Validation Of EUCAST Methods By Pathology QLD
Validation of EUCASTmethods by Pathology QLDNarelle GEORGESupervising Scientist, MicrobiologyCentral 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 QLDLaboratories Metropolitan Tertiary(METRO) Group Co-ordinating(GCL) District RegionalRoll out across26/34 Labs
Metropolitan Tertiary (METRO)Full range MIC and disc testing for routine anduncommon microbes with extended range ofantimicrobial susceptibility MIC testing at Central(Automated Vitek2, Disc Diffusion, ETest MIC)Group Co-ordinating (GCL)Full range MIC and disc testing capability for routineclinical isolates with referral for extended testing(Automated Vitek2, Disc Diffusion, Limited ETest MIC)District RegionalLimited testing with referral toGCL for routine testing of a varietyof different clinical isolates(Disc Diffusion only)District RegionalLimited testing for a variety ofdifferent clinical isolates with referralto GCL for wider range of testing(Automated Vitek2, Disc Diffusion)
Prelude to validation? Decide HOW you intend to roll-out EUCAST acrossmultiple laboratories with both manual and automatedmethodsØØWhat is the Implementation process?Who will be involved in the Implementation team Senior management need to decideØHow to manage§ Organisms calibrated in current CLSI based database – notcalibrated in EUCAST§ Antibiotics calibrated in current CLSI based interpretations – notcalibrated in EUCAST§ Inadequate MIC range of antimicrobials in current Vitek2 ASTpanels vs range required for EUCASTØOptions§§§Retain CLSI interpretations (M45-A2, M100-S24)Test an alternative antibiotic orDo not test
What is the validation strategy?Validation Strategy Integral part of any new method implementation NATA/ISO/TGA requirements (verification notvalidation) Needs to be cost effective (restricted budgets, staffand time for validation activities Centralise for maximum efficiency (possible withstatewide standardised methods) Identify areas where centralised management is notapplicable (i.e. staff training and competency testing)– ? standardise training and competency assessmentprotocols and training records Identify what needs to be validated and who will beresponsible
What next?Validation plan Series of plans– Individual validation plan for each area that requiresvalidation Include the SCOPE of the validation– What is being validated and why Specify the QC organisms to be usedSpecify the number of tests to be performedSpecify the test method/sDocument the performance criteria to be assessedAssign the acceptance criteria– QC within specified range and one repeat allowed toachieve 100% correlation with standard Who will perform the testing– Specify individual roles– Variable skill set (bench scientist, quality, clinicians) Set the timeframe
Management EssentialsSenior ManagementReview the validation plan and approveDirector of MicrobiologyBe available to make the “tough decisions”when requiredReview and approve the final MethodValidation/Implementation Report
Impact of Changing Method from CLSI to EUCASTDisc TestingVitek2 Users Minimal impact to workflow New BPs & interpretationsset within instruments Need to standardise theconfiguration of softwareand rollout across multipleinstruments Less antibiotics reported Less computerised changesNew disc concentrationsNew zone diametersNew media for XV/PNExtensive update ofdocumentation New computer codes fordata entry or QC(other reading templates) Multi-skilled regionalscientists
What needs to be validated? Vitek2– New MIC breakpoints– MIC range for non EUCAST calibrated organism:antibioticcombinations 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
VALIDATION OF AUTOMATED MICTESTING USING VITEK2
Organisms calibrated – Vitek295% workloadCLSIEUCASTEnterobacteriaceaePPPseudomonas speciesPPStaphylococcus aureus / CoNSPPEnterococcusPPStreptococcus pneumoniaePPVibrio cholerae / AeromonasBurkholderiaPNon-fermenting GNBPUSE CLSI M45-A2 or CLSI M100-S24
Antibiotics Tested not calibrated by EUCASTStaphylococcus aureusNitrofurantoin(urines)EUCAST BPs applied (MICdistributionsPseudomonas aeruginosaNorfloxacinEUCAST statesInappropriate for OrganismUse CLSI MIC interpretationStenotrophomonas maltophiliaTicarcillinclavulanateEUCAST has no calibrationUse CLSI MICsStenotrophomonas maltophiliaCeftazidimeEUCAST has no calibrationUse CLSI MICsAcinetobacter speciesAmp, Aug, TCC EUCAST states testing unreliable.Pip-Taz,3GC, Use CLSI MIC se CLSI MICsEnterobacteriaceaeESBL Screen Notrecommended byEUCASTStill test report ifpositive, if negAdd comment
Problem Antimicrobial MIC rangesAST-P612Vitek 060.5Set as CLSI breakpoints
Problem Antimicrobial MIC oncentrations not low enoughDo not report except for urine
Lots of decisions that need to made before the Vitek2validation work now begins!
