Clinical laboratory testing and in vitro diagnostic test systems - Broth micro-dilution reference method for testing the in vitro activity of antimicrobial agents against yeast fungi involved in infectious diseases (ISO 16256:2021)

This document 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 Blastomyces dermatitidis
and/or Histoplasma capsulatum variety capsulatum. Moreover, testing filamentous fungi (moulds)
introduces several additional problems in standardization not addressed by the current procedure.
Those methods are beyond the scope of this document.
This document describes the broth micro-dilution reference method, which can be implemented by
either of two pathways. One pathway involves visual determination of MICs (CLSI method)[1][5]
; the
second pathway involves spectrophotometric determination of MICs (EUCAST method)[2][10]. 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. 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 document;
interpretive category breakpoints specific to the CLSI- and EUCAST-derived methods can be found by
consulting the latest interpretive tables provided by the organizations[5][15]. Routine susceptibility
testing methods or diagnostic test devices can be compared with this reference method in order to
ensure comparable and reliable results for validation or registration purposes.

Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme - Referenzmethode zur Testung der In-vitro-Aktivität von antimikrobiellen Substanzen gegen Pilze, die Infektionskrankheiten verursachen (ISO 16256:2021)

Dieses Dokument beschreibt eine Methode zur Empfindlichkeitsprüfung von Sprosspilzen gegen Antimykotika, einschließlich Candida spp. und Cryptococcus neoformans, die Infektionen verursachen. Das hier beschriebene Referenzverfahren wurde nicht bei Untersuchungen der Hefeformen dimorpher Pilze, wie z. B. Blastomyces dermatitidis und/oder Histoplasma capsulatum Varietät capsulatum angewendet. Außerdem führt die Prüfung von fadenförmigen Pilzen (Schimmelpilzen) zu verschiedenen zusätzlichen Problemen hinsichtlich der Standardisierung, die in dem vorliegenden Verfahren nicht behandelt werden. Diese Methoden liegen außerhalb des Anwendungsbereiches dieses Dokuments.
Dieses Dokument beschreibt die Referenzmethode der Bouillonmikrodilution, die auf eine von zwei Weisen umgesetzt werden kann. Eine Weise umfasst die visuelle Bestimmung von MHK (CLSI-Methode) [1, 5]; die zweite die spektrophotometrische Bestimmung von MHK (EUCAST-Methode) [2, 10]. Die MHK widerspiegelt die Aktivität des Wirkstoffs unter den beschriebenen Prüfbedingungen und kann unter Berücksichtigung anderer Faktoren, wie z. B. der Pharmakologie des Wirkstoffs oder antimykotischer Resistenzmechanismen, zur Interpretation für klinische Zwecke angewendet werden. Die Verteilung der MHK-Werte kann zudem dazu dienen, Wildtyp-Pilzpopulationen von Nicht-Wildtyp-Pilzpopulationen zu unterscheiden. Die klinische Interpretation der MHK-Werte fällt nicht in den Anwendungsbereich dieses Dokuments; erläuternde Grenzwerte für die Kategorien, die für die von CLSI und EUCAST abgeleiteten Methoden spezifisch sind, können durch Hinzuziehen der von den Organisationen bereitgestellten aktuellen erläuternden Tabellen [5, 15] ermittelt werden. Es ist ratsam, die Routinemethoden oder Diagnosegeräte zur Empfindlichkeitsprüfung mit dieser Referenzmethode zu vergleichen, um vergleichbare und zuverlässige Ergebnisse für Validierungs- oder Registrierungszwecke sicherzustellen.

Laboratoires d’analyses de biologie médicale et systèmes de diagnostic in vitro - Méthode de référence de microdilution en milieu liquide pour soumettre à essai l’activité in vitro des agents antimicrobiens par rapport aux levures impliquées dans les maladies infectieuses (ISO 16256:2021)

