In vitro diagnostic test systems - Requirements and recommendations for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid amplification methods (ISO/TS 5798:2022)

This document provides requirements and recommendations for the design, development, verification, validation and implementation of analytical tests for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using nucleic acid amplification. It addresses pre-examination, examination and post-examination process steps for human specimens.
This document is applicable to medical laboratories. It is also intended to be used by in vitro diagnostic developers and manufacturers, as well as by institutions and organizations supporting SARS-CoV-2 research and diagnostics.
This document does not apply to environmental samples.

In-vitro-Diagnostika-Systeme - Anforderungen und Empfehlungen für Qualitätsverfahren für den Nachweis des Coronavirus 2 des Schweren Akuten Respiratorischen Syndroms (SARS-CoV-2) mittels Nukleinsäureamplifikation (ISO/TS 5798:2022)

Systèmes d’essai pour diagnostic in vitro - Exigences et recommandations pour la détection du coronavirus 2 associé au syndrome respiratoire aigu sévère (SARS-CoV-2) par des méthodes d’amplification des acides nucléiques (ISO/TS 5798:2022)

Le présent document fournit des exigences et des recommandations pour la conception, le développement, la vérification, la validation et la mise en œuvre d’analyses afin de détecter le coronavirus 2 du syndrome respiratoire aigu sévère (SARS‑CoV‑2) au moyen de l’amplification de l’acide nucléique. Il aborde les étapes du processus préanalytique, de l’analyse et du processus postanalytique pour des spécimens humains.
Le présent document s’applique aux laboratoires de biologie médicale. Il est également destiné aux concepteurs et fabricants de diagnostics in vitro, ainsi qu’aux institutions et aux organisations soutenant la recherche et le diagnostic du SARS‑CoV‑2.
Le présent document ne s’applique pas aux échantillons environnementaux.

Diagnostični preskusni sistemi in vitro - Zahteve in priporočila za odkrivanje koronavirusa (SARS-CoV-2) z metodami amplifikacije nukleinskih kislin (ISO/TS 5798:2022)

General Information

Status
Published
Public Enquiry End Date
16-Oct-2022
Publication Date
11-Jan-2023
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Jan-2023
Due Date
13-Mar-2023
Completion Date
12-Jan-2023

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SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 5798:2023
01-februar-2023
Diagnostični preskusni sistemi in vitro - Zahteve in priporočila za odkrivanje
koronavirusa (SARS-CoV-2) z metodami amplifikacije nukleinskih kislin (ISO/TS
5798:2022)

In vitro diagnostic test systems - Requirements and recommendations for detection of

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid
amplification methods (ISO/TS 5798:2022)

In-vitro-Diagnostika-Systeme - Anforderungen und Empfehlungen für Qualitätsverfahren

für den Nachweis des Coronavirus 2 des Schweren Akuten Respiratorischen Syndroms
(SARS-CoV-2) mittels Nukleinsäureamplifikation (ISO/TS 5798:2022)
Systèmes d’essai pour diagnostic in vitro - Exigences et recommandations pour la

détection du coronavirus 2 associé au syndrome respiratoire aigu sévère (SARS-CoV-2)

par des méthodes d’amplification des acides nucléiques (ISO/TS 5798:2022)
Ta slovenski standard je istoveten z: CEN ISO/TS 5798:2022
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST-TS CEN ISO/TS 5798:2023 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST-TS CEN ISO/TS 5798:2023
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SIST-TS CEN ISO/TS 5798:2023
CEN ISO/TS 5798
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
November 2022
TECHNISCHE SPEZIFIKATION
ICS 11.100.01
English Version
In vitro diagnostic test systems - Requirements and
recommendations for detection of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid
amplification methods (ISO/TS 5798:2022)

Systèmes d'essai pour diagnostic in vitro - Exigences et In-vitro-Diagnostika-Systeme - Anforderungen und

recommandations pour la détection du coronavirus 2 Empfehlungen für Qualitätsverfahren für den

associé au syndrome respiratoire aigu sévère (SARS- Nachweis des Coronavirus 2 des Schweren Akuten

CoV-2) par des méthodes d'amplification des acides Respiratorischen Syndroms (SARS-CoV-2) mittels

nucléiques (ISO/TS 5798:2022) Nukleinsäureamplifikation (ISO/TS 5798:2022)

