DIN EN ISO 16256-2013 Clinical laboratory testing and in vitro diagnostic test systems - Reference method for testing the in vitro activity of antimicrobial agents against yeast of6.pdf

1、April 2013 Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS

2、11.100.10!$A“1999330www.din.deDDIN EN ISO 16256Clinical laboratory testing and in vitro diagnostic test systems Reference method for testing the in vitro activity of antimicrobial agentsagainst yeast fungi involved in infectious diseases (ISO 16256:2012);English version EN ISO 16256:2012,English tra

3、nslation of DIN EN ISO 16256:2013-04Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme Referenzmethode zur Testung der In-vitro-Aktivitt von antimikrobiellen Substanzengegen Pilze, die Infektionskrankheiten verursachen (ISO 16256:2012);Englische Fassung EN ISO 16256:2012,Englische ber

4、setzung von DIN EN ISO 16256:2013-04Essais de laboratoire clinique et systmes de diagnostic in vitro Mthode de rfrence pour soumettre essai lactivit in vitro des agents antimicrobienspar rapport aux levures impliques dans les maladies infectieuses (ISO 16256:2012);Version anglaise EN ISO 16256:2012,

5、Traduction anglaise de DIN EN ISO 16256:2013-04www.beuth.deDocument comprises 24 pagesIn case of doubt, the German-language original shall be considered authoritative.03.13DIN EN ISO 16256:2013-04 2 A comma is used as the decimal marker. National foreword This document (EN ISO 16256:2012) has been p

6、repared by Technical Committee ISO/TC 212 “Clinical laboratory testing and in vitro diagnostic test systems” (Secretariat: ANSI, USA) in collaboration with Technical Committee CEN/TC 140 “In vitro diagnostic medical devices” (Secretariat: DIN, Germany). The responsible German body involved in its pr

7、eparation was the Normenausschuss Medizin (Medical Standards Committee), Working Committee NA 063-05-10 AA Chemotherapeutische Untersuchungsmethoden. EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16256 December 2012 ICS 11.100.10 English Version Clinical laboratory testing and in vitro di

8、agnostic test systems - Reference method for testing the in vitro activity of antimicrobial agents against yeast fungi involved in infectious diseases (ISO 16256:2012) Essais de laboratoire clinique et systmes de diagnostic in vitro - Mthode de rfrence pour soumettre essai lactivit in vitro des agen

9、ts antimicrobiens par rapport aux levures impliques dans les maladies infectieuses (ISO 16256:2012) Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme - Referenzmethode zur Testung der In-vitro-Aktivitt von antimikrobiellen Substanzen gegen Pilze, die Infektionskrankheiten verursachen

10、 (ISO 16256:2012) This European Standard was approved by CEN on 30 November 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and

11、bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the re

12、sponsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav

13、Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE N

14、ORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16256:2012: EContents PageForeword .Introduction .1 Scope 2 Terms and definit

15、ions 3 Test procedures 3.1 General 3.2 Medium .3.3 Antifungal agents .3.4 Storage of microdilution trays .3.5 Preparation of inoculum General .3.6 Inoculation of microdilution trays .3.7 Incubation of microdilution trays .3.8 Reading MIC results 3.9 Interpretation of MICs 144 Quality control (QC) 14

16、Annex A (informative) RPMI-1640 medium .17Annex B (informative) McFarland 0,5 barium sulfate turbidity standard Annex C (informative) Acceptable reading times for MIC interpretations using the visual MIC reading procedure Bibliography 345588891112121313192021EN ISO 16256:2012 (E) DIN EN ISO 16256:20

17、13-04 2Foreword This document (EN ISO 16256:2012) has been prepared by Technical Committee ISO/TC 212 “Clinical laboratory testing and in vitro diagnostic test systems“ in collaboration with Technical Committee CEN/TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN

18、. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 2013, and conflicting national standards shall be withdrawn at the latest by December 2015. Attention is drawn to the possibility that some o

19、f the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to impleme

20、nt this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, S

21、lovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 16256:2012 has been approved by CEN as a EN ISO 16256:2012 without any modification. EN ISO 16256:2012 (E) DIN EN ISO 16256:2013-04 3 IntroductionIn vitro susceptibility tests are perform

22、ed on microorganisms suspected of causing disease, particularly if the organism is thought to belong to a species that may exhibit acquired resistance to frequently used antimicrobial agents. The tests are also important in resistance surveillance, epidemiological studies of susceptibility and in co

23、mparisons of new and existing agents.Dilution procedures are used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents and represent the reference method for antifungal susceptibility testing. MIC methods are used in resistance surveillance, comparative testing of new ag

24、ents for research or registration purposes, to establish the susceptibility of organisms that give equivocal results in routine tests, for tests with organisms where routine tests may be unreliable and when a quantitative result is needed for clinical management. In dilution tests, microorganisms ar

25、e tested for their ability to produce discernible growth on a series of agar plates (agar dilution) or in broth (broth dilution) containing serial dilutions of the antimicrobial agent.The lowest concentration of an antimicrobial agent (in mg/l) that, under defined in vitro test conditions, reduces v

26、isible or optically measurable growth of a microorganism within a defined period of time is known as the MIC. The MIC is a guide for the clinician to the susceptibility of the organism to the antimicrobial agent and aids treatment decisions. Careful control and standardization is required for intra-

27、 and inter-laboratory reproducibility, as results may be influenced by the method used. It is generally accepted that broth MIC tests are reproducible to within one doubling dilution of the true end point (i.e. 1 well or tube in a doubling dilution series).Broth dilution is a technique in which cont

