CONF-4-Lionel-Pineau.pdf

1、Comparison of endoscope sampling and culturing methodsLionel PINEAUEurofins Hospital HygieneDisclosure of Conflicts of Interest:Eurofins Biotech Germandereports having been consulted and having received financialsupport from medical device manufacturers to performed studieson the efficacy of medical

2、 devices.Introduction2Endoscope contamination rateMicrobial contaminationInfectious risk?What is the microbiological quality of our endoscopes?Is there a risk for the patients?IntroductionIn 2021,in France,13%of endoscopes are at the action level and 8.1%are at the alert level,meaning that the conta

3、mination level of 21.1%of endoscopes exceeds what has been defined as a maximum acceptable value.Pineau Lionel.Endoscope reprocessing:Retrospective analysis of 90,311 samples.Endosc In t Open 2023;11:E247E257010203040506070809010020002005201020152020%of non-compliant endoscopesSaviuc2015Minebois2016

4、Studies published in the literature indicate that the non-compliance rate of ready to use endoscopes varies from 0.4%to 49.0%Chiu2012Gillespie 2008Rauwers2018Bader 2001Saliou2010Okamoto2022Chomel2009Mark2020Moses 2003Alfa 2002Osborne 2007IntroductionBader L et al,2002 In 2 test periods,endoscopes re

5、ady for use were found contaminated at high rates:Period 1:49%of 152 endoscopes;Period 2:39%of 154 endoscopes).0,9%NaClSuction(20 ml)Biopsy(20 ml)Total viable countPlate-Count agar plates(20C/37C for 48 hours)Sampling and culturing method1ml10 mlPathogensMembrane filtration Brain-Heart-Infusion agar

6、(37C for 48 h)Swab on 5%Blood-Columbia agar and MacConkey agar(37C for 48 h)Air/water(20 ml)Water bottle Distilled water,tap water or sterile water60 mlGillespie et al,2007Blood agar&MacConkey(35C/48 hours+28C for 3 days)2 x 0,1 ml10 ml10 ml in each channelSterile water40 ml1 ml9 ml withdrawn40 CFU

7、There were 2374 screening tests performed during the 5-year period,including 287 AFER,631 bronchoscopes for mycobacteria and 1456 endoscope bacterial screens.There were no positive results of the AER or bronchscopes for mycobacteria.Of the 1456 endoscopic bacterialsamples,6 were positive.i.e.0,4%Cen

8、trifugationSampling and culturing methodIntroductionAlfa MJ,Singh H.Contaminated flexible endoscopes:Review of impact of channel sampling methods on culture results and recommendations for root-cause analysis.Infect Control Hosp Epidemiol.2021 May 7:1-16.doi:10.1017/ice.2021.128Remaining gaps in the

9、 guidelines include ensuring that optimal endoscope-channel sample methods are used and ensuring effective root-cause analysis and remediation when contamination is detected.So many different methods!.Recent CDC and FDA recommendations focus on reducing“exogenous”infection transmission and specifica

10、lly recommend the culture of patient-ready endoscopes to detect contamination with organisms of concern.Alfa MJ,Singh H.Contaminated flexible endoscopes:Review of impact of channel sampling methods on culture results and recommendations for root-cause analysis.Infect Control Hosp Epidemiol.2021 May

11、7:1-16.doi:10.1017/ice.2021.128IntroductionC.Aumeran,E.Thibert,F.A.Chapelle,C.Hennequin,O.Lesens and O.Traora.Assessment on experimental Bacterial Biofilms and in Clinical Pracdce of the Efficacy of Sampling Soludons for Microbiological Tesdng of Endoscopes.Journal of Clinical Microbiology.March 201

12、2.Volume 50.Number 3.938942 A single flushing of internal channels with saline solution removes only a very small number of bacteria.Importance of sampling solution.In conclusion,the efficiency and therefore the value of the monitoring of endoscope reprocessing by microbiological cultures is depende

