A roadmap to better COVID-19 testing from the Coronavirus Standards Working Group

Abstract

Testing has been central to our response to the COVID-19 pandemic. However, the accuracy of testing relies on standards, including reference materials, proficiency testing schemes, and information and reporting guidelines. The use of standards is a simple, inexpensive, and effective method to ensure reliable test results that inform clinical and public health decisions. Here we describe the central role of standards during the COVID-19 pandemic, where they have enabled population-scale screening, genomic surveillance and measures of immune protection measures. Given these benefits, the Coronavirus Standards Working Group (CSWG) was formed to coordinate standards in SARS-CoV-2 testing. This network of scientists has developed best-practices, reference materials, and conducted proficiency studies to harmonize laboratory performance. We propose that this coordinated development of standards should be prioritized as a key early step in the public health response to future pandemics that is necessary for reliable, large-scale testing for infectious disease.

Full text

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A roadmap to better COVID-19 testing from the
Coronavirus Standards Working Group
Tim Mercer
The University of Queensland
Neil Almond
National Institute for Biological Standards and Control
Patrick Chain
Los Alamos National Laboratory https://orcid.org/0000-0003-3949-3634
Michael Crone
Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London
https://orcid.org/0000-0002-5675-0172
Alina Deshpande
Los Alamos National Laboratory
Deepa Eveleigh
Asuragen, Inc.
Paul Freemont
Imperial College London https://orcid.org/0000-0002-5658-8486
Sebastien Fuchs
Western Univeristy of Health Sciences, College of Osteopathic Medicine of the Pacic
Russell Garlick
LGC Clinical Diagnostics
Jim Huggett
National Measurement Laboratory, LGC
Martin Kammel
INSTAND https://orcid.org/0000-0002-2011-086X
Po-E Li
Los Alamos National Laboratory
Mojca Milavec
National Institute of Biology https://orcid.org/0000-0002-5794-2109
Elizabeth Marlowe
Quest Diagnostic Infectious Disease
Denise O’Sullivan
National Measurement Laboratory, LGC
Mark Page
National Institute for Biological Standards and Control

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Gary Pestano
Biodesix, Inc.
Sara Suliman
Zuckerberg San Francisco General Hospital, Division of Experimental Medicine, University of California
San Francisco https://orcid.org/0000-0002-5154-576X
Birgitte Simen
Ginkgo Bioworks, Inc.
John Sninsky
SPARK Stanford University
Lynne Sopchak
SPARK Stanford University
Cristina Tato
CZ Biohub https://orcid.org/0000-0002-0614-756X
Jo Vandesompele
Biogazelle
Peter Vallone
National Institute of Standards and Technology https://orcid.org/0000-0002-8019-6204
Thomas White
Human Rights Center, School of Law, University of California, Berkeley
Heinz Zeichhardt
INSTAND e.V., Duesseldorf, and IQVD GmbH Institut fuer Qualitaetssicherung in der Virusdiagnostik
Marc Salit (  msalit@stanford.edu )
Stanford University
no mistake
none
Article
Keywords: COVID-19, SARS-CoV-2 testing, standards
Posted Date: November 18th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-1066221/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Testing has been central to our response to the COVID-19 pandemic. However, the accuracy of testing
relies on standards, including reference materials, prociency testing schemes, and information and
reporting guidelines. The use of standards is a simple, inexpensive, and effective method to ensure
reliable test results that inform clinical and public health decisions. Here we describe the central role of
standards during the COVID-19 pandemic, where they have enabled population-scale screening, genomic
surveillance and measures of immune protection measures. Given these benets, the Coronavirus
Standards Working Group (CSWG) was formed to coordinate standards in SARS-CoV-2 testing. This
network of scientists has developed best-practices, reference materials, and conducted prociency
studies to harmonize laboratory performance. We propose that this coordinated development of
standards should be prioritized as a key early step in the public health response to future pandemics that
is necessary for reliable, large-scale testing for infectious disease.
Introduction
The scale and diversity of testing performed in response to the COVID-19 pandemic is unprecedented.
Testing has been needed to identify infectious individuals, diagnose patients, and measure the immune
protection elicited by vaccines1.The pandemic has prompted innovations in molecular, antigen, and
serology testing, and the global deployment of genomic surveillance.Given these key roles,SARS-CoV-2
testinghas been established at unprecedented urgency and scale, and will likelyremain established in
future clinical diagnostics and public health testing.
Standards are required to ensure that testing is accurate and reliable. Standards include well-
characterized reference materials that ensure a test is calibrated and t-for-purpose, prociency testing
schemes that evaluate laboratory performance, and information standards for clear communication of
test performance and results. Together, these standards underpin reliable and robust testing, however,
despite their importance the development and implementation of SARS-CoV-2 standards has received
little attention during the pandemic2.
This importance of standards was recognized early during the pandemic by an
ad hoc
consortium of
concerned scientists that formed the
Coronavirus Standards Working Group
(CSWG)2. This diverse group
of scientists fullled differing roles during the pandemic, and represented a range of commercial,
government, academic and other organizations. Together, this group has advocated for the importance of
standards in SARS-CoV-2 testing, and led the development of reference materials, conducted prociency
studies, and critical consideration of testing methods.
This article describes the collective expertise, experiences and recommendations of the CSWG during the
COVID-19 pandemic. The CSWG proposes that the development and dissemination of standards is a
cost-effective strategy that can broadly improve SARS-CoV-2 testing worldwide, and is needed to address
the evolving needs of testing and preparation for future public-health emergencies. The development and

