the challenge of antimicrobial susceptibility testing in iran
TRANSCRIPT
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
7/25/2017
7/25/2017
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.
7/25/2017
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.
7/25/2017
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.
7/25/2017
Fig .Microdilution
7/25/2017
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
7/25/2017
A Staphylococcus aureus isolate tested by the Etest gradient diffusion method with vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar.
7/25/2017
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.
7/25/2017
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
Dr.T.V.Rao MD 14
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 .
7/25/2017
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
Dr.T.V.Rao MD 17
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
7/25/2017
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.
7/25/2017
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
7/25/2017
Antimicrobial disks and QC strains
7/25/2017
Manufacturers and disk lots First study, 2014
7/25/2017
1 2 3 4 5 6 7 8 9
Results first study, 2014
7/25/2017
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
7/25/2017
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 .
7/25/2017
SELECTION OF DRUGS FOR ROUTINE TESTING
• 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.
7/25/2017
SELECTION OF DRUGS FOR ROUTINE TESTING
• 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 ……..)
7/25/2017
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.
DR.T.V.RAO MD 34
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
Dr.T.V.Rao MD35
35
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.
7/25/2017
• 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
7/25/2017
INTERPRETATION OF SUSCEPTIBILITY TEST RESULTS
• 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
• ies 7/25/2017
INTERPRETATION OF SUSCEPTIBILITY TEST RESULTS
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
DR.T.V.RAO MD 39
• 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
DR.T.V.RAO MD 40
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.
7/25/2017
WHAT IS THE ACCEPTABLE ACCURACY OF A SUSCEPTIBILITY TEST METHOD
• 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.
7/25/2017
WHAT IS THE ACCEPTABLE ACCURACY OF A SUSCEPTIBILITY TEST METHOD
• 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
7/25/2017
There is not a defined break point for someantibiotics
There is not method of susceptibility testing forsome microorganism ( Bacillus spp,….)
7/25/2017
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.,
DR.T.V.RAO MD 45
WHAT IS THE ROLE OF MICROBIOLOGY
DEPARTMENTS