CHAPTER 1.1.1.

PRINCIPLES AND METHODS OF

VALIDATION OF DIAGNOSTIC ASSAYS
FOR INFECTIOUS DISEASES

INTRODUCTION1

Validation is a process that determines the fitness of an assay2, which has been properly developed,
optimised and standardised, for an intended purpose. All diagnostic assays (laboratory and field assays)
should be validated for the species in which they will be used. Validation includes estimates of the
analytical and diagnostic performance characteristics of a test. In the context of this chapter, an assay that
has completed the first three stages of the validation pathway (see Figure 1 below), including performance
characterisation, can be designated as “validated for the original intended purpose(s)”. To maintain a
validated assay status, however, it is necessary to carefully monitor the assay's performance under
conditions of routine use, often by tracking the behaviour of assay controls over time. This ensures that
the assay, as originally validated, consistently maintains its performance characteristics. Should it no
longer produce results consistent with the original validation data, the assay may be rendered unfit for its
intended purpose(s). Thus, a validated assay is continuously assessed to assure it maintains its fitness
for purpose through both the assessment of results of the assay controls included with each run and
through on-going assessment during routine use in the targeted population.
Assays applied to individuals or populations have many purposes, such as aiding in: documenting
freedom from disease in a country or region, preventing spread of disease through trade, contributing to
eradication of an infection from a region or country, confirming diagnosis of clinical cases, estimating
infection prevalence to facilitate risk analysis, identifying infected animals toward implementation of
control measures, and classifying animals for herd health or immune status post-vaccination. A single
assay may be validated for one or more intended purposes by optimising its performance characteristics
for each purpose, e.g. setting diagnostic sensitivity (DSe) high, with associated lower diagnostic specificity
(DSp) for a screening assay, or conversely, setting DSp high with associated lower DSe for a confirmatory
assay.
The ever-changing repertoire of new and unique diagnostic reagents coupled with many novel assay
platforms and protocols has precipitated discussions about how to properly validate these assays. It is no
longer sufficient to offer simple examples from serological assays, such as the enzyme-linked
immunosorbent assay, to guide assay developers in validating the more complex assays, such as nucleic
acid detection tests. In order to bring coherence to the validation process for all types of assays, this
chapter focuses on the criteria that must be fulfilled during assay development and validation of all assay
types. The inclusion of assay development as part of the assay validation process may seem
counterintuitive, but in reality, three of the required validation criteria (definition of intended purpose[s],
optimisation, and standardisation) that must be assessed in order to achieve a validated assay, comprise
steps in the assay development process. Accordingly the assay development process seamlessly leads
into an assay validation pathway, both of which require fulfilment of validation criteria. Further, more
detailed guidance is provided in a series of Recommendations for validation of diagnostic tests3 that are
tailored for several fundamentally different types of assay (e.g. detection of nucleic acids, antibodies, or
antigens) and provide more information on specific issues related to the validation of diagnostic assays.
For specific information for wildlife species, refer to Chapter 3.6.74 Principles and methods for the
validation of diagnostic tests for infectious diseases applicable to wildlife of the Manual of Diagnostic Tests
and Vaccines for Terrestrial Animals. The information provided in Chapter 3.6.7, which is specific to
wildlife species, might also be useful for domestic animal test validation, for example, where the number
or availability of samples is limited.

1 NB: Version adopted by the World Assembly of Delegates of the OIE in May 2013.
2 Assay, test method and test are synonymous terms for purposes of this chapter, and therefore are used interchangeably
3 Available at: http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.6.00_INTRODUCTION.pdf
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PRELIMINARY CONSIDERATIONS IN ASSAY DEVELOPMENT AND VALIDATION

All laboratories should comply with the requirements of Chapter 1.1.1 (Aquatic Manual) or Chapter 1.1.5 (Terrestrial
Manual) on Quality management in veterinary testing laboratories. This will minimise the influence of factors that do not
depend on the test itself such as instrumentation, operator error, reagent choice (both chemical and biological) and
calibration, reaction vessels and platforms, water quality, pH and ionicity of buffers and diluents, incubation temperatures
and durations, and errors in the technical performance of the assay.

The first step in assay development is to define the purpose of the assay, because this guides all subsequent steps in
the validation process. Assay validation criteria are the characterising traits of an assay that represent decisive factors,
measures or standards upon which a judgment or decision may be based. By considering the variables that affect an
assay's performance, the criteria that must be addressed in assay validation become clearer. The variables can be
grouped into categories such as: (a) the sample – individual or pooled, matrix composition, and host/organism
interactions affecting the target analyte quantitatively or qualitatively; (b) the assay system – physical, chemical,
biological and operator-related factors affecting the capacity of the assay to detect a specific analyte in the sample; and
(c) the test result interpretation - the capacity of a test result, derived from the assay system, to predict accurately the
status of the individual or population relative to the purpose for which the assay is applied.