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implementation of standards should be prioritized within the pandemic response, and recognized as the
necessary foundation for the development of a robust and reliable testing enterprise.
Main Text
The SARS CoV-2 testing process
Testing measures the presence of an analyte in a sample, such as the presence of the SARS-CoV-2 RNA
genome within a respiratory sample. However, like any measurement, there is uncertainty in this process,
and standards are needed to estimate and manage this uncertainty. Testing can also involve a
quantitative measurement to determine the abundance of an analyte in a sample. However, viral load is
rarely considered during SARS-CoV-2 testing, and quantitative measurements may be reduced to a simple
presence or absence result when viral abundance exceeds a threshold3.
The testing process can be usefully divided into three steps; the pre-analytical phase which includes
preparing the sample specimen, the analytical phase which includes the actual testing for the analyte in
the sample, and the post-analytical phase, which includes interpreting the data for an actionable result
(see Figure 1). Testing performance, including the sensitivity (see Glossary) and specicity for detecting
the analyte, is inuenced by the performance of all these steps. While many manufacturer studies may
focus on test performance during the analytical step, in practice, variables in the pre-analytical phase,
such as sample source and amount, collection, transport and storage often markedly impact test
performance and can be the least-controllable steps in the testing process4–6.
Reference materials for SARS CoV-2 testing
Standards include well-characterized reference materials with known properties that can be used to
evaluate whether tests are t-for-purpose (see Figure 1). Primary reference standards are issued by a
recognized authority, and are assigned qualitative and quantitative properties without reference to other
standards. For example, the WHO Expert Committee on Biological Standardization established primary
International Standards for SARS-CoV-2 testing in December 2020, and these standards dene the
International Units7,8. These primary standards are then used to calibrate secondary reference materials
which are more routinely used during test and laboratory validation. 
Natural reference materials are derived from natural sources, such as clinical patient samples, that have
been well-characterized using differing methods. Whilst natural materials match the complexity and
challenges of a patient sample, they are often nite in quantity, and dicult to reliably manufacture at
scale9. Natural reference materials should ideally match nal use cases and target populations, given
that viral and antibody titers can vary widely between individuals and across the course of infection,
resulting in natural reference materials with varying properties10,11. SARS-CoV-2 tests that were originally

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validated using hospitalized patient samples with high viral titers performed markedly worse when used
to screen mildly or asymptomatic individuals for which there are few available reference materials12.
During initial stages of the pandemic, laboratories faced a major challenge in sourcing reference
materials to evaluate testing, with patient samples also needed for competing research and therapeutic
needs. While some large laboratories could leverage established clinical collaborations to source patient
reference materials, the majority of laboratories faced diculties sourcing patient reference materials
needed to verify tests13. The coordinated and equitable dissemination of reference patient materials
would have accelerated the deployment of tests, and also provide an early opportunity to harmonize test
performance amongst laboratories.
The rapid development of synthetic reference materials can provide an interim solution in the absence of
reference patient materials. Synthetic reference materials, including synthetic SARS-CoV-2 DNA and RNA
genomes and recombinant proteins, were rapidly developed following the publication of the SARS-CoV-2
reference genome, and enabled the analytical validation of tests in countries even before the rst reported
cases of SARS CoV-2 virus had emerged14–16. However, synthetic materials may be poor surrogates for
assessing pre-analytical variables, and may need to be further spiked into buffer or negative respiratory
specimens to contrive a specimen-like matrix that can recapitulate RNA extraction steps. While regulatory
agencies, such as the Food and Drug Administration (FDA) and European Medicines Agency (EMA)
permitted the use of synthetic reference material to validate tests at early stages of the pandemic, they
now require natural reference materials for verication and approval17,18.
Molecular testing for SARS-CoV-2 genes.
Molecular testing involves the detection of the SARS-CoV-2 RNA genome using methods such as reverse
transcription quantitative (RT-qPCR) and digital PCR , loop-mediatedisothermalamplication, next-
generation sequencing, and other nucleic amplication methods19. Molecular tests use respiratory
samples that are particularly susceptible to pre-analytical variables, including specimen type, collection
media and transport conditions20,21.
Numerous synthetic genomes and natural reference materials have been developed toverify molecular
testing protocols. However, given the sensitivity of molecular tests, laboratories must take care to
implement best practice guidance and negative controls, with false-positive test results due to
contamination by synthetic SARS-CoV-2 reagents or previous tests leading to false-positive results22–25.
At early stages of the pandemic the US Centre for Disease Control (CDC) distributed RT-qPCR tests that
included faulty negative controls, ultimately requiring new test kits to be developed and issued that
resulted in delays during critical early stages of the pandemic21.
Whilst molecular tests have the potential to measure viral abundance, they must be rst calibrated to
reference materials to realize this quantitative potential. For example, although the RT-qPCR cycling

