Diagnosis of Viral Infections Flashcards
risk group 1
no or low individual and community risk
ex. adeno-associated virus
risk group 2
moderate individual risk, low community risk; risk of spread of infection limited
ex. herpes viruses, foot-and-mouth disease
risk group 3
high individual risk, low community risk; doesn’t ordinarily spread from one infected individual to another; effective tx available
ex. HIV, hepatitis B, hantaviruses
risk group 4
high individual and community risk; effective tx and preventative measure not usually available
ex. Lassa fever, smallpox, influenza
What requirements exist for BSL-4 labs?
- positively air pressured suit
- negative air pressure in lab room
- HEPA filtered air
- double autoclave
- suit decontamination shower
What risk group is handled at BSL4 labs?
risk group 4 (E.g. Ebola)
biohazard
biological substances that pose a threat to the health of living organisms
biosafety
containment principles, technologies and practices that are implemented to prevent the unintentional exposure to pathogens and toxins, or their accidental release
aerosol
very small droplets of fluid that can spread via air
biosafety cabinets (BSC)
enclosed, ventilated laboratory workspace for safely working with materials contaminated with pathogens requiring a defined biosafety level
biosecurity
protection, control and accountability for valuable biological materials within labs, in order to prevent their unauthorized access, loss, theft, misuse, diversion or intentional release
successful detection of viruses from a sample depends on:
- collection of sample from right site
- at right time
- from most appropriate animal
- proper transport and storage of sample
- performing correct diagnostic test
- proper interpretation of results
When is the chance of viral recovery best?
during first 3 days after onset; greatly reduced beyond 5 days
When should blood samples be collected for serological tests?
one during acute phase of illness and one during convalescence period (10-14 days after 1st)
When should specimens for PCR be collected?
during early part of the illness
Where should samples be collected for respiratory and ocular disease?
live: nasal and conjunctival swabs, blood
postmortem: tissues from affected system, lymph nodes
Where should samples be collected for skin disease and lesions of mucous membranes?
live: scrapings of lesion, swab affected area, blood
postmortem: tisues from affected system, LN
Where should samples be collected for gastroenteritis?
live: feces, blood
postmortem: tissues from affected system, LN, intestinal contents
Where should samples be collected for systemic disease ?
live: blood, nasal and urogenital swabs, feces
postmortem: tissues from various organs
Where should samples be collected for CNS disease?
live: blood, CSF, feces, nasal and urogenital swabs
postmortem: tissues from affected system, LN
Where should samples be collected for disease of urinary tract ?
live: urogenital swab, urine, blood
postmortem: tissues from affected system, LN
Where should samples be collected for abortion?
live: blood from dam, vaginal mucus
postmortem: tissues from placenta and fetus; blood from fetal heart; intestinal contents
How much blood should be sampled for serological testing?
10-20 mL for large animals and 5-10 mL for small animals
*clotted sample for serology and sample with anticoagulant for other tests
Should you freeze samples?
NO refrigerate at 2-8 C; if they have to be frozen, freeze rapidly at -20 C or - 70C
T/F specimens for histo examination should never be frozen
T
How should histo samples be fixed?
10% buffered fomalin or fixatives
viral transport medium (VTM)
prevents specimen from drying, helps maintain viral viability and retards growth of microbial contaminants; consists of buffered salt solution with added protein and antimicrobials
3 potential hazards associated with transportation of pathogens
- breakage of containers
- exposure to possible infection
- delays in package delivery to diagnostic lab
Whats the best way to prevent spillage?
basic triple packaging system
processing samples
- tissue homogenization (Ten Broeck grinder, mortar and pestle)
- vortex mixer and centrifuge for feces and swabs
negative stain electron microscopy
virus sample mixed with solution of heavy metal salt that is highly opaque to electrons (sodium phosphotungstate or uranyl acetate) then spread on thin layer on carbon-coated copper grid and dried
How many virions must fluid matrix contain for virus particles to be detected by negative stain electron microscopy?
