Dr.T.V.Rao MD
QUALITY ASSURANCE IN
ANTIBIOTIC SENSITIVITY TESTING DISC DIFFUSION AND E-TESTS
DR.T.V.RAO MD 1
• The goal of antimicrobial susceptibility testing is to
predict the in vivo success or failure of antibiotic
therapy. Tests are performed in vitro, and measure the
growth response of an isolated organism to a
particular drug or drugs. The tests are performed
under standardized conditions so that the results are
reproducible. The test results should be used to guide
antibiotic choice. The results of antimicrobial
susceptibility testing should be combined with clinical
information and experience when selecting the most
appropriate antibiotic for our patients.
DR.T.V.RAO MD 2
WHAT IS THE GOAL OF ANTIBIOTIC
SENSITIVITY TESTING?
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ANTIMICROBIAL SUSCEPTIBILITY TESTS
Provide information for selection of an appropriate agent for antimicrobial therapy
COMPONENTS OF ANTIBIOTIC
SENSITIVITY TESTING
• 1.The identification of relevant pathogens in exudates and body fluids collected from patients
• 2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs
• 3. Assay of the concentration of an administered drug in the blood or body fluid of patient required to control the schedule of dosage.
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• Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant
• Micro dilution and agar gradient diffusion methods provide a quantitative result, a minimum inhibitory concentration
AST METHODS INTERPRETATION
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Dr.T.V.Rao MD 6
AST Methods
ANTIMICROBIAL RESISTANCE
Results from misuse, overuse, under/ inadequate use of
antimicrobials
• Costs money, lives and undermines effectiveness of
health delivery programs
• Threat to global stability and national security
WHO Global Strategy for Containment of Antimicrobial
Resistance:
• Intervention framework to slow emergence and reduce
the spread of antimicrobial resistant microorganisms
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ANTIBIOTIC RESISTANT INFECTIONS
Diseases Agent Resistances
Pneumonia S pneumoniae Penicillin
Dysentery S dysenteriae Multiple resistances
Typhoid S typhi Multiple resistances
Gonorrhea N gonorrhoeae Penicillin and
tetracycline
Tuberculosis M tuberculosis Rifampicine and INH
Nosocomial infections S aureus Methicillin, vancomycin
E species Vancomycin
Klebsiella,
Pseudomonas
Multiple resistances
GENETIC EXCHANGE OF ANTIMICROBIAL
RESISTANCE GENES
Enterobacteriaceae Enterococci
Staphylococci Pseudomonas
Campylobacter
Vibrio cholerae Pneumococci
Streptococci
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ANTIMICROBIAL SUSCEPTIBILITY TESTS
Minimum inhibitory concentration [MIC]
• The smallest concentration of antibiotic that inhibits the
growth of organism
Liquid media (dilution) allows MIC estimation
Solid media (diffusion)
• Disk diffusion (Kirby-Bauer)
• E-tests
• Allows MIC estimation
Beta lactamase production: quick screening method
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CRITICAL POINTS IN QUALITY ASSURANCE
1. Culture media: Muller-Hinton
2. Reagents: disks
3. Size of the inoculums
4. Incubation condition
5. Control with reference strains
6. Reading inhibition diameters (accurate measurement)
7. Knowledge of staff
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THE HANDS AND HEADS -
PERSONNEL • Essential that everyone is aware
of the importance of QC
• Training of personnel in correct technique
• Storage of discs
• Preparation of a standard inoculum
• Swabbing of plates
• Choice and storage of media
• Timing and methods of incubation of plates
• Measurement of zone sizes
• Recording of results
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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
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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
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QUALITY ASSURANCE IN ANTIBIOTIC SUSCEPTIBILITY TESTING WITH CONTROL STRAINS
• Susceptibility test with quality
control strains
for every new batch of Mueller-
Hinton agar
• Staphylococcus aureus
(ATCC 25923)
• Escherichia coli (ATCC
25922)
• Pseudomonas aeruginosa
(ATCC 27853 )
SUSCEPTIBILITY TESTING METHODS
Inoculate
MH plate
Place disks
on agar plate
Incubate plate
18-24 hr, 35 C
Measure and record
zone of inhibition
around each disk
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Disc Diffusion Method
Measurement of the diameters of inhibition zone
Measure from the edge where the growth stats, BUT there
are three exceptions
With sulfonamides and co-trimoxazole, ignore slight growth within
the zone
Certain Proteus spp. may swarm into the area of inhibition
When beta-lactamase producing Streptococci are tested, zone of
inhibition are produced with a heaped-up, clearly defined edge,
regardless of the size of the inhibition zone, they should be
reported as resistant
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SELECTION OF A COLONY TO TEST
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DISK DIFFUSION TEST
Select colonies Prepare inoculum
suspension
Prepare inoculum
suspension
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MCFARLAND 0.5 AND
ADJUSTED TEST ORGANISM
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PREPARE THE MATERIAL FOR
INOCULATION
Standardize inoculum
Suspension as per Mac farland standard Mix well
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SWAB THE PLATE WITH OPTIMAL SAMPLE
Remove sample Swab plate
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ANTIBIOTIC DISCS
• Stored and handled correctly
• Refrigeration – taken out 1
hour before use
• Expiry dates noted
• Discs at room temperature
before use
• Avoid condensation
• Placing of discs within 15
minutes of swabbing
SELECT THE DISKS AND APPLY
Select disks
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INCUBATE OVERNIGHT
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DISC DIFFUSION METHOD
Place the appropriate
drug-impregnated disc
on the surface of the
inoculated agar plate
Invert the plates and incubate them at 35 oC, o/n (18-24 h)
Measure the diameters
of inhibition zone in
mm
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DR.T.V.RAO MD 27
LOOK AT THE CHARTS FOR ESTABLISHING THE
ZONES OF SENSITIVITY
• The zone sizes are
looked up on a
standardized chart to
give a result of
sensitive, resistant, or
intermediate. Many
charts have a
corresponding column
that also gives the MIC
(minimal inhibitory
concentration) for that
drug.