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threshold (Ct, also known as Cq or Cp) score can be used to estimate viral abundance, this abundance
can vary up ~1000-fold for a given Ct value between instruments and laboratories3. Accordingly,
calibration to reference materials is needed for quantitative comparisons of RT-qPCR results7, and viral
load cannot be considered in clinical stratication and the assessment of analytical sensitivity due to the
current lack of harmonized reporting of RT-qPCR results26,27.
Reference materials must also be regularly updated toreect the diversity of SARS-CoV-2 variants
circulating within a population. For example, new genetic variants can interfere with RT-qPCR primer or
probe binding and result in false-negative testing results28,29. Whilst multiplex testing for several gene
targets can mitigate the impact of a single variant, probes and primers require ongoing verication to
ensure continued validity of a molecular test, and the FDA routinely monitors the predicted impact of
variants on the performance of EUA approved tests30.
Molecular testing can be suciently sensitive to detect the presence of SARS-CoV-2 RNA in wastewater.
This can enable surveillance for SARS-CoV-2 in sewage collected across a large catchment area, and
provide leading indication of an outbreak31. However, the sensitive detection and interpretation of
wastewater testing is challenging, and there is a pressing need to standardize the sampling, methods and
analysis used to detect and quantify SARS-CoV-2 in sewage. These standards are needed to ensure
comparability and consistency between different municipalities and across time, and support
surveillance measures that inform a public health response.
Antigen testing for SARS-CoV-2 proteins
Antigen tests employ lateral-ow or enzyme-linked immunosorbent assays to directly detect the presence
of viral proteins. These antigen tests are typically less sensitive than molecular tests, and detect SARS
CoV-2 across a narrower window during the viral infectious course (although repeated serial testing may
mitigate this lower sensitivity)32,33. However, antigen tests are inexpensive to manufacture, can be
performed at point-of-care and quicklyreturn results. Given this convenience, antigen tests can be sold
direct to consumers, and are often used for rapid screening of individuals during travel, or for attendance
to schools, workplaces or community events.
Many laboratories reported that the performance of antigen tests differed markedly from the
manufacturers declarations, and independent validation with reference standards was needed to conrm
test performance34. Antigen tests can be evaluated using inactivated viruses or recombinant expressed
proteins, however, their performance is more typically measured by comparison to results from previously
authorized RT-qPCR tests18. However, relying on evaluation by positive agreement to a comparator test
can be problematic, as it can propagate inaccuracies, differences or limitations present in the benchmark
RT-qPCR method35. 

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Antigen tests have been promoted as a viable method to realize massive population-scale testing;
however, this proposal remains controversial36. An antigen test widely used in a pilot program to evaluate
whether population-scale testing could curb rates of infection in Liverpool, UK was criticized for poor
sensitivity, and found to miss almost half of individuals who otherwise tested positive using RT-qPCR37–
39. This eld performance of this antigen test was markedly lower than the manufacturer’s declaration,
and demonstrated the need to independently verify test performance with appropriate reference materials
to understand limitations.
Serology testing for a COVID-19 immune response
Serology testing measures the presence of antibodies in an individual’s blood that are reactive to SARS-
CoV-2 proteins. A range of serology tests have been designed using different methods (including lateral
ow, enzyme-linked, and chemiluminescent immunoassays) that detect antibody isotypes (IgM, IgA, IgG
and total) elicited by previous infection or vaccination40,41. Serology tests can measure the avidity,
duration and composition of reactive antibody responseto SARS-CoV-2 infection.However, serology
assays must be standardized and calibrated to enable quantitative comparisons of antibody
measurements between individuals, across time and in response to differing variants42.
Reference materials for COVID-19 serology tests are largely derived from convalescent patient serum. The
WHO International Standard, prepared and supplied by National Institute for Biological Standards and
Control (NIBSC), comprises a pool of convalescentplasma from recovered COVID-19 patients, with
plasma from healthy donors collected before the pandemic to serve as a negative control8.The WHO
assigned anarbitrary unitageto the reference standards to establish international units forneutralizing
antibodies (e.g. IU/mL) and binding assays (e.g. BAU/mL). Calibration to these international units can
standardize quantitative serology measurements used in clinical trials, can dene consensus antibody
titer thresholds, and can establish comparable correlates of protection against COVID-1943,44.
Standardization for serological testing also underpins reproducible research in epidemiology, and the
development of vaccines and therapeutics. Large-scale studies have used serology testing to understand
the transmission of SARS-CoV-2 though populations, as well as the impact of vaccination on this
transmission45–47. However, without standardizing these serology tests, comparison among datasets and
populations can be dicult, resulting in a lost opportunity for interoperable research. Indeed,
standardization of key research methods, such as cell-based assays used to measure neutralizing
antibodies, would is a next step for reproducible research results48.
Prociency testing schemes are used to harmonize testing
across laboratories.

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Prociency studies (or external quality assessment studies) share samples amongst participating
laboratories for testing, who then report their results back for evaluation and comparison. Prociency
testing can evaluate the performance of individual laboratories, and provide collective appraisal of
diverse SARS CoV-2 testing across different laboratories.
Given the scale and diversity of COVID-19 testing, prociency testing schemes are needed to harmonize
results between laboratories with different capabilities, particularly given many laboratories were
established or re-purposed for COVID-19 testing with little previous clinical experience. Systematic
prociency testing schemes also provide an opportunity to calibrate international standards and units
amongst participating laboratories, and provide the technical basis forguidance documents for
harmonizing results49.
Prociency testing schemes can also evaluate the ongoing performance of tests following their initial
validation for regulatory approval. Numerous prociency studies were launched at early stages of the
pandemic for both genome detection and serology testing50–52.These studies proved particularly
important given that many SARS CoV-2 tests were given accelerated regulatory approval (such as
emergency use authorization, EUAs) with little demonstrated performance under real-world
settings.These studies proved key in providing an independent validation of testing methods, with results
shared widely among laboratories and organizations using the results from those labs.
Information standards are needed to describe materials,
procedures, results and performance.
Information standards are rules or guidelines that dene how test performances, processes and results
should be described. Information standards ensure these descriptions use consistent, transparent and
harmonized terminology that enables the necessary information to be clearly communicated and
interpreted by labs, clinical, and public health authorities53. With information standards, a practitioner can
reliably select the appropriate test according to their requirements.
Information standards provide a consistent basis to compare amongst SARS-CoV-2 tests, and promote
communication, consistency and transparency in results shared between laboratories54. A key standard
for describing and reporting SARS-CoV-2 RT-qPCR assays is the Minimum Information for Publication of
Quantitative Real-Time PCR Experiments (MIQE) guideline, which provide a checklist for the disclosure of
all reagents, sequences and methods necessary for other laboratories to reproduce RT-qPCR methods and
results55,56.
Information standards can ensure that results and data-sets are harmonized, and accompanied by meta-
data that ensure results can be easily searched, accessed and analyzed. Meta-data typically describes
useful accompanying information about samples (such as patient clinical status, location, gender and
age) to support downstream integrated data analysis. The CSWG encourages scientists, clinicians,