Le présent document décrit une méthode d’essai de la sensibilité aux agents antifongiques des levures pathogènes, dont Candida spp. et Cryptococcus neoformans. La méthode de référence décrite ici n’a pas été utilisée dans des études sur les phases levures de champignons dimorphes tels que Blastomyces dermatitidis et/ou Histoplasma capsulatum variété capsulatum. En outre, les essais sur les champignons filamenteux (moisissures) posent d’autres problèmes de normalisation qui ne sont pas traités par le présent mode opératoire. Ces méthodes vont au-delà du domaine d’application du présent document.
Le présent document décrit la méthode de référence de microdilution en milieu liquide qui peut être réalisée de deux façons. La première implique une détermination visuelle de la CMI (méthode CLSI)[1][5]; la seconde implique une détermination spectrophotométrique de la CMI (méthode EUCAST)[2][10]. La CMI reflète l’activité de l’agent antimicrobien dans les conditions d’essai décrites et peut, avec d’autres facteurs tels que la pharmacologie de l’agent ou les mécanismes de résistance antifongique, servir dans la prise de décision de traitement clinique. Par ailleurs, les CMI peuvent servir à définir les populations en types sauvage ou non sauvage. L’interprétation clinique de la CMI sort du domaine d’application du présent document. Des critères d’interprétation catégoriels spécifiques aux méthodes du CLSI et de l’EUCAST sont donnés dans les derniers tableaux d’interprétation édités par ces organismes[5][15]. Dans le cadre d’une validation ou d’un enregistrement, les méthodes d’essai de sensibilité de routine ou les dispositifs de diagnostic peuvent être comparés avec la présente méthode de référence, afin de garantir des résultats comparables et fiables.

Klinično laboratorijsko preskušanje ter diagnostični preskusni sistemi in vitro - Referenčna metoda za preskušanje aktivnosti in vitro antimikrobnih snovi proti glivam kvasovkam, ki povzročajo infekcijske bolezni (ISO 16256:2021)

General Information

Status
Published
Public Enquiry End Date
19-Apr-2021
Publication Date
15-Nov-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-Nov-2021
Due Date
09-Jan-2022
Completion Date
16-Nov-2021

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SLOVENSKI STANDARD
SIST EN ISO 16256:2021
01-december-2021
Nadomešča:
SIST EN ISO 16256:2013
Klinično laboratorijsko preskušanje ter diagnostični preskusni sistemi in vitro -
Referenčna metoda za preskušanje aktivnosti in vitro antimikrobnih snovi proti
glivam kvasovkam, ki povzročajo infekcijske bolezni (ISO 16256:2021)
Clinical laboratory testing and in vitro diagnostic test systems - Broth micro-dilution
reference method for testing the in vitro activity of antimicrobial agents against yeast
fungi involved in infectious diseases (ISO 16256:2021)
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme -
Referenzmethode zur Testung der In-vitro-Aktivität von antimikrobiellen Substanzen
gegen Pilze, die Infektionskrankheiten verursachen (ISO 16256:2021)
Laboratoires d’analyses de biologie médicale et systèmes de diagnostic in vitro -
Méthode de référence de microdilution en milieu liquide pour soumettre à essai l’activité
in vitro des agents antimicrobiens par rapport aux levures impliquées dans les maladies
infectieuses (ISO 16256:2021)
Ta slovenski standard je istoveten z: EN ISO 16256:2021
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST EN ISO 16256:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16256:2021

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SIST EN ISO 16256:2021


EN ISO 16256
EUROPEAN STANDARD

NORME EUROPÉENNE

October 2021
EUROPÄISCHE NORM
ICS 11.100.10 Supersedes EN ISO 16256:2012
English Version

Clinical laboratory testing and in vitro diagnostic test
systems - Broth micro-dilution reference method for
testing the in vitro activity of antimicrobial agents against
yeast fungi involved in infectious diseases (ISO
16256:2021)
Laboratoires d'analyses de biologie médicale et Labormedizinische Untersuchungen und In-vitro-
systèmes de diagnostic in vitro - Méthode de référence Diagnostika-Systeme - Referenzmethode zur Testung
de microdilution en milieu liquide pour soumettre à der In-vitro-Aktivität von antimikrobiellen Substanzen
essai l'activité in vitro des agents antimicrobiens par gegen Pilze, die Infektionskrankheiten verursachen
rapport aux levures impliquées dans les maladies (ISO 16256:2021)
infectieuses (ISO 16256:2021)
This European Standard was approved by CEN on 2 August 2021.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16256:2021 E
worldwide for CEN national Members.

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SIST EN ISO 16256:2021
EN ISO 16256:2021 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 16256:2021
EN ISO 16256:2021 (E)
European foreword
This document (EN ISO 16256:2021) 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 April 2022, and conflicting national standards shall be
withdrawn at the latest by October 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 16256:2012.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 16256:2021 has been approved by CEN as EN ISO 16256:2021 without any modification.