Selection, collection, preparation, preservation and management of samples are critical variables in design and
development of an assay to ensure valid test results. Other variables such as transport, chain of custody, tracking of
samples, and the laboratory information management system are also key sources of variation/error that become
especially important when the assay is implemented for routine testing. Integrity of experimental outcomes during assay
development and validation is only as good as the quality of the samples used. Anticipating the factors that can
negatively impact sample quality must precede launching an assay validation effort. Reference samples used in assay
development and validation should be in the same matrix that is to be used in the assay (e.g. serum, tissue, whole blood)
and representative of the species to be tested by the assay. The reference materials should appropriately represent the
range of analyte concentration to be detected by the assay. Information on sample collection, preparation, preservation,
management, and transport is available in chapters 1.1.2 and 1.1.3 of the Terrestrial Manual.

The matrix in which the targeted analyte is found (serum, faeces, tissue, etc.) may contain endogenous or exogenous
inhibitors that prevent some assays from working. This is of particular concern for enzyme-dependent tests such as
polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA). Other factors that affect the
concentration and composition of the target analyte (particularly antibody) in the sample may be mainly attributable to
the host and are either inherent (e.g. age, sex, breed, nutritional status, pregnancy, immunological responsiveness) or
acquired (e.g. passively acquired antibody, active immunity elicited by vaccination or infection). Non-host factors, such
as contamination or deterioration of the sample, also potentially affect the ability of the assay to detect the specific
targeted analyte in the sample. It is also important that biological reagents are free of extraneous agents that might
otherwise lead to erroneous results.

THE CRITERIA OF ASSAY DEVELOPMENT AND VALIDATION

Assay performance is affected by many factors beginning with optimisation of the assay. After initial optimisation for an
intended purpose, characteristics of the performance of the assay will be tested. The assay may need additional
optimisation or may be found to be fit for purpose based on the results of the validation work.

Criteria for assay development and validation

i) Definition of the intended purpose(s)

ii) Optimisation
iii) Standardisation
iv) Repeatability
v) Analytical sensitivity
vi) Analytical specificity
vii) Thresholds (cut-offs)
viii) Diagnostic sensitivity
ix) Diagnostic specificity

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 Chapter 1.1.1. - Principles and methods of validation of diagnostic assays for infectious diseases

x) Reproducibility
xi) Fitness for intended purpose(s)

1. ASSAY DEVELOPMENT PATHWAY

1.1. Definition of the intended purpose(s) for an assay

The OIE Standard for Management and Technical Requirements for Laboratories Conducting Tests for Infectious
Diseases (World Organisation for Animal Health (OIE), 2008)5 states that test methods and related procedures
must be appropriate for specific diagnostic applications in order for the test results to be of relevance. In other
words, the assay must be “fit for purpose”. The qualitative and quantitative assessment of capacity of a positive or
negative test result to predict accurately the infection or exposure status of the animal or population of animals is
the ultimate consideration of assay validation. This capacity is dependent on development of a carefully optimised
and standardised (Section 1.2.5) assay that, through accrual of validation data, provides confidence in the assay’s
ability to perform according to the intended purpose. In order to ensure that test results provide useful diagnostic
inferences about animals or populations with regard to the intended purpose, the validation process encompasses
initial development and assay performance documentation, as well as on-going assessment of quality control and
quality assurance programmes. Figure 1 shows the assay validation process, from assay design through the
development and validation pathways to implementation, deployment, and maintenance of the assay.

The first step of assay development is selection of an assay type that is appropriate and that likely can be validated
for a particular use (fitness for purpose).

The most common purposes are to:

1. Contribute to the demonstration of freedom from infection in a defined population

(country/zone/compartment/herd) (prevalence apparently zero):

a) 'Free' with and/or without vaccination,

b) Re-establishment of freedom after outbreaks

2. Certify freedom from infection or presence of the agent in individual animals or products for trade/movement
purposes.

3. Contribute to the eradication of disease or elimination of infection from defined populations.

4. Confirm diagnosis of suspect or clinical cases (includes confirmation of positive screening test).

5. Estimate prevalence of infection or exposure to facilitate risk analysis (surveys, herd health status, disease
control measures).