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journals, and peer reviewers to request appropriate standardized annotation and reported test results in
International Units where possible.
Information standards are needed to provide unambiguous descriptions of test performance that can be
independently vered53. Manufacturers of SARS-CoV-2 tests often used secondary reference materials
without traceability to a primary reference that can be dicult to independently verify. Many metrics, such
as sensitivity and specicity, are also not xed test properties, and need to be considered in the context of
the clinical samples or secondary reference material used57. As a result, comparisons of manufacturer’s
declarations of test performance often showed diverged markedly from independent real-world
evaluations58.
Genome surveillance of new SARS-CoV-2 variants
Novel SARS-CoV-2 variants impact the tness of the virus, allowing the virus to spread more easily, cause
more severe disease, or escape the body’s immune response59. Genomic surveillance has proven useful
in monitoring the emergence and circulation of variants of concern and informing public health response.
Many countries now sequence a targeted fraction of positive samples to measure SARS-CoV-2 variants
circulating within a population60. Sequencing is also used in genomic epidemiology where the presence
of shared mutations can infer chains and clusters of transmission between individuals. This can assist
contact tracing of infected individuals in an outbreak, and track the spread of SARS-CoV-2 strains across
the world61.
Genomic surveillance will likely become an established feature of global testing, with a requirement to
monitor novel, seasonal or resistant strains. Accordingly, there is a pressing need to develop reference
materials and bioinformatic standards to ensure quality and comparability of results between national
and international surveillance laboratories. This includes the collection of reference materials for different
variant strains in biorepositories, or the rapid synthesis of variant genomes under safe biosecurity
restrictions62.
Information standards are also needed to ensure that sequence data-sets, which can be large and
complex, are standardized to enable subsequent querying and analysis. More than one million SARS-2-
CoV genome sequences have been submitted to databases such as GISAID63. The assignment of
consistent meta-data to these genome sequences facilitates data integration, accessibility and the re-use
of data for insightful analysis in future studies64. The nomenclature used to describe different SARS-CoV-
2 strains has also been standardized to consolidate naming schemas and avoid the stigma of naming
variants according to origin65.
The analysis of genomic data, from raw sequence data into actionable information, is often complex and
lengthy66,67. The diverse range of sequencing technologies, bioinformatic tools and data formats used
within the analytical workow must be harmonized to ensure interoperability and best-practice across the

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surveillance network64. Stable versioning, data freezes and workow management tools can standardise
bioinformatic protocols, data outputs and reference les. Reference genomic data-sets can also be used
in bioinformatic prociency schemes to test the ability of genomic surveillance networks to detect novel
variants.
Coordinated efforts amongst organizations for SARS CoV-2
testing and standards.
The pandemic response has demanded close cooperation between commercial, academic, non-
governmental, and governmental organizations. The scale, urgency and uncertainty of the pandemic has
required organizations to assume new roles, share information and pool resources to build testing
capacity, and this coordination has been considered key to the success of pandemic responses13.
Government and regulatory organizations were able to leverage reference laboratories to develop
standards, evaluate tests, and disseminate information and best practices. TheWHO and its
collaborating reference laboratories at the Paul Ehrlich Institute (Germany), the FDA’s Centre for Biologics
Evaluation and Research (CBER) and the UK’s National Institute for Biological Standards and Control
(NIBSC), quickly initiated development of international standards7,8. However, while these international
standards constitute a valuable global resource, their dissemination and adoption needs ongoing support
from regional organizations to calibrate secondary standards to these international standards, and
promote widespread harmonization of testing68.
At early stages of the pandemic, many regulatory bodies allowed expedited validation of COVID-19 tests
due to concerns about access and scaling of testing, and prior to widespread availability of reference
standards18. While this accelerated the deployment of tests, it has also resulted in uneven testing
performance. The FDA developed a reference panel that wasavailable to test developers intending to
submit tests forEUA submissionto assess limit of detection69. Additionally, laboratories or organizations,
such as FIND70, were needed to independently evaluate test performance with interim reference
materials.
In addition to developing reference materials, global organizations also provided expertise and guidance
to countries that may lack their own established regulatory or standards organizations.Global
dissemination of reference materials is needed to support the implementation of testing in low- and
middle-income countrieswithgreater dependence on point-of-care tests performed under heterogenous
conditions. One innovation proposed dissemination of materials for decentralized production of “open
source” secondary standards that could be provided by plasmid repositories and distributed under open-
source terms to empower regional centers to manufacture their own secondary standardsto validate
local testing workows71. Global imbalances in vaccination and testing have contributed to a global
disparity in the impact of COVID-19; standards have a key role in mitigating these imbalances, and
ensuring that testing is performed effectively and eciently worldwide.