3

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SIST EN ISO 16256:2021

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SIST EN ISO 16256:2021
INTERNATIONAL ISO
STANDARD 16256
Second edition
2021-10
Clinical laboratory testing and in
vitro diagnostic test systems — Broth
micro-dilution reference method
for testing the in vitro activity of
antimicrobial agents against yeast
fungi involved in infectious diseases
Laboratoires d’analyses de biologie médicale et systèmes de
diagnostic in vitro — Méthode de référence de microdilution en
milieu liquide pour soumettre à essai l’activité in vitro des agents
antimicrobiens par rapport aux levures impliquées dans les maladies
infectieuses
Reference number
ISO 16256:2021(E)
© ISO 2021

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SIST EN ISO 16256:2021
ISO 16256:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
  © ISO 2021 – All rights reserved

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SIST EN ISO 16256:2021
ISO 16256:2021(E)
Contents Page
Foreword .iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test procedures . 3
4.1 General . 3
4.1.1 Trays and method . 3
4.1.2 Conditions for use of disposable micro-dilution trays . 3
4.2 Medium . 3
4.2.1 General . 3
4.2.2 Visual reading pathway. 3
4.2.3 Spectrophotometric reading pathway . 4
4.3 Antifungal agents . 4
4.3.1 General . 4
4.3.2 Preparation of stock solutions . 4
4.3.3 Preparation of working solutions . 5
4.4 Preparation of broth micro-dilution trays . 6
4.4.1 Preparation for tests read visually – Visual reading pathway . 6
4.4.2 Preparation for tests read by spectrophotometer - Spectrophometric
reading pathway . 6
4.5 Storage of micro-dilution trays. 6
4.6 Preparation of inoculum . 7
4.6.1 General . 7
4.6.2 Preparation of inoculum for visual test reading . 7
4.6.3 Preparation of inoculum for spectrophotometric test reading . 7
4.7 Inoculation of micro-dilution trays . 7
4.8 Incubation of micro-dilution trays . 8
4.8.1 General . 8
4.8.2 Visual pathway . 8
4.8.3 Spectrophotometric pathway . 8
4.9 Reading MIC results . 8
4.9.1 General . 8
4.9.2 Visual reading method . 8
4.9.3 Spectrophotometric reading methods . 8
4.10 Interpretation of MICs . 9
5 Quality Control (QC) . 9
Annex A (informative) RPMI-1640 medium .12
Annex B (informative) McFarland 0,5 barium sulfate turbidity standard .14
Bibliography .15
iii
© ISO 2021 – All rights reserved

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SIST EN ISO 16256:2021
ISO 16256:2021(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 140, In vitro diagnostic medical devices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 16256:2012), which has been technically
revised.
The main changes are as follows:
— addition of “broth micro-dilution” to the title;
— removal of 48 h reading for Candida species by the visual reading method;
— removal of definitions for susceptibility and resistance that are beyond the scope of this test
performance document;
— inclusion of considerations for antifungal testing of yeast species with micro-dilution trays “treated”
by manufacturers of the trays prior to use in the tests;
— updating of viable count testing methods for visual and spectrophotometer test pathways.
— addition of new antifungals (isavuconazole, rezafungin) to the testing and quality control range
tables;
— detailed characterization of the components of one formulation of RPMI-1640 known to provide
reproducible results of antifungal susceptibility tests for Candida species and Cryptococcus
neoformans;
— reassigning of annexes;
— update of bibliography to more relevant information about performance of antifungal susceptibility
testing for yeast fungi.
iv
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SIST EN ISO 16256:2021
ISO 16256:2021(E)
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2021 – All rights reserved

