EN ISO 16256:2012

SLOVENSKI STANDARD
01-marec-2013
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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)
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme -
Referenzmethode zur Testung der In-vitro-Aktivität von antimikrobiellen Substanzen
gegen Pilze, die Infektionskrankheiten verursachen (ISO 16256:2012)
Essais de laboratoire clinique et systèmes de diagnostic in vitro - Méthode de référence
pour soumettre à essai l'activité in vitro des agents antimicrobiens par rapport aux
levures impliquées dans les maladies infectieuses (ISO 16256:2012)
Ta slovenski standard je istoveten z: EN ISO 16256:2012
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 16256
NORME EUROPÉENNE
EUROPÄISCHE NORM
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 laboratoire clinique et systèmes de diagnostic in Labormedizinische Untersuchungen und In-vitro-
vitro - Méthode de référence pour soumettre à essai Diagnostika-Systeme - Referenzmethode zur Testung der
l'activité in vitro des agents antimicrobiens par rapport aux In-vitro-Aktivität von antimikrobiellen Substanzen gegen
levures impliquées dans les maladies infectieuses (ISO Pilze, die Infektionskrankheiten verursachen (ISO
16256:2012) 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 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 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 bodies 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, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16256:2012: E
worldwide for CEN national Members.

Contents Page
Foreword . 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/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 withdrawn 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, 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, Latvia, 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.

INTERNATIONAL ISO
STANDARD 16256
First edition
2012-12-01
Clinical 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 diseases
Essais de laboratoire clinique et systèmes de diagnostic in vitro —
Méthode de référence pour soumettre à essai l’activité in vitro des
agents antimicrobiens par rapport aux levures impliquées dans les
maladies infectieuses
Reference number
ISO 16256:2012(E)
©
ISO 2012
ISO 16256:2012(E)
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any
means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the
address below or ISO’s member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 16256:2012(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Test procedures . 4
3.1 General . 4
3.2 Medium . 4
3.3 Antifungal agents . 5
3.4 Storage of microdilution trays . 7
3.5 Preparation of inoculum — General . 8
3.6 Inoculation of microdilution trays . 8
3.7 Incubation of microdilution trays . 9
3.8 Reading MIC results . 9
3.9 Interpretation of MICs .10
4 Quality control (QC) .10
Annex A (informative) RPMI-1640 medium .13
Annex B (informative) McFarland 0,5 barium sulfate turbidity standard .15
Annex C (informative) Acceptable reading times for MIC interpretations using the visual MIC
reading procedure .16
Bibliography .17
ISO 16256:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards 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, also 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 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 possibility 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 – All rights reserved

ISO 16256:2012(E)
Introduction
In 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. 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 antifungal 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 unreliable 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 lowest 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
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 true 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.
Broth 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
Standard are essentially the same as those described by the Clinical and Laboratory Standards Institute
[1] [2]
(CLSI) and by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) . These
methods have been shown to provide MICs of fluconazole that are essentially the same, if not identical
[3]
up to 2 mg/l . 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 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.
INTERNATIONAL STANDARD ISO 16256:2012(E)
Clinical 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 diseases
WARNING — The use of this International Standard may involve hazardous materials, operations
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
limitations prior to use.
1 Scope
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. 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 developed
[4][5][6][7][8]
and are now available as CLSI document M38 and EUCAST document E.DEF 9.1 .
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] [2]
; the second pathway involves spectrophotometric determination of MICs (EUCAST method) . 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 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 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
[2][9]
organizations . It is advisable to compare routine susceptibility testing methods or diagnostic test
devices with this reference method in order to ensure comparable and reliable results for validation or
registration purposes.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
antifungal agent
substance of biological, semi-synthetic or synthetic origin that inhibits the growth of or kills fungi, and
is thus of potential use in the treatment of infections
NOTE Disinfectants, antiseptics and preservatives are not included in this definition.
ISO 16256:2012(E)
2.2
antifungal agents — properties
2.2.1
potency
active fraction of a test substance, determined in a bioassay against a reference powder of the same substance
NOTE 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 amount-of-
substance concentration (mass fraction) in mole per litre of ingredients in the test substance.
2.2.2
concentration
amount of an antifungal agent in a defined volume of liquid
NOTE 1 The concentration is expressed as mg/l.
NOTE 2 mg/l = µg/ml but use of the unit µg/ml is not recommended.
2.3
stock solution
initial solution used for further dilutions
2.4
minimum inhibitory concentration
MIC
lowest concentration that, under defined in vitro test conditions, reduces growth by an agreed amount
within a defined period of time
NOTE The MIC is expressed in mg/l.
2.5
breakpoint
BP
specific 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 latest publications of organizations
[1][2][9]
employing the reference method (e.g. CLSI and EUCAST) .
2.5.1
visual reading pathway
2.5.1.1
susceptible
S
fungal strain inhibited in vitro by a concentration of an antifungal agent that is associated with a high
likelihood of therapeutic success
NOTE 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,
emergence of new resistance mechanisms).
2.5.1.2
susceptible dose-dependent
S-DD
fungal strain inhibited in vitro by a concentration of an antifungal agent that may be achieved in vivo by
using higher than normal doses of the agent when such dosage schedules can be safely employed
NOTE 1 Fungal strains are categorized as susceptible dose-dependent by applying the appropriate breakpoints
in a defined phenotypic test system.
2 © ISO 2012 – All rights reserved