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Conclusions
The standardization of SARS-CoV-2 testing remains an ongoing priority, and is part of thenormalisation
ofepidemic prevention and control.Standards are needed for evolving testing methods and the
increasing use of serological testing, genomic and wastewater surveillance. Worldwide testing must also
be calibrated to international primary standards to harmonize performance and results across countries,
enabling the global research enterprise to meaningfully share results. All of the standards must be
maintained and updated in response to the emergence of novel SARS-CoV-2 variants. Whilst SARS-CoV-2
testing will likely remain a feature of public health, it is likely to become reduced in scope to seasonal and
targeted testing of outbreaks, vulnerable populations or for international travel.
An independent review of the processes by which the FDA authorizes tests in the pandemic has
recommended establishing a framework to validate test performance in preparation for and during a
public health emergency13. This framework includes strengthening the communication between
regulatory, government organizations and test developers, developing an independent capability to
evaluate test performance quickly, and the better development and deployment of reference standards,
such as clinical samples that were needed for test validation. During the pandemic, the CSWG contributed
to many to these capabilities, and we recommend that this expertise andexperiencebe institutionalized
as part of pandemic preparedness(see Box 1). An institutionalized capacity to respond to standards
needs willimprove our pandemic response and establish the foundation for a reliable testing
infrastructure for emerging diseases.
Many of these recommendations for standards are generalizable, and could similarly benet the testing
ofpathogens, such as inuenza, that are currently monitored for seasonal variants.These proposals can
also be extended to developing standards for monitoring and responding to viral outbreaks in agriculture
and livestock populations, which can further act as reservoirs for SARs-CoV-2 and other viruses that
undergo zoonotic transfer72.
The public are theultimate beneciaries of better standards. Standards ensure that patients will receive
consistent and reliable results that inform their treatment, regardless of how and where they are tested.
Standards permit informed evaluation of consistent measures of test performance, which may be
otherwise considered secondary to cost and convenience. During the pandemic, numerous governments
awarded contracts to the manufacturers of tests that were subsequently shown to perform poorly when
independently validated73. Reliable testing and surveillance are also needed to inform policy, potentially
avoiding the costs of alternative non-pharmaceutical interventions, and ultimately reducing healthcare
costs. Previous research has demonstrated that standards are a cost-effective solution to improve testing
and health outcomes that ultimately benet the broader economy74.
The pandemic has focused media, government and community attention on the importance of testing.
However, while extensive resources have been invested in new testing methods75, relatively fewer
resources have been invested in the development of standards, despite their proven effectiveness. There

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is an opportunity to ensure that the new widespread appreciation of testing for public health is
accompanied by a matched appreciation of standards. Accordingly, we propose similar consideration
and investment be afforded to standards, commensurate with their strategic, far-reaching and impactful
benets. Standards are a simple, proven method to assure test performance and a robust, reliable, and
effective testing enterprise at the massive scale and diversity we have witnessed during the COVID-19
pandemic.
Boxes
Box 1.
Emerging Infectious Disease Standards Working Group
We propose to establish an enduring working group, termed the
Emerging Infectious Disease Standards
Working Group
(EIDSWG) to advocate for testing standards in emerging infectious diseases and future
public health emergencies. The proposed Terms of Reference are:
Scope and objective.
The EIDSWG serves as an independent, international, standing network of members that coordinate
efforts and advocate for standards in testing undertaken in preparation or response to an infectious
disease public health emergency.
Membership.
The EIDSWG comprises general members that are self-selected and unrestricted. Membership
includes representatives from differing technical backgrounds and expertise, including research and
clinical scientists, government, public-health organizations, regulatory authorities, non-prot
organizations and companies developing tests or reference standards. The EIDWG strives to ensure
an open, inclusive diverse membership needed to respond to the global challenges of a public health
emergency.
The EIDSWG also comprises core members selected on technical expertise that represent an
established network of laboratories readied to develop, implement and disseminate standards in
preparation for and immediately in response to the emergence of a public health emergency. In
addition, the EIDWSG may establish sub-groups dedicated to specic objectives as required.
The EIDSW will work closely and partner with regional and international third-party partners involved
in the development, implementation or validation of tests or standards.
Roles.
Establish standing relationships between key stakeholders to coordinate the development and
implementation of standards in preparation for and immediately in response to the emergence of a
public health emergency.

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Develop and communicate policy and recommendations relating to standards and their use in test
validation.
Provide representatives to champion use of standards within the local, national, and international
testing organizations.
Support the development and use of standards (including reference materials, bioinformatic and
information standards) by members as well as third-party organizations. This includes conducting
harmonization studies to propagate international standards and units.
Provide training and education on the development, role and adoption of standards. This includes
the dissemination of standards best practices for the use of standards in test and laboratory
validation.
Prepare for and coordinate the equitable dissemination of standards in response to the emergence of
a public health emergency. This includes dissemination of clinical specimens that are needed for
optimal test validation.
Support the development and use of interim reference materials in the absence of an international
standard, and support the harmonization of interim references to international standard when
available.
Identify appropriate partners and funding to support the roles of the EIDSWG.
Meetings.
The EIDSWG will regularly convene its membership with annual meetings, seminars, conferences
and communications amongst members.
Box 2. GLOSSARY
Diagnostic performance – evaluates the ability of a test to discriminate between two binary outcomes. In
the case of testing for infectious disease, diagnostic performance is typically the ability of a test to
distinguish between the presence or absence of an analyte (such as viral genome, protein, or reactive
antibodies) within a sample. For quantitative tests, the binary discrimination between presence or
absence of a biomarker is based on the abundance of the biomarker exceeding a decision threshold (for
example, a positive RT-qPCR result corresponds to a Ct level exceeding a given threshold).
Sensitivity – the probability that the test result is positive when the analyte is present (also referred to as
the true positive rate). 
Specicity – the probability that the test result Is negative when the analyte is absent (also referred to as
the true negative rate). 
Analytical performance – evaluates the ability of a test to measure the analyte of interest. This
evaluation can be quantitative, and considers the difference between the result and the target reference