28、ainers holding identical volumes of broth with antimicrobial agent solutions in incrementally (usually twofold) increasing concentrations are inoculated with a known number of microorganisms.Broth microdilution denotes the performance of the broth dilution test in microdilution trays.The reference m

29、ethods described in this International Standard are intended for the testing of pure cultures of yeast fungi. The broth microdilution methods described in this part of this International Standard are essentially the same as those described by the Clinical and Laboratory Standards Institute (CLSI)1an

30、d by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)2. These methods have been shown to provide MICs of fluconazole that are essentially the same, if not identical up to 2 mg/l3. Studies with various other antifungal agents are planned or under way. The laboratory that wishes

31、 to use this International Standard for conducting studies of newer antifungal agents, or as a reference method for comparison to MICs generated by a diagnostic device, should select which of the procedure options to use based upon the choice of MIC reading determined by visual inspection (CLSI meth

32、od) or by use of a spectrophotometer (EUCAST method). In either case, the procedural details for that option are to be followed explicitly.EN ISO 16256:2012 (E) DIN EN ISO 16256:2013-04 4WARNING The use of this International Standard may involve hazardous materials, operations and equipment. This In

33、ternational Standard does not purport to address all of the safety problems associated with its use. It is the responsibility of the user of this International Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.1 Scope

34、This International Standard describes a method for testing the susceptibility to antifungal agents of yeasts, including Candida spp. and Cryptococcus neoformans, that cause infections. The reference method described here has not been used in studies of the yeast forms of dimorphic fungi, such as B.

35、dermatitidis and/or H. capsulatum variety capsulatum. Moreover, testing filamentous fungi (moulds) introduces several additional problems in standardization not addressed by the current procedure. Reference methods for broth dilution antifungal susceptibility testing of filamentous fungi have been d

36、eveloped and are now available as CLSI document M38 and EUCAST document E.DEF 9.145678.This International Standard describes the broth microdilution reference method which can be implemented by either of two pathways. One pathway involves visual determination of MICs (CLSI method)1; the second pathw

37、ay involves spectrophotometric determination of MICs (EUCAST method)2. The MIC reflects the activity of the drug under the described test conditions and can be interpreted for clinical management purposes by taking into account other factors, such as drug pharmacology or antifungal resistance mechan

38、isms. MICs can be categorized as “susceptible” (S), “susceptible dose-dependent” (S-DD), “intermediate” (I), “non-susceptible” (NS) or “resistant” (R). In addition, MIC distributions can be used to define wild type or non-wild type fungal populations. Clinical interpretation of the MIC value is beyo

39、nd the scope of this International Standard; interpretive category breakpoints specific to the CLSI- and EUCAST-derived methods can be found by consulting the latest interpretive tables provided by the organizations29. It is advisable to compare routine susceptibility testing methods or diagnostic t

40、est devices with this reference method in order to ensure comparable and reliable results for validation or registration purposes.2 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.2.1antifungal agentsubstance of biological, semi-synthetic or syntheti

41、c origin that inhibits the growth of or kills fungi, and is thus of potential use in the treatment of infectionsNOTE Disinfectants, antiseptics and preservatives are not included in this definition.EN ISO 16256:2012 (E) DIN EN ISO 16256:2013-04 5 2.2antifungal agents properties2.2.1potencyactive fra

42、ction of a test substance, determined in a bioassay against a reference powder of the same substanceNOTE The potency is expressed as mass fraction in milligrams per gram (mg/g), or as activity content in International Units (IU) per gram, or as a volume fraction or mass fraction in percent, or as an

43、 amount-of-substance concentration (mass fraction) in mole per litre of ingredients in the test substance.2.2.2concentrationamount of an antifungal agent in a defined volume of liquidNOTE 1 The concentration is expressed as mg/l.NOTE 2 mg/l = g/ml but use of the unit g/ml is not recommended.2.3stock

44、 solutioninitial solution used for further dilutions2.4minimum inhibitory concentrationMIClowest concentration that, under defined in vitro test conditions, reduces growth by an agreed amount within a defined period of timeNOTE The MIC is expressed in mg/l.2.5breakpointBPspecific MIC values that can

45、 be used to assign fungi to the clinical categories “susceptible”, “susceptible dose-dependent,” “intermediate,” “nonsusceptible” and “resistant”NOTE For current interpretive breakpoints, reference can be made to the latest publications of organizations employing the reference method (e.g. CLSI and

46、EUCAST)129.2.5.1visual reading pathway2.5.1.1susceptibleSfungal strain inhibited in vitro by a concentration of an antifungal agent that is associated with a high likelihood of therapeutic successNOTE 1 Fungal strains are categorized as susceptible by applying the appropriate breakpoints in a define

47、d phenotypic test system.NOTE 2 This breakpoint can be altered in certain circumstances (e.g. changes in commonly used drug dosages, emergence of new resistance mechanisms).2.5.1.2susceptible dose-dependentS-DDfungal strain inhibited in vitro by a concentration of an antifungal agent that may be ach

48、ieved in vivo by using higher than normal doses of the agent when such dosage schedules can be safely employedNOTE 1 Fungal strains are categorized as susceptible dose-dependent by applying the appropriate breakpoints in a defined phenotypic test system.EN ISO 16256:2012 (E) DIN EN ISO 16256:2013-04

49、 6NOTE 2 This class of susceptibility implies that an infection due to the isolate can be appropriately treated in body sites where the drugs are physiologically concentrated or when a high dosage of drug can be used.NOTE 3 This breakpoint can be altered in certain circumstances (e.g. changes in commonly used drug dosages, emergence of new resistance mechanisms)