Clinical Lab Assays 2

Question 1

Antibiotic Spectrum of Activity - Penicillin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 1

little end of gram-, all of gram+, little beginning of chlamydiae

Question 2

Antibiotic Spectrum of Activity - Sulfonamides, cephalosporins, quinolones, carbapenems spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 2

half of gram-, most of gram+

Question 3

Antibiotic Spectrum of Activity - streptomycin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 3

most of mycobacteria, most of gram-

Question 4

Antibiotic Spectrum of Activity - tetracyclines spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 4

most of gram-. all of gram+, chlamydiae, rickettsiae

Question 5

Antibiotic Spectrum of Activity - Isoniazid spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 5

only mycobacteria

Question 6

Antibiotic Spectrum of Activity - Polymyxin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 6

only gram -

Question 7

Antibiotic Spectrum of Activity - Vancomycin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)

Answer 7

only gram +

Question 8

Sites of action for antibiotics: what are agents that inhibit DNA and RNA polymerase in cells?

Answer 8

DNA: fluoroquinolones
RNA: rifampin

Question 9

Sites of action for antibiotics: what are agents that inhibit cell wall synthesis

Answer 9

Penicillin, cephalosporins, carbapenems, glycopeptides (vancomycin)

Question 10

Sites of action for antibiotics: what are agents that bind to ribosomes and inhibit protein synthesis?

Answer 10

Aminoglycosides, tetracyclines, macrolides, clindamycin, cloramphenicol, linezolid

Question 11

Mechanisms of resistance for antibiotics in a cell: 4 of these main mechanisms

Answer 11

Permeability barriers, efflux pump, antibiotic target modification (altered penicillin-binding proteins, altered DNA gyrase), inactivating enzymes (beta-lactamase, aminoglycoside-modifying enzymes)

Question 12

What is AST, and what are the 3 responsibilities of the lab for AST

Answer 12

Antimicrobial Susceptibility Testing: A laboratory procedure used to determine what antibiotics a bacterium is susceptible to.
It is the laboratory’s responsibility to:
 Provide technically accurate information
 Provide reproducible results
 Report in easily interpreted format

Question 13

When should a laboratory perform susceptibility tests?

Answer 13

 When a pathogen is considered clinically significant
 With pathogens for which standardized methods are available
 With pathogens where resistance is known to be a clinical problem
 Should NOT performed on normal flora

Question 14

WHAT IS THE MIC?

Answer 14

Minimum Inhibitory Concentration: Lowest concentration of an antibiotic in μg/mL that prevents in vitro growth of bacteria

Question 15

What are the available qualitative (1x) and quantitative (3x) susceptibility testing methods?

Answer 15

Quality: Disk diffusion (Kirby-Bauer)
Quantity: 
Broth dilution (BMD)
 micro
 macro
Gradient diffusion method
Agar dilution

Question 16

What is the disk diffusion method and the procedure?

Answer 16

 Manual method using an antibiotic or antifungal impregnated disc.
 Disc is placed on a lawn of bacteria.
 Plates are incubated.
 Antibiotic diffuses away from the disc at decreasing concentration.
 The “Zone of Inhibition” (mm) correlates to an
interpretation.
 Provides qualitative results (ex. S/I/R).

Question 17

What is the macrobroth dilution method and the procedure?

Answer 17

 One of the first AST methods developed
 Test tubes contain serially diluted concentrations of antibiotic or antifungal
 One tube serves as a positive control (no antibiotic)
 Tubes are incubated.
 MIC is recorded and correlates to an interpretation.

Question 18

What is the microbroth dilution method and the procedure?

Answer 18

 Scaled down macrobroth dilution test which allows higher throughput.
 Allows for testing a panel of different antibiotics at one time.
 Uses smaller volumes of reagents.
 Panels are commercially available.
 Still very manual.

Question 19

What is the automated BMD method and the procedure?

Answer 19

Can be automated
 Primary method used by labs
 High throughput
Cards or panels are setup manually, but the process is automated thereafter.
Software reads the MICs and also interprets the results per user dictated guidelines.
Results automatically transfer into the culture report.

Question 20

what is the Gradient Method (Etest)?

Answer 20

 Applies the same principle as the disc diffusion test
except a gradient of antibiotic/antifungal is
impregnated into a strip.
 The strip directly lists the concentrations of
antibiotics.
 MIC = where bacterial growth intersects the strip.

Question 21

Practical Use of Gradient and Disk Diffusion Methods? (3x)

Answer 21

Used for organisms not validated by automated method
 Organisms require special growth factors
 Certain organisms do not “behave” in broth (ex. Haemophilus, Strep)
Used for antibiotics not on automated panels
 Not first-line agents
 Not on formulary
Used to verify rare results
 Ex. daptomycin non-susceptible Enterococcus

Question 22

what is the Agar Dilution?

Answer 22

 A standard suspension of bacteria is inoculated onto a series of agar plates each containing a different
concentration of antibiotic.
 Plates are incubated.
 The MIC is recorded and correlates to an interpretation.

Question 23

What is the CLSI and what do they do?

Answer 23

Clinical and Laboratory Standards Insititute, founded in 1968, Nonprofit, educational organization for development, promotion and use of national and international standards

 Represent the clinical laboratory testing community
 Volunteers participate from all areas: clinical laboratories, industry, government agencies
 Reproducible results are achieved if labs adhere to
recommended protocols
 Most common standard used in North America.

Question 24

CLSI Guidelines tell us which categories to follow standards for?

Answer 24

 Guidelines for standardized testing
 What drugs to test for each organism
 Rules on site-specific testing
 How to interpret results
   -Clinical Breakpoints
   -Epidemiologic Cutoff Values
 Quality Control Ranges

Question 25

What is EUCAST

Answer 25

European Committee on Antimicrobial Susceptibility testing

Question 26

Interpretation Definitions for the 4 interpretation result categories:

Answer 26

 Susceptible: Organism should respond to treatment with recommended dose of antibiotic
 Susceptible Dose Dependent: Organisms should respond to treatment with higher or more frequent dosing as recommended
 Intermediate: Organism may or may not respond to treatment with usual dose of antibiotic: buffer zone to prevent major discrepancies
 Resistant: Organism will not respond to usual dose of
antibiotic

Question 27

Clinical Breakpoints throughout process: (5x)

Answer 27

 In vitro activity: MIC distributions, Disk diffusion zone diameter scattergrams
 In vitro and animal model PD
 Human PD
 Clinical outcome studies
 Final choice of which data to emphasize made by standard setting committees and groups

Question 28

What are the parameters that have Standardized Guidelines? What is the standard for each?

Answer 28

 Growth Media:Organism Dependent: Enterobacterales: Mueller Hinton Agar or Broth +/- blood Yeast: RPMI Broth/Agar
 pH: 7.2-7.4 at room temperature
 Cation Concentration
 Atmosphere
 Temperature
 Inoculum
 Time
 Antibiotic Preparation and Concentrations
 Quality Control