Diagnosis of viral infections

Question 1

Risk group 1

Answer 1

no or low individual and community risk

Question 2

risk group 2

Answer 2

moderate individual risk, low community risk

Question 3

risk group 3

Answer 3

high individual risk, low community risk
-pathogen causes serious disease but is not spread often, treatment is available

Question 4

risk group 4

Answer 4

high individual risk and high community risk
-pathogen causes disease and can be easily transmitted, no treatment available

Question 5

BSL 4

Answer 5

maximum containment lab, handle dangerous pathogens EX ebola virus
has neagtive air pressure that is contained, HEPA filter for air, steralization through double autoclaving door, decontamination shower

Question 6

biohazard
biosafety
aerosol
biosecurity

Answer 6

substances that pose a threat to health

protocols to prevent external contamination

very small droplets of fluids that can spread via air

protection, control and accountability for valuable biological materials to prevent loss, theft, etc.

Question 7

timing of sample collection:
virus isolation vs. serological test

general rule for morphological diagnostic

Answer 7

virus isolation: collect sample as soon as symptoms start, chance of viral recovery is best during first 3 days and declines after 5 days

serological test: collect 2 blood specimens, one during acute phase and one during convalescence period

morph. diagnostic: for PCR, obtain during early part of illness

Question 8

transporting sample

Answer 8

put swabs into viral transport medium and use triple packing system

Question 9

diagnosis of viral infection (3 ways)

Answer 9

clinical signs, necropsy, histopathology

Question 10

detection of virus by cultivation (2 ways)

Answer 10

in cell culture and inoculation in eggs

Question 11

diagnosis method - EM

Answer 11

1. electron microscopy: see characteristic morph.
   use negative stain EM, bombard with electron beam, the stain absorbs electrons in much higher amounts than the sample, parts of the viral particles that are not penetrated appear as electron lucent (low affinity, less density), areas on opaque background, to detect: matrix must have 10^6 - 10^7 virions per mL

Question 12

2 types of EM

Answer 12

1. scanning EM: to see surface and topography, scattered electrons, produces 3D images but lower magnification and resolution compared to TEM
2. transmission EM: based on transmitted electrons, to see through virus and beyond surface, higher magnification and greater resolution but produces 2D images

Question 13

assay

gold standard test

Answer 13

qualitative or quantitative measurement of a target entity/analyte such as a drug or biomolecule

diagnostic test that is considered to be the most accurate and best available under a particular condition or set of condition

Question 14

sensitivity

specificity

Answer 14

probability of cases with the infection that will have a positive result using the test under evaluation

probability that cases without the infection will have a negative result using the test under evaluation

Question 15

collection of serum - what color tube top?

Answer 15

RED top vacutainer tube

Question 16

collection of plasma - what color tube top?

Answer 16

lavender top EDTA tube (has anti coagulant)

Question 17

Direct Enzyme linked immunosorbent assay (ELISA)

Answer 17

1. AG coated in a well
2. add AB tagged with an enzyme
3. AG binds to enzyme-AB
4. wash excess unbound AB
5. add substrate
6. enzyme tagged to AB which is bound to AG will change color of substrate
   - intensity of color indicates more positive reaction

-AG immobilized and enzyme - conjugated primary AB are used to detect or quantify AG concentration, specificity of primary AB is very important

Question 18

indirect ELISA

Answer 18

primary AB are not labelled, but detected instead with enzyme conjugated secondary AB that recognize the primary AB
known AG, unknown AB

Question 19

sandwich ELISA

Answer 19

AG to be measured is bound between a layer of capture AB and a layer of detection AB - the 2 AB must be very critically chosen to prevent cross reactivity or competition of binding sites - 2 AB are known, AG is unknown

Question 20

Competitive ELISA

Answer 20

decrease in signal when compared to assay wells with AG alone = presence of AG in sample
-WEAKER SIGNAL = PRESENCE OF AG IN SAMPLE = + ve result

Question 21

fluorescence antibody test (FAT)
direct vs. indirect

Answer 21

direct: labelled AB are added to the sample (AG), visible fluorescence appears at the binding sites of the specific AB

indirect: IFTA employs a secondary AB labelled with a marker that recognizes the primary AB bound to an AG

Question 22

Immunohistochemistry

Answer 22

AB is tagged with enzyme, generally horseradish peroxidase, enzyme reacts with a substrate to produce a colored product that can be visualized in the infected cells with a standard light microscope

Question 23

immunochromatography (lateral flow devices)
what does POC mean

Answer 23

form of POC (point of care) test that is simple to perform, easy to carry and does not require specialized equipment

Question 24

agglutination

Answer 24

method using the property of specific AB to bind many AG (AG on pathogen, or AG coated particles - latex beads) into single clumps thereby forming large complexes, which are easily precipitated, precipitation can be seen macroscopically or microscopically

Question 25

Hemagglutination and hemagglutination inhibition test

Answer 25

method relies on property of some pathogens (viruses) to nonspecifically agglutinate erythrocytes
- if have AB = AB binds to virus and neutralizes = no agglutination = POSITIVE RXN because have AB

-if dont have AB = RBC destroyed and clumping = can see clumping because no AB to destroy virus = NEGATIVE RXN

Question 26

agar gel immunodiffusion test

Answer 26

AB and AB placed in separate wells of agar gel –> diffuse toward each other –> thin white line is formed due to precipitation of AG/AB complex

Question 27

complement fixation test - pos vs. neg reaction

Answer 27

EX: serum from patient A has AB against virus A –> intact sheep RBC settle at bottom = POSITIVE RXN

EX: serum from patient is negative to virus A, no AB –> hemolysis of RBC, destruction of sheep RBC because no AB to neutralize virus = NEGATIVE RXN

Question 28

neutralization assay
-neutralization of a virus is ?
-presence of unneutralized virus may be detected?

Answer 28

-loss of infectivity through reaction of the virus with specific AB
-detected by reaction such as CPE, hemagglutination, plaque formation, disease
-when virus and serum (AB) are mixed under appropriate condition and then inoculated into cell culture, eggs or animals –> AG-AB reaction

AB bound virus (neutralized) becomes non infectious and cannot produce desired effects in eggs, cell lines or animals