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Validation of EUCAST methods by Pathology QLD
Narelle GEORGESupervising Scientist, Microbiology
Central Laboratory, Herston Hospitals Complex
Overview
§ Background to Pathology QLD§ Organisation of Microbiological Testing
§ CLSI change to EUCAST – Validation strategy/plan§ What needs to be validated§ Automated AST methods (VItek2)§ Disc testing method
§ Methods used for validation of § Vitek2 EUCSAT AES configuration file§ EUCAST discs and EUCAST zone diameter interpretation
§ Staff training and competency assessment
Pathology QLD Laboratories
• Metropolitan Tertiary (METRO)
• Group Co-ordinating (GCL)
• District Regional
Roll out across 26/34 Labs
Metropolitan Tertiary (METRO)Full range MIC and disc testing for routine and uncommon microbes with extended range of
antimicrobial susceptibility MIC testing at Central(Automated Vitek2, Disc Diffusion, ETest MIC)
Group Co-ordinating (GCL)Full range MIC and disc testing capability for routine
clinical isolates with referral for extended testing(Automated Vitek2, Disc Diffusion, Limited ETest MIC)
District RegionalLimited testing with referral to
GCL for routine testing of a variety of different clinical isolates
(Disc Diffusion only)
District RegionalLimited testing for a variety of
different clinical isolates with referral to GCL for wider range of testing
(Automated Vitek2, Disc Diffusion)
Prelude to validation?• Decide HOW you intend to roll-out EUCAST across
multiple laboratories with both manual and automated methods Ø What is the Implementation process?Ø Who will be involved in the Implementation team
• Senior management need to decideØ How to manage§ Organisms calibrated in current CLSI based database – not
calibrated in EUCAST§ Antibiotics calibrated in current CLSI based interpretations – not
calibrated in EUCAST§ Inadequate MIC range of antimicrobials in current Vitek2 AST
panels vs range required for EUCASTØ Options
§ Retain CLSI interpretations (M45-A2, M100-S24)§ Test an alternative antibiotic or§ Do not test
What is the validation strategy?Validation Strategy • Integral part of any new method implementation
• NATA/ISO/TGA requirements (verification not validation)
• Needs to be cost effective (restricted budgets, staff and time for validation activities
• Centralise for maximum efficiency (possible with statewide standardised methods)
• Identify areas where centralised management is not applicable (i.e. staff training and competency testing) – ? standardise training and competency assessment
protocols and training records
• Identify what needs to be validated and who will be responsible
What next?Validation plan • Series of plans
– Individual validation plan for each area that requires validation
• Include the SCOPE of the validation– What is being validated and why
• Specify the QC organisms to be used• Specify the number of tests to be performed• Specify the test method/s• Document the performance criteria to be assessed• Assign the acceptance criteria
– QC within specified range and one repeat allowed to achieve 100% correlation with standard
• Who will perform the testing – Specify individual roles– Variable skill set (bench scientist, quality, clinicians)
• Set the timeframe
Management Essentials
Review the validation plan and approve
Be available to make the “tough decisions” when required
Review and approve the final Method Validation/Implementation Report
Senior Management
Director of Microbiology
Impact of Changing Method from CLSI to EUCAST
Vitek2 Users• Minimal impact to workflow• New BPs & interpretations
