Chapter 17- infection diagnosis

Question 1

Direct fluorescent antibody [DFA]

Answer 1

*highlights the presence of microbes with fluorescently labeled antibodies

*Get antibodies (Y-shaped proteins) and bind them to fluorescent molecules > mix sample with these specific antibodies > microscope slide > Add monoclonal antibody > wash it > if you see glowing bacteria = positive diagnosis

*ex) diagnosing syphilis

Question 2

Rapid biochemical tests

Answer 2

*[phenotypic analysis]- initial isolation > introduce microbe to various wells on a strip > look at unique color combo reaction > based on this, identify microbe [tests for different metabolic pathways, the material contains assay media/assays]

*Ex) testing for catalase, amylase, TSIA, fermentation of sugars, citric acid fermentation, etc…

*Computers can scan for quick analysis/results

Question 3

Serology

Answer 3

branch of immunology that deals with in vitro diagnostic testing of serum

Question 4

Western blot

Answer 4

procedure for separating and identifying antigen or antibody mixtures by 2-d electrophoresis in polyacrylamide gel, followed by immune labeling

*named as a joke from Dr. southerns last name
*use proteins run through a protein gel; antibodies are fluorescentally labeled/ or radiation somehow labeled, placed into paper where proteins were transferred and washed over to see if antibodies bind
*used as verification for HIV after ELISA

Question 5

ELISA

Answer 5

Enzyme-linked immunosorbent assay; a very sensitive serological test used to detect antibodies in diseases like AIDS

2 types: indirect, capture

Question 6

rapid biochemical tests analyze:

Answer 6

presence of enzymes

[looking at metabolic pathways]

[NOT involving DNA or antigens]

Question 7

which test analyzes a sample for the presence of a specific gene?

Answer 7

PCR

Question 8

All of the following are methods to diagnose viral infections, except

-Western Blot.

-detection of viral nucleic acid using specific probes.

-cells taken from patient are examined for evidence of viral infection.

-signs and symptoms.

-the light microscope.

Answer 8

the light microscope.

Question 9

Serological tests should have ____ sensitivity and specificity.

Answer 9

high

Question 10

Tests that take place in the lab, such as in a test tube or Petri-dish, outside of a living host, are referred to as ________ whereas tests that take place in a living host, such as in a patient, are described as ________.

Answer 10

In vitro; In vivo

Question 11

Serological testing always involves reactions between specific :

Answer 11

antibody and antigen.

Question 12

The property of a test to detect only a certain antibody or antigen, and not to react with any others, is

Answer 12

specificity.

Question 13

Some diseases are diagnosed without identification or observation of the microbe itself in a patient’s specimen.

true or false?

Answer 13

true

Question 14

Which method would allow for direct observation of a specimen?

Answer 14

Gram stain

Question 15

Soluble antigens are detected in which type of test?

Answer 15

precipitation

Question 16

The indirect ELISA test detects ________ in a patient’s blood.

Answer 16

antibody

Question 17

When we describe a test by saying even small or trace amounts of antibodies or antigens can be detected, we are describing its:

Answer 17

sensitivity

Question 18

A single patient sample can be loaded into multiple PCR tubes to test for different things. The ingredient that would be altered to diagnose for different infections given the same patient sample would be:

Answer 18

the primers

Question 19

A rising antibody titer a few days apart indicates

Answer 19

the patient has a current infection.

Question 20

Antigens attached to the surface of cells are detected in which type of test?

Answer 20

agglutination

Question 21

The tuberculin test is an example of an ____________ test.

Answer 21

in vivo serological

Question 22

Maggie was told she has a positive antibody titer to measles. Which of the following could explain this?

Answer 22

Any of these V

She had measles sometime in her life.

She was vaccinated against measles.

The test was a false positive.

She has measles now.

Question 23

Host cells grown in vitro can be used for:

Answer 23

the cultivation of viruses

Question 24

If trying to isolate a specific pathogen from amongst a variety of normal microbiota, which type of media would be most helpful?

Answer 24

selective and differential agar

Question 25

Culture of patient specimens to detect pathogens is usually done on blood agar plates only.

True or false?

Answer 25

False

Question 26

*FISH test: [genotyping]

Answer 26

fluorescent in situ hybridization

*take sample on microscope slide; take intact microbe and add fluorescent oligonucleotide to sample [similar result to DFA but binding fluorescent oligo to heat fixed DNA sequences]; specific, sensitive; looking for DNA sequences

Question 27

*PFGE: [genotyping]

Answer 27

post field gel electrophoresis

*extract DNA, cut up DNA from sample with restriction enzymes, run RFLP analysis; useful for epidemics to compare pathogens from various ind. With same sx to see if microbes are same.