This Technical Specification (CEN/TS) was approved by CEN on 21 November 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Türkiye 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

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 5798:2022 E

worldwide for CEN national Members.
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SIST-TS CEN ISO/TS 5798:2023
CEN ISO/TS 5798:2022 (E)
Contents Page

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

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SIST-TS CEN ISO/TS 5798:2023
CEN ISO/TS 5798:2022 (E)
European foreword

The text of ISO/TS 5798:2022 has been prepared by Technical Committee ISO/TC 212 "Clinical

laboratory testing and in vitro diagnostic test systems” of the International Organization for

Standardization (ISO) and has been taken over as CEN ISO/TS 5798:2022 by Technical Committee

CEN/TC 140 “In vitro diagnostic medical devices” the secretariat of which is held by DIN.

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.

Any feedback and questions on this document should be directed to the users’ national standards body.

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 announce this Technical Specification: 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, Türkiye and the

United Kingdom.
Endorsement notice

The text of ISO/TS 5798:2022 has been approved by CEN as CEN ISO/TS 5798:2022 without any

modification.
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SIST-TS CEN ISO/TS 5798:2023
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SIST-TS CEN ISO/TS 5798:2023
TECHNICAL ISO/TS
SPECIFICATION 5798
First edition
2022-04
In vitro diagnostic test systems —
Requirements and recommendations
for detection of severe acute
respiratory syndrome coronavirus 2
(SARS-CoV-2) by nucleic acid
amplification methods
Systèmes d’essai pour diagnostic in vitro — Exigences et
recommandations pour la détection du coronavirus 2 associé au
syndrome respiratoire aigu sévère (SARS-CoV-2) par des méthodes
d’amplification des acides nucléiques
Reference number
ISO/TS 5798:2022(E)
© ISO 2022
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Overview ....................................................................................................................................................................................................................... 7

4.1 SARS-CoV-2 ................................................................................................................................................................................................ 7

4.1.1 General ........................................................................................................................................................................................ 7

4.1.2 Pre-examination ................................................................................................................................................................. 9

4.1.3 Examination — Overview .......................................................................................................................................... 9

4.1.4 Post-examination ............................................................................................................................................................ 11

4.2 Nucleic acid amplification methods .................................................................................................................................. 11

4.2.1 Reverse transcription qPCR (RT-qPCR) ...................................................................................................... 11

4.2.2 Reverse transcription digital PCR (RT-dPCR) .......................................................................................12

4.2.3 Isothermal amplification methods ..................................................................................................................12

5 Laboratory requirements .......................................................................................................................................................................12

5.1 General ........................................................................................................................................................................................................12

5.2 Biosafety requirements ............................................................................................................................................................... 13

5.2.1 Laboratory area ............................................................................................................................................................... 13

5.2.2 Risk control .......................................................................................................................................................................... 13

5.2.3 Personal protective equipment (PPE) .......................................................................................................... 13

5.3 General laboratory set-up ..........................................................................................................................................................13

5.4 Instrumentation ................................................................................................................................................................................. 14

5.5 Laboratory personnel .................................................................................................................................................................... 14

6 Design and development ..........................................................................................................................................................................14

6.1 Customer, patient and stakeholder needs ................................................................................................................... 14

6.2 Intended use of analytical test .............................................................................................................................................. 14

6.3 Institutional guideline strategy ........................................................................................................................................... 15

6.3.1 Laboratory developed tests (LDTs) versus in vitro diagnostic medical

devices (IVD medical devices) ............................................................................................................................ 15

6.3.2 Emergency use authorization .............................................................................................................................. 15

6.4 Clinical strategy ................................................................................................................................................................................. 15

6.5 Design and development planning ..................................................................................................................................... 16

6.5.1 Pre-examination of respiratory specimens for SARS-CoV-2 testing................................. 16

6.5.2 Examination design specifications (analytical test specifications) ..................................22

6.5.3 Design risk management .......................................................................................................................................... 27

6.6 Optimization of reagents and methods .........................................................................................................................28

6.6.1 Selection of SARS-CoV-2 target sequences ...............................................................................................28

6.6.2 Potential impact of variants of concern (VOCs) on the quality of NAAT

diagnostic methods for detecting SARS-CoV-2.....................................................................................28

6.6.3 Selection of amplification methods ................................................................................................................28

6.6.4 Design and selection of primers ........................................................................................................................28