Setting the Vitek2 to EUCASTWhat do you need?– New AES configuration file to interpret MIC values inaccordance with EUCAST breakpoints
Setting the Vitek2 to EUCAST – NEW AES File CLSI and EUCAST breakpoints are “hard-wired” in Vitek2 AESconfigurationPathology QLD has a copy of the CLSI file that is modified to useour 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 breakpointsand CLSI interpretations for non-calibrated antibiotics to beroutinely reported– Modify this to include§ Current EUCAST MIC breakpoints§ Add CLSI breakpoints for non EUCAST calibratedanitibioitics/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 fileagainst EUCAST MIC Breakpoints Standard document– Correct file and print– Sign and date checked file– Maintain this paper copy for the validation fileAND Check Instrument application of AES file– Check that the instrument is applying the correct EUCASTbreakpoints for interpretation of MIC results– HOW?
Range of OrganismsStaphylococcus aureus MSSAEnterobacter cloacaeStaphylococcus aureus nmMRSAProteus mirabilisStaphylococcus aureus MRSAProvidencia speciesStaphylococcus epidermidisMorganella morganiiEnterococcus faecalisSerratia marcescensEnterococcus faeciumSalmonella speciesEnterococcus faecium vanBPseudomonas aeruginosa (S)Enterococcus faecium vanAPseudomonas aeruginosa (CRP)Escherichia coliBurkholderia cepaciaEscherichia coli ESBLStenotrophomonas maltophiliaKlebsiella pneumoniaeAcinetobacter baumanniiKlebsiella pneumoniae ESBLAcinetobacter lwoffiKlebsiella oxytocaAchromobacter (other non fermenters)
Range of Organism PhenotypesStaphylococcus aureus MSSAEnterobacter cloacaeStaphylococcus aureus nmMRSAProteus mirabilisStaphylococcus aureus MRSAProvidencia speciesStaphylococcus epidermidisMorganella morganiiEnterococcus faecalisSerratia marcescensEnterococcus faeciumSalmonella species (ampC)Enterococcus faecium vanBPseudomonas aeruginosa (S)Enterococcus faecium vanAPseudomonas aeruginosa (CRP)Escherichia coliBurkholderia cepaciaEscherichia coli ESBLStenotrophomonas maltophiliaKlebsiella pneumoniaeAcinetobacter baumanniiKlebsiella pneumoniae ESBLAcinetobacter lwoffiKlebsiella oxytocaAchromobacter (other non fermenter)
How to Manage Vitek2 File - Statewide Generate new EUCAST file within the Vitek2instrument located in the Central Laboratory Export a copy of this file on USB drive Forward file or USB to all Vitek2 laboratorieswithin 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 fileand save.
