-2D images
-ADV: high magnification & greater resolution - SEM
-scattered electrons
-focuses on surface & composition
-ADV: 3D images
Describe sensitivity VS specificity.
- Sensitivity
-probability (%) that cases w the inf (determined by the result of the reference or gold standard test) will have a pos result using the test under eval - Specificity
-probability (%) that cases w/o inf (determined by result of the reference or gold standard test) will have a neg result using the test under eval
Describe collection of serum VS plasma.
- Serum = red top vacutainer tube
- Plasma = lavender top EDTA vacutainer tube
Describe the different types of serological assays for detection of viral infections.
- Enzyme linked immunosorbent assay (ELISA)
-Direct ELISA
-Indirect ELISA
-Sandwich ELISA
-Competitive ELISA - Fluorescence antibody test (FAT)
- Immunohistochemistry
- Immunochromatography (lateral flow device)
- Agglutination
- Hemagglutination & inhibition test
- Agar gel immunodiffusion test
- Complement fixation test
- Neutralization assay
Describe the steps of ELISA.
- Antigen coated in a well
- Add antibody tagged w enzyme
- Antigen binds to enzyme tagged antibody
- Wash excess unbound antibodies
- Add substrate
- Enzyme tagged to antibody which is bound to antigen will change color of substrate
>intensity of color indicates more pos reaction
Describe direct ELISA.
-antigens are immobilized & enzyme conjugated primary antibodies used to detect or quantify antigen conc
-specificity of primary antibody is imp
Describe indirect ELISA.
-primary antibodies aren’t labeled but instead detected w enzyme conjugated 2ndary antibodies that recog primary antibodies
Describe sandwich ELISA.
-antigen (meat) to be measured is bound between a layer of capture antibodies & a layer of detection antibodies
-2 antibodies (buns) critically chosen to prevent cross reactivity or competitions of binding sites
Describe fluorescence antibody test.
- Direct FAT
-labelled antibodies added onto sample (antigen)
-visible fluorescence appears at the binding sites of the specific antibodies (antigen-antibody binding) - Indirect FAT (IFAT)
-secondary antibody labeled w fluorescent marker that recog the primary antibody bound to antigen
Describe immunohistochemistry.
-antibody tagged w enzyme (horseradish peroxidase)
-enzyme reacts w substrate to make colored prod that can be visualized in the inf cell w light microscope
Describe agglutination.
-using the property of specific antibodies to bind many antigens (antigens on pathogen, or antigen coated particles - latex beads) into single clumps to form lg complexes = easily precipitated
-precipitation can be microscopically or macroscopically visible
Describe hemagglutination & hemagglutination inhibition test.
-relies on property of some pathogens (viruses) to non specifically agglutinate erythrocytes
Describe agar gel immunodiffusion test.
Describe the complement fixation test.
- Positive reaction
-serum from patient has antibodies against virus A -> intact sheep RBCs settle at bottom = pos reaction - Negative reaction
-serum from patient is neg to virus A = it has no antibodies
-hemolysis/destruction of sheep RBC = neg reaction
Describe microarrays.
-known DNA (probes), amplified by PCR/RT-PCR spotted onto glass or silicon chip
-target/sample DNA fluorescently labeled & hybridized/added to chip w DNA probes
-+ reactions between probe DNA & sample DNA (hybridization) gen a fluorescent signal from spot where probe DNA spotted in chip
*ADV: detect 100s of path in surveillance study & can be screened for simultaneously using single microarray chip