Page 14/22
value. Analytical performance includes considerations such as the accuracy, precision, limit of detection
of the test, as well as the reproducibility of test performance.
Reference materials – physical materials with well-characterized properties that can be used to validate
or calibrate a testing process.
Information standards – Guidelines and rules that dene how information (about process, performance,
or result) is dened and described.
Primary standards - Reference materials whose metrological qualities are assigned without reference to
other standards. 
Secondary standards – reference materials whose metrological qualities are assigned by comparison to
a primary standard. Secondary standards are more routinely used for within laboratory.
Natural materials – Reference samples that are derived from a natural source, such as a patient
respiratory or blood samples, whose meteorological properties have been established through extensive
characterization.
Synthetic materials – reference materials that have been manufactured to mimic patient sample
properties using an articial process. For example, the production of synthetic of RNA or DNA genomes,
of the preparation of contrived sample matrices.
Prociency (or external quality assurance) study – study conducted by external agency to evaluate the
performance of one or more laboratories.
Accreditation - Formal evaluation and recognition that a testing laboratory is suciently competent to
carry out specic tests.
TYPES OF REFERENCE MATERIALS
International reference standard - reference materials that are issued by an authorized body (such as
WHO, NIST, JRC, NMI Australia, or other national and international bodies), and whose whose
metrological qualities (possibly including the denitions of a quantitative unit) are assigned without
reference to other standards and thereby provides the highest level of traceability.
Certied Reference Material – reference material accompanied by a certicate that provides the value of
the specied property, its associated uncertainty, and a statement of metrological traceability, typically to
a higher order standard (such as an SI unit or international reference material). Certied reference
materials are produced and disseminated in accordance with ISO17034 and ISO17025 guidance.
Reference Material – a t-for-purpose material used to verify, validate, or calibrate a test procedure.
Reference materials are often produced and disseminated by an accredited reference material producer

Page 15/22
(according to ISO17034). Certicates of Analysis are often provided according to ISO guidelines as best
practice.
Declarations
DISCLAIMER.
Points of view in this document are those of the authors and do not necessarily represent the ocial
position or policies of listed aliated organizations. Specically, the manuscript does not o not
necessarily represent the ocial position or policies of National Institute of Standards and Technology or
the U.S. Department of Commerce. 
Certain commercial equipment, instruments, and materials are identied in order to specify experimental
procedures as completely as possible. In no case does such identication imply a recommendation or
endorsement by authors or aliated organizations, nor does it imply that any of the materials,
instruments, or equipment identied are necessarily the best available for the purpose.
References
1.    Mercer, T. R. & Salit, M. Testing at scale during the COVID-19 pandemic.
Nature Reviews Genetics
vol. 22 415–426 (2021).
2.    Page, M., Almond, N., Rose, N. J. & Schneider, C. K. Diagnostics and the coronavirus: don’t let the
standards slip.
Nature Biotechnology
vol. 38 673–674 (2020).
3.    Evans, D.
et al.
The dangers of using Cq to quantify nucleic acid in biological samples; a lesson
from COVID-19.
Clin. Chem.
(2021) doi:10.1093/CLINCHEM/HVAB219.
4.    Lippi, G., Simundic, A. M. & Plebani, M. Potential preanalytical and analytical vulnerabilities in the
laboratory diagnosis of coronavirus disease 2019 (COVID-19).
Clinical Chemistry and Laboratory
Medicine
vol. 58 1070–1076 (2020).
5.    Engelmann, I.
et al.
Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-
PCR Testing: Should Test Results Include These?
ACS Omega
6, 6528–6536 (2021).
6.    Göhring, K., Dietz, K., Hartleif, S., Jahn, G. & Hamprecht, K. Inuence of different extraction
methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) lter
cards.
J. Clin. Virol.
48, 278–281 (2010).
7.    WHO/BS.2020.2402 Collaborative Study for the Establishment of a WHO International Standard
for SARS-CoV-2 RNA. https://www.who.int/publications/m/item/WHO-BS-2020.2402.

Page 16/22
8.    Kristiansen, P. A.
et al.
WHO International Standard for anti-SARS-CoV-2 immunoglobulin.
The
Lancet
vol. 397 1347–1348 (2021).
9.    Bossuyt, P. M. Testing COVID-19 tests faces methodological challenges.
Journal of Clinical
Epidemiology
vol. 126 172–176 (2020).
10.   Woloshin, S., Patel, N. & Kesselheim, A. S. False Negative Tests for SARS-CoV-2 Infection —
Challenges and Implications.
N. Engl. J. Med.
383, e38 (2020).
11.   Iacobucci, G. Covid-19: Tests must be more rigorously regulated to protect public, say
statisticians.
BMJ
373, (2021).
12.   Takahashi, S., Greenhouse, B. & Rodríguez-Barraquer, I. Are Seroprevalence Estimates for Severe
Acute Respiratory Syndrome Coronavirus 2 Biased?
J. Infect. Dis.
222, 1772–1775 (2020).
13.   Emergency Use Authorization of COVID-19 Tests: Independent Assessment of the FDA’s Response
| FDA. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/emergency-use-
authorization-covid-19-tests-independent-assessment-fdas-response.
14.   Madala, B. S., Reis, A. L. M., Deveson, I. W., Rawlinson, W. & Mercer, T. R. Chimeric synthetic
reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular
tests.
Sci. Rep.
11, 2636 (2021).
15.   Corman, V. M.
et al.
Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
Eurosurveillance
25, 2000045 (2020).
16.   Molecular assays to diagnose COVID-19: Summary table of available protocols.
https://www.who.int/publications/m/item/molecular-assays-to-diagnose-covid-19-summary-table-of-
available-protocols.
17.   Ravi, N., Cortade, D. L., Ng, E. & Wang, S. X. Diagnostics for SARS-CoV-2 detection: A
comprehensive review of the FDA-EUA COVID-19 testing landscape.
Biosens. Bioelectron.
165, (2020).
18.   Mitchell, S. L.
et al.
Understanding, verifying, and implementing emergency use authorization
molecular diagnostics for the detection of sars-cov-2 RNA.
Journal of Clinical Microbiology
vol. 58
(2020).
19.   Esbin, M. N.
et al.
Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid
testing approaches for COVID-19 detection.
RNA
vol. 26 771–783 (2020).
20.   Butler-Laporte, G.
et al.
Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid
Amplication Testing for Detection of SARS-CoV-2: A Systematic Review and Meta-analysis.
JAMA Intern.
Med.
181, 353–360 (2021).