6. Determine immune status of individual animals or populations (post-vaccination).

These purposes are broadly inclusive of many narrower and more specific applications of assays (see Section 3.6
of the OIE Terrestrial Manual: Recommendations for validation of diagnostic tests [footnote 3]) for each assay type
for details). Such specific applications and their unique purposes need to be clearly defined within the context of a
fully validated assay.

Further to the intended purpose, the assay needs to be defined in terms of target animal species, target
pathogen(s) or condition, and sampling matrix.

5 This is a specific interpretation of the more generally stated requirements of the ISO/IEC 17025:2005 international quality
standard for testing laboratories (2005). This publication further states that for a test method to be considered appropriate, it must
be properly validated and that this validation must respect the principles outlined in this document, the OIE Validation Standard.

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Chapter 1.1.1. - Principles and methods of validation of diagnostic assays for infectious diseases

Fig. 1. The assay development and validation pathways with assay validation criteria
highlighted in bold typescript within shadowed boxes.

1.2. Assay development - the experimental studies

1.2.1. Test method design and proof of concept

Prior knowledge, thought and planning need to go into designing all steps of a new assay destined for
validation, or an existing assay that is being modified. Assistance is offered in Section 3.6 of the OIE
Terrestrial Manual Recommendations for validation of diagnostic tests (see footnote 3), which cover best
practices for development and validation of assays for detection of various analytes (e.g. antibody, antigen,
and nucleic acid detection).

Development of any assay is dependent on analyte reference samples that reflect the target analyte, the
matrix in which the analyte is found, and the population for which the assay is intended to be used. The
reference samples may be sera, fluids (including meat juices) or tissues that contain the analyte of interest or
a genomic construct consistent with the target analyte. These reference materials are used in experiments
conducted throughout the development process and carried over into the validation of the assay.

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1.2.2. Operating range of the assay

The operating range of an assay is the interval of analyte concentrations or titres over which the method
provides suitable accuracy and precision. Accuracy is the closeness of a test value to the expected (true)
value (mean or median) for a reference standard reagent of known concentration or titre. Precision6 is the
degree of dispersion (variance, standard deviation or coefficient of variation) within a series of measurements
of the same sample tested under specified conditions. During development of the assay, the lower and upper
limits of the operating range are determined. To formally determine this range, a high positive reference
sample is selected (ideally, this sample will be the same one from among the three samples described under
Section 1.2.3 Standardisation and optimisation below). This high positive sample is serially diluted to
extinction of the assay’s response in an analyte-negative matrix of the same constitution as the sample matrix
from animals in the population targeted by the assay. The results are plotted as a ‘response-curve’, with the
response (e.g. optical density, cycle threshold, counts, etc.) a function of analyte concentration (amount). The
curve establishes the working range of the assay. If the range is found to be unacceptable for the intended
purpose, additional optimisation may be needed. The typical calibration curve for most assays is sigmoidal in
shape. The data are transformed to approximate a linear relationship between response and concentration
using a suitable algorithm (Findlay & Dillard, 2007).

1.2.3. Standardisation and optimisation

Optimisation is the process by which the most important physical, chemical and biological parameters of an
assay are evaluated and adjusted to ensure that the performance characteristics of the assay are best suited
to the intended application. It is useful to select at least three well-defined reference samples, representing
the analyte ranging from high positive to negative (e.g. high, low positive and negative). These samples
ideally should represent known infected and uninfected animals from the population that will become the
target of the assay. Obtaining such reference samples, however, is not always possible, particularly for
nucleic acid and antigen detection assays. The alternative of preparing reference samples spiked with
cultured agents or positive sera is inferior as these samples do not truly represent the naturally occurring
matrix-agent interaction (see also Terrestrial Manual Chapter 3.6.67 Selection and use of reference samples
and panels). When no other alternative exists, spiking a sample with a known amount of the analyte or agent
derived from culture, or diluting a high positive serum in negative serum of the same species may be all that
is available. In either case, it is imperative that the matrix, into which the analyte is placed or diluted, is
identical to, or resembles as closely as possible the samples that ultimately will be tested in the assay. Ideally,
reference samples have been well characterised by one or preferably at least two alternate methodologies.
These samples can be used in experiments to determine if the assay is able to distinguish between varying
quantities of analyte, distinguish the target from closely related analytes, and for optimising the reagent
concentrations and perfecting the protocol. In principle, for all assay types, it is highly desirable to prepare
and store a sufficient amount of each reference sample in aliquots for use in every run of the candidate assay
as it is evaluated through the entire development and validation process. Switching reference samples during
the validation process introduces an intractable variable that can severely undermine interpretation of
experimental data and, therefore, the integrity of the development and validation process.