6.6.5 Optimization of the reaction system .............................................................................................................29

6.6.6 Determination of cut-off values .........................................................................................................................29

6.6.7 Verification and validation of test design .................................................................................................29

7 Verification for patient care .................................................................................................................................................................31

7.1 General ...................................................................................................................................................................................................... 31

7.2 Confirmation of analytical performance characteristics .............................................................................. 31

7.2.1 Accuracy ................................................................................................................................................................................. 31

7.2.2 Limit of detection (LOD) ........................................................................................................................................... 31

7.2.3 Inclusivity .............................................................................................................................................................................. 32

7.2.4 Specificity .............................................................................................................................................................................. 32

iii
© ISO 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(E)

7.2.5 Robustness ................................... ........................................................................................................ ................................. 32

7.3 Clinical evidence ................................................................................................................................................................................ 33

8 Validation for patient care ........................................................................................................................................... ...........................33

8.1 General consideration ................................................................................................................................................................... 33

8.2 Clarification of the intended use ......................................................................................................................................... 33

8.3 Performance with clinical specimens or samples ................................................................................................34

9 Design transfer to production ............................................................................................................................................................34

10 Implementation and use in the laboratory and reporting of results ........................................................34

10.1 Implementation and use in the laboratory .................................................................................................................34

10.2 Reporting and interpretation of results ....................................................................................................................... 35

11 Quality assurance ............................................................................................................................................................................................36

11.1 Performance monitoring ............................................................................................................................................................ 36

11.2 Design change including optimization of analytical test ...............................................................................36

11.3 Interlaboratory comparison .................................................................................................................................................... 37

Annex A (informative) Nucleic acid amplification techniques ..............................................................................................38

Bibliography .............................................................................................................................................................................................................................41

© ISO 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(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 Technical Committee ISO/TC 276, Biotechnology.

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 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(E)
Introduction

Coronaviruses are enveloped RNA viruses that are broadly distributed in the animal kingdom. They

have been identified in humans, other mammals, and birds. Coronaviruses were named because the

spike proteins known to facilitate viral attachment and cell entry appear like a halo on the virus surface

when viewed under an electron microscope. Coronaviruses are roughly spherical with a diameter

ranging from 118 nm to 136 nm. The coronavirus genome, which ranges from 26 kb to 32 kb, is the

largest among all RNA viruses, including RNA viruses that have segmented genomes. Until 2019, six

coronaviruses have been associated with human diseases:
— severe acute respiratory syndrome-related coronavirus (SARS-CoV),
— Middle East respiratory syndrome coronavirus (MERS-CoV),
— human coronavirus 229E (HCoV-229E),
— human coronavirus OC43 (HCoV-OC43),
— human coronavirus NL63 (HCoV-NL63), and
[1]
— human coronavirus HKU1 (HCoV-HKU1) .

In 2019, a cluster of patients presenting with a respiratory disease were shown, by sequencing, to be

[2]

infected with a novel coronavirus . The coronavirus associated with this cluster was subsequently

named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee

[3]

on Taxonomy of Viruses . SARS-CoV-2 is the seventh coronavirus known to infect humans. The disease

caused by SARS-CoV-2 was designated as coronavirus infectious disease 2019 (COVID-19) by the World

[4]
Health Organization (WHO) .

The host range for SARS-CoV-2 is not yet fully defined. SARS-CoV-2 is a beta-coronavirus. The receptor

for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2). ACE2 is a cell-surface, zinc-binding

carboxypeptidase involved in regulation of cardiac function and blood pressure. It is expressed in

epithelial cells of the lung and the small intestine, which are the primary targets of SARS-CoV-2, as well

as the heart, kidney, and other tissues.

SARS-CoV-2 replicates in the upper and lower respiratory tracts and is transmitted by droplets and

aerosols and most likely other contact with asymptomatic and symptomatic infected persons. The basic

reproduction number (R ) of the original variant is between 2 and 3, but significantly more contagious

[5]

variants have developed. The median incubation period is 5,7 (range 2 to 14) days . Similarly to SARS

and MERS, superspreading events have been reported, with a dispersion parameter (kappa) estimated

at 0,1. Most infections are uncomplicated, and 5 % to 10 % of patients are hospitalized mainly due to

pneumonia with severe inflammation. However, complications include respiratory and multiorgan

failures. Risk factors for the complicated disease increase with age and include hypertension, diabetes,

chronic cardiovascular and chronic pulmonary diseases, and immunodeficiency.