ISO 16256:2012(E)
NOTE 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).
2.5.1.3
intermediate
I
micro-organism having a level of antimicrobial agent activity associated with uncertain therapeutic effect
NOTE This implies that an infection due to the isolate may be appropriately treated in body sites where the
drugs are physically concentrated or when a high dosage of drug can be used; it also indicates a buffer zone that
should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations.
2.5.1.4
nonsusceptible
NS
category used for yeast fungi that currently have only a susceptible interpretive category, but not
susceptible dose-dependent, intermediate or resistant interpretive categories (i.e. susceptible-only
interpretive category)
NOTE This category is often given to new antifungal agents for which no resistant isolates have yet been
encountered.
2.5.1.5
resistant
R
fungal strain inhibited in vitro by a concentration of an antifungal agent that is associated with a high
likelihood of therapeutic failure
NOTE 1 Fungal strains are categorized as resistant 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,
emergence of new resistance mechanisms).
2.5.2
spectrophotometric reading pathway
2.5.2.1
susceptible
S
micro-organism having a level of antimicrobial activity associated with a high likelihood of
therapeutic success
2.5.2.2
intermediate
I
micro-organism having a level of antimicrobial agent activity associated with uncertain therapeutic effect
NOTE This implies that an infection due to the isolate may be appropriately treated in body sites where the
drugs are physically concentrated or when a high dosage of drug can be used; it also indicates a buffer zone that
should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations.
2.5.2.3
resistant
R
micro-organism having a level of antimicrobial activity associated with a high likelihood of
therapeutic failure
ISO 16256:2012(E)
2.6
wild type
absence of acquired resistance mechanisms to the antifungal agent in a given fungal strain
2.7
reference strain
catalogued, well-characterized fungal strain with stable, defined antifungal susceptibility phenotypes
and/or genotypes
NOTE Reference strains are kept as stock cultures, from which working cultures are derived. They are
obtainable from culture collections and used for quality control.
2.8
susceptibility testing method
2.8.1
broth dilution
technique in which containers are filled with appropriate volumes of an antifungal solution, employing
incrementally (usually two-fold) increasing concentrations of the antifungal agent and appropriate
volumes of broth with a defined inoculum
NOTE The aim of this method is the determination of the MIC.
2.8.2
microdilution
performance of broth dilution in microdilution trays with a capacity of ≤ 300 µl per well
2.9
broth
fluid medium used for the in vitro growth of yeast fungi
2.10
inoculum
number of yeast in a suspension, calculated with respect to the final volume
NOTE The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
2.11
inoculum effect
change in MIC related to change in inoculum
3 Test procedures
3.1 General
The tests are performed in plastic disposable microdilution trays. The method is based on the preparation
of double strength antifungal agent working solutions in 100 µl volumes per well with the addition of an
inoculum also in a volume of 100 µl.
3.2 Medium
3.2.1 General
RPMI-1640 broth shall be used (see Appendix A for details for preparation of the two versions of RPMI-
1640 glucose broth).
4 © ISO 2012 – All rights reserved

ISO 16256:2012(E)
3.2.2 Visual reading pathway
The RPMI-1640 medium should contain 0,2 % glucose. The RPMI-1640 broth is prepared and dispensed
at single strength with double strength antifungal agent dilutions and the inoculum is delivered in equal
volumes of RPMI-1640 broth containing the adjusted yeast inoculum suspension.
3.2.3 Spectrophotometric reading pathway
The RPMI-1640 medium should contain 2 % glucose. The RPMI-1640 broth and antifungal agents are
both prepared at double strength with the inoculum subsequently added in an equal volume of sterile
distilled water.
3.3 Antifungal agents
3.3.1 General
Antifungal agents shall be obtained directly from the manufacturer or from reliable commercial sources;
pharmaceutical preparations for clinical use are not acceptable. The antifungal agents shall be supplied
with a lot number, potency, an expiry date and details of recommended storage conditions. Substances
shall be stored in tightly closed containers in the dark, at −20 °C, with a desiccant unless otherwise
recommended by the manufacturer. Hygroscopic agents should be dispensed into aliquots, one of which
is used on each test occasion.
Allow containers to warm to room temperature before opening them in order to avoid condensation and
loss of potency.
3.3.2 Preparation of stock solutions
The use of a calibrated analytical balance is required for weighing antifungal agents. Allowance for the
potency of the pow
...