Diagnostic Tests - Overview + Serology

Question 1

Screening vs. Diagnosis clinical question

Answer 1

Screening: test to ID early disease states or risk factors in apparently healthy/asymptomatic subjects

Diagnosis: test to determine the presence or absence of a specific entitiy in a symptomatic patient

Question 2

formula for sensitivity

Answer 2

[true +] / [(true +) + (false -)]

proportion of patients w/ dz and + test result

Question 3

formula for specificity

Answer 3

[true -] / [(true -) + (false +)]

proportion of patients w/out dz and a negative test result

Question 4

PPV vs. NPV

Answer 4

PPV: proportion of pts w/ (+) test that actually have disease

NPV: proportion of pts w/ (-) test that do NOT have disease

Requires knowledge on incidence & prevalence of disease in a population.

Question 5

A negative test rules out disease:

Answer 5

SnNout: test w/ high sensitivity (produces very few false negatives)
- prone to false positives

Question 6

A positive test rules in disease:

Answer 6

SpPIN: test w/ high specificity (produces very few false positives)
- prone to false negatives

Question 7

What dx methods directly visualize viruses, parasites, and bacteria?

Answer 7

Light or electron microscopy

Question 8

What dx methods detect antigen surface proteins?

Answer 8

Antibody-Antigen Based Tests
1. Enzyme-Linked Immunoabsorbent Assay (ELISA-Sandwich)
2. Immunofluorescence (IFA)
3. Immunohistochemistry (IHC)

detect ANTIGENS b/c of their surface proteins

Question 9

What dx methods (3) detect DNA or RNA genomes?

Answer 9

1. PCR & RT-PCR
2. NGS
3. In-situ hybridization

Question 10

How does ELISA work?

Answer 10

Antigen immobilized in well -> detection antibody w/ enzyme attached, forming Ag-Ab complex -> enzyme-specific substrate generates signal => color reaction

Capture-Antigen

Question 11

How do Direct IFA tests work?

Answer 11

Cells with high viral load fixed on slide -> antibody with fluorescent tag applied and links with antigen (conjugated) -> fluorescence microscope used to visualize complex

Question 12

How does IHC work?

Answer 12

tissues fixed to slide -> antibody with enzyme applied -> enzyme-specific substrate generates color reaction if antigen present in the lesion or specific cells

Question 13

How can IHC be used in neoplasia dx?

Answer 13

Tumor markers can have antibodies -> IHC detects antigens revealing info on cell of origin, proliferation stage, and prognostic indicators

Question 14

In RT-PCR, how do you know if the sample has a low viral load?

Answer 14

A high Cycle Threshold (Ct)

More amplification cycles required to detect DNA/RNA

Ct value = cycle # at which signal passes a fixed threshold

Question 15

How can PCR be used to dx neoplasia?

Answer 15

ID clonal lymphocyte populations; detect chromosomal translocations, deletions, duplications; detect mutations in MCTs

Question 16

What is NGS used for?

Answer 16

Unknown viruses are detected by sequencing the whole genome

Question 17

What is in-situ hybridization used for?

Answer 17

To visualize pathologic virus in the tissue or at the site of infection

Question 18

What is an indirect way to detect presence of a virus that you can’t do with other pathogens?

Answer 18

ID Cytopathic effect in cell culture

Cytopathic effect refers to structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to replicate.

Question 19

When are tests detecting organisms vs detecting antibodies most useful?

Answer 19

Detecting…
- Organisms: ID cause of acute disease, or determine cell of origin of a neoplasm for prognosis of tx
- Antibodies: ID organisms circulating within a population

Question 20

Methods to detect antibodies using serology (7)

Answer 20

1. Detect host response (IgM and IgG)
2. ELISA (Direct and Indirect)
3. Agar Gel Immunodiffusion (AGID)
4. Virus Neutralization
5. IFA
6. IHC
7. Agglutination

Question 21

What is detected in the FIV/FeLV Snap Test?

Answer 21

1. FIV Ab
2. FeLV Ag
3. (+) and (-) Controls

FIV antigen can go into latency -> preference is detecting antigen over antibody to determine clinical infection (Snap test only detects FeLV antigen).

SNAP tests = type of ELISA

Question 22

How does AGID work?

Answer 22

Center well = antigen, test serum loaded in periphery -> a (+) charge is added to the gel and Abs move toward Ag, precipitating a band

Question 23

How does Virus Neutralization work?

Answer 23

Used to determine viral antibody titer:
- serum from infected patient is cultured & loaded in wells -> diluted w/ Ab until virus neutralized
- highest dilution of serum that prevents cytopathic effect = Ab titer reported (e.g., canine distemper, BVDV)

Question 24

Molecular diagnostics are direct detectors of what?

Answer 24

DNA or RNA genomes

Question 24

What are 3 scenarios that can produce antibodies?

Answer 24

1. Infection 2. Exposure but no clinical disease 3. Vaccination

Question 25

Serological diagnostics are indirect detectors of what?

Answer 25

IgG or IgM antibodies (and antigens)

Question 26

Purpose of PCR

Answer 26

to synthesize and amplify specific target genome sequence to determine pathogen load

Question 27

IgG and IgM

Answer 27

IgG = most abundant; smallest; sticks to/neutralizes antigens IgM = appears 1st in circulation; largest; sticks to/neutralizes antigens | IgG is the only Ab that can penetrate placenta & blood vessels

Question 28

When are IgG levels highest?

Answer 28

During secondary response/after secondary antigen exposure (e.g., after booster shot)

Question 29

Serostatus vs Serocoversion

Answer 29

**Serostatus**: specifies whether sample contains Ab/Ag (sero+) or no Ab (sero-) **Seroconversion**: indicates a change from sero- to sero+ (or 4-fold increase in titer b/w acute & a convalescent sample taken 2-4 weeks later) | Sero- does NOT mean patient is NOT infected!! Only that no Ab present

Question 30

Can ELISA tests differentiate b/w Ab produced from infection vs. vaccination?

Answer 30

No (i.e., FIV)

Question 31

Direct vs. Indirect IFA

Answer 31

**Direct**: detects antigen; poor sensitivity - Giardia & Cryptosporidium (feces) - Rabies (brain tissue) **Indirect**: tests antibody and helps establish serum antibody titer; more sensitive - Anaplasma & Ehrlichia

Question 32

How does IHC differ from IFA?

Answer 32

IHC: antibody is conjugated with horseradish peroxidase; visualized with light microscope IFA: antibody is conjugated with a fluorescent tag; visualized with fluorescent microscope

Question 33

How does Agglutination work?

Answer 33

- Detects Ab or Ag in serum - relies on Ag-Ab complex bivalence property to produce assessable clumps - protocol = series of dilutions of pt's blood serum mixed w/ live organisms of pathogen -> highest titer = where ≥ 50% of Ag-Ab still cross-linked - brucellosis, lepto, crypto, blood typing

Question 34

How are antibody titers interpreted?

Answer 34

Highest dilution of Ab that will detectably interact w/ Ag - aka lowest [Ab] ## Footnote 1 part serum, 160 parts saline -> it would take 160 parts of viral load for there to be no detectable antibodies left (aka, animal would need to be severely, severely infected to be clinically affected).