Clinical management of COVID-19 and control of infections and spread of SARS-CoV-2 require effective

and efficient in vitro diagnostics. There are a number of tests and kits in use for the detection of SARS-

CoV-2 and the number of methods will continue to increase. Acceptable design, development and

establishment of quality SARS-CoV-2 diagnostics based on nucleic acid detection methods is critical

to ensure COVID-19 infection control. Establishing indices for conducting comprehensive quality

evaluation of these methods and kits both during development and in routine application will ensure

the accuracy of the test results and support epidemic prevention and control. This document provides

requirements and recommendations to consider for the quality practice of SARS-CoV-2 nucleic acid

amplification methods.
© ISO 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
TECHNICAL SPECIFICATION ISO/TS 5798:2022(E)
In vitro diagnostic test systems — Requirements and
recommendations for detection of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid
amplification methods
1 Scope

This document provides requirements and recommendations for the design, development, verification,

validation and implementation of analytical tests for detecting the severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2) using nucleic acid amplification. It addresses pre-examination, examination

and post-examination process steps for human specimens.

This document is applicable to medical laboratories. It is also intended to be used by in vitro diagnostic

developers and manufacturers, as well as by institutions and organizations supporting SARS-CoV-2

research and diagnostics.
This document does not apply to environmental samples.
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
severe acute respiratory syndrome coronavirus 2
SARS-CoV-2
virus that causes coronavirus infectious disease 2019 (COVID-19)
3.2
specimen
primary sample

discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or

more quantities or properties assumed to apply for the whole

Note 1 to entry: The Global Harmonisation Task Force (GHTF) uses the term specimen in its harmonized guidance

documents to mean a sample of biological origin intended for examination by a medical laboratory.

Note 2 to entry: In some countries, the term “specimen” is used instead of “primary sample” (or a subsample

of it), which is the sample prepared for sending to, or as received by, the laboratory and which is intended for

examination.
[6]

[SOURCE: ISO 15189:2012, 3.16 modified — Note 2 to entry was removed and Note 3 to entry was

renumbered as Note 2 to entry.]
© ISO 2022 – All rights reserved
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SIST-TS CEN ISO/TS 5798:2023
ISO/TS 5798:2022(E)
3.3
sample
one or more parts taken from a primary sample (3.2)
EXAMPLE A volume of serum taken from a larger volume of serum.
[6]
[SOURCE: ISO 15189:2012, 3.24 ]
3.4
reverse transcription

process of making complementary DNA [cDNA (3.6)] from an RNA (3.20) template (3.22), using the

enzymatic activity of a reverse transcriptase associated with one or more oligonucleotide primers

under a suitable set of conditions
[7]

[SOURCE: ISO 16577:2016, 3.180 , modified — Replaced “DNA” with “complementary DNA (cDNA)”.]

3.5
deoxyribonucleic acid
DNA

polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)

form
[8]
[SOURCE: ISO 22174:2005, 3.1.2 ]
3.6
complementary DNA
cDNA

single-stranded DNA (3.5), complementary to a given RNA (3.20) and synthesised in the presence of

reverse transcriptase to serve as a template (3.22) for DNA amplification
[9]
[SOURCE: ISO 20395:2019, 3.5 ]
3.7
analytical specificity

capability of a measuring system, using a specified measurement procedure, to provide measurement

results for one or more measurands which do not depend on each other nor on any other quantity in the

system undergoing measurement

Note 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1:2009, A.3.2).

[10]
[SOURCE: ISO 18113-1:2009, A.3.4 ]
3.8
limit of detection
LOD

measured quantity value, obtained by a given measurement procedure, for which the probability of

falsely claiming the absence of a component in a material is 0,05, given a probability of 0,05 of falsely

claiming its presence
[11]

[SOURCE: ISO/IEC Guide 99:2007, 4.18 , modified — “β, given a probability α” was replaced by “0,05,

given a probability of 0,05” and Notes 1 to 3 to entry were deleted.]
3.9
verification
provision of objective evidence that a given item fulfils specified requirements
[11]

[SOURCE: ISO/IEC Guide 99:2007, 2.44 , modified — EXAMPLES 1 to 3 and Notes 1 to 6 to entry were

deleted.]
© ISO 2022 – Al
...

