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Antimicrobial Susceptibility Tests. Antimicrobial Susceptibility Tests. provide information for selection of an appropriate agent for antimicrobial therapy. 2. Antimicrobial susceptibility tests. Minimum inhibitory concentration [MIC] - PowerPoint PPT Presentation
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Antimicrobial Susceptibility Tests
QC Antimicrobial Susceptibility Testing - Module 8 22
Antimicrobial Susceptibility Tests
provide information for selection ofan appropriate agent for antimicrobial therapy
QC Antimicrobial Susceptibility Testing - Module 8 3
Antimicrobial susceptibility testsMinimum inhibitory concentration [MIC]
The smallest concentration of antibiotic that inhibits the growth of organism
Liquid media (dilution) allows MIC estimationSolid media (diffusion)
Disk diffusion (Kirby-Bauer)
E-tests
Allows MIC estimation Beta lactamase production: quick screening method
QC Antimicrobial Susceptibility Testing - Module 8 4
AST Methods Interpretation
agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant
microdilution and agar gradient diffusion methods provide a quantitative result, a minimum inhibitory concentration
QC Antimicrobial Susceptibility Testing - Module 8 5
AST Methods
QC Antimicrobial Susceptibility Testing - Module 8 6
Dilution in liquid broth Tubes containing increasing antibiotic concentrations Incubation during 18 hr at 37°C
0 (Control) 0,25 0,50 1 2 4 8 mg/l
MICBacterial growth Inhibition
QC Antimicrobial Susceptibility Testing - Module 8 7
Kirby-Bauer disc testingAntibiotic-impregnated discs placed on an agar plate at theinterface between test organism and susceptible control
organismResulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables)
QC Antimicrobial Susceptibility Testing - Module 8 8
E-testPlastic strips with a predefined
gradient of One antibiotic One antifungal
Only one manufacturer One strip per antibioticWide range of antibioticsEasy to useStorage at -20°CShort shelf life, expensive
QC Antimicrobial Susceptibility Testing - Module 8 9
Reading E-tests
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin for Yersinia pestis
Intermediate 1-4 ug/ml
Upper reading
QC Antimicrobial Susceptibility Testing - Module 8 1010
Where errors can occur in susceptibility testing
media antimicrobials inoculum incubation equipment interpretation
QC Antimicrobial Susceptibility Testing - Module 8 1111
Agar disk diffusion method
Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4
Antibiotic storage -20oC minimum disks temperature
Inoculum McFarland 0.5 (108 bacteria/mL)
Incubator temperature 35oC
atmosphere ambient air
QC Antimicrobial Susceptibility Testing - Module 8 1212
Reference Strains
E. coli ATCC 25922 S. aureus ATCC 25923 P. aeruginosa ATCC 27853
QC organisms must be obtained from reputable source
Use specific QC organisms to test different groups of “drug-bug” combinations
QC Antimicrobial Susceptibility Testing - Module 8 13
QC Antimicrobial Susceptibility Testing - Module 8 14
QC Antimicrobial Susceptibility Testing - Module 8 15
BAP are enriched, differential media used to isolate fastidiousorganisms and detect hemolytic activity. β-hemolytic activity will show lysis and complete digestion of red blood cell contents surrounding colony
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Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= non-hemolytic, S. salivarius)
17Macconkey agar with lactose (left) and non-lactose (right) fermenters
QC Antimicrobial Susceptibility Testing - Module 8 18
Selection of a Colony to Test
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QC Antimicrobial Susceptibility Testing - Module 8 19
MacFarland 0.5 and Adjusted Test Organism
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QC Antimicrobial Susceptibility Testing - Module 8 2020
Disk Susceptibility Testing Problems
QC Antimicrobial Susceptibility Testing - Module 8 21
Disk Susceptibility Testing Problems
QC Antimicrobial Susceptibility Testing - Module 8 2222
Measuring Conditions
RulerCalipers
read with good light, and from the back of the platezone size reading is drug specific magnification may helpmillimeters matter
QC Antimicrobial Susceptibility Testing - Module 8 23
Etest – antimicrobial gradient method
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QC Antimicrobial Susceptibility Testing - Module 8 24
Patient results may be incorrect if:
the organism was misidentified a clerical error was made inappropriate choice of antimicrobials
were tested and reported the wrong patient’s sample was examined the wrong test was ordered the sample was not preserved properly
QC Antimicrobial Susceptibility Testing - Module 8 25
Critical points in quality assurance1. Culture media: Muller-Hinton2. Reagents: disks3. Size of the inoculums4. Incubation condition5. Control with reference strains6. Reading inhibition diameters (accurate