Vitek2 – Validation Not Required Routine Vitek2 Quality Control for 4 weeks– QC organisms for CLSI and EUCAST identical for therange of organisms tested within the Vitek2 system– QC testing is based on the MIC determined NOT theinterpretation (i.e. EUCAST or CLSI) Interface between V2 and LIS– Validation not required if only interpreted results aretransmitted (i.e. S or R)– Verification of data transfer may be required if MICvalues as well as interpreted results transmitted– For interface verification, use QC organisms as testisolates for Dummy patient ID set up in LIS
Don’t forget Check your BioART rules– Particularly those using MIC values to prompt foradditional testing EUCAST breakpoint interpretation standardchanges annually– Need to update AES configuration file regularly– Audit regularly as part of laboratory quality system
VALIDATION OF DISC DIFFUSION TESTING
What needs to be validated? Validation required for– New fastidious test media MHF– New (low potency) discs– Ability to apply the EUCASTmethod and– New QC organism (H influenzaeNCTC 8468) Centralise validation forefficiency & cost effectiveness Provide validation data to otherlaboratories Verify inter-laboratoryperformance
Validation of Disc Testing – New medium New medium MHF agar Commercial or local manufacture Local manufacture (Central laboratory, PQ)––––NATA Biological validation requiredMultiple 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 againstcommercially produced MHF agar Non-metro PQ laboratories use all commerciallyproduced media Test protocols– 3 scientists set up all QC organisms on MH and MHF weekly for5 weeks– 1 scientist continues to set up all QC organisms on MH and MHFweekly for 4 weeks Data compared using excel Acceptance parameters – 100% within publishedEUCAST zone diameter range for both media types
Media validation for Enterococcus faecalis ATCC 29212 & ampicillin on Inhouse and commercial media
Media validation for Streptococcus pneumoniae ATCC 49616 & oxacillin onIn-house and commercial media
Tips and Tricks Local manufacture of MHA andMHF aim to use the same MHagar baseHaemophilus influenzae Fuzzy zones to SXT Medium formulation (OXOID) CXM zones too small Age of culture not medium Streptococcus pneumoniae Indistinct Oxacillin zone edgeresulting in zone reading problem Best Medium formulation (MAST)but poor growth of EnterococcusALL MH AGAR BASES areNOT the SAME
Validation of New AST procedure Be practical- validation needs to be efficient and cost effective forthe laboratoryCurrently apply CLSI recommendation for validation of AST methodsRequire minimum of 4 weeks testing and multiple staffComparison to involve both media types (MHEU and MHF agars),new disc concentrations, new QC organismUtilising at least 5scientists at Central to set up over 5 consecutive days for 4 weeksUse resource staff for inter-laboratory performance (5 scientists – 5days)Capture data using Excel program allows graphical comparison ofdata 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 29213MH EUPEN 1, FOX 30, ERY 15, DA 2, CN 10,RD 5, FD10, CIP5, C30, SXT 25Escherichia coliATCC 25922MH EUAMP 10, AMC 30, CN10, CTX 5, SXT25, TOB 10, AK30, CAZ 10, CIP 5,MEM 10, TIM 85, TAZ 60, F 100Ps aeruginosaATCC 27863MH EUCN10, TOB 10, AK 30, TIM 85, TAZ 60,MEM 10, CAZ 10, CIP 5,Enterococcus faecalisATCC 29212MH-FAMP 2, VA 5, TEC 30, CN 30, F100Strep pneumoniaeATCC 49619MH-FOX 1, ERY 15, VA 5, C30, SXT 25, TET30Haem influenzaeATCC 8468MH-FAMP2 AMC 3, CTX 5, CIP 5, SXT 25,C30
Enterococci Validation Data - OP118Zone (mm)16VA5 Zone14VA5 MinVA5 MaxVA5 Target121081 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Ope ratorEUCAST method and vancomycin 5 disc validation dataover 4 weeks for Operator No 1
What about Staff Training and CompetencyAssessment?