SLOVENSKI STANDARD
kSIST-TS FprCEN ISO/TS 5798:2022
01-oktober-2022
Diagnostični preskusni sistemi in vitro - Zahteve in priporočila za odkrivanje
koronavirusa (SARS-CoV-2) z metodami amplifikacije nukleinskih kislin (ISO/TS
5798:2022)

In vitro diagnostic test systems - Requirements and recommendations for detection of

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid
amplification methods (ISO/TS 5798:2022)

In-vitro-Diagnostika-Systeme - Anforderungen und Empfehlungen für Qualitätsverfahren

für den Nachweis des Coronavirus 2 des Schweren Akuten Respiratorischen Syndroms
(SARS-CoV-2) mittels Nukleinsäureamplifikation (ISO/TS 5798:2022)
Systèmes d’essai pour diagnostic in vitro - Exigences et recommandations pour la

détection du coronavirus 2 associé au syndrome respiratoire aigu sévère (SARS-CoV-2)

par des méthodes d’amplification des acides nucléiques (ISO/TS 5798:2022)
Ta slovenski standard je istoveten z: FprCEN ISO/TS 5798
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
kSIST-TS FprCEN ISO/TS 5798:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN ISO/TS 5798:2022
---------------------- Page: 2 ----------------------
kSIST-TS FprCEN ISO/TS 5798:2022
TECHNICAL ISO/TS
SPECIFICATION 5798
First edition
2022-04
In vitro diagnostic test systems —
Requirements and recommendations
for detection of severe acute
respiratory syndrome coronavirus 2
(SARS-CoV-2) by nucleic acid
amplification methods
Systèmes d’essai pour diagnostic in vitro — Exigences et
recommandations pour la détection du coronavirus 2 associé au
syndrome respiratoire aigu sévère (SARS-CoV-2) par des méthodes
d’amplification des acides nucléiques
Reference number
ISO/TS 5798:2022(E)
© ISO 2022
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN ISO/TS 5798:2022
ISO/TS 5798:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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 2022 – All rights reserved
---------------------- Page: 4 ----------------------
kSIST-TS FprCEN ISO/TS 5798:2022
ISO/TS 5798:2022(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Overview ....................................................................................................................................................................................................................... 7

4.1 SARS-CoV-2 ................................................................................................................................................................................................ 7

4.1.1 General ........................................................................................................................................................................................ 7

4.1.2 Pre-examination ................................................................................................................................................................. 9

4.1.3 Examination — Overview .......................................................................................................................................... 9

4.1.4 Post-examination ............................................................................................................................................................ 11

4.2 Nucleic acid amplification methods .................................................................................................................................. 11

4.2.1 Reverse transcription qPCR (RT-qPCR) ...................................................................................................... 11

4.2.2 Reverse transcription digital PCR (RT-dPCR) .......................................................................................12

4.2.3 Isothermal amplification methods ..................................................................................................................12

5 Laboratory requirements .......................................................................................................................................................................12

5.1 General ........................................................................................................................................................................................................12

5.2 Biosafety requirements ............................................................................................................................................................... 13

5.2.1 Laboratory area ............................................................................................................................................................... 13

5.2.2 Risk control .......................................................................................................................................................................... 13

5.2.3 Personal protective equipment (PPE) .......................................................................................................... 13

5.3 General laboratory set-up ..........................................................................................................................................................13

5.4 Instrumentation ................................................................................................................................................................................. 14

5.5 Laboratory personnel .................................................................................................................................................................... 14

6 Design and development ..........................................................................................................................................................................14

6.1 Customer, patient and stakeholder needs ................................................................................................................... 14

6.2 Intended use of analytical test .............................................................................................................................................. 14

6.3 Institutional guideline strategy ........................................................................................................................................... 15

6.3.1 Laboratory developed tests (LDTs) versus in vitro diagnostic medical

devices (IVD medical devices) ............................................................................................................................ 15

6.3.2 Emergency use authorization .............................................................................................................................. 15

6.4 Clinical strategy ................................................................................................................................................................................. 15

6.5 Design and development planning ..................................................................................................................................... 16

6.5.1 Pre-examination of respiratory specimens for SARS-CoV-2 testing................................. 16

6.5.2 Examination design specifications (analytical test specifications) ..................................22

6.5.3 Design risk management .......................................................................................................................................... 27