set within instruments• Need to standardise the
configuration of software and rollout across multiple instruments
• Less antibiotics reported• Less computerised changes
Disc Testing• New disc concentrations• New zone diameters• New media for XV/PN• Extensive update of
documentation• New computer codes for
data entry or QC (other reading templates)
• Multi-skilled regional scientists
What needs to be validated?• Vitek2
– New MIC breakpoints– MIC range for non EUCAST calibrated organism:antibiotic
combinations• Disc Testing
– New medium for Fastidious organisms§ Mueller Hinton Fastidious Agar with 5% horse blood + NAD
– New disc concentrations (lower than CLSI)§ Vancomycin 5, Cefotaxime 5
– New zone diameter interpretations§ No intermediate zones§ <= for S categories, > for R categories
– Staff competency in EUCAST test method
Organisms calibrated – Vitek2
CLSI EUCASTEnterobacteriaceae P P
Pseudomonas species P P
Staphylococcus aureus / CoNS P P
Enterococcus P P
Streptococcus pneumoniae P P
Vibrio cholerae / Aeromonas Burkholderia
P
Non-fermenting GNB P
95% workload
USE CLSI M45-A2 or CLSI M100-S24
Antibiotics Tested not calibrated by EUCAST
Staphylococcus aureus Nitrofurantoin(urines)
EUCAST BPs applied (MIC distributions
Pseudomonas aeruginosa Norfloxacin EUCAST statesInappropriate for Organism Use CLSI MIC interpretation
Stenotrophomonas maltophilia Ticarcillinclavulanate
EUCAST has no calibrationUse CLSI MICs
Stenotrophomonas maltophilia Ceftazidime EUCAST has no calibrationUse CLSI MICs
Acinetobacter species Amp, Aug, TCCPip-Taz, 3GC,cefepime
EUCAST states testing unreliable. Use CLSI MIC interpretation
Enterobacteriaceae Cefazolin Use CLSI MICs
Enterobacteriaceae ESBL Screen Not recommended by EUCAST
Still test report ifpositive, if neg Add comment
Problem Antimicrobial MIC ranges
AST-P612
AntimicrobialVitek 2 Panel CLSI EUCAST
CodeConcentratio
ns S R S R
Mupirocin MUP 1 n/c n/c 1 256
Rifampicin RA 0.25,0.5,2 1 4 0.06 0.5
Set as CLSI breakpoints
Problem Antimicrobial MIC ranges
AST-N256
CLSI EUCASTCode Concentrations S R S R
Cefazolin CZ 4,16,64 2 8 nil nil
Norfloxacin NOR 1,8,32 4 16 0.5 1
Concentrations not low enough
Do not report except for urine
Setting the Vitek2 to EUCAST
What do you need?– New AES configuration file to interpret MIC values in
accordance with EUCAST breakpoints
Setting the Vitek2 to EUCAST – NEW AES File• CLSI and EUCAST breakpoints are “hard-wired” in Vitek2 AES
configuration• Pathology QLD has a copy of the CLSI file that is modified to use
our own “user defined breakpoints” (No Intermediate category)• Process will be
– Copy the Global European file within Vitek2§ Use the Natural Resistance file not the Phenotype file§ Using the new copy, enter current EUCAST MIC breakpoints
and CLSI interpretations for non-calibrated antibiotics to be routinely reported
– Modify this to include§ Current EUCAST MIC breakpoints§ Add CLSI breakpoints for non EUCAST calibrated
anitibioitics/organisms§ Modify any Intermediate interpretation to Resistant
(Pathology QLD requirement only)
Validation of New EUCAST File• Manual checking cannot be avoided
– Print a hard copy of the AES Breakpoint file– Assign 2 members of the validation team to check the file
against EUCAST MIC Breakpoints Standard document– Correct file and print– Sign and date checked file– Maintain this paper copy for the validation file
AND• Check Instrument application of AES file
– Check that the instrument is applying the correct EUCAST breakpoints for interpretation of MIC results
– HOW?