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SIST EN ISO 16256:2021
ISO 16256:2021(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 can 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 can 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 are required
for intra- and inter-laboratory reproducibility, as results can 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 two-fold) increasing concentrations are inoculated with a
known number of microorganisms.
Broth micro-dilution denotes the performance of the broth dilution test in micro-dilution trays.
The reference methods described in this document are intended for the testing of pure cultures of yeast
fungi. The broth micro-dilution methods described in this document are the same as those described
[1][5]
by the Clinical and Laboratory Standards Institute (CLSI) and by the European Committee on
[2][10]
Antimicrobial Susceptibility Testing (EUCAST) . These methods were initially shown to provide
[3]
MICs of fluconazole that were similar, if not identical up to 2 mg/l . Further the methods have been
shown to provide MICs for two quality control strains of licensed antifungal agents that are similar
as described in this document although quality control results for the spectrophotometer can trend
slightly lower than for the visual reading method. The laboratory that wishes to use this document
for conducting studies of newer antifungal agents, or as a reference method for comparison to MICs
generated by a diagnostic device, can select which of the procedure options to use based upon the choice
[5]
of MIC reading determined by visual inspection (CLSI method) or by use of a spectrophotometer
[2][10]
(EUCAST method) . In either case, the procedural details for that option should be followed
explicitly. In the first edition of this document, i.e. ISO 16256:2012, the reported quality control
tests were performed using broth micro-dilution trays that were not treated in some way by the
manufacturers of the plastic trays for either the visual or spectrophotometer method.
In this document the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— ”may” indicates a permission;
— “can” indicates a possibility or a capability.
vi
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SIST EN ISO 16256:2021
INTERNATIONAL STANDARD ISO 16256:2021(E)
Clinical laboratory testing and in vitro diagnostic test
systems — Broth micro-dilution reference method for
testing the in vitro activity of antimicrobial agents against
yeast fungi involved in infectious diseases
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all of the safety problems associated
with its use. It is the responsibility of the user of this document to establish appropriate safety
and health practices and determine the applicability of regulatory limitations prior to use.
1 Scope
This document 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 Blastomyces dermatitidis
and/or Histoplasma capsulatum variety capsulatum. Moreover, testing filamentous fungi (moulds)
introduces several additional problems in standardization not addressed by the current procedure.
Those methods are beyond the scope of this document.
This document describes the broth micro-dilution reference method, which can be implemented by
[1][5]
either of two pathways. One pathway involves visual determination of MICs (CLSI method) ; the
[2][10]
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. 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 document;
interpretive category breakpoints specific to the CLSI- and EUCAST-derived methods can be found by
[5][15]
consulting the latest interpretive tables provided by the organizations . Routine susceptibility
testing methods or diagnostic test devices can be compared with this reference method in order to
ensure comparable and reliable results for validation or registration purposes.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
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 1 to entry: Disinfectants, antiseptics and preservatives are not included in this definition.
3.2 Antifungal agents — properties
1
© ISO 2021 – All rights reserved

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SIST EN ISO 16256:2021
ISO 16256:2021(E)
3.2.1
potency
active fraction of a test substance, determined in a bioassay against a reference powder of the same
substance
Note 1 to entry: 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.
3.2.2
concentration
amount of an antifungal agent (3.1) in a specified volume of liquid
Note 1 to entry: The concentration is expressed as mg/l.
Note 2 to entry: mg/l = µg/ml but use of the unit µg/ml is not recommended.
3.3
stock solution
initial solution used for further dilutions
3.4
minimum inhibitory concentration
MIC
lowest concentration (3.2.2) that, under specified in vitro test conditions, reduces growth by an agreed
amount within a specified period of time
Note 1 to entry: The MIC is expressed in mg/l.
3.5
wild type
absence of phenotypically-detectable acquired resistance mechanisms to the antifungal agent (3.1) in a
given fungal strain
3.6
reference strain
catalogued, well-characterized fungal strain with stable, specified antifungal susceptibility phenotypes
and/or genotypes
Note 1 to entry: Reference strains are kept as stock cultures, from which working cultures are derived. They are
obtainable from culture collections and used for quality control.
3.7 Susceptibility testing method
3.7.1
broth dilution
technique in which containers are filled with appropriate volumes of an antifungal solution, employing
incrementally (usually two-fold) increasing concentrations (3.2.2) of the antifungal agent (3.1) and
appropriate volumes of broth (3.8) with a specified inoculum (3.9)
Note 1 to entry: The aim of this method is the determination of the minimum inhibitory concentration (3.4).
3.7.2
broth micro-dilution
performance of broth dilution (3.7.1) in micro-dilution trays with a capacity of ≤300 µl per well
3.8
broth
fluid medium used for the in vitro growth of yeast fungi
2
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SIST EN ISO 16256:2021
ISO 16256:2021(E)
3.9
inoculum
number of colony-forming units of yeast in a suspension, calculated with respect to the final volume
Note 1 to entry: The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
4 Test procedures
4.1 General
4.1.1 Trays and method
The tests are performed in plastic disposable micro-dilution 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.
4.1.2 Conditions for use of disposable micro-dilution trays
The tests were originally performed in broth micro-dilution trays that have had no additional
treatment by the manufacturer. Quality control data by manufacturers of untreated trays (and on
which this document was originally based) have shown that quality control results are consistently
in specification for all antifungal agents tested. In some jurisdictions there has been a suggestion that
results can be more consistent using treatment of the plastic trays. Treatment of the plastic, either
by coating or corona discharge to impart an electrical charge to the plastic, is used in tissue culture
studies and allows the tissue cells to adhere to the plastic. It is unknown if this process has been
standardized for all micro-dilution tray manufacturers. It is known that with some antifungal agents
the treated trays can result in elevated MICs compared to untreated trays. Such treatment can affect
[13]
the reporting of results for those agents . Those laboratories that use “treated” micro-dilution trays
and read by spectrophotometer should ensure that the treated trays being utilized in testing provide
the same quality control results as those indicated in Table 5. Tho
...