6.6 Optimization of reagents and methods .........................................................................................................................28

6.6.1 Selection of SARS-CoV-2 target sequences ...............................................................................................28

6.6.2 Potential impact of variants of concern (VOCs) on the quality of NAAT

diagnostic methods for detecting SARS-CoV-2.....................................................................................28

6.6.3 Selection of amplification methods ................................................................................................................28

6.6.4 Design and selection of primers ........................................................................................................................28

6.6.5 Optimization of the reaction system .............................................................................................................29

6.6.6 Determination of cut-off values .........................................................................................................................29

6.6.7 Verification and validation of test design .................................................................................................29

7 Verification for patient care .................................................................................................................................................................31

7.1 General ...................................................................................................................................................................................................... 31

7.2 Confirmation of analytical performance characteristics .............................................................................. 31

7.2.1 Accuracy ................................................................................................................................................................................. 31

7.2.2 Limit of detection (LOD) ........................................................................................................................................... 31

7.2.3 Inclusivity .............................................................................................................................................................................. 32

7.2.4 Specificity .............................................................................................................................................................................. 32

iii
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ISO/TS 5798:2022(E)

7.2.5 Robustness ................................... ........................................................................................................ ................................. 32

7.3 Clinical evidence ................................................................................................................................................................................ 33

8 Validation for patient care ........................................................................................................................................... ...........................33

8.1 General consideration ................................................................................................................................................................... 33

8.2 Clarification of the intended use ......................................................................................................................................... 33

8.3 Performance with clinical specimens or samples ................................................................................................34

9 Design transfer to production ............................................................................................................................................................34

10 Implementation and use in the laboratory and reporting of results ........................................................34

10.1 Implementation and use in the laboratory .................................................................................................................34

10.2 Reporting and interpretation of results ....................................................................................................................... 35

11 Quality assurance ............................................................................................................................................................................................36

11.1 Performance monitoring ............................................................................................................................................................ 36

11.2 Design change including optimization of analytical test ...............................................................................36

11.3 Interlaboratory comparison .................................................................................................................................................... 37

Annex A (informative) Nucleic acid amplification techniques ..............................................................................................38

Bibliography .............................................................................................................................................................................................................................41

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kSIST-TS FprCEN ISO/TS 5798:2022
ISO/TS 5798:2022(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 Technical Committee ISO/TC 276, Biotechnology.

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/TS 5798:2022(E)
Introduction

Coronaviruses are enveloped RNA viruses that are broadly distributed in the animal kingdom. They

have been identified in humans, other mammals, and birds. Coronaviruses were named because the

spike proteins known to facilitate viral attachment and cell entry appear like a halo on the virus surface

when viewed under an electron microscope. Coronaviruses are roughly spherical with a diameter

ranging from 118 nm to 136 nm. The coronavirus genome, which ranges from 26 kb to 32 kb, is the

largest among all RNA viruses, including RNA viruses that have segmented genomes. Until 2019, six

coronaviruses have been associated with human diseases:
— severe acute respiratory syndrome-related coronavirus (SARS-CoV),
— Middle East respiratory syndrome coronavirus (MERS-CoV),
— human coronavirus 229E (HCoV-229E),
— human coronavirus OC43 (HCoV-OC43),
— human coronavirus NL63 (HCoV-NL63), and
[1]
— human coronavirus HKU1 (HCoV-HKU1) .

In 2019, a cluster of patients presenting with a respiratory disease were shown, by sequencing, to be

[2]

infected with a novel coronavirus . The coronavirus associated with this cluster was subsequently

named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee

[3]

on Taxonomy of Viruses . SARS-CoV-2 is the seventh coronavirus known to infect humans. The disease

caused by SARS-CoV-2 was designated as coronavirus infectious disease 2019 (COVID-19) by the World

[4]
Health Organization (WHO) .

The host range for SARS-CoV-2 is not yet fully defined. SARS-CoV-2 is a beta-coronavirus. The receptor

for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2). ACE2 is a cell-surface, zinc-binding

carboxypeptidase involved in regulation of cardiac function and blood pressure. It is expressed in

epithelial cells of the lung and the small intestine, which are the primary targets of SARS-CoV-2, as well

as the heart, kidney, and other tissues.