Training and Competency Assessment Cannot be centralised but staff trainingand competency assessment can bestandardisedDependent on standardised methodsDisc vs Automated MIC§ Minimal for automated AST§ More intensive manual training for disctestingCLSI vs EUCAST§ Only minimal changes for mostorganisms§ Major changes with fastidious organisms(Haemophilus influenzae) Dependent on skill set of scientific staff– Muliskilled scientists in regionallaboratories (disc testing)
The Process One Narelle can be everywhere!Train “resource staff” within GCLsForward QC organisms and testunknowns for hands-on training ofresource staff (Metro/GCL)Develop training file for recordingof test results for assessmentPhotograph test plates andforward with zone diameter resultsto Central laboratory forassessment of technique andaccuracy in application of theEUCAST methodsResource Staff training recordssigned off by Central TrainersSend in photographs of your plate set-up
Results for 2 Resource Staff MembersGuess the problem here?
WRONG MEDIUM 24 HR SUBCULTUREMHSB (CLSI)Results for 2 Resource Staff MembersGuess the problem here?
Pathology QLD EUCAST Training Summary Large organisations Training methods “Hands on” set up (QC)Unknown isolateCompetency Utilise resource personnelTrain the Trainer processResource staff to trainindividual staff within labs ordistrict (GCL)Assess media set upFinal written exam with85% pass markResource Documents USB for each Resourceperson with lectures,documents, referencedocuments, interpretationtables etc
In Summary For large organisations, centralise validation toensure cost effective utilisation of resources butstandardised methods required Validation of disc diffusion testing more labourintensive due to new medium and different discpotencies Staff training and competency testing needs tobe managed locally Multi-skilled Team effort required
Validation of newmethods can feellike this . .
But with a little thoughtful planning andeffort, the results can be rewarding .
Acknowledgements Pathology QLD EUCAST Implementation Team– Dr Sally Appleton, Haakon Bergh, Greg Flohr, Michael Caffery,Cathy Engler Dr Graeme Nimmo, Director of Microbiology, PathologyQLD Members of the Pathology QLD, Microbiology DisciplineWorking 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 Songuanand staff in Central Laboratory, Herston Hospitals Complex.
– 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 .
contents of the tool box" used for validation. The methods and techniques listed in the report are grouped as - review - models - analysis - dynamic methods - methods regarding formality - development methods The validation methods have to be combined together in a validation plan. The plan shall list requirements and validation methods.
contents of the tool box" used for validation. The methods and techniques listed in the report are grouped as - review - models - analysis - dynamic methods - methods regarding formality - development methods The validation methods have to be combined together in a validation plan. The plan shall list requirements and validation methods.
Cleaning validation Process validation Analytical method validation Computer system validation Similarly, the activity of qualifying systems and . Keywords: Process validation, validation protocol, pharmaceutical process control. Nitish Maini*, Saroj Jain, Satish ABSTRACTABSTRACT Sardana Hindu College of Pharmacy, J. Adv. Pharm. Edu. & Res.
Validation of standardized methods (ISO 17468) described the rules for validation or re-validation of standardized (ISO or CEN) methods. Based on principles described in ISO 16140-2. -Single lab validation . describes the validation against a reference method or without a reference method using a classical approach or a factorial design approach.
Dipl.-Ing. Becker EN ISO 13849-1 validation EN ISO 13849-2: Validation START Design consideration validation-plan validation-principles documents criteria for fault exclusions faults-lists testing is the testing complete? Validation record end 05/28/13 Seite 4 Analysis category 2,3,4 all
Pharmaceutical Engineers (ISPE) GAMP 5. Our validation service is executed in accordance with GxP standards producing a validation library that features the following documents: Validation and Compliance Plan The Validation and Compliance Plan (VCP) defines the methodology, deliverables, and responsibilities for the validation of Qualer.
heard. These goals relate closely to the Validation principles. Validation Principles and Group Work The following eleven axioms are the Validation Principles as revised in 2007. I have tried to find various ways of incorporating the principles into teaching Group Validation and by doing so, anchoring group work to theory. 1.
The grade 10 ELA Reading Comprehension test included three separate test sessions. Sessions 1 and . 2 were both administered on the same day, and Session 3 was administered on the following day. Each session included reading passages, followed by multiple-choice and open-response questions. Common reading passages and test items are shown on the following pages as they appeared in test .
