The Challenge of Antimicrobial Susceptibility Testing in Iran

The Challenge of Antimicrobial Susceptibility Testing in Iran

Mohammad Rahbar(Ph.D)

P Arofessor of Clinical Microbiology

Tehran April 23, 2017

Introduction

• Antimicrobial resistance is a critical publichealth and safety dilemma. We havewitnessed a seemingly inexorable increase inthe number of infections due to carbapenem-resistant Enterobacteriaceae, multidrug-resistant (MDR) Pseudomonas aeruginosa,MDR Acinetobacter baumannii, andvancomycin-resistant Enterococcus faecium.

Introduction

The performance of antimicrobialsusceptibility testing by the clinicalmicrobiology laboratory is importantto confirm susceptibility to chosenempirical antimicrobial agents, or todetect resistance in individualbacterial isolates.

Continue..

• Susceptibility testing of individualisolates is important with species thatmay possess resistance mechanisms (eg,members of the Enterobacteriaceae,Pseudomonas species, Staphylococcusspecies, Enterococcus species, andStreptococcus pneumoniae. In otherhand susceptibility an important issue indrug resistance surveillance

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Methods of susceptibility testing.• Phenotypic methods which is widely used in

clinical microbiology methods.• Genotypic methods. It is only used for certain

microorganisms for example defection ofmecA in Staphylocococus aurues or detectionof cetaingenes susc as coding ESBLs ,KPC and... . Molecular is difficult for standardized antnot applicable in many microbiologylaboratories. This method is very expensiveand needs special facilities and expertise staff.

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Phenotypic methods• Broth dilution tests: This method is used for

detection of minimum inhibitoryconcentration (MIC) and is one of the earliestantimicrobial susceptibility testing methodswas the macrobroth or tube-dilution methodThis procedure involved preparing two-folddilutions of antibiotics (eg, 1, 2, 4, 8, and 16µg/mL) in a liquid growth medium dispensedin test tubes.

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Microdilution • The miniaturization and mechanization of

the test by use of small, disposable, plastic“microdilution” trays .It has made brothdilution testing practical and popular.Standard trays contain 96 wells, eachcontaining a volume of 0.1 mL that allowsapproximately 12 antibiotics to be tested in arange of 8 two-fold dilutions in a single tray.

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Fig .Microdilution

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Antimicrobial gradient method• The antimicrobial gradient diffusion method

uses the principle of establishment of anantimicrobial concentration gradient in anagar medium as a means of determiningsusceptibility. The Etest (bioMérieux ABBIODISK

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A Staphylococcus aureus isolate tested by the Etest gradient diffusion method with vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar.

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Disk Diffusion• The disk diffusion method is the

most widely used technique in the

World and is suitable for testing

rapidly growing and certain

fastidious bacterial pathogens.

Dr.T.V.Rao MD 12

Automation Systemes

• Integrated in the VITEK 2 system is the Advanced Expert System (AES™), a software which validates and interprets susceptibility test results, and detects antibiotic resistance mechanisms. The AES Expert System is the most developed software system in this field, and is capable of identifying even emerging and low-level resistance.

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Godliness for Susceptibility testing1-Clinical laboratory Standards Institutes ( CLSI)

Formerly CLSI 2006

2- European Committee on AntimicrobialSusceptibility Testing – EUCAST

In Our country nearly all microbiologyLaboratories use CLSI guidelines.

Critical Challnes in AST

1. Pure isolate

2. Culture media: Muller-Hinton

3. Reagents: disks

4. Size of the inoculums

5. Incubation condition

6. Control with reference strains

7. Reading inhibition diameters (accurate measurement)

8. Knowledge of staff

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Patient results may be incorrect if

• The organism was misidentified

• Misidentification leads to chose wrong antibiotic disks for susceptibility testing

• If mix microorganisms was tested

• Mixed organism testing results different zone of inhibition or growth inside of zone of inhibition .

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Mueller-Hinton Agar

• It shows acceptable batch-to-batch

reproducibility for susceptibility testing.

• It is low in sulfonamide, trimethoprim, and

tetracycline inhibitors.

• It gives satisfactory growth of most

nonfastidious pathogens.

MHA

Unfortunately some of MHA culture mediain our country have not good quality and alluser should provide it from approvedcompanies and sources.

performance of quality control MHA is alsomandatory

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Antibiotic disks

• Stored and handled correctly

– Refrigeration – taken out 1 hour before use

• Expiry dates noted

• Disks at room temperature before use

– Avoid condensation

• Placing of disks within 15 minutes of swabbing

Reading Plates and Interpreting

Results

If cefoxitin (35 C) is being tested

against Staphylococcus spp. or

vancomycin against Enterococcus

spp., 24 hours of incubation are

required before reporting as

susceptible; other agents can be read

and reported at 16 to 18 hours ..

A question

Which companies produce good quality Antibiotic disks

Wide variation in disk quality in 16 selected disks from nine manufacturers.

EUCAST Development Laboratory (EDL)

Växjö

Sweden

23 October 2015

Notice

The aim of our for showing these results isnot confirmation or rejection of companies ,forthis reason the name of manufactures has beendeleted and shown as 1,2 …………..9

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Disk diffusion testing

• Nine manufacturers were asked to supplydisks for the 16 selected antimicrobial agentsand nominal disk potencies. Not allmanufacturers supplied all disks. Diskdiffusion testing was performed by the EDL inApril – September 2014. The results from thisstudy was presented as a poster at ECCMID2015 (P1239), with manufacturersanonymized.