measurement)7. Knowledge of staff
QC Antimicrobial Susceptibility Testing - Module 8 26
Standard strains for quality assurance
Precision and accuracy ensured through control strainsKnown susceptibility to antimicrobial agents
Standard strains includeStaplylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853
QC Antimicrobial Susceptibility Testing - Module 8 27
The main concept is the “clinical categorisation" Strains are sorted according to level of Minimal
Inhibitory Concentration (MIC) versus reference breakpoints
c and C are the minor and major breakpoints
Susceptible Intermediate
Resistant
MIC < c ≤ MIC < C ≤ MIC
Interpretation
QC Antimicrobial Susceptibility Testing - Module 8 28
Understanding breakpointsWords of laboratory specialists
It is not possible to work aloneBreakpoints are the expression of a consensus
among the scientific community at a given time in a country
Breakpoints are determined using two approaches
Pharmacological concept Epidemiological concept
QC Antimicrobial Susceptibility Testing - Module 8 29
0
10
20
30
40
50
60
0.01 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128
MICc
Wild type
Inherited resistance
mechanism
C
The epidemiological concept for breakpoints
QC Antimicrobial Susceptibility Testing - Module 8 30
The pharmacological concept for breakpoints
The concentration range tested for a drug and theinterpretative criteria for various categories are based onextensive studies that correlate with
Serum achievable levels for each antimicrobial agent Particular resistance mechanisms Successful therapeutic outcome
In practice situations the entire range may not be used fordecision making and therefore the concept of breakpointconcentration
QC Antimicrobial Susceptibility Testing - Module 8 31
From breakpoints to interpretation
Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problemDefining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring
MIC ≤ c Sensitive strain
MIC > C Intermediate strain
c < MIC ≤ C Resistant strain
QC Antimicrobial Susceptibility Testing - Module 8 32
Host factors affecting treatment
Diffusion in tissuesSerum protein bindingDrug interactionsImmune systemMultiple simultaneous infectionsVirulence of organismSite and severity of infection
QC Antimicrobial Susceptibility Testing - Module 8 33
Interpreting intermediate resistance
Sometime the agent can still be usedHigher doses required to ensure efficacy Agent may be efficacious if concentrated in
vivo in an infected body fluid (e.g., urine)
Sometimes there is uncertainty Intermediate resistance may represent a
“buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant
QC Antimicrobial Susceptibility Testing - Module 8 34
Common interpretation problemsResults depends on the technique used Many factors influence results
Lack of standardization of the inoculumsThickness and quality of the culture
mediaQuality and conservation of the disksWuality control with standardized strains Condition and duration of incubation
QC Antimicrobial Susceptibility Testing - Module 8 35
An agar gel that is too thick leads to smaller zones
Common interpretation problems
Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/
QC Antimicrobial Susceptibility Testing - Module 8 36
Common interpretation problems
Problem with the size of the inoculums
Solution: Use McFarland 0.5 photometer Scale -> same tubes
QC Antimicrobial Susceptibility Testing - Module 8 37
Common interpretation problems
Contamination with another organism
QC Antimicrobial Susceptibility Testing - Module 8 38
Common interpretation problems
Bad manipulationInoculation of the
Muller Hinton Swabbing Not by flooding
QC Antimicrobial Susceptibility Testing - Module 8 39
Problems with E-test reading
Common interpretation problems
QC Antimicrobial Susceptibility Testing - Module 8 40
Cost of anti-microbial resistanceCheap antimicrobials become ineffectiveIndividual treatment failureProlonged illness, hospitalizationNeed to switch to more expensive, complex drugs that
areoften not even available in resource-poor settingsNeed to develop new antimicrobialsGood antimicrobial susceptibility testing saves lives andmoney
QC Antimicrobial Susceptibility Testing - Module 8 41
WHO Global Principles For The Containment of AntimicrobialResistance In Animals Intended for Food
Department for Communicable Diseases Surveillance and Response
World Health Organization
Report of a WHO Consultation
with the participation of the Food and Agriulture Organization and the
Office International des Epizooties
Geneva, Switzerland
5 – 9 June 2000
WHO/CDS/CSR/APH/2000.4
Distr. : General
English only
42
Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:
European Program for Intervention Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
Antimicrobial resistance
QC Antimicrobial Susceptibility Testing - Module 8 43
References Manual for the laboratory Identification and
Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/