Chapter 20

Question 1

why take the time to identify an infectious agent

Answer 1

• many bacteria are resistant to certain antibiotics
• antibiotic-resistant bacteria and viruses are spreading across the world
• specific pathogens are associated with secondary disease complications
• tracking the spread of disease can lead to its source

Question 2

classification

Answer 2

placing organisms in groups of related species
- lists of characteristics of known organisms

Question 3

identification

Answer 3

• matching characteristics of an “unknown” organism to lists of known organisms
• clinical lab identification

Question 4

problem solving algorithms to identify bacteria

Answer 4

• step by step problem solving procedures
• often, lab technicians will inoculate several tests at once to speed up identification
• they will interpret the results in a set sequence

Question 5

microorganism identification method

Answer 5

1. isolate bacterium from patient
2. pure culture- cell and colony morphology
3. gram stain
4. biochemical pathways/properties (metabolism, cell properties, behavior)

Question 6

dichotomous key

Answer 6

• has paired statements in the form of “either-or”
• followed by statements to go to another pair of statements

Question 7

RFLP analysis

Answer 7

DNA is digested with restriction enzymes, run on a gel, and stained with fluorescent or radioactive probe

Question 8

Polymerase Chain Reaction (PCR)

Answer 8

• most widely used molecular method in the clinical laboratory
• DNA primers can be made for specific pathogens–> multiple sets of DNA primers can identify individual genes from a pathogen for more specific typing
• useful for pathogens that are hard to grow or slow to grow

Question 9

qRT-PCR

Answer 9

• fluorescent dye binds to double stranded DNA
• more fluorescence= higher mRNA present

Question 10

direct ELISA

Answer 10

• antigens are immobilized in the well of a microtiter plate
• antibody specific for the antigen is conjugated to an enzyme and added to each well
• if antigen is present, then the antibody will bind
• after washing to remove any unbound antibodies, a colorless substrate is added
• presence of enzyme converts the substrate into a colored end product
• lower sensitivity

Question 11

sandwich ELISA

Answer 11

• use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood/urine
  1. add primary antibody to wells (sticks to plastic)
  2. unbound antibody washed away
  3. primary antibody captures antigen
  4. wash
  5. secondary antibody is added which is a polyclonal antibody conjugated to an enzyme
  6. colorless substrate is added and enzyme converts it into a colored end product
• color intensity is measured with a spectrophotometer
• amount of color produced is proportional to captured antigen

Question 12

indirect ELISA

Answer 12

• quantify antigen-specific antibody rather than antigen
• detects antibodies against many types of pathogens
• attach known antigen, if antibodies are present, they will bind to antigen
• after washing, secondary antibody is added
• secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced

Question 13

fluorescent antibody staining

Answer 13

• antibody specific to particular pathogen types is tagged with a fluorescent tag

Question 14

point of care laboratory tests

Answer 14

• used directly at the sire of patient care
• the result is obtained quickly in order to make appropriate treatment decisions
• important to make sure POC tests are accurate and reliable
• good POC tests have high specificity and high sensitivity

Question 15

sensitivity

Answer 15

how small of a sample a test can detect

Question 16

specificity

Answer 16

how well a test can distinguish positives and negatives

Question 17

commercial POC tests

Answer 17

• many tests involve immunochromatographic assays (ICT)
• newer tests have higher specificity and sensitivity but require more advanced instrumentation

Question 18

Advantages of point of care laboratory tests

Answer 18

• no culturing is required
• clinician can immediately prescribe the right antibiotic
• patients can avoid taking unnecessary antibiotics
• clinicians can quickly determine a chain of infection in patients with similar symptoms
• clinicians can notify patients who are hard to reach once they have left the clinician’s office while they are still present

Question 19

disadvantages to POC laboratory tests

Answer 19

• no data about pathogen antibiotic sensitivity
• increased risk of clinician becoming infected
• multiple infections may be overlooked if the initial test is positive