Page 17/22
21.   Lee, R. A., Herigon, J. C., Benedetti, A., Pollock, N. R. & Denkinger, C. M. Performance of saliva,
oropharyngeal swabs, and nasal swabs for SARS-CoV-2 molecular detection: A systematic review and
meta-analysis.
J. Clin. Microbiol.
59, (2021).
22.   Mögling, R.
et al.
Delayed laboratory response to COVID-19 caused by molecular diagnostic
contamination.
Emerg. Infect. Dis.
26, 1944–1946 (2020).
23.   Wang, C. Y. T., Buckley, C., Bletchly, C., Harris, P. & Whiley, D. Contamination of SARS-CoV-2 RT-PCR
probes at the oligonucleotide manufacturer.
Pathology
vol. 52 814 (2020).
24.   Davidi, D.
et al.
Amplicon residues in research laboratories masquerade as COVID-19 in
surveillance tests.
Cell Reports Methods
1, 100005 (2021).
25.   Huggett, J. F.
et al.
Cautionary Note on Contamination of Reagents Used for Molecular Detection
of SARS-CoV-2.
Clin. Chem.
66, 1369–1372 (2020).
26.   Fajnzylber, J.
et al.
SARS-CoV-2 viral load is associated with increased disease severity and
mortality.
Nat. Commun. 2020 111
11, 1–9 (2020).
27.   Goyal, A., Reeves, D. B., Fabian Cardozo-Ojeda, E., Schiffer, J. T. & Mayer, B. T. Viral load and
contact heterogeneity predict sars-cov-2 transmission and super-spreading events.
Elife
10, 1–63 (2021).
28.   SARS-CoV-2 Viral Mutations: Impact on COVID-19 Tests | FDA. https://www.fda.gov/medical-
devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-viral-mutations-impact-covid-19-tests.
29.   Vogels, C. B. F.
et al.
Multiplex qPCR discriminates variants of concern to enhance global
surveillance of SARS-CoV-2.
PLoS Biol.
19, e3001236 (2021).
30.   SARS-CoV-2 Viral Mutations: Impact on COVID-19 Tests | FDA. https://www.fda.gov/medical-
devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-viral-mutations-impact-covid-19-tests.
31.   Peccia, J.
et al.
Measurement of SARS-CoV-2 RNA in wastewater tracks community infection
dynamics.
Nat. Biotechnol.
38, 1164–1167 (2020).
32.   Mina, M. J., Parker, R. & Larremore, D. B. Rethinking Covid-19 Test Sensitivity — A Strategy for
Containment.
N. Engl. J. Med.
383, e120 (2020).
33.   NR, P.
et al.
Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen
Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts.
J. Clin. Microbiol.
59, (2021).
34.   Fitzpatrick, M. C., Pandey, A., Wells, C. R., Sah, P. & Galvani, A. P. Buyer beware: inated claims of
sensitivity for rapid COVID-19 tests.
The Lancet
vol. 397 24–25 (2021).

Page 18/22
35.   Bossuyt, P. M., Irwig, L., Craig, J. & Glasziou, P. Comparative accuracy: Assessing new tests
against existing diagnostic pathways.
Br. Med. J.
332, 1089–1092 (2006).
36.   Mina, M. J., Parker, R. & Larremore, D. B. Rethinking Covid-19 Test Sensitivity — A Strategy for
Containment.
N. Engl. J. Med.
(2020) doi:10.1056/nejmp2025631.
37.   Crozier, A., Rajan, S., Buchan, I. & McKee, M. Put to the test: Use of rapid testing technologies for
Covid-19.
BMJ
372, (2021).
38.   Wise, J. Covid-19: Lateral ow tests miss over half of cases, Liverpool pilot data show.
BMJ
371,
m4848 (2020).
39.   Mahase, E. Covid-19: Innova lateral ow test is not t for “test and release” strategy, say experts.
BMJ
(2020) doi:10.1136/bmj.m4469.
40.   Lisboa Bastos, M.
et al.
Diagnostic accuracy of serological tests for covid-19: Systematic review
and meta-analysis.
BMJ
370, 2516 (2020).
41.   Whitman, J. D.
et al.
Evaluation of SARS-CoV-2 serology assays reveals a range of test
performance.
Nat. Biotechnol.
38, 1174–1183 (2020).
42.   Lisboa Bastos, M.
et al.
Diagnostic accuracy of serological tests for covid-19: systematic review
and meta-analysis.
BMJ
370, 2516 (2020).
43.   Earle, K. A.
et al.
Evidence for antibody as a protective correlate for COVID-19 vaccines.
Vaccine
39, 4423–4428 (2021).
44.   Khoury, D. S.
et al.
Neutralizing antibody levels are highly predictive of immune protection from
symptomatic SARS-CoV-2 infection.
Nat. Med. 2021 277
27, 1205–1211 (2021).
45.   Alter, G. & Seder, R. The Power of Antibody-Based Surveillance.
N. Engl. J. Med.
(2020)
doi:10.1056/nejme2028079.
46.   Gudbjartsson, D. F.
et al.
Spread of SARS-CoV-2 in the Icelandic Population.
N. Engl. J. Med.
(2020) doi:10.1056/nejmoa2006100.
47.   Bonanni, P.
et al.
The Role of Serology Testing to Strengthen Vaccination Initiatives and Policies
for COVID-19 in Europe.
COVID 2021, Vol. 1, Pages 20-38
1, 20–38 (2021).
48.   Muruato, A. E.
et al.
A high-throughput neutralizing antibody assay for COVID-19 diagnosis and
vaccine evaluation.
Nat. Commun.
11, (2020).
49.   Milavec, M.
et al.
The performance of human cytomegalovirus digital PCR reference
measurement procedure in seven external quality assessment schemes over four years.
Methods
(2021)
doi:10.1016/J.YMETH.2021.03.016.