Range of Organisms Staphylococcus aureus MSSA Enterobacter cloacae
Staphylococcus aureus nmMRSA Proteus mirabilis
Staphylococcus aureus MRSA Providencia species
Staphylococcus epidermidis Morganella morganii
Enterococcus faecalis Serratia marcescens
Enterococcus faecium Salmonella species
Enterococcus faecium vanB Pseudomonas aeruginosa (S)
Enterococcus faecium vanA Pseudomonas aeruginosa (CRP)
Escherichia coli Burkholderia cepacia
Escherichia coli ESBL Stenotrophomonas maltophilia
Klebsiella pneumoniae Acinetobacter baumannii
Klebsiella pneumoniae ESBL Acinetobacter lwoffi
Klebsiella oxytoca Achromobacter (other non fermenters)
Range of Organism Phenotypes Staphylococcus aureus MSSA Enterobacter cloacae
Staphylococcus aureus nmMRSA Proteus mirabilis
Staphylococcus aureus MRSA Providencia species
Staphylococcus epidermidis Morganella morganii
Enterococcus faecalis Serratia marcescens
Enterococcus faecium Salmonella species (ampC)
Enterococcus faecium vanB Pseudomonas aeruginosa (S)
Enterococcus faecium vanA Pseudomonas aeruginosa (CRP)
Escherichia coli Burkholderia cepacia
Escherichia coli ESBL Stenotrophomonas maltophilia
Klebsiella pneumoniae Acinetobacter baumannii
Klebsiella pneumoniae ESBL Acinetobacter lwoffi
Klebsiella oxytoca Achromobacter (other non fermenter)
How to Manage Vitek2 File - Statewide
• Generate new EUCAST file within the Vitek2 instrument located in the Central Laboratory
• Export a copy of this file on USB drive• Forward file or USB to all Vitek2 laboratories
within Pathology QLD• Provide instructions for uploading of new file
(copy current instrument file first to act as backup)• File loaded prior to GO LIVE date• On day of changeover, simply select the new file
and save.
Vitek2 – Validation Not Required
• Routine Vitek2 Quality Control for 4 weeks– QC organisms for CLSI and EUCAST identical for the
range of organisms tested within the Vitek2 system– QC testing is based on the MIC determined NOT the
interpretation (i.e. EUCAST or CLSI)• Interface between V2 and LIS
– Validation not required if only interpreted results are transmitted (i.e. S or R)
– Verification of data transfer may be required if MIC values as well as interpreted results transmitted
– For interface verification, use QC organisms as test isolates for Dummy patient ID set up in LIS
Don’t forget
• Check your BioART rules– Particularly those using MIC values to prompt for
additional testing• EUCAST breakpoint interpretation standard
changes annually– Need to update AES configuration file regularly – Audit regularly as part of laboratory quality system
What needs to be validated?• Validation required for
– New fastidious test media MHF– New (low potency) discs– Ability to apply the EUCAST
method and – New QC organism (H influenzae
NCTC 8468)
• Centralise validation for efficiency & cost effectiveness
• Provide validation data to other laboratories
• Verify inter-laboratory performance
Validation of Disc Testing – New medium• New medium MHF agar• Commercial or local manufacture• Local manufacture (Central laboratory, PQ)
– NATA Biological validation required– Multiple lots to be manufactured on different days (3 lots)– Daily testing of QC organisms on each lot (10 days)– Shelf life validations§ pH, water content (w/v), QC performance§ Weekly for 12 weeks § Each individual lot tested
• Commercial supply– Routine QC testing on different lots required– How manage this for state?
Validation of Commercial vs In House MHF
• Locally produced MHF media validated against commercially produced MHF agar
• Non-metro PQ laboratories use all commercially produced media
• Test protocols– 3 scientists set up all QC organisms on MH and MHF weekly for
5 weeks– 1 scientist continues to set up all QC organisms on MH and MHF
weekly for 4 weeks
• Data compared using excel• Acceptance parameters – 100% within published
EUCAST zone diameter range for both media types
Media validation for Streptococcus pneumoniae ATCC 49616 & oxacillin on In-house and commercial media
Tips and Tricks • Local manufacture of MHA and
MHF aim to use the same MH agar base
• Haemophilus influenzae• Fuzzy zones to SXT
• Medium formulation (OXOID)• CXM zones too small
