Transcript M-100

Developed by
JALAL SHEIKH, Ph.D.
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Acknowledgements
The presenters would like to thank:
 NIH/DAIDS -Daniella Livnat and Mike Ussery
This project has been funded in whole or in part with Federal funds from the Division of AIDS (DAIDS),
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health
and Human Services, under contract No. HHSN266200500001C, titled Patient Safety Monitoring in
International Laboratories.
 Johns Hopkins University
 Dr. Robert Miller - Principal Investigator
 Barbara Parsons - Operations Manager
 Kurt Michael - Project Manager
 SMILE Staff
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Basic components of this
presentation
 Basics of Antimicrobial Susceptibility Testing (AST)
 Different classes of antibiotics and antimicrobial agents
based on their mechanisms of actions
 A practical guide: steps associated with AST methods
 QA/QC on AST
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Objectives of this presentation:
 Decisions of performing AST
 Different steps associated with AST
 Effective reporting of antimicrobial
agents/antibiotics
 Why CLSI?
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Questions to ask before initiating AST
 What is the organism?
 What was the site of infection?
 What antibiotics/Antimicrobial agents need to be
included/tested?
 What method of AST will be implemented?
 How to report the results?
 How to ensure the accuracy of AST results (QA/QC)?
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Microorganisms and choice of
Antibiotics/antimicrobial agents
 Single drug of choice: No AST required
 Commensal or opportunistic pathogen: special request
from physician
 Multiple choice of drugs: AST required
 Antibiotics selection for testing and reporting: CLSI guidelines
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Difference between Antibiotics and
antimicrobial agents
 Antibiotics: Organic compounds produced by
microorganisms that kills or inhibits the growth of
other microorganisms.
 Antimicrobial agents: Synthetic chemical
compounds that kills or inhibits the growth of other
microorganisms.
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Mechanisms of antimicrobial activity
•
•
•
•
Inhibits cell-wall synthesis
Inhibits protein synthesis
Inhibits nucleic acid (RNA/DNA) synthesis
Inhibits synthesis of essential metabolites
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Bacterial Structure: Targets of
antimicrobial agents
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Different classes of antibiotics based on
mechanisms of actions (1)
•Inhibitors of cell-wall synthesis:
- Penicillin (natural and semi-synthetic)
- Cephalosporins (1st/2nd/3rd/4th and 5th
generation)
- Bacitracin
- Vancomycin
- Isoniazid (INH)
- Ethambutol
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Different classes of antibiotics based on
mechanisms of actions (2)
• Inhibitors of protein synthesis:
- Chloramphenicol
- Aminoglycosides (Streptomycin, neomycin,
gentamicin
- Tetracycline
- Macrolides (Erythromycin for gram-positives)
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Different classes of antibiotics based on
mechanisms of actions (3)
•Inhibitors of Nucleic Acid synthesis:
- Rifamycin
- Quinolones
- Fluoroquinolones
•Inhibitor of folic acid synthesis:
- Sulfonamides (Sulfa drugs)
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Different methods of AST
1. Diffusion method: Kirby-Bauer Disk Diffusion
2. Dilution method: MIC (Minimal Inhibitory Concentration)
3. Combination of diffusion and Dilution method: E-test
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Kirby-Bauer Disk Diffusion method (1)
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Preparation of inoculum
•
Standardize inoculum suspension
•
Inoculate Mueller Hinton Agar (MHA)
•
Dispense antibiotic disks
•
Incubate overnight at 37°C
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Kirby-Bauer Disk Diffusion method (2)
•Measure zone sizes
•Interpret the zone sizes with established chart:
- Sensitive (S)
- intermediate Sensitive (I) and
- Resistant (R)
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QC/QA: Kirby-Bauer Disk Diffusion:
Factors affecting the zone of inhibition (1)
1. Thickness of medium: should be 4 mm
- Thicker medium – narrow zone
- Thin medium – larger zone
2. pH of medium: should be neutral pH
- Acidic pH: larger zone size for
Tetracycline, Methicillin and Novobiocin
- Alkaline pH: larger zone size for
Aminoglycosides and Erythromycin
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QC/QA: Kirby-Bauer Disk Diffusion:
Factors affecting the zone of inhibition (2)
3. Potency of antibiotic disks: Right concentration
- Zone size narrower if antibiotic
concentration not maintained
4. Inoculum density: based on McFarland standard
-0.5 (mostly non-fastidious gram-negatives)
-1.0 (mostly fastidious slow-growers)
- Light inoculum: Large zone size
- Heavy inoculum: Small zone size
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QC/QA: Kirby-Bauer Disk Diffusion:
Factors affecting the zone of inhibition (2)
5. Placing the disks: immediately
6. Spacing the disks: 2.5 cm
7. Incubation time: 16-18 hours
8. Incubation temperature: 35°C-37°C
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Procedure for Dilution method:
Broth / Agar dilution (1)
•Quantitative method
•Microbroth dilution: MIC
(Minimal Inhibitory Concentration)
•Agar dilution method
•MIC: The lowest concentration of any
antibiotics that inhibits the growth of a
microorganism.
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Procedure for Dilution method: Broth
dilution
•Making dilutions of antibiotics in 96-well plate or tubes
•Standardize inoculum preparation
•Inoculation of MIC tray or tubes
•Incubate at 35°C-37°C for overnight (16-18 h)
•Read the turbidity under the light box or automated
reader
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Combining diffusion and dilution
method: E-test (AB Biodisk, Solna, Sweden)
•The E test, consisting of a continuous stable
gradient of antimicrobial agent provides 15
twofold dilutions on a strip
•The E-test strip is placed on the surface of an
agar plate inoculated with test organisms
•Incubate overnight at 35°C-37°C
•MIC is measured on the test strip scale where
the zone of inhibition intersect the strip
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Selecting and reporting of antimicrobial
agents: Why CLSI ?
 CLSI: Clinical Laboratory Standards Institute
 To improve patient testing and health care services
 International, interdisciplinary, nonprofit, standards-developing,
educational organization
 Recognized internationally worldwide
* CAP, Accutest and other EQA-providers follow CLSI guidelines.
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CLSI publications
CLSI published three types of publications:
•Standard
•Guideline
•Report
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CLSI Subcommittee on AST
Reviews data from a variety of sources and studies
•To establish AST methods
•To establish interpretive criteria
•To establish QC parameters
This process is continually refined and updated annually.
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CLSI document: M100-S21
• Performance Standards for Antimicrobial
Susceptibility Testing
• CLSI reference methods for AST:
1. Disk Diffusion method (Kirby-Bauer)
2. Dilution method: MIC Testing
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How to use CLSI
M100-S21 Document ?
A detailed presentation will be available on
pSMILE resources.
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If you have any
questions please
contact SMILE
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