The labour-intensive process of optimising an assay is fundamental and critical to achieving a reliable and
predictable assay performance. Scientific judgment and use of best scientific practices, as provided in Section
3.6 of the Terrestrial Manual (see footnote 3), are recommended to guide optimisation of all elements of assay
development and validation. The approach outlined provides a solid foundation for development of a reliable
assay. Often, prototype assays are developed using reagents and equipment at hand in the laboratory.
However, if the assay is intended for routine diagnostic use in multiple laboratories, standardisation becomes
critical. Every chemical and buffer formulation must be fully described. All reagents must be defined with
respect to purity and grade (including water). Acceptable working ranges must be established and
documented for parameters such as pH, molarity, etc. Standards for quality, purity, concentration and
reactivity of biologicals must be defined. Shelf lives and storage conditions must also be considered for both
chemicals and biologicals. Acceptable ranges for reaction times and temperatures also need to be
established. Essential equipment critical to assay performance must be described in detail, including
operational specifications and calibration. Process (quality) control should be an integral part of optimisation
and considered from the very beginning rather than, as is often the case at the end of assay development. In
addition to the above, downstream aspects such as data capture, data manipulation and interpretation may

6 Laboratory sources of variation that affect assay precision include: 1) within a single test run, 2) between concurrent runs, 3a)
between assay runs at different times in the same day or on different days under similar conditions, 3b) between assay runs on
different days with different operators, 4) between laboratories. In this chapter, categories 1–3 are estimates of repeatability, and
category 4 is synonymous with reproducibility.
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also require standardisation and optimisation. Finally, all of these parameters, once optimised, must be fully
described in the test method protocol.

During optimisation of an assay, it is important to take note of procedural steps and assay parameters that
have a narrow range in which the assay performs optimally, as these are the critical points that ultimately
affect an assay’s reliability (see Section 1.2.7). For some assay types, specific steps in the procedure may
have more impact than other steps on the final assay performance (see Section 2.5below and Terrestrial
Manual Chapter 3.6.88 Comparability of assays after minor changes in a validated test method for additional
information on establishing comparability when reagents or processes are changed).

A variety of analyte reference samples and other process controls that are routinely included in any assay
system are identified in the following sections. These provide critical assay monitoring functions that require
special attention during assay optimisation. In addition, attention must be paid to the proper preparation and
storage of all biological reagents and reference materials to ensure stability (see Chapter 1.1.2 of the
Terrestrial Manual).

1.2.4. Inhibitory factors in sample matrix

Each different matrix to be used in an assay must be used in the validation process. Some sample matrices
include inhibitory factors that interfere with the performance of specific types of assays. Serum, particularly if
haemolysed, may contain factors toxic to the cells used in viral neutralisation assays, while endogenous
substances found in some tissues and fluids can interfere with or inhibit ligand-binding and enzymatic-based
assays such as ELISAs. Faeces, autolysed tissues and semen samples tend to contain more interfering
substances and are therefore more problematic for assay performance than are serum, blood or fresh tissues.

1.2.5. Robustness

Robustness refers to an assay’s capacity to remain unaffected by minor variations in test situations that may
occur over the course of testing in a single laboratory. Assessment of robustness should begin during assay
development and optimisation stages. The deliberate variations in method parameters may be addressed in
experiments after optimal conditions for an assay are established. However, when multi-factorial titrations of
reagents are used for optimising the assay, indications of a compromised robustness may surface. If slight
differences in conditions or reagent concentrations cause unacceptable variability, the assay most likely will
not be robust. Early knowledge of this situation elicits a critical decision point for determining whether to
continue with validation of the assay would be worthwhile, because if an assay is not robust within one
laboratory under rather ideal conditions, it is unlikely to be reproducible when transferred to other laboratories.

The factors most likely to affect assay robustness include pH, temperature batch of reagents or brand of
microtitre plates and aqueous or organic matrix factors (Dejaegher & Vander Heyden, 2006). Once
optimisation is complete, the robustness of the assay becomes part of the assessment of repeatability.

1.2.6. Calibration of the assay to standard reagents

1.2.6.1. International and national reference standards

Ideally, OIE or other international reference standards, containing a known concentration or titre of analyte,
are the reagents to which all assays are standardised (see OIE Guide 39 and also Terrestrial Manual
Chapter 3.6.6 [footnote 7]). Such standards are prepared and distributed by OIE Reference Laboratories or
other international reference laboratories. National reference standards are calibrated by comparison with
an international reference standard whenever possible; they are prepared and distributed by a national
reference laboratory. In the absence of an international reference standard, a national reference standard
becomes the standard of comparison for the candidate assay. These standards are highly characterised
through extensive analysis, and preferably the methods for their characterisation, preparation, and storage
have been published in peer-reviewed publications.

