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)

Resolution BT C87/2011 (extension of DOW): DOW = DAV + 36 months

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)

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.

---------------------- Page: 1 ----------------------
SIST EN ISO 16256:2021
---------------------- Page: 2 ----------------------
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.
---------------------- Page: 3 ----------------------
SIST EN ISO 16256:2021
EN ISO 16256:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
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.

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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
---------------------- Page: 7 ----------------------
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
© ISO 2021 – All rights reserved
---------------------- Page: 8 ----------------------
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
---------------------- Page: 9 ----------------------
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.
© ISO 2021 – All rights reserved
---------------------- Page: 10 ----------------------
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.
© ISO 2021 – All rights reserved
---------------------- Page: 11 ----------------------
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.
© ISO 2021 – All rights reserved
---------------------- Page: 12 ----------------------
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
© ISO 2021 – All rights reserved
---------------------- Page: 13 ----------------------
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
© ISO 2021 – All rights reserved
---------------------- Page: 14 ----------------------
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.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 16256:2021
---------------------- Page: 2 ----------------------
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
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oSIST prEN ISO 16256:2021
ISO/DIS 16256:2021(E)
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ii © ISO 2021 – All rights reserved
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oSIST prEN ISO 16256:2021
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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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oSIST prEN ISO 16256:2021
ISO/DIS 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.

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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oSIST prEN ISO 16256:2021
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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)
mP×
V = (2)
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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