13、nt on the sampling solutions used.A sampling solution with a tensioactive action is more efficient than saline in detecting biofilm contamination of endoscopes.Introduction10 Since the recent outbreaks associated with duodenoscopes,the interest of endoscope sampling to assess regularly the adequacy

14、of endoscope reprocessing,is well accepted.Studies published in the literature indicate that the contamination level(or non-compliance rate)of ready to use endoscopes varies from 0.4%to 49.0%Differences observed between these studies regarding,the sampling method(flush vs flush-brush-flush,one chann

15、el vs all channels,),the nature of the sampling solution(water,0.9%NaCl,neutralizer,),the sample culturing protocols(filtration vs centrifugation,),the interpretation criteria and the limited number of samples analysed,make difficult the comparison and the interpretation of these values.What is the

16、most appropriate sampling&culturing method to be used as a quality indicator?Objectives Compare the efficacy of 5 endoscope sampling and culturing methods.Define the critical parameters for an endoscope sampling and culturing method.11Principle12ContaminationTransportCultureAnalysis1 endoscope:1 duo

17、denoscope(TJF-Q180V),3 microbial strains:E.coli,S.aureus and P.aeruginosa,3 microbial concentrations are tested:10 CFU/scope,100 CFU/scope and 1000 CFU/scope,5 sampling methods are compared:Germany,Netherland,France,Australia and FDA,2 transportation times:1 and 24 hours,RR:Recovery ratio(ISO 11737-

18、1)6 assays are performed per conditions i.e.6 x 2 x 3 x 3=108 assays per sampling method SamplingRREfficacy of the sampling/culturing methodsIf my endoscope contains100 CFU,how many bacteriawill I be able to collect withmy sampling method?Validation by repeated sampling to establish the relationship

19、 between the number of microorganisms recovered and the actual number of microorganisms present on the product.Recovery ratioN5=0 CFUNumber of microorganismsin the endoscopen0=130 CFUN1=110 CFUN2=8 CFUN3=2 CFUN4=2 CFUNumber of microorganismsin the endoscope after 1stsamplingn1=20 CFUNumber of microo

20、rganismsin the endoscope after 2ndsamplingn2=12 CFUNumber of microorganismsin the endoscope after 3rdsamplingn3=10 CFUNumber of microorganismsin the endoscope after 4thsamplingn4=8 CFU=110/(110+8+2+2)=110/122=90%Sampling solution-!#$=0=1/-!#%Number of microorganismsin the endoscope after 5thsampling

21、n5=8 CFUAll microorganisms initially present in the endoscope have been sampledSampling methodsFRA:France,USA:United States of America,AUST:Australia,GER:Germany,NL:The Netherlands(a)FSF:Flush-Suction-Flush,(b):FBF:Flush-Brush-Flush,(c):FB:Flush-Brush,(d)F:Flush,(e):FSBF:Flush-Suction-Brush-Flush.Y:

22、Yes,N:No,NDP+thio:Neutralizing Pharmacopeia Diluent plus thiosulfate.x:figures in square brackets define the chronology in which channels/sites were sampled.COUNTRY:FRA AUSTNLUSAGERSample sitesInstrument channelY(FSFa)2Y(FBc)1Y(FSBFe)2Y(FBFb)2Y(F)2Suction/instrument channelY(FSF)3Y(Fd)3Y(FSBF)3NY(F)

23、3Air/water channelY(FSF)4Y(Fd)2Y(FSF)4NY(F)4Elevator recess(distal end)with brush or swabY 1NY 1Y(1Y 1Sampling solutionNDP+thioSterile waterNaCl 0.9%Sterile water NaCl 0.9%Addition of neutralizer to extracted sampleNo(NDP+thio used for sampling)NoNoY(NDP+thio)Y(NDP+thio)two time concentratedSample v

