Diagnostic Testing for Viruses and Bacteria

What is used for the detection and identification of virus particles in specimens?

electron microscope

What is used for the detection of a whole infectious virus?

virus isolation

What is used for the detection of viral nucleic acid in clinical specimens?

PCR

What is used for the detection of viral antigen in clinical specimens?

ELISA

What is used for the detection of viral antigen in tissues/cell?

Immunohistocytology

What is used for the detection of viral nucleic acid in tissue/cells?

In Situ Hybridization

What is used for the detection of host immune response?

antibody serology

What is a direct detection method?

detect the virus or its components; pathogen

What is a indirect detection method?

detect the host immune response (Serology)

What should be avoided with swabs?

calcium alginate and wooden handles

True or False: Whole blood should never be frozen.

True

What blood tube is not suitable for virus isolation and PCR?

green top-heparin

Why collect blood for laboratory testing?

viremia (virus in blood) for virus isolation and can also measure humoral (antibody) and cellular immune responses

What are paired samples?

two samples taken 2-3 weeks apart during acute and convalescent phases

What would cause a highly accurate test to still come back negative?

collected at the wrong time

What temperature should be avoided when storing samples due to its ability to kill viruses?

-20 °C

What can be used to preserve DNA and RNA at room temperature for storage and shipment?

molecular transport medium (MTM)

What direct methods are used for the detection of viruses?

electron microscopy (EM) and virus isolation (VI)

What direct methods are used for the detection of viral antigens?

Immunofluorescence assay (IHC, ICC) and point-of-care (POC) assay (immunochromatography and immunofiltration)

What direct methods are used for the detection of viral nucleic acids (Molecular Diagnostic Assays)?

PCR, In Situ Hybridization, Next Generation Sequencing

What indirect methods are used for the demonstration of virus-specific antibodies?

serum virus neutralization test, plaque reduction test, ELISA, hemagglutination inhibition, complement fixation, agar gel, western blotting

What are the two methods of electron microscopy?

negative stain and thin section

What is negative stain electron microscopy?

virus particles in fluid

What is thin section electron microscopy?

thin sections of tissues

Due to viruses not being able to grow on microbiological broths or on agar plates, what must you do for virus isolation?

culture inside suitable host cells (requires living cells)

What are the three major cell culture systems?

primary cells, continuous cell lines (immortalized), and organ cultures

What are cytopathic effects?

morphological changes to viral monolayer; virus changes cells

What does cytopathic effect (CPE) tell you in virus isolation?

suggest viral replication but cannot identify the virus alone

Why does CPE need to be confirmed with other techniques?

lack specificity

What is the key to detecting viral antigens?

antibodies

What is required for an immunofluorescence assay?

microscope with UV light source and fluorescein isothiocyanate or texas red dyes

What is a direct fluorescent antibody test?

the primary antibody specific for the antigen is directly labels with the fluorescence dye

What is an indirect fluorescent antibody test?

the secondary antiglobulin antibody is labeled with fluorescence dye

What are immunoenzyme assays?

enzymes conjugated to immunoglobulins identify specific antigens in tissue sections

What are the two types of immunoenzyme assays?

immunohistochemistry (IHC) and immunocytochemistry (ICC)

What is a direct IHC/ICC?

the primary antibody specific for the antigen is directly labeled with the enzyme horseradish peroxidase

What is an indirect IHC/ICC?

the secondary antiglobulin antibody is labeled with the enzyme horseradish peroxidase

What do immunoenzyme assays (IHC/ICC) tell you?

distribution and localization of specific cells or viral proteins within cells and within proper histological context

What is the difference between immunocytochemistry and immunohistochemistry?

ICC is performed on cell monolayers instead of histology sections

What can be used for the detection of noncytopathic effect viruses?

immunofluorescence or immunocytochemistry

What are enzyme-linked immunosorbent assays? (ELISA)

detect antigens or antibodies with moderate sensitivity

What are the basic principles of direct sandwich ELISA?

immobilization of antigen (capture antibody), primary antibody binding with enzyme, ± secondary enzyme-labeled antibody, substrate addition (color development), and detection

How can you increase the sensitivity of an ELISA?

addition of biotin-avidin streptavidin

What are the three steps of PCR amplification?

denaturation, annealing, extension

Due to detection occurring after amplification in PCR rather then continuously during the reaction, what can you do to detect in real time?

fluorescence probes (TaqMan)

What is the threshold cycle (C_T) of TaqMan?

calculated fractional cycle number at which fluorescence crosses a defined threshold

C_T correlates with the initial amount of target temple. What does a lower C_T indicate?

more target nucleic acid; sooner to be detected

C_T correlates with the initial amount of target temple. What does a higher C_T indicate?

less target nucleic acid; longer to be detected

What allows you to measure two or more genes in one reagent mix?

multiplex qPCR because multiple amplified in same reaction

What does qPCR rely on?

every amplification cycle to determine the relative amount of the target

What does dPCR rely on?

Poisson statistics to determine the absolute target quantity following an end-point amplification (using negative partitions)

Can PCR detect infectious virus?

can only be detected by VI

Which of the following lists the diagnostic tests in order from least to most sensitivity and specificity?

EM, VI, IF/IHC/ICC, PCR

What is In Situ Hybridization (ISH)?

DNA probes used like antibodies to detect, locate, and quantitate specific viral nucleic acid sequences in tissue sections but you need to know the sequence you are testing for

What are the two types of In Situ Hybridization (ISH)?

chromogenic ISH and by fluorescence detection (FISH)

What does serology tell you?

confirmed past or active exposure to viruses (bacteria or fungi) by detecting the host immune response; immune system has encountered an antigen

What is an “antibody titer“?

quantification of the antibody response; greatest dilution of the serum sample that retains detectable activity

What is 1:4 to 1:64?

4-fold increase

What is the factors that influence serological tests?

vaccines because cannot distinguish antibodies generated against inactivated and modified live vaccines from natural infection of a particular disease agent and maternal antibodies

What ELISAs are used for Ag detection?

direct and sandwich

What ELISAs are used for Ab detection?

indirect and competitive

What ELISAs are used for early detection of IgM Ab?

IgM capture

What is a virus neutralization test?

uses cells as an indicator system to identify serum virus-neutralizing (SVN) antibodies

What is a SVN test used with?

known virus and unknown serum to be tested for Abs

What is a VNT test used with?

unknown virus and known virus-specific anti-serum

What is a plaque reduction neutralization test (PRNT)?

used to quantify the titer of neutralizing antibody for a virus

What is a hemagglutination inhibition test?

antibodies against certain viruses can be detected by their ability to interfere with viral hemagglutination

What is an agar gel immunodiffusion test?

antigen and antibodies are incubated at room temperature for 24-48 hours and they diffuse through the gel to form precipitation lines (positive test result)

What is the most common agar gel immunodiffusion test used in veterinary medicine?

coggins test in horses for equine infectious anemia

What samples should you never refrigerate?

blood culture specimens, cerebrospinal fluid, specimens submitted for anaerobic culture

What temperature range is best for bacterial samples?

2 °C to 8 °C

What are the three medium used for anaerobic bacteria storage and transport?

amies agar, thioglycolate, anaerobic transport medium