1.2.6.2. In-house standard

An in-house reference standard generally should be calibrated against an international or national standard.
In the absence of either of these calibrators and to the extent possible, the in-house standard is highly
characterised in the same manner as international and national standards (see Terrestrial Manual Chapter
3.6.6 [footnote 7]). This local in-house standard therefore becomes the best available standard, and is

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retained in sufficient aliquotted volumes for periodic use as the standard to which working standards are
calibrated.

1.2.6.3. Working standard

One or more working standards, commonly known as analyte or process controls, are calibrated to an
international, national, or in-house standard, and are prepared in large quantities, aliquotted and stored for
routine use in each diagnostic run of the assay.

1.2.7. “Normalising” test results to a working standard

Due to the inherent variation in raw test results that are often observed between test runs of the same assay
or among laboratories using the same or similar assays, it is almost impossible to compare directly (semi-)
quantitative data. To improve markedly the comparability of test results both within and between laboratories,
one or more working standard reagent(s) are included in each run of an assay. Raw test values for each test
sample can then be converted to units of activity relative to the working standard(s) by a process called
“normalisation”. The “normalised” values may be expressed in many ways, such as a per cent of a positive
control (e.g. in an ELISA), or as the estimated concentration or titre of an analyte derived from a standard
curve. It is good practice to include working standards in all runs of the assay during assay development and
validation because this allows “normalisation” of data, which provides a valid means for direct comparison of
results between runs of an assay. It is mandatory to control the (absolute) variation of the normalisation
standards as otherwise normalisation can introduce a bias. For more information, see Terrestrial Manual
Chapter 3.6.110 Development and optimisation of antibody detection assays, 3.6.211 Development and
optimisation of antigen detection assays and 3.6.312 Development and optimisation of nucleic acid detection
assays.

1.2.8. Preliminary study of the repeatability

Assessment of repeatability should begin during assay development and optimisation stages. Early
knowledge of this situation elicits a critical decision point for determining whether it is worthwhile to continue
with validation of the assay.

Repeatability is further verified during Stage 1 of assay validation (Section 2.1.1). When the optimised test is
run under routine laboratory or field conditions (Stage 4 of assay validation), repeatability is continually
monitored as part of process control procedures for the duration of the life of the assay (see Section 2.5.1).

2. ASSAY VALIDATION PATHWAY

“Validation” is a process that determines the fitness of an assay that has been properly developed, optimised and
standardised for an intended purpose(s). Validation includes estimates of the analytical and diagnostic performance
characteristics of a test. In the context of this document, an assay that has completed the first three stages of the
validation pathway (Figure 1), including performance characterisation, can be designated as “validated for the original
intended purpose(s)”.

2.1. Stage 1 - Analytical performance characteristics

Ideally, the design of studies outlined in the following sections should be done with assistance of a statistician and
a disease expert to ensure that the sample size and experimental approach are valid. It is possible to design
experiments that efficiently provide information on likely within- and between-laboratory sources of variation in
assay precision (see footnote 6in Section 1.2.2, above), which will define the performance characteristics of the
assay. The choice of organisms, strains or serotypes to assess analytical sensitivity and specificity should reflect
current knowledge and therefore inform the best possible experimental design for targeting specific analytes.

2.1.1. Repeatability

Repeatability is the level of agreement between results of replicates of a sample both within and between runs
of the same test method in a given laboratory. Repeatability is estimated by evaluating variation in results of
replicates. The number of replicates should preferably be determined in consultation with a statistician with a
suggested minimum of three samples representing analyte activity within the operating range of the assay.

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Each of these samples is then aliquoted into the appropriate number of individual vessels as identical
replicates of the original sample containing the original analyte and matrix concentration (see Terrestrial
Manual Chapter 3.6.6 [footnote 7]). Each replicate is then run through all steps of the assay, including creating
the working dilution, as though it were a test sample derived from the population targeted by the assay. It is
not acceptable to prepare a final working dilution of a sample in a single tube from which diluted aliquots are
pipetted into reaction vessels, or to create replicates from one extraction of nucleic acid rather than to extract
each replicate before dilution into the reaction vessels. Such 'samples' do not constitute valid replicates for
repeatability studies. Between-run variation is determined by using the same samples in multiple runs
involving two or more operators, done on multiple days. The variation in replicate results can be expressed
as standard deviations, coefficients of variation (standard deviation ÷ mean of replicates), or other possible
options (see Terrestrial Manual Chapter 3.6.413 Measurement uncertainty for assessments of repeatability).