24、olume(sampling solution+neutralizer)100 mL(distal end)+130 mL(channels)30 mL60 mL82 mL3 x 50 mLFriction for Instrument channel(bristle brush)NYYYNNumber of samples2(all channels pooled&distal end)1(all channels pooled)2(all channels pooled&distal end)1(Instrument channel&distal end pooled)4(All chan

25、nels separately&distal end)MethodSampling of the Air/water channels using the flush methodABMH 944 cleaning adaptorInjection of the sampling solution into the air/water channel using a syringe connected to air connector(A)while the valve cylinders were closed with the MH-944 connector(B).Channel wer

26、e then purged with air.MH 946 cleaning adaptorFlushMethodSampling of the suction/instrument channel using the flush-suction-flush methodBiopsy valve MB-358ABMH 944 cleaning adaptorInjection of the sampling solution from the suction connector(A)while the valve cylinders were closed with the MH-944 co

27、nnector(B)(Flush).The plunger of the syringe was pulled up(Suction)and down(Flush)and the channel was purged with air.Flush-Suction-FlushMethodSampling of the instrument channel using the flush-brush-flush methodInjection of the sampling solution in the instrument channel followed by an air purge,Br

28、ushing of the channel,New injection of sampling solution and air purge.Flush-Brush-FlushNote:for Australian method repeat all stages on suction and suction/instrument channel.Sampling methodSampling of the duodenoscope distal end1.Swabbing along the seam between the distal cap and the distal end 2.E

29、levator recess flush elevator down and up3.Elevator brush(large brush)4.Elevator brush(small brush)20Instruction n DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative au traitement des endoscopes souples thermosensibles canaux au sein des lieux de soins.Available at:https:/www.legifrance.gouv.fr/c

30、irculaire/id/41172.Last accessed 11/10/2023.GESA-Gastroenterological Society of Australia.Infection control in endoscopy 2nd Edition.2003.Available at https:/ Last accessed 11/10/2023.Gillespie E,Despina Kotsanas D,Stuart RL.Microbiological monitoring of endoscopes:5-year review.J Gastroenterol Hepa

31、tol 2008;23:10691074Rauwers AW,Voor Int Holt AF,Buijs JG et al.High prevalence rate of digestive tract bacteria in duodenoscopes:a nationwide study.Gut 2018;67:16371645.FDA/CDC/ASM.Duodenoscope Surveillance Sampling and Culturing Protocols.2018 Available at:https:/www.fda.gov/media/111081/download.L

32、ast accessed 23/04/2023.Hygiene Requirements for the Reprocessing of Medical Devices.Bundesgesundheitsbl 2012 55:12441310.Available at https:/www.rki.de/DE/Content/Infekt/Krankenhaushygiene/Kommission/Downloads/Hygiene_Requirements_Medical_Devices_2012.pdf?_blob=publicationFile.Last accessed 24/05/2

33、023.21(a)F:Filtration.(b)C:centrifugationTested MethodFRAAUST NL USA GER Culture methodFiltrationCentrifugationFiltrationFiltrationCentrifugationFiltrationTotal volume used for sample extraction230 mL30 mL60 mL82 mL82 mL3 x 50 mLTotal sample volume analyzed 230 mL0.2 mL60 mL82 mL82 mL3 x 50 mL%of sa

34、mple volume analyzed100%6.6%100%100%100%100%Culture mediumTrypticase soy agar(TSA)Blood+MacConkey agarR2A AgarBlood agarBlood agarBlood agarIncubation time5 days5 days3 days3 days3 days2 daysIncubation temperature30C35C then 28C35C35C to 37C35C to 37C36CResult expression according to sourceCFU/endos

35、copeCFU/mLCFU/20 mLCFU/endoscopeCFU/endoscopeCFU/channelCulturing methodsMembrane filtrationhttps:/plantlet.org/general-methods-of-microbial-isolation/A vacuum is created in the receiving flask.The air pressure forces the liquid through the filter.The microorganisms are retained on the filter surfac