SLOVENSKI STANDARD
oSIST prEN ISO 16256:2021
01-april-2021
Klinično laboratorijsko preskušanje ter dignostični preskusni sistemi in vitro -
Referenčna metoda za preskušanje aktivnosti in vitro antimikrobnih snovi proti
gobam kvasovkam, ki povzročajo infekcijske bolezni (ISO/DIS 16256:2021)
Clinical laboratory testing and in vitro diagnostic test systems - Broth micro-dilution
reference method for testing the in vitro activity of antimicrobial agents against yeast
fungi involved in infectious diseases (ISO/DIS 16256:2021)
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme -
Referenzmethode zur Testung der In-vitro-Aktivität von antimikrobiellen Substanzen
gegen Pilze, die Infektionskrankheiten verursachen (ISO/DIS 16256:2021)
Laboratoires d’analyses de biologie médicale et systèmes de diagnostic in vitro -
Méthode de référence de microdilution en milieu liquide pour soumettre à essai l’activité
in vitro des agents antimicrobiens par rapport aux levures impliquées dans les maladies
infectieuses (ISO/DIS 16256:2021)
Ta slovenski standard je istoveten z: prEN ISO 16256
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
oSIST prEN ISO 16256:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 16256:2021

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oSIST prEN ISO 16256:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16256
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
2021-02-11 2021-05-06
Clinical laboratory testing and in vitro diagnostic test
systems — Broth micro-dilution 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 de micro-dilution
en bouillon pour soumettre à essai l'activité in vitro des agents antimicrobiens par rapport aux levures
impliquées dans les maladies infectieuses
ICS: 11.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 16256:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2021

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COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved

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Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test procedures . 3
4.1 General . 3
4.1.1 4.1.1 Trays and method . 3
4.1.2 Conditions for use of disposable micro-dilution trays . 3
4.2 Medium . 3
4.2.1 General. 3
4.2.2 Visual reading pathway . 3
4.2.3 Spectrophotometric reading pathway. 4
4.3 Antifungal agents . 4
4.3.1 General. 4
4.3.2 Preparation of stock solutions . 4
4.3.3 Preparation of working solutions . 5
4.4 Preparation of broth micro-dilution trays . 6
4.4.1 Preparation for tests to be read visually – Visual reading pathway . 6
4.4.2 Preparation for tests to be read by spectrophotometer - Spectrophometric
reading pathway . 6
4.5 Storage of microdilution trays . 6
4.6 Preparation of inoculum . 7
4.6.1 General. 7
4.6.2 Preparation of inoculum for visual test reading . 7
4.6.3 Preparation of inoculum for spectrophotometric test reading . 7
4.7 Inoculation of micro-dilution trays . 7
4.8 Incubation of micro-dilution trays . 8
4.8.1 General. 8
4.8.2 Visual pathway . 8
4.8.3 Spectrophotometric pathway . 8
4.9 Reading MIC results . 8
4.9.1 General. 8
4.9.2 Visual reading method . 8
4.9.3 Spectrophotometric reading methods . 8
4.10 Interpretation of MICs . 9
5 Quality Control (QC) . 9
Annex A (informative) RPMI-1640 medium .12
Annex B (informative) McFarland 0,5 barium sulfate turbidity standard .14
Bibliography .15
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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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems.
This second edition cancels and replaces the first edition (ISO 16256:2012), which has been technically
revised.
The main changes compared to the previous edition are as follows:
— Addition of “broth micro-dilution” to the title (English and French);
— Removal of 48 h reading for Candida species by the visual reading method;
— Removal of definitions for susceptibility and resistance that are beyond the scope of this test
performance document;
— Inclusion of considerations for antifungal testing of yeast species with micro-dilution trays “treated”
by manufacturers of the trays prior to use in the tests;
— Updating of viable count testing methods for visual and spectrophotometer test pathways.
— Addition of new antifungals (e.g. isavuconazole, rezafungin) to the testing and quality control
range tables;
— Detailed characterization of the components of one formulation of RPMI-1640 known to provide
reproducible results of antifungal susceptibility tests for Candida species and Cryptococcus
neoformans;
— Reassigning of Annexes;
— Updating of bibliography to more relevant information about performance of antifungal susceptibility
testing for yeast fungi.
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Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
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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 can 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 can 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 are required
for intra- and inter-laboratory reproducibility, as results can 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 two-fold) increasing concentrations are inoculated with a
known number of microorganisms.
Broth micro-dilution denotes the performance of the broth dilution test in microdilution trays.
The reference methods described in this document are intended for the testing of pure cultures of
yeast fungi. The broth micro-dilution methods described in document are the same as those described
by the Clinical and Laboratory Standards Institute (CLSI)[1,5] and by the European Committee on
Antimicrobial Susceptibility Testing (EUCAST)[2,10]. These methods were initially shown to provide
MICs of fluconazole that were similar, if not identical up to 2 mg/l [3]. Further the methods have
been shown to provide MICs for two quality control strains of licensed antifungal agents that are
similar as described in this document although quality control results for the spectrophotometer
can trend slightly lower than for the visual reading method. The laboratory that wishes to use this
document 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)[5] or by use of a
spectrophotometer (EUCAST method)[2,10]. In either case, the procedural details for that option are to
be followed explicitly. In the original ISO 16256:2012 document, the reported quality control tests were
performed using broth micro-dilution trays that were not treated in some way by the manufacturers of
the plastic trays for either the visual or spectrophotometer method.
In this document the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— ”may” indicates a permission;
— “can” indicates a possibility or a capability.
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oSIST prEN ISO 16256:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 16256:2021(E)
Clinical laboratory testing and in vitro diagnostic test
systems — Broth micro-dilution reference method for
testing the in vitro activity of antimicrobial agents against
yeast fungi involved in infectious diseases
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all of the safety problems associated
with its use. It is the responsibility of the user of this doocument to establish appropriate safety
and health practices and determine the applicability of regulatory limitations prior to use.
1 Scope
This document 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 Blastomyces dermatitidis
and/or Histoplasma capsulatum variety capsulatum. Moreover, testing filamentous fungi (moulds)
introduces several additional problems in standardization not addressed by the current procedure.
Those methods are beyond the scope of this document.
This document describes the broth micro-dilution reference method which can be implemented by
either of two pathways. One pathway involves visual determination of MICs (CLSI method)[1,5]; the
second pathway involves spectrophotometric determination of MICs (EUCAST method)[2,10]. 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. 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 document;
interpretive category breakpoints specific to the CLSI- and EUCAST-derived methods can be found by
consulting the latest interpretive tables provided by the organizations[5,15]. 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 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
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 1 to entry: Disinfectants, antiseptics and preservatives are not included in this definition.
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3.2
antifungal agents — properties

3.2.1
potency
active fraction of a test substance, determined in a bioassay against a reference powder of the same
substance
Note 1 to entry: 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.
3.2.2
concentration
amount of an antifungal agent (3.1) in a defined volume of liquid
Note 1 to entry: The concentration is expressed as mg/l.
Note 2 to entry: mg/l = µg/ml but use of the unit µg/ml is not recommended.
3.3
stock solution
initial solution used for further dilutions
3.4
minimum inhibitory concentration
MIC
lowest concentration (3.2.2) that, under defined in vitro test conditions, reduces growth by an agreed
amount within a defined period of time
Note 1 to entry: The MIC is expressed in mg/l.
3.5
wild type
absence of phenotypically-detectable acquired resistance mechanisms to the antifungal agent (3.1) in a
given fungal strain
3.6
reference strain
catalogued, well-characterized fungal strain with stable, defined antifungal susceptibility phenotypes
and/or genotypes
Note 1 to entry: Reference strains are kept as stock cultures, from which working cultures are derived. They are
obtainable from culture collections and used for quality control.
3.7
susceptibility testing method