Page 19/22
50.   Matheeussen, V.
et al.
International external quality assessment for SARSCoV-2 molecular
detection and survey on clinical laboratory preparedness during the COVID-19 pandemic.
Eurosurveillance
25, (2020).
51.   Edson, D. C., Casey, D. L., Harmer, S. E. & Downes, F. P. Identication of SARS-CoV-2 in a
Prociency Testing Program.
Am. J. Clin. Pathol.
154, 475–478 (2020).
52.   Tacker, D. H.
et al.
Interlaboratory agreement of anti–severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) serologic assays in the expedited College of American Pathologists
prociency testing program.
Arch. Pathol. Lab. Med.
145, 536–542 (2021).
53.   Cohen, J. F.
et al.
STARD 2015 guidelines for reporting diagnostic accuracy studies: Explanation
and elaboration.
BMJ Open
6, e012799 (2016).
54.   Doust, J. A.
et al.
Guidance for the design and reporting of studies evaluating the clinical
performance of tests for present or past SARS-CoV-2 infection.
BMJ
372, (2021).
55.   SA, B.
et al.
The MIQE guidelines: minimum information for publication of quantitative real-time
PCR experiments.
Clin. Chem.
55, 611–622 (2009).
56.   ISO - ISO 20395:2019 - Biotechnology — Requirements for evaluating the performance of
quantication methods for nucleic acid target sequences — qPCR and dPCR.
https://www.iso.org/standard/67893.html.
57.   Irwig, L., Bossuyt, P., Glasziou, P., Gatsonis, C. & Lijmer, J. Evidence base of clinical diagnosis:
Designing studies to ensure that estimates of test accuracy are transferable.
British Medical Journal
vol.
324 669–671 (2002).
58.   MacKay, M. J.
et al.
The COVID-19 XPRIZE and the need for scalable, fast, and widespread
testing.
Nature Biotechnology
(2020) doi:10.1038/s41587-020-0655-4.
59.   Krause, P. R.
et al.
SARS-CoV-2 Variants and Vaccines.
N. Engl. J. Med.
(2021)
doi:10.1056/NEJMsr2105280.
60.   Rockett, R. J.
et al.
Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome
sequencing and agent-based modeling.
Nat. Med.
(2020) doi:10.1038/s41591-020-1000-7.
61.   Gardy, J. L. & Loman, N. J. Towards a genomics-informed, real-time, global pathogen surveillance
system.
Nature Reviews Genetics
(2018) doi:10.1038/nrg.2017.88.
62.   West, R. & Gronvall, G. K. California shows the way for biosecurity in commercial gene synthesis.
Nature Biotechnology
vol. 38 1021 (2020).

Page 20/22
63.   Elbe, S. & Buckland-Merrett, G. Data, disease and diplomacy: GISAID’s innovative contribution to
global health.
Glob. Challenges
1, 33–46 (2017).
64.   Griths, E. J.
et al.
The PHA4GE SARS-CoV-2 Contextual Data Specication for Open Genomic
Epidemiology. (2020) doi:10.20944/PREPRINTS202008.0220.V1.
65.   Konings, F.
et al.
SARS-CoV-2 Variants of Interest and Concern naming scheme conducive for
global discourse.
Nature Microbiology
vol. 6 821–823 (2021).
66.   Black, A., MacCannell, D. R., Sibley, T. R. & Bedford, T. Ten recommendations for supporting open
pathogen genomic analysis in public health.
Nat. Med. 2020 266
26, 832–841 (2020).
67.   Gwinn, M., MacCannell, D. & Armstrong, G. L. Next-Generation Sequencing of Infectious
Pathogens.
JAMA
321, 893–894 (2019).
68.   ENGLISH ONLY 4 5 MANUAL FOR THE PREPARATION OF SECONDARY REFERENCE MATERIALS
FOR IN VITRO DIAGNOSTIC ASSAYS DESIGNED FOR. (2016).
69.   SARS-CoV-2 Reference Panel Comparative Data | FDA. https://www.fda.gov/medical-
devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-reference-panel-comparative-data.
70.   FIND evaluations of SARS-CoV-2 assays - FIND. https://www.nddx.org/covid-19/sarscov2-eval/.
71.   Kahl, L.
et al.
Opening options for material transfer.
Nat. Biotechnol. 2018 3610
36, 923–927
(2018).
72.   Munnink, B. B. O.
et al.
Transmission of SARS-CoV-2 on mink farms between humans and mink
and back to humans.
Science (80-. ).
371, 172–177 (2021).
73.   Covid-19: government must urgently rethink lateral ow test roll out - The BMJ.
https://blogs.bmj.com/bmj/2021/01/12/covid-19-government-must-urgently-rethink-lateral-ow-test-roll-
out/.
74.   Leech, D. P. & Belmont, P. A. The Economic Impacts of NIST’S Cholesterol Standards Program.
(2000).
75.   Tromberg, B. J.
et al.
Rapid Scaling Up of Covid-19 Diagnostic Testing in the United States — The
NIH RADx Initiative.
N. Engl. J. Med.
(2020) doi:10.1056/nejmsr2022263.
Figures

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Figure 1
Standards needed for the SARS-CoV-2 testing process. Schematic diagram illustrating the steps in the
molecular (a) and serological (b) testing process which can be classied into pre-analytical (yellow),
analytical (green) and post-analytical (blue) stages. Additional test development (yellow) is performed
prior be manufacturers. Lower panel describes the range of standards available for validating test
development, processes and results.

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Figure 2
Schematic diagram illustrates key milestones in the development of testing and standards during the
COVID-19 pandemic. Understanding these milestones can assist in preparing for current and future public
health emergencies.