36、e.This filter is then transferred to a petri dish containing a pre-poured set medium,where colonies arise from the bacteria on the surface of the filter.Centrifugationhttps:/ of culture mediaThe centrifuging and washing method is used to concentrate microorganisms in a small volume,but a number of q

37、uestions remain unanswered:the sensitivity of the microorganisms to centrifugation,the recovery efficiency of the methodResults Comparison of the efficacy of 5 endoscope sampling methods.What are the critical parameters for an endoscope sampling and culturing method.241%24%31%39%29%12%0%5%10%15%20%2

38、5%30%35%40%45%50%AUSTUSA centrifugationUSA filtrationFRAGERNLRECOVERY RATIO(%)ResultsEfficacy of the sampling/culturing methods 25No neutralizer Only 2.5%of the solution injected was analysedNo neutralizerAddition of neutralizerAddition of neutralizerSampling solution=neutralizer260%18%28%24%21%7%0%

39、31%35%70%22%21%2%23%21%23%29%8%0%10%20%30%40%50%60%70%80%AUSTUS CentrifugationUS FiltrationFRAGERNLE.coliPs.aeruginosaS.aureusThe efficacy of the sampling/culturing method varies according to the nature of the microorganisms present in endoscope channels.RECOVERY RATIO(%)ResultsInfluence of the micr

40、oorganism27No differencebetween 1h and 24h transportation time for US and FRA sampling methods.For the GER and NL method a decreaseof the recovery ratio is observed if the sample is analysed24h after sampling.0%22%29%36%16%41%1%25%34%43%8%18%0%5%10%15%20%25%30%35%40%45%AUSTUSA centrifugationUSA filt

41、rationFRANLGER1h24hRECOVERY RATIO(%)ResultsInfluence of the transportation time28The efficacy of the sampling and culturing methoddecreases of about 10%when the endoscope contamination levelvaries from 1000 CFU to 10 CFU/endoscope.0%5%10%15%20%25%30%35%40%45%AUSTUS CentriUS FiltraFRAGERNL10 CFU100 C

42、FU1000 CFURECOVERY RATIO(%)ResultsInfluence of the initial contamination level29Comparison of results obtained after sampling and culturing of an endoscope containing initially 100 CFU with the different methods tested.Sampling solution150 ml100 CFUCentrifugation+platting of the pellet100 CFU66 CFU1

43、50 ml100 CFUFiltration 1 CFU(2)Direct inoculation100 ml150 ml100 l100 CFU10 CFU(1)6,6%100%25 CFU25%(1)40 CFU/endoscope (2)20 CFU per working channel.M.Wehrl et al.Elution of working channels with the flush-brush-flush-method for microbiological testing of reprocessed endoscopes2022.Zentralsterilizat

44、ion,Volume 30,2772-277Importance of brushingTurbulent flow“The novel Turbulent Fluid Flow(TFF)method for extraction of samples from colonoscope channels is a more effective method than the existing FBF and F methods”Sohn S Y,Alfa M J,Lai R,Tabani Y,Labib M E Turbulent Fluid Flow is a novel closed-sy

45、stem sample extraction method for flexible endoscope channels of various inner diameters J Microbiol Methods.2020 January;168:105782ConclusionEndoscope sampling and culturing practices need to be harmonized1.The sampling solution shall include neutralizing as well as a tensioactive agents.2.All chan

46、nels in the endoscope should be sampled.3.Ideally use of friction during sample collection for all channels.4.Ensuring that 80%of the total sample injected into the channels is collected.5.Ensuring that the sampling solution maintains microbial viability for 24 hours at refrigeration temperature(4C).6.The entire sample collected should be concentrated by 0.45 m(or 0.2m)membrane filtration and cultured on agar medium.7.Harmonized interpretation criteria shall be defined.Thank you for your attention!