10^6 - 10^7 virions /mL
TEM
based on transmitted electrons; 2D images with higher mag and greater resolution than SEM
SEM
based on scattered electrons; 3D image of the surface
assay
qualitative or quantitative measurement of a target entity/analyte
gold standard test
diagnostic test that is considered to be the most accurate and best available under a particular condition or set of conditions
negative predictive value (NPV)
probability that a negative test result accurately indicates the absence of infection
positive predictive value (PPV)
probability of a positive result accurately indicating presence of infection
sensitivity
probability that cases with the infection will have a positive result using the test under evaluation
specificity
probability that cases without the infection will have a negative result using the test under evaluation
serum
clear yellowish fluid obtained upon separating whole blood into its solid and liquid components after it has been allowed to clot
*put in red top vacutainer tube
plasma
produced when whole blood is collected in tubes that are treated with an anticoagulant; cells removed by centrifugation
*put in lavender-top EDTA vacutainer tube
Whats the make up of blood ?
- plasma 55% total blood
- buffy coat (leukocytes and platelets)
What indicates a positive reaction in an ELISA?
intensity of color
direct ELISA
antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration; specificity of primary antibody is very important
indirect ELISA
primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary antibodies that recognize the primary antibodies
sandwich ELISA
antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies; two antibodies must be critically chosen to prevent cross-reactivity or competition of binding sites
competitive ELISA
antigen of interest from the sample and purified immobilized antigen compete for binding to the 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 labelled with a fluorescent dye (FITC or rhodamine) so that visible fluorescence appears following Ag-Ab reaction
can be direct or indirect (IFAT)
immunohistochemistry
antibody tagged with enzyme, generally horseradish preoxidase; enzyme reacts with substrate to produce a colored product that can be visualized in the infected cells with a standard light microscope
*direct or indirect
immunochromatography
aka lateral flow devices
point of care test that is simple to perform, easy to carry, and doesn’t require specialized equipment
*HIV test, pregnancy test
2 lines = positive, 1 line = control
point of care (POC)
diagnostic testing performed at or near the patient’s site of care
With immunochromatography, what are the antibodies labeled with?
colloidal gold
agglutination
method using property of specific antibodies to bind many antigens into single clumps thereby forming large complexes which are easily precipitated (visible macroscopically or microscopically)
hemagglutination
relies on property of some pathogens (mainly viruses) to nonspecifically agglutinate erythrocytes
What has the hemagglutination inhibition test been used for?
detection of serotype specific antibodies against avian influenze and peste des petits ruminants (PPR)
hemagglutination inhibition test
virus will bind to antibodies instead of RBC so no agglutination
agar gel immunodiffusion test
antigen and antibody placed in separate wells of an agar gel -> diffuse toward each other -> thin white line formed due to precipitation of ag-ab complex
When is agar gel immunodiffusion test used?
to detect antibodies against avian influenza (matrix antibodies), EIA (coggins) and enzootic bovine leukosis
complement fixation test
reactive: RBC settle in pellet -> no lysis
nonreactive: RBCs lysed by unbound complement
immunoblotting
antigen samples separated via gel (smaller go farther) and blotted onto nitrocellulose sheet -> specific antigen bind to corresponding labelled antibody -> autoradiography -> antigen bands visualized
hemadsorption
glycoproteins inserted into host cell membrane at sites of budding of enveloped viruses -> allows monolayer cells to adsorb RBC on their cell membranes
hemadsorption-inhibition assay
infected monolayer cells incubated with known specific antibody -> antibodies bind to viral glycoproteins in cell membrane -> wash away antibodies that aren’t bound to viral proteins -> pretreated monolayer cells are incubated with RBCs -> RBC binding inhibited
neutralization assay
antibody bound virus (neutralized) becomes non-infectious and can’t produce desired effects in eggs, cell-lines or animals
How can unneutralized virus be detected?
CPE, hemadsorption, hemagglutination, plaque formation, disease in animals
What is indicative of recent infection?
IgM antibodies -> appear early after infection but drop to low levels within 1-2 months and disappear within 3 months