2.1.2. Analytical specificity

Analytical specificity (ASp) is the ability of the assay to distinguish the target analyte (e.g. antibody, organism
or genomic sequence) from non-target analytes, including matrix components. The assessment is qualitative
and the choice and sources of sample types, organisms and sequences for the ASp evaluation should reflect
test purpose and assay type. See Terrestrial Manual Chapters 3.6.1, 3.6.2 and 3.6.3 (footnotes 10, 11and 12)
for guidance for antibody, antigen and nucleic acid assays, respectively. ASp is documented during Stage 1
validation, and cross-reactions identified. Cross-reactivity (ASp less 100%) may be acceptable depending on
the proposed use of the assay. The impact of cross-reactivity is further documented during Stage 2
(establishment of DSp) and assessed at Stage 4 implementation.

2.1.2.1. Selectivity

Selectivity refers to the extent to which a method can accurately quantify the targeted analyte in the
presence of: 1) interferents such as matrix components (e.g. inhibitors of enzymes in the reaction mix);
2) degradants (e.g. toxic factors); 3) nonspecific binding of reactants to a solid phase (e.g. conjugate of an
ELISA adsorbed to well of microtitre plate); 4) antibodies to vaccination that may be confused with
antibodies to active infection. Such interferents may cause falsely reduced or elevated responses in the
assay that negatively affect its analytical specificity. Vessman et al. (Vessman et al., 2001) is a useful
overview of selectivity as defined for analytical chemistry from which a modification described herein was
deduced for application to diagnostic tests.

2.1.2.2. Exclusivity

Exclusivity is the capacity of the assay to detect an analyte or genomic sequence that is unique to a targeted
organism, and excludes all other known organisms that are potentially cross-reactive. This would also
define a confirmatory assay.

2.1.2.3. Inclusivity

Inclusivity is the capacity of an assay to detect several strains or serovars of a species, several species of
a genus, or a similar grouping of closely related organisms or antibodies thereto. It characterises the scope
of action for a screening assay.

2.1.3. Analytical sensitivity

The limit of detection (LOD) is a measure of the analytical sensitivity (ASe) of an assay. The LOD is the
estimated amount of analyte in a specified matrix that would produce a positive result at least a specified
percent of the time. Typically, estimated LOD will be based on spiking of the analyte into the target matrix.
The choice of analyte(s) (e.g. species, strains) is part of the ASe definition and should be reported properly.
These experiments may be designed for precise and accurate estimation of the probability point (e.g. 50% or
100%), but in some circumstances a conservative estimate of the LOD (e.g. 100%) may be acceptable. For
example, in a titration using tenfold dilutions all replicates at all dilutions might show either 100% or 0%
response. There are two choices at that point. The last dilution showing 100% response may be accepted as
a conservative estimate of the lower limit of detection. A more accurate estimate may be obtained by a second
stage experiment using narrower intervals in the dilution scheme focusing on the region between 100% and
0%. Methods for statistical evaluation of LOD data are in the Terrestrial Manual Chapter 3.6.514 Statistical
approaches to validation.

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2.1.4. Analytical accuracy of adjunct tests or procedures

Some test methods or procedures may be qualified for use as analytical tools in the diagnostic laboratory.
These usually are secondary adjunct tests or procedures that are applied to an analyte that has been detected
in a primary assay. The purpose of such analytical tools is to further characterise the analyte detected in the
primary assay. Examples of such adjunct tests include virus neutralisation to type an isolated virus, and
molecular sequencing.

Such adjunct tests must be validated for analytical performance characteristics (Sections 1.2through 2.1.3,
above). However, they differ from diagnostic tests because they do not require validation for diagnostic
performance characteristics (Sections 2.2through 2.4, below) if their results are not used to establish a final
diagnosis with regard to the intended purpose. The analytical accuracy of these tools may be defined by
comparison with a reference reagent standard, or by characteristics inherent in the tool itself (such as
endpoint titration). In all of these examples, the targeted analyte is further characterised quantitatively or
qualitatively by the analytical tool.

