1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 16256:2012Clinical laboratory testingand in vitro diagnostic testsystems Reference methodfor testing the in vitro activityof antimicrobial agents againstyeast of fungi
2、involved ininfectious diseases (ISO16256:2012)BS EN ISO 16256:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO16256:2012.The UK participation in its preparation was entrusted to TechnicalCommittee CH/212, IVDs.A list of organizations represented on this
3、committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 73551 6ICS 11.10
4、0.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 December 2012.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPIS
5、CHE NORM EN ISO 16256 December 2012 ICS 11.100.10 English Version Clinical laboratory testing and in vitro diagnostic test systems - Reference method for testing the in vitro activity of antimicrobial agents against yeast of fungi involved in infectious diseases (ISO 16256:2012) Essais de laboratoir
6、e clinique et systmes de diagnostic in vitro - Mthode de rfrence pour soumettre essai lactivit in vitro des agents antimicrobiens par rapport aux levures impliques dans les maladies infectieuses (ISO 16256:2012) Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme - Referenzmethode zur
7、Testung der In-vitro-Aktivitt von antimikrobiellen Substanzen gegen Pilze, die Infektionskrankheiten verursachen (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 f
8、or giving this European Standard the status of a national standard without any alteration. Up-to-date lists and 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 thr
9、ee official versions (English, French, German). A version in any other language made by translation under the responsibility 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 bo
10、dies of 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, Slovakia, Slovenia,
11、Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION 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
12、 national Members. Ref. No. EN ISO 16256:2012: EBS EN ISO 16256:2012EN ISO 16256:2012 (E) 3 Foreword 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/
13、TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN. 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 withdr
14、awn at the latest by December 2015. Attention is drawn to the possibility that some of 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,
15、 the national standards organisations of the following countries are bound to implement 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, La
16、tvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, 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. BS EN ISO 16256:2
17、012ISO 16256:2012(E) ISO 2012 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope . 12 Terms and definitions . 13 Test procedures . 43.1 General . 43.2 Medium 43.3 Antifungal agents 53.4 Storage of microdilution trays . 73.5 Preparation of inoculum General . 83.6 Inoculation of micr
18、odilution trays . 83.7 Incubation of microdilution trays . 93.8 Reading MIC results 93.9 Interpretation of MICs . 104 Quality control (QC) .10Annex A (informative) RPMI-1640 medium 13Annex B (informative) McFarland 0,5 barium sulfate turbidity standard .15Annex C (informative) Acceptable reading tim
19、es for MIC interpretations using the visual MIC reading procedure .16Bibliography .17BS EN ISO 16256:2012ISO 16256:2012(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International S
20、tandards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, als
21、o take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees
22、 is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.Attention is drawn to the possibil
23、ity that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights.ISO 16256 was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in vitro diagnostic test systems.iv ISO 2012 A
24、ll rights reservedBS EN ISO 16256:2012ISO 16256:2012(E)IntroductionIn vitro susceptibility tests are performed 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.
25、The tests are also important in resistance surveillance, epidemiological studies of susceptibility and in comparisons 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 antif
26、ungal susceptibility testing. MIC methods are used in resistance surveillance, comparative testing of new agents 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 unrel
27、iable and when a quantitative result is needed for clinical management. In dilution tests, microorganisms are 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 lowe
28、st concentration of an antimicrobial agent (in mg/l) that, under defined in vitro test conditions, reduces visible 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
29、antimicrobial agent and aids treatment decisions. Careful control and standardization is required for intra- 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 tr
30、ue end point (i.e. 1 well or tube in a doubling dilution series).Broth dilution is a technique in which containers holding identical volumes of broth with antimicrobial agent solutions in incrementally (usually twofold) increasing concentrations are inoculated with a known number of microorganisms.B
31、roth microdilution denotes the performance of the broth dilution test in microdilution trays.The reference methods 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 Stand
32、ard are essentially the same as those described by the Clinical and Laboratory Standards Institute (CLSI)1and 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 t
33、o 2 mg/l3. Studies with various other antifungal agents are planned or under way. The laboratory that wishes 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
34、the procedure options to use based upon the choice of MIC reading determined by visual inspection (CLSI method) or by use of a spectrophotometer (EUCAST method). In either case, the procedural details for that option are to be followed explicitly. ISO 2012 All rights reserved vBS EN ISO 16256:2012BS
35、 EN ISO 16256:2012Clinical laboratory testing and in vitro diagnostic test systems Reference method for testing the in vitro activity of antimicrobial agents against yeast fungi involved in infectious diseasesWARNING The use of this International Standard may involve hazardous materials, operations
36、and equipment. This International 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 limitation
37、s prior to use.1 ScopeThis 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 dimorp
38、hic fungi, such as B. 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 filamen
39、tous fungi have been developed 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 meth
40、od)1; the second pathway 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 antifu
41、ngal resistance mechanisms. 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
42、 the MIC value is beyond 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 m
43、ethods or diagnostic test 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
44、-synthetic or synthetic 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.INTERNATIONAL STANDARD ISO 16256:2012(E) ISO 2012 All rights reserved 1BS EN ISO
45、 16256:2012ISO 16256:2012(E)2.2antifungal agents properties2.2.1potencyactive fraction 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 U
46、nits (IU) per gram, or as a volume fraction or mass fraction in percent, or as an 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 expresse
47、d as mg/l.NOTE 2 mg/l = g/ml but use of the unit g/ml is not recommended.2.3stock 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
48、timeNOTE The MIC is expressed in mg/l.2.5breakpointBPspecific MIC values that can 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 la
49、test publications of organizations employing the reference method (e.g. CLSI and 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 defined phenotypic test system.NOTE 2 This breakpoint can be altered in certain circumstances (e.g. changes in commonly used drug dosages, emergenc