• Age of culture not medium
• Streptococcus pneumoniae• Indistinct Oxacillin zone edge
resulting in zone reading problem • Best Medium formulation (MAST)
but poor growth of Enterococcus
ALL MH AGAR BASES are NOT the SAME
Validation of New AST procedure• Be practical- validation needs to be efficient and cost effective for
the laboratory• Currently apply CLSI recommendation for validation of AST methods• Require minimum of 4 weeks testing and multiple staff• Comparison to involve both media types (MHEU and MHF agars),
new disc concentrations, new QC organismUtilising at least 5 scientists at Central to set up over 5 consecutive days for 4 weeks
• Use resource staff for inter-laboratory performance (5 scientists – 5 days)
• Capture data using Excel program allows graphical comparison of data to demonstrate– Reproducibility of testing – Staff competency– Determination of test variables (e.g SD values)for ongoing routine QC of disc tests
Test ParametersStaph aureusATCC 29213
MH EU PEN 1, FOX 30, ERY 15, DA 2, CN 10, RD 5, FD10, CIP5, C30, SXT 25
Escherichia coliATCC 25922
MH EU AMP 10, AMC 30, CN10, CTX 5, SXT 25, TOB 10, AK30, CAZ 10, CIP 5, MEM 10, TIM 85, TAZ 60, F 100
Ps aeruginosaATCC 27863
MH EU CN10, TOB 10, AK 30, TIM 85, TAZ 60, MEM 10, CAZ 10, CIP 5,
Enterococcus faecalisATCC 29212
MH-F AMP 2, VA 5, TEC 30, CN 30, F100
Strep pneumoniaeATCC 49619
MH-F OX 1, ERY 15, VA 5, C30, SXT 25, TET 30
Haem influenzaeATCC 8468
MH-F AMP2 AMC 3, CTX 5, CIP 5, SXT 25, C30
Enterococci Validation Data - OP1
8
10
12
14
16
18
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Operator
Zone
(mm
) VA5 Zone
VA5 Min
VA5 Max
VA5 Target
EUCAST method and vancomycin 5 disc validation data over 4 weeks for Operator No 1
Training and Competency Assessment• Cannot be centralised but staff training
and competency assessment can be standardised
• Dependent on standardised methodsDisc vs Automated MIC
§ Minimal for automated AST § More intensive manual training for disc
testingCLSI vs EUCAST
§ Only minimal changes for most organisms
§ Major changes with fastidious organisms (Haemophilus influenzae)
• Dependent on skill set of scientific staff– Muliskilled scientists in regional
laboratories (disc testing)
The Process• Train “resource staff” within GCLs• Forward QC organisms and test
unknowns for hands-on training of resource staff (Metro/GCL)
• Develop training file for recordingof test results for assessment
• Photograph test plates and forward with zone diameter results to Central laboratory for assessment of technique and accuracy in application of the EUCAST methods
• Resource Staff training records signed off by Central Trainers
One Narelle can be everywhere!
Send in photographs of your plate set-up
Results for 2 Resource Staff Members Guess the problem here?
WRONG MEDIUM
MHSB (CLSI)>24 HR SUBCULTURE
Pathology QLD EUCAST Training Summary• Large organisations
• Utilise resource personnel• Train the Trainer process• Resource staff to train
individual staff within labs or district (GCL)
• Training methods• “Hands on” set up (QC)• Unknown isolate
• Competency• Assess media set up• Final written exam with
85% pass mark• Resource Documents
• USB for each Resource person with lectures, documents, reference documents, interpretation tables etc
In Summary
• For large organisations, centralise validation to ensure cost effective utilisation of resources but standardised methods required
• Validation of disc diffusion testing more labour intensive due to new medium and different disc potencies
• Staff training and competency testing needs to be managed locally
• Multi-skilled Team effort required
Acknowledgements• Pathology QLD EUCAST Implementation Team
– Dr Sally Appleton, Haakon Bergh, Greg Flohr, Michael Caffery, Cathy Engler
• Dr Graeme Nimmo, Director of Microbiology, Pathology QLD
• Members of the Pathology QLD, Microbiology Discipline Working Party
• EUCAST Resource Staff, Microbiology, Pathology QLD – Russell Enbom, Richard Lord, Karen Griffiths, David Stranger,
Sharon Dal-Cin, Jennine Hay, Mila Golmayo• EUCAST “Helpers”
– Dr Claire Heney, Katrina Lawrence, Anna Jones, Ron Songuan and staff in Central Laboratory, Herston Hospitals Complex.