2.2. Stage 2 - Diagnostic performance of the assay

Estimates of DSe (proportion of samples from known infected reference animals that test positive in an assay) and
DSp (the proportion of samples from known uninfected reference animals that test negative in an assay) are the
primary performance indicators established during validation of an assay. These estimates are the basis for
calculation of other parameters from which inferences are made about test results (e.g. predictive values of positive
and negative test results). Therefore, it is imperative that estimates of DSe and DSp are as accurate as possible.
Ideally, they are derived from testing a panel of samples from reference animals, of known history and infection
status relative to the disease/infection in question and relevant to the country or region in which the test is to be
used. An estimate of the area under the receiver operating characteristic (ROC) curve is a useful adjunct to DSe
and DSp estimates for a quantitative diagnostic test because it assesses its global accuracy across all possible
assay values (Greiner et al., 2000; Zweig & Campbell, 1993). This approach is described in Terrestrial Manual
Chapter 3.6.5 (footnote 14).

The designated number of known positive and known negative samples will depend on the likely values of DSe
and DSp of the candidate assay and the desired confidence level for the estimates (Table 2.1.and Jacobson, 1998).
Table 2.1.provides two panels of the theoretical number of samples required, when either a 5% or 2% error is
allowed in the estimates of DSe or DSp. Many samples are required to achieve a high confidence (typically 95%)
in the estimates of DSe and DSp when a small error margin in the estimate is desired. For example comparison of
a 2% vs 5% error for a likely DSe or Dse of 90% and 95% confidence shows a considerable increase (864 vs 138)
in the number of samples required. Logistical and financial limitations may require that less than the statistically
required sample size will be evaluated, in which case the confidence interval calculated for DSe and DSp will
indicate less diagnostic confidence in the results. Sample size also may be limited by the fact that reference
populations and OIE reference standards may be lacking (see Terrestrial ManualChapter 3.6.5 [footnote 14] for
further details). It may, therefore, be necessary to use a suboptimal number of samples initially. It is, however,
highly desirable to enhance confidence and reduce error margin in the DSe and DSp estimates by adding more
samples (of equivalent status to the original panel) as they become available.

Table 2.1. Theoretical number of samples from animals of known infection status required for
establishing diagnostic sensitivity (DSe) and specificity (DSp) estimates depending on likely
value of DSe or DSp and desired error margin and confidence

2% error allowed in estimate of DSe and DSp 5% error allowed in estimate of DSe and DSp

Confidence Confidence

Estimated DSe or DSp 90% 95% 99% 90% 95% 99%

90% 610 864 1493 98 138 239

92% 466 707 1221 75 113 195

94% 382 542 935 61 87 150

95% 372 456 788 60 73 126

96% 260 369 637 42 59 102

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Chapter 1.1.1. - Principles and methods of validation of diagnostic assays for infectious diseases

Table 2.1. Theoretical number of samples from animals of known infection status required for
establishing diagnostic sensitivity (DSe) and specificity (DSp) estimates depending on likely
value of DSe or DSp and desired error margin and confidence

2% error allowed in estimate of DSe and DSp 5% error allowed in estimate of DSe and DSp

Confidence Confidence

97% 197 279 483 32 45 77

98% 133 188 325 21 30 52

99% 67 95 164 11 15 26

The following are examples of reference populations and methodologies that may aid in determining performance
characteristics of the test being validated.

2.2.1. Reference animal populations

Ideally, selection of reference animals requires that important host variables in the target population are
represented in animals chosen for being infected with or exposed to the target agent, or that have never been
infected or exposed. The variables to be noted include but are not limited to species, age, sex, breed, stage
of infection, vaccination history, and relevant herd disease history (for further details see Terrestrial Manual
Chapter 3.6.6 [footnote 7]).

2.2.1.1. Negative reference samples

True negative samples, from animals that have had no possible infection or exposure to the agent, may be
difficult to locate. It is often possible to obtain these samples from countries or zones that have eradicated
or have never had the disease in question. Such samples may be useful as long as the targeted population
for the assay is sufficiently similar to the sample-source population.

2.2.1.2. Positive reference samples

It is generally problematic to find sufficient numbers of true positive reference animals, as determined by
isolation of the pathogen. It may be necessary to resort to samples from animals that have been identified
by another test of sufficiently high accuracy, such as a validated nucleic acid detection assay. The candidate
test is applied to these reference samples and results (positive and negative) are cross-classified in a 2 ×
2 table. This has been called the “gold standard model” as it assumes the reference standard is perfect. A
sample calculation is shown in Table 2.2.in Section 2.2.5).

