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?

Dr.T.V.Rao MD 3

3

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.

DR.T.V.RAO MD 4

• 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

DR.T.V.RAO MD 5

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

DR.T.V.RAO MD 7

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

DR.T.V.RAO MD 9

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

DR.T.V.RAO MD 10

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

DR.T.V.RAO MD 11

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

Dr.T.V.Rao MD 13

13

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

Dr.T.V.Rao MD 14

14

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

DR.T.V.RAO MD 15

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

DR.T.V.RAO MD 17

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

Dr.T.V.Rao MD 18

SELECTION OF A COLONY TO TEST

18

DISK DIFFUSION TEST

Select colonies Prepare inoculum

suspension

Prepare inoculum

suspension

DR.T.V.RAO MD 19

Dr.T.V.Rao MD 20

MCFARLAND 0.5 AND

ADJUSTED TEST ORGANISM

20

PREPARE THE MATERIAL FOR

INOCULATION

Standardize inoculum

Suspension as per Mac farland standard Mix well

DR.T.V.RAO MD 21

SWAB THE PLATE WITH OPTIMAL SAMPLE

Remove sample Swab plate

DR.T.V.RAO MD 22

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

DR.T.V.RAO MD 24

INCUBATE OVERNIGHT

DR.T.V.RAO MD 25

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

DR.T.V.RAO MD 26

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

DR.T.V.RAO MD 28

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

DR.T.V.RAO MD 30

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

DR.T.V.RAO MD 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 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

DR.T.V.RAO MD 32

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

DR.T.V.RAO MD 33

Dr.T.V.Rao MD 34

34

DISK SUSCEPTIBILITY TESTING

PROBLEMS

Dr.T.V.Rao MD 35

DISK SUSCEPTIBILITY TESTING

PROBLEMS

Dr.T.V.Rao MD 36

36

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

DR.T.V.RAO MD 37

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/

DR.T.V.RAO MD 38

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

DR.T.V.RAO MD 40

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

Dr.T.V.Rao MD 44

ETEST – ANTIMICROBIAL GRADIENT

METHOD

44

READING E-TESTS

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for

Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

DR.T.V.RAO MD 45

DR.T.V.RAO MD 46

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

DR.T.V.RAO MD 48

48

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

DR.T.V.RAO MD 49

Dr.T.V.Rao MD 50

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

DR.T.V.RAO MD 51

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

DR.T.V.RAO MD 54

• 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

DR.T.V.RAO MD 55

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

DR.T.V.RAO MD 56

• 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

DR.T.V.RAO MD 57

WHAT IS THE ROLE OF MICROBIOLOGY

DEPARTMENTS

• Created by Dr.T.V.Rao MD for „e‟ learning

resources for Microbiologists in

Developing World • Email

• doctortvrao@gmail.com

DR.T.V.RAO MD 58