ISO/PRF 16256

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

General Information

• Status: Published
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: ISO/TC 212 - Clinical laboratory testing and in vitro diagnostic test systems
• Drafting Committee: ISO/TC 212/WG 4 - Microbiology and molecular diagnostics

RELATIONS

• Revised: ISO 16256:2012 - Clinical laboratory testing and in vitro diagnostic test systems -- Reference method for testing the in vitro activity of antimicrobial agents against yeast fungi involved in infectious diseases
• Effective Date: 23-Apr-2020

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 16256
Second edition
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
PROOF/ÉPREUVE
Reference number
ISO 16256:2021(E)
ISO 2021
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ISO 16256:2021(E)
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© ISO 2021

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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 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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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 compared to the previous edition 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.
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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.
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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 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

[1][5]

Clinical and Laboratory Standards Institute (CLSI) and by the European Committee on Antimicrobial

[2][10]

Susceptibility Testing (EUCAST) . These methods were initially shown to provide MICs of

[3]

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 of MIC reading

[5]

determined by visual inspection (CLSI method) or by use of a spectrophotometer (EUCAST method)

[2][10]

. 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.
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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 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.

3.2 Antifungal agents — properties
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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
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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-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

[13]

agents . 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

[10] [2]

using untreated tray and for treated trays , with the exception of caspofungin (see Table 5).

[5]

Comparative MIC observations for clinical isolates provided by the visual reading method and those

[2]

spectrophotometer readings using treated plate for both testing methods should be interpreted with

caution.
4.2 Medium
4.2.1 General

RPMI-1640 broth shall be used (see 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.

1) ATCC is the registered trademark of a product supplied by the American Type Culture Collection. This information

is given for the convenience of users of this document and does not constitute an endorsement by ISO of the product

named. Equivalent products may be used if they can be shown to lead to the same results.

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ISO 16256:2021(E)
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 Formulae (1) and (2) 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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ISO 16256:2021(E)

Table 1 — Solvents and diluents for preparation of stock solutions of antifungal agents

Antifungal agent Solvent Diluent
(Full strength and intermediate solutions)
Amphotericin B DMSO Medium
Anidulafungin DMSO Medium
Caspofungin DMSO Medium
Flucytosine DMSO Medium
Fluconazole DMSO Medium
Isavuconazole DMSO Medium
Itraconazole DMSO Medium
Ketoconazole DMSO Medium
Micafungin DMSO Medium
Posaconazole DMSO Medium
Ravuconazole DMSO Medium
Rezafungin DMSO Medium
Voriconazole DMSO Medium
DMSO (dimethyl sulfoxide) is potentially toxic.
4.3.3 Preparation of working solutions

The interval of concentrations selected for testing depends on the organisms and antifungal agent. The

chosen range shall allow full end point MIC determination for appropriate reference strains. A two-fold

dilution series based on 1 mg/l is prepared in RPMI-1640 glucose broth. The procedure outlined in

Tables 2 and 3 are known to reliably produce a satisfactory dilution series and should be followed unless

an alternative method is carefully validated. For example, it has been reported that serial dilutions of

[6]

the more hydrophilic compounds can produce acceptable results . Working solutions shall be used

the same day unless information is available from the manufacturer on stability of the solutions under

specified storage conditions.

Table 2 — Scheme for preparing dilutions of water-soluble antifungal agents used in broth

dilution susceptibility tests
Antifungal solution
Step Concentration Source Volume + Medium = Intermediate = Final Log
Concentration Concentration
at 1:10
mg/l ml ml mg/l mg/l
1 5 120 Stock 1,0 3,0 1 280 128 7
2 5 120 Stock 1,0 7,0 640 64 6
3 640 Step 2 1,0 1,0 320 32 5
4 640 Step 2 1,0 3,0 160 16 4
5 160 Step 4 1,0 1,0 80 8 3
6 160 Step 4 0,5 1,5 40 4 2
7 160 Step 4 0,5 3,5 20 2 1
8 20 Step 7 1,0 1,0 10 1 0
9 20 Step 7 0,5 1,5 5 0,5 -1
10 20 Step 7 0,5 3,5 2,5 0,25 -2
11 2,5 Step 10 1,0 1,0 1,25 0,125 -3
12 2,5 Step 10 0,5 1,5 0,625 0,062 5 -4
13 2,5 Step 10 0,5 3,5 0,312 5 0,031 25 -5
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