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
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UNDERSTANDING BREAKPOINTS Words of laboratory specialists
• It is not possible to work alone
• Breakpoints 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 DR.T.V.RAO MD 29
MIC c
Wild type Inherited resistance
mechanism
C
THE EPIDEMIOLOGICAL CONCEPT FOR
BREAKPOINTS
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THE PHARMACOLOGICAL CONCEPT
FOR BREAKPOINTS
The concentration range tested for a drug and the
interpretative criteria for various categories are based on
extensive 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 for
decision making and therefore the concept of breakpoint
concentration
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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 problem
Defining 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
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INTERPRETING INTERMEDIATE RESISTANCE
Sometime the agent can still be used
• Higher 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
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DISK SUSCEPTIBILITY TESTING
PROBLEMS
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DISK SUSCEPTIBILITY TESTING
PROBLEMS
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MEASURING CONDITIONS
Ruler Calipers
read with good light, and from the back of the plate zone size reading is drug specific magnification may help millimeters matter
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Factors Affecting Size of Zone of Inhibition
Potency of antibiotic discs
Composition of medium
Acidic pH of medium
Alkaline pH of medium
Reading of zones
Deterioration in contents leads to reduced size
Affects rate of growth, diffusion of antibiotics and activity of antibiotics
Tetracycline, novobiocin, methicillin zones are larger
Aminoglycosides, erythromycin zones are larger
Subjective errors in determining the clear edge
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/
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COMMON INTERPRETATION PROBLEMS
Problem with the size of the inoculums
Solution:
• Use McFarland 0.5 photometer
• Scale -> same tubes
Contaminati
on with
another
organism
COMMON INTERPRETATION
PROBLEMS
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COMMON INTERPRETATION PROBLEMS
Bad manipulation
Inoculation of the
Muller Hinton
• Swabbing
• Not by flooding
E-TEST Plastic strips with a predefined gradient of
• One antibiotic
• One antifungal
Only one manufacturer
One strip per antibiotic
Wide range of antibiotics
Easy to use
Storage at -20°C
Short shelf life, expensive
MIC on a strip abbiodisk.com
Be familiar with
Instructions
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ETEST – ANTIMICROBIAL GRADIENT
METHOD
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READING E-TESTS
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin for
Yersinia pestis
Intermediate 1-4 ug/ml
Upper reading
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E TEST – MIC REPORTS ARE HELPFUL
IN CRITICAL MANAGEMENT DECISIONS
• Quantitative MIC data is a
prerequisite for the
management of critical
infections, including
sepsis, especially among
critical care patients. Etest
is particularly valuable in
such situations, when on-
scale MICs are needed for
treatment decisions.
ANTIMICROBIAL GRADIENT TESTING
E-TEST®
Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale
• media
• antimicrobials
• inoculum
• incubation
• equipment
• interpretation
WHERE ERRORS CAN OCCUR IN
SUSCEPTIBILITY TESTING
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COMMON INTERPRETATION
PROBLEMS Results depends on the technique used
Many factors influence results
• Lack of standardization of the inoculums
• Thickness and quality of the culture media
• Quality and conservation of the disks
• Quality control with standardized strains
• Condition and duration of incubation
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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
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Quality Assurance in Antibiotic Susceptibility Test
Salient features of quality control
Use antibiotic discs of 6 mm diameter
Use correct content of antimicrobial agent per disc
Store supply of antimicrobial discs at -20 oC
Use Mueller-Hinton medium for antibiotic sensitivity
determination
Use appropriate control cultures
Use standard methodology for the test
WHEN THINGS GO WRONG…
• Questions to ask?
• Is the procedure correct?
• Check test materials including test strains
• Check equipment
• Fridges
• Incubators
• Freezers
• Review technique of personnel
Culture of general QC in lab essential
INVESTIGATION ON ERRORS
• Quality of media should be investigated
• pH will affect macrolides, tetracycline's and aminoglycoside
• In this case the pH was too low – acidic
• Ideal pH 7.2-7.4
• TMP-SMX is affected by the amount of thymidine in media
• This QC may indicate the presence of excess thymidine in the agar which will allow the bacteria to bypass the inhibitory effects of TMP-SMX
• Corrective action should be taken in house or with the manufacturer and QC repeated with a new batch
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• Modified Methods in Disc diffusion for Antibiotic sensitivity testing to be used for detections of following bacterial isolates
• 1 MRSA
• 2 ESBL
• 3 Enterobacteriaceae and Gram negative bacteria and Carbapenems resistant using Modified Hodge test
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NEED FOR MODIFIED METHODS
• AST QC needs a culture of general QC in the laboratory
• Systems for performing, recording and troubleshooting should be documented in writing
• Any errors should be investigated timeously and systematically
• Results can then be reported with confidence and permit appropriate and safe antimicrobial use
SELF CORRECTION OF ERRORS
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• Each laboratory should have a staff member with the time,
interest, and expertise to provide leadership in antibiotic testing
and resistance. This person would read relevant publications,
network with other laboratories, and evaluate potentially useful
tests to detect new forms of resistance before new CLSI-
recommended tests become available”
• - Ken Thomson, Emerging Infect. Dis., 2001
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WHAT IS THE ROLE OF MICROBIOLOGY
DEPARTMENTS
• Created by Dr.T.V.Rao MD for „e‟ learning
resources for Microbiologists in
Developing World • Email
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