Question 28

*PCR test: [genotyping]

Answer 28

common in hospitals, fast, specific, important for covid testing; take sample, may exteact DNA or not, add primers specific to microbe you think you have and observe for amplified bands = positive microbe

Question 29

*Real time PCR

Answer 29

analyzes DNA during cycling process ; analyzes amplified product throughout the cycle; fast result within an hour or less

Question 30

*RT-PCR: [genotyping]

Answer 30

reverse transcriptase PCR

*Uses reverse transcriptase enzyme instead of tac polymerase; take RNA and make DNA copies of it; used on RNA sample; useful for RNA viruses like Covid & for genetic engineering [making DNA copy of RNA/mRNA strand, then can easily clone, store, manipulate

Question 31

*Nucleic acid sequencing/ Whole genome sequencing

Answer 31

easy, cheap, uses PCR and computer program to copy many fragments; looks for overlapping regions, pastes together = whole reconstructed genome > detailed info about microbe; more commonly used in research

Question 32

Agglutination

Answer 32

clumping of whole cells; antigen is on the cell & when you add antibodies, it causes clumping of whole cells > causing cell to precipitate and fall to bottom of test tube for example.

*used for diagnosing infectious diseases: salmonella, syphyllis.. Also commonly used for blood banks to determine blood type

Question 33

Precipitation

Answer 33

clumping molecules instead of cells; not easily seen with naked eye

Question 34

What is complement fixation and how does it work?

Answer 34

*not commonly used in hospitals; ELISA more common
*look for either antibodies or antigens
*need sheeps blood or blood with lysins, complement proteins
*take patient’s serum, add antigen, if they have the antibody, it will form a complex, then add complement proteins, if the complex has formed, then proteins will bind to this, forming a complement fixed antibody antigen complex. Occurs when patient has the antibody

*if serum did not contain the antibodies you are looking for, complement proteins free to bind to RBC specifically lysins on the sheep blood cells, causing hemolysis, leading to destruction of RBC you can see with naked eye

Question 35

Direct Fluorescent Antibody (DFA) Testing:

Answer 35

use monoclonal antibodies bound to fluorescent molecule - test for presence of unknown antigen; id specific pathogens in tissues; easily see since glows in dark; used for syphilis, gonorrhea, pertussis, legionnaires diseases, meningitis, listeria

Question 36

Indirect Fluorescent Antibody (IFA) Testing:

Answer 36

using unknown antibodies with a known antigen; fluorescenrt antibodies are secondary antibodies that bind to presence of antibody you are looking for

Question 37

Difference in steps between indirect & Capture ELISA tests

Answer 37

Indirect ELISA for Antibody Detection:
Known antigens are attached to the surface of small wells.
Patient’s serum (containing antibodies) is added to the wells.
If the patient’s antibodies bind to the antigens, they will stay in the wells.
The wells are then washed to remove any unbound antibodies.
A secondary antibody, linked to an enzyme, is added. It binds to the patient’s antibodies if they are present in the well.
The wells are washed again to remove any unbound secondary antibodies.
A substrate is added, and if a chemical reaction occurs, it indicates the presence of the enzyme, which in turn indicates the presence of the secondary antibody and the primary antibody of interest in the patient’s sample.

Capture/Sandwich ELISA for Antigen Detection:
Antibodies are attached to the surface of the wells.
The patient’s sample is washed over the wells.
If the sample contains the antigen of interest, it binds to the antibodies in the well.
A secondary antibody, linked to an enzyme, is added. It binds to the antigens in the well, forming a “sandwich” structure.
A substrate is added, and if a chemical reaction occurs, it indicates the presence of the enzyme, which indicates the presence of the antigen in the patient’s sample.

Question 38

Describe the TB test and explain what this can tell you.

Answer 38

Immunologic test done on human body

*TB test: small amount of purified tuberculin protein from microbe is injected just underneath skin to see if there is a local immune reaction from specific immune system, if antigen is recognized by antibodies already in body, you will see a red raised bump, large.
~Leave a day to get the inflammatory response to be cleared so you are seeing the immune reaction from antibodies recognizing antigen
~This indicates either you have TB or had it at one point or you had a vaccine outside of the US.

Question 39

RAST (Radioallergosorbent test)-

Answer 39

type of blood test used to detect and measure specific IgE antibodies in response to allergens. It is primarily used to diagnose allergies and determine the specific allergens that trigger an individual’s allergic reactions.