QUALITY ASSURANCE IN ANTIBIOTIC SENSITIVITY TESTING

DISC DIFFUSION AND E-TESTS

Dr.T.V.Rao MD

DR.T.V.RAO MD

WHAT IS THE GOAL OF ANTIBIOTIC SENSITIVITY TESTING?

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

ANTIMICROBIAL SUSCEPTIBILITY TESTS

Provide information for selection of an appropriate agent for antimicrobial therapy

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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.

DR.T.V.RAO MD

AST METHODS INTERPRETATION

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
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AST Methods

Dr.T.V.Rao MD

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
Pneumonia

Agent
S pneumoniae

Resistances
Penicillin

Dysentery
Typhoid Gonorrhea Tuberculosis Nosocomial infections

S dysenteriae
S typhi N gonorrhoeae M tuberculosis S aureus E species Klebsiella, Pseudomonas

Multiple resistances
Multiple resistances Penicillin and tetracycline Rifampicine and INH Methicillin, vancomycin Vancomycin Multiple resistances

GENETIC EXCHANGE OF ANTIMICROBIAL RESISTANCE GENES

Staphylococci

Pseudomonas

Enterobacteriaceae

Enterococci

Vibrio cholerae Campylobacter

DR.T.V.RAO MD

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 metho d
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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

AGAR DISK DIFFUSION METHOD

Medium Antibiotic disks Inoculum Incubator atmosphere Mueller Hinton storage temperature McFarland 0.5 temperature 4 mm thickness pH 7.2 to 7.4 -20oC minimum

(108 bacteria/mL) 35oC 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 MuellerHinton agar Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Pseudomonas aeruginosa (ATCC 27853 )
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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

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

Prepare inoculum suspension
Prepare inoculum Select colonies 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

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.
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INTERPRETATION
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 <

MIC <

MIC

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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
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THE EPIDEMIOLOGICAL CONCEPT FOR BREAKPOINTS

Wild type Inherited resistance mechanism

MIC
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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

MIC c
MIC > C
Sensitive strain
Intermediate strain

c < MIC C

Resistant strain

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
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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

Calipers

Ruler

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 Deterioration in contents leads to

reduced size

Affects rate of growth, diffusion of

Acidic pH of medium

antibiotics and activity of antibiotics zones are larger zones are larger clear edge

Tetracycline, novobiocin, methicillin Alkaline pH of medium Reading of zones

Aminoglycosides, erythromycin
Subjective errors in determining the

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COMMON INTERPRETATION PROBLEMS

An agar gel that is too thick leads to smaller zones

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

COMMON INTERPRETATION PROBLEMS

Contaminati on with another organism

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COMMON INTERPRETATION PROBLEMS

Bad manipulation

Inoculation of the Muller Hinton Swabbing

Not by flooding

Plastic strips with a predefined gradient of

E-TEST

One antibiotic
One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics

Easy to use
Storage at -20C Short shelf life, expensive

MIC on a strip
abbiodisk.com Be familiar with Instructions

ETEST ANTIMICROBIAL GRADIENT METHOD

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READING E-TESTS

Ciprofloxacin for Yersinia pestis

Resistant > 4 ug/ml

Intermediate 1-4 ug/ml

Susceptible < 1

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 onscale MICs are needed for treatment decisions.

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ANTIMICROBIAL GRADIENT TESTING E-TEST

Read plates after recommended Incubation Read MIC where elipse intersects scale

WHERE ERRORS CAN OCCUR IN SUSCEPTIBILITY TESTING

media antimicrobials inoculum incubation equipment interpretation
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Results depends on the technique used Many factors influence results

COMMON INTERPRETATION PROBLEMS

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 patients sample was examined The wrong test was ordered The sample was not preserved properly
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Dr.T.V.Rao MD

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

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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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NEED FOR MODIFIED METHODS

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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SELF CORRECTION OF ERRORS

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
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WHAT IS THE ROLE OF MICROBIOLOGY DEPARTMENTS

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 CLSIrecommended tests become available - Ken Thomson, Emerging Infect. Dis., 2001

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 Created by Dr.T.V.Rao MD for e learning resources for Microbiologists in Developing World

Email

doctortvrao@gmail.com

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