Clinical Lab Assays 2 Flashcards
Antibiotic Spectrum of Activity - Penicillin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
little end of gram-, all of gram+, little beginning of chlamydiae
Antibiotic Spectrum of Activity - Sulfonamides, cephalosporins, quinolones, carbapenems spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
half of gram-, most of gram+
Antibiotic Spectrum of Activity - streptomycin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
most of mycobacteria, most of gram-
Antibiotic Spectrum of Activity - tetracyclines spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
most of gram-. all of gram+, chlamydiae, rickettsiae
Antibiotic Spectrum of Activity - Isoniazid spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
only mycobacteria
Antibiotic Spectrum of Activity - Polymyxin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
only gram -
Antibiotic Spectrum of Activity - Vancomycin spectrum covers which of (mycobacteria, gram-, gram+, chlamydiae, rickettsiae)
only gram +
Sites of action for antibiotics: what are agents that inhibit DNA and RNA polymerase in cells?
DNA: fluoroquinolones
RNA: rifampin
Sites of action for antibiotics: what are agents that inhibit cell wall synthesis
Penicillin, cephalosporins, carbapenems, glycopeptides (vancomycin)
Sites of action for antibiotics: what are agents that bind to ribosomes and inhibit protein synthesis?
Aminoglycosides, tetracyclines, macrolides, clindamycin, cloramphenicol, linezolid
Mechanisms of resistance for antibiotics in a cell: 4 of these main mechanisms
Permeability barriers, efflux pump, antibiotic target modification (altered penicillin-binding proteins, altered DNA gyrase), inactivating enzymes (beta-lactamase, aminoglycoside-modifying enzymes)
What is AST, and what are the 3 responsibilities of the lab for AST
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
When should a laboratory perform susceptibility tests?
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
WHAT IS THE MIC?
Minimum Inhibitory Concentration: Lowest concentration of an antibiotic in μg/mL that prevents in vitro growth of bacteria
What are the available qualitative (1x) and quantitative (3x) susceptibility testing methods?
Quality: Disk diffusion (Kirby-Bauer) Quantity: Broth dilution (BMD) micro macro Gradient diffusion method Agar dilution
What is the disk diffusion method and the procedure?
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).
What is the macrobroth dilution method and the procedure?
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.
What is the microbroth dilution method and the procedure?
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.
What is the automated BMD method and the procedure?
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.
what is the Gradient Method (Etest)?
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.
Practical Use of Gradient and Disk Diffusion Methods? (3x)
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
what is the Agar Dilution?
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.
What is the CLSI and what do they do?
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.
CLSI Guidelines tell us which categories to follow standards for?
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
What is EUCAST
European Committee on Antimicrobial Susceptibility testing
Interpretation Definitions for the 4 interpretation result categories:
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
Clinical Breakpoints throughout process: (5x)
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
What are the parameters that have Standardized Guidelines? What is the standard for each?
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