SARS-CoV-2 replicates in the upper and lower respiratory tracts and is transmitted by droplets and

aerosols and most likely other contact with asymptomatic and symptomatic infected persons. The basic

reproduction number (R ) of the original variant is between 2 and 3, but significantly more contagious

[5]

variants have developed. The median incubation period is 5,7 (range 2 to 14) days . Similarly to SARS

and MERS, superspreading events have been reported, with a dispersion parameter (kappa) estimated

at 0,1. Most infections are uncomplicated, and 5 % to 10 % of patients are hospitalized mainly due to

pneumonia with severe inflammation. However, complications include respiratory and multiorgan

failures. Risk factors for the complicated disease increase with age and include hypertension, diabetes,

chronic cardiovascular and chronic pulmonary diseases, and immunodeficiency.

Clinical management of COVID-19 and control of infections and spread of SARS-CoV-2 require effective

and efficient in vitro diagnostics. There are a number of tests and kits in use for the detection of SARS-

CoV-2 and the number of methods will continue to increase. Acceptable design, development and

establishment of quality SARS-CoV-2 diagnostics based on nucleic acid detection methods is critical

to ensure COVID-19 infection control. Establishing indices for conducting comprehensive quality

evaluation of these methods and kits both during development and in routine application will ensure

the accuracy of the test results and support epidemic prevention and control. This document provides

requirements and recommendations to consider for the quality practice of SARS-CoV-2 nucleic acid

amplification methods.
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kSIST-TS FprCEN ISO/TS 5798:2022
TECHNICAL SPECIFICATION ISO/TS 5798:2022(E)
In vitro diagnostic test systems — Requirements and
recommendations for detection of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) by nucleic acid
amplification methods
1 Scope

This document provides requirements and recommendations for the design, development, verification,

validation and implementation of analytical tests for detecting the severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2) using nucleic acid amplification. It addresses pre-examination, examination

and post-examination process steps for human specimens.

This document is applicable to medical laboratories. It is also intended to be used by in vitro diagnostic

developers and manufacturers, as well as by institutions and organizations supporting SARS-CoV-2

research and diagnostics.
This document does not apply to environmental samples.
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
severe acute respiratory syndrome coronavirus 2
SARS-CoV-2
virus that causes coronavirus infectious disease 2019 (COVID-19)
3.2
specimen
primary sample

discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or

more quantities or properties assumed to apply for the whole

Note 1 to entry: The Global Harmonisation Task Force (GHTF) uses the term specimen in its harmonized guidance

documents to mean a sample of biological origin intended for examination by a medical laboratory.

Note 2 to entry: In some countries, the term “specimen” is used instead of “primary sample” (or a subsample

of it), which is the sample prepared for sending to, or as received by, the laboratory and which is intended for

examination.
[6]

[SOURCE: ISO 15189:2012, 3.16 modified — Note 2 to entry was removed and Note 3 to entry was

renumbered as Note 2 to entry.]
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kSIST-TS FprCEN ISO/TS 5798:2022
ISO/TS 5798:2022(E)
3.3
sample
one or more parts taken from a primary sample (3.2)
EXAMPLE A volume of serum taken from a larger volume of serum.
[6]
[SOURCE: ISO 15189:2012, 3.24 ]
3.4
reverse transcription

process of making complementary DNA [cDNA (3.6)] from an RNA (3.20) template (3.22), using the

enzymatic activity of a reverse transcriptase associated with one or more oligonucleotide primers

under a suitable set of conditions
[7]

[SOURCE: ISO 16577:2016, 3.180 , modified — Replaced “DNA” with “complementary DNA (cDNA)”.]

3.5
deoxyribonucleic acid
DNA

polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)

form
[8]
[SOURCE: ISO 22174:2005, 3.1.2 ]
3.6
complementary DNA
cDNA

single-stranded DNA (3.5), complementary to a given RNA (3.20) and synthesised in the presence of

reverse transcriptase to serve as a template (3.22) for DNA amplification
[9]
[SOURCE: ISO 20395:2019, 3.5 ]
3.7
analytical specificity

capability of a measuring system, using a specified measurement procedure, to provide measurement

results for one or more measurands which do not depend on each other nor on any other quantity in the

system undergoing measurement

Note 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1:2009, A.3.2).