3.7.1
broth dilution
technique in which containers are filled with appropriate volumes of an antifungal solution, employing
incrementally (usually two-fold) increasing concentrations (3.2.2) of the antifungal agent (3.1) and
appropriate volumes of broth (3.8) with a defined inoculum (3.9)
Note 1 to entry: The aim of this method is the determination of the MIC (3.4).
3.7.2
broth micro-dilution
performance of broth dilution (3.7.1) in micro-dilution trays with a capacity of ≤ 300 µl per well
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3.8
broth
fluid medium used for the in vitro growth of yeast fungi
3.9
inoculum
number of colony-forming units of yeast in a suspension, calculated with respect to the final volume
Note 1 to entry: The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
4 Test procedures
4.1 General
4.1.1 4.1.1 Trays and method
The tests are performed in plastic disposable micro-dilution 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.
4.1.2 Conditions for use of disposable micro-dilution trays
The tests were originally performed in broth-microdilution trays that have had no additional treatment
by the manufacturer. Quality control data by manufacturers of untreated trays (and on which this
document was originally based) have shown that quality control results are consistently in specification
for all antifungal agents tested. In some jurisdictions there has been a suggestion that results can be
more consistent using treatment of the plastic trays. Treatment of the plastic, either by coating or corona
discharge to impart an electrical charge to the plastic, is used in tissue culture studies and allows the
tissue cells to adhere to the plastic. It is unknown if this process has been standardized for all micro-
dilution tray manufacturers. It is known that with some antifungal agents the treated trays can result in
elevated MICs compared to untreated trays. Such treatment can affect the reporting of results for those
agents[13]. Those laboratories that use “treated” microdilution trays and read by spectrophotometer
should ensure that the treated trays being utilized in testing provide the same quality control results
as those indicated in Table 5. Those quality control ranges were originally performed with untreated
trays, The data indicates that for almost all antifungal agents, the quality control ranges for the
two standard strains listed in this document (Candida parapsilosis ATCC®22019 and Candida krusei
ATCC®6258) are the within one log2 dilution for both testing/reading methods. Comparative quality
control ranges for those strains for the spectrophotometer method are the same as originally reported
using untreated trays[10] and for treated trays[2], with the exception of caspofungin (see Table 5).
Comparative MIC observations for clinical isolates provided by the visual reading method[5] and those
spectrophotometer readings using treated plates[2] for both testing methods should be interpreted
with caution.
4.2 Medium
4.2.1 General
RPMI-1640 broth shall be used (see Annex A Table A.2 for details for preparation of the two complete
product versions of RPMI-1640 glucose broth) for both reading methods.
4.2.2 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.
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4.2.3 Spectrophotometric reading pathway
The RPMI-1640 medium should contain 2,0 % 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.
4.3 Antifungal agents
4.3.1 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.
4.3.2 Preparation of stock solutions
The use of a calibrated analytical balance is required for weighing antifungal agents. Allowance for the
potency of the powder shall be made by use of the following formula to obtain the amount of antifungal
agent substance or the volume of diluent needed for a standard solution:
V ×ρ
m = (1)
P
mP×
V = (2)
ρ
where
ρ is the concentration of the stock solution, in mg/l;
m is mass of the antifungal agent (powder), in g;
P is the potency of the antifungal agent (powder), in mg/g;
V is the volume of diluent, in l.
Concentrations of stock solutions should be 1 000 mg/l or greater, although the solubility of some agents
is a limiting factor. The actual concentrations of stock solutions depend on the method of preparing
working solutions (serial dilutions). Some agents require alternative solvents (see Table 1). Sterilization
of solutions is not usually necessary. If required, sterilization should be done by membrane filtration
and samples before and after sterilization should be compared by assay to ensure that adsorption has
not occurred.
Unless information is available on stability of stock solutions under specified storage conditions, they
should be prepared fresh for each test batch.
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Table 1 — Solvents and diluents for preparation of stock solutions of antifungal agents
Antifungal agent Solvent Diluent
(Full strength and intermediate solut
...

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