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Disk diffusion was performed according to EUCASTmethodology.

Each disk was tested against all QC strains with targets andranges to the agent in the EUCAST Quality Control Tables v. 5.0.

For each agent, all disks supplied for the study were placed on asingle 150mm circular agar plate, except for meropenem 10 µg,for which two plates were used due to large inhibition zones.

Each disk-strain combination was tested in triplicate (threeindividually prepared inoculum suspensions) on the same day.

All tests were performed on in-house prepared Mueller-Hintonagar plates (MH and MH-F as recommended) using MH agarfrom two manufacturers.

Disk diffusion testing

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Antimicrobial disks and QC strains

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Manufacturers and disk lots First study, 2014

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1 2 3 4 5 6 7 8 9

Results first study, 2014

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2 3 4 5 6 7 8 91

Conclusion

• The results from this study show that thereare significant differences in antimicrobialactivity in disks from different manufacture.

• Disk manufacturers must continuously reviewtheir disk manufacturing process.

• User should perform quality control diskroundly as recommended by CLSI or EUCAST

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QC Antimicrobial Susceptibility Testing - Module 8 29

29

Reference Strains for QC of antibiotic disks

E. coli ATCC 25922

S. aureus ATCC 25923

P. aeruginosa ATCC 27853

QC organisms must be obtained from reputable source

SELECTION OF DRUGS FOR ROUTINE TESTING

• The laboratory must test and report theantimicrobial agents that are mostappropriate for the isolated organism andsite of the infection, and the institution’sformulary .

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• The CLSI provides tables that list the agentsappropriate for testing members of theEnterobacteriaceae, Pseudomonas, and othergram-negative ,nonfermenters, staphylococci,enterococci, streptococci, Haemophilusspecies, etc. .The listings includerecommendations for agents that areimportant to test routinely, and those thatmay be tested or reported selectively basedon the institution’s formulary.

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• The availability of antimicrobial agents fortesting by the laboratory’s routine testingmethodology must next be determined. Thedisk diffusion and gradient diffusionprocedures offer the greatest flexibilityincluding testing of newly available drugs. Inour country .disk of some antibiotics are notavailable for testing.( Linozolid ,daptomycinazteronam ……..)

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Reading Plates and Interpreting

Results

Zones are measured to the nearest

whole millimeter, using sliding calipers

or a ruler, which is held on the back of

the inverted petri plate. The petri plate

is held a few inches above a black,

nonreflecting background andilluminated with reflected light.

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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

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MEASURING CONDITIONS

RulerCalipers

read with good light, and from the back of the platezone size reading is drug specific magnification may helpmillimeters matter

INTERPRETATION OF SUSCEPTIBILITY TEST RESULTS

• The results of a susceptibility test must beinterpreted by the laboratory prior tocommunicating a report to a patient’sphysician. Optimal interpretation of MICsrequires knowledge of the pharmacokineticsof the drug in humans, and information on thelikely success of a particular drug ineradicating bacteria at various body sites.

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• This is best accomplished by referring to anexpert source such as the CLSI or EUCASTwhich publishes interpretive criteria for MICsof all relevant antibiotics for most bacterialgenera .Indeed, both MIC values and diskdiffusion zone diameters must be interpretedusing a table of values that relate to provenclinical efficacy of each antibiotic and forvarious bacterial species

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• The CLSI zone size and MIC interpretivecriteria are established by analysis of 3 kindsof data: (1.) microbiologic data, including acomparison of MICs and zone sizes on a largenumber of bacterial strains, including thosewith known mechanismsofresistancethathavebeen defined either phenotypically orgenotypically; (2) pharmacokinetic andpharmacodynamic data; and (3) clinicalstudies

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COMMON INTERPRETATION

PROBLEMSResults 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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• Modified Methods in Disk 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

WHAT IS THE ACCEPTABLE ACCURACY OF A SUSCEPTIBILITY TEST METHOD

• It is important that the tables used forsusceptibility test interpretations representthe most current criteria. Indeed, the CLSIdocuments are reviewed and updatedfrequently, usually once per year. Use of oldor outdated information from the originaleditions of FDA-approved drug labels or olderCLSI tables could represent a seriousshortcoming in the reporting of patients’results.

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• When assessing the accuracy of varioussusceptibility testing methods as compared tostandard reference methods, the terms verymajor and major errors have been used todescribe false susceptible or false-resistantresults, respectively.

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• In evaluations of new susceptibility testingmethods it is important to examine arepresentative number of strains that areresistant to various drugs to verify the abilityof the new test to detect resistance and totest a number of susceptible strains todetermine the rate of major errors that mightbe expected in a typical clinical laboratorysetting

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There is not a defined break point for someantibiotics

There is not method of susceptibility testing forsome microorganism ( Bacillus spp,….)

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Lack of break point for some microorganisms

• 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.,

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WHAT IS THE ROLE OF MICROBIOLOGY

DEPARTMENTS

The Challenge of Antimicrobial Susceptibility Testing in Iran

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