[10]
[SOURCE: ISO 18113-1:2009, A.3.4 ]
3.8
limit of detection
LOD

measured quantity value, obtained by a given measurement procedure, for which the probability of

falsely claiming the absence of a component in a material is 0,05, given a probability of 0,05 of falsely

claiming its presence
[11]

[SOURCE: ISO/IEC Guide 99:2007, 4.18 , modified — “β, given a probability α” was replaced by “0,05,

given a probability of 0,05” and Notes 1 to 3 to entry were deleted.]
3.9
verification
provision of objective evidence that a given item fulfils specified requirements
[11]

[SOURCE: ISO/IEC Guide 99:2007, 2.44 , modified — EXAMPLES 1 to 3 and Notes 1 to 6 to entry were

deleted.]
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ISO/TS 5798:2022(E)
3.10
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use or application have been fulfilled

Note 1 to entry: The word “validated” is used to designate the corresponding status.

[12]

[SOURCE: ISO 9000:2015, 3.8.13 , modified — Notes 1 and 3 to entry were deleted and Note 2 to

entry was renamed Note 1 to entry.]
3.11
amplicon

specific DNA (3.5) fragment produced by a DNA-amplification technology, such as the polymerase chain

reaction (PCR) (3.12)
[13]
[SOURCE: ISO 13495:2013, 3.3.1 ]
3.12
polymerase chain reaction
PCR

enzymatic procedure which allows in vitro amplification of DNA (3.5) or RNA (3.20)

[8]

[SOURCE: ISO 22174:2005, 3.4.1 , modified — “or RNA” added to the end of the definition and “in vitro”

has been unitalicized in accordance with the ISO House Style.]
3.13
reference material

material, sufficiently homogeneous and stable with reference to specified properties, which has been

established to be fit for its intended use in measurement or in examination of nominal properties

[11]

[SOURCE: ISO/IEC Guide 99:2007, 5.13 , modified — Notes 1 to 8 to entry and EXAMPLES 1 to 5 were

deleted.]
3.14
pseudo-virus

virus or virus-like particle that can integrate the envelope glycoprotein of another virus to form a virus

with an exogenous viral envelope, and the genome retains the characteristics of the retrovirus itself

3.15
digital PCR
dPCR

procedure in which nucleic acid templates (3.22) are distributed across multiple partitions of nominally

equivalent volume, such that some partitions contain template and others do not, followed by PCR

(3.12) amplification of target sequences and detection of specific PCR products, providing a count of the

number of partitions with a positive and negative signal for the target template

Note 1 to entry: Nucleic acid target sequences are assumed to be randomly and independently distributed across

the partitions during the partitioning process.

Note 2 to entry: The count of positive and negative partitions is normally based on end point detection of PCR

products following thermal cycling, however real-time qPCR (3.16) monitoring of PCR product accumulation is

additionally possible for some dPCR platforms.
[9]
[SOURCE: ISO 20395:2019, 3.10 ]
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ISO/TS 5798:2022(E)
3.16
quantitative real-time PCR
qPCR

enzymatic procedure which combines the in vitro amplification of specific DNA (3.5) or RNA (3.20)

segments with the detection and quantification of specific PCR (3.12) products during the amplification

process

Note 1 to entry: While the PCR is producing copies of the relevant DNA sequence, the fluorescent marker

fluoresces in direct proportion to the amount of DNA present, which can theoretically be back-calculated to infer

the original amount of that particular DNA present in a sample (3.3) prior to initiation of PCR.

[9]
[SOURCE: ISO 20395:2019, 3.25 , modified — “RNA” was added.]
3.17
quantification cycle

quantitative real-time PCR (qPCR) (3.16) cycle at which the fluorescence from the reaction crosses a

specified threshold level at which the signal can be distinguished from background levels

Note 1 to entry: Quantification cycle is a generic term which includes cycle threshold (C ), crossing point (C ),

t p

take off point and all other instrument specific terms referring to the fractional cycle which is proportional to

the concentration of target in the qPCR assay.

Note 2 to entry: The quantification cycle is based either on a threshold applied to all samples (3.3) or on a

regression analysis of the signal, for each sample.

Note 3 to entry: The quantification cycle is a measure with poor reproducibility and cannot be used when

comparing kit performance.

Note 4 to entry: Laboratory based considerations sometimes lead to selection of a cut-off for the cycle number.

The cut-off cannot be chosen not to have a detrimental influence on available limit of detection (3.8).

Note 5 to entry: C does not apply for digital PCR (3.15) and isotherma
...

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