2.2.2. Samples from animals of unknown status

When the so-called reference standard is imperfect, which is the rule with any diagnostic tests, estimates of
DSe and DSp for the candidate assay based on this standard will be flawed. A way to overcome this problem
is to perform a latent class analysis of the joint results of the two tests assuming neither test is perfect.

Latent-class models do not rely on the assumption of a perfect reference test but rather estimate the accuracy
of the candidate test and the reference standard with the joint test results (Branscum et al., 2005; Enøe et al.,
2000; Georgiadis et al., 2003; Hui & Walter, 1980). If a Bayesian latent class analysis is used, prior knowledge
about the performance of the reference test and the candidate test can be incorporated into the analysis.

Because these statistical models are complex and require critical assumptions, statistical assistance should
be sought to help guide the analysis and describe the sampling from the target population(s), the
characteristics of other tests included in the analysis, the appropriate choice of model and the estimation
methods based on peer-reviewed literature (see Terrestrial Manual Chapter 3.6.5 [footnote 14] for details).

2.2.3. Experimentally infected or vaccinated reference animals

Samples obtained sequentially from experimentally infected or vaccinated animals are useful for determining
the kinetics of antibody responses or the presence/absence of antigen or organisms in samples from such
animals. However, multiple serially acquired pre- and post-exposure results from individual animals are not
acceptable for establishing estimates of DSe and DSp because the statistical requirement of independent
observations is violated. Single time-point sampling of individual experimental animals can be acceptable
(e.g. one sample randomly chosen from each animal). Nevertheless it should be noted that for indirect

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methods of analyte detection, exposure to organisms under experimental conditions, or vaccination, may elicit
antibody responses that are not quantitatively and qualitatively typical of natural infection in the target
population (Jacobson, 1998). The strain of organism, dose, and route of administration to experimental
animals are examples of variables that may introduce error when extrapolating DSe and DSp estimates to the
target population. In cases when the near-impossibility of obtaining suitable reference samples from naturally
exposed animals necessitates the use of samples from experimental animals for validation studies, the
resulting DSe and DSp measures should be considered as less than ideal estimates of the true DSp and DSe.

2.2.4. Cut-off (threshold) determination

To obtain DSe and DSp estimates of the candidate assay, which is measured on a continuous scale, the test
results first must be reduced to two (positive or negative) or three (positive, intermediate [doubtful] or
negative) categories of test results. This is accomplished by insertion of one or two cut-off points (threshold
or decision limits) on the scale of test results. The selection of the cut-off(s) should reflect the intended
purpose of the assay and its application, and must support the required DSe and DSp of the assay. Options
and descriptive methods for determining the best way to express DSe and DSp are available (Branscum et
al., 2005; Georgiadis et al., 2003; Greiner et al., 1995; Greiner et al., 2000; Jacobson, 1998; Zweig &
Campbell, 1993; and Terrestrial Manual Chapter 3.6.5 [footnote 14]). If considerable overlap occurs in the
distributions of test values from known infected and uninfected animals, it is impossible to select a single
cut-off that will accurately classify these animals according to their infection status. Rather than a single
cut-off, two cut-offs can be selected that define a high DSe (e.g. inclusion of 99% of the values from infected
animals), and a high DSp (e.g. 99% of the values from uninfected animals) (Greiner et al., 1995).

The main difficulty in establishing cut-offs based on diagnostic performance characteristics is the lack of
availability of the required number of well-characterised samples. Alternatives are discussed in Section 2.2.6.
on provisional acceptance of an assay during accrual of data to enhance estimates of DSe and DSp.

2.2.5. Calculation of DSe and DSp based on test results of reference samples

A typical method for determining DSe and DSp estimates is to test the reference samples in the new assay,
and cross tabulate the categorical test results in a 2 × 2 table. In a hypothetical example, assume the test
developer has selected a sample size for DSe and DSp for the new assay under the assumption that the most
likely values are 97% (DSe) and 99% (DSp), respectively, with a desired confidence of 95% for both
estimates. The desired error margin in the estimates was set at 2%. Table 2.1.indicates that 279 samples from
known infected animals are required for the DSe assessment, and 95 known negative samples are needed
for establishing the DSp estimate. The samples were then run in the new assay. Table 2.2.is a hypothetical
set of results from which DSe and DSp estimates have been obtained.

In this example, the DSe estimate is as anticipated, but the DSp is much lower (92%) than the anticipated
value of 99%. As a consequence, the width of the confidence interval for DSp is greater than expected.
Re-inspection of Table 2.1.indicates that 707 samples are necessary to achieve an error margin of ± 2% at a
DSP of 92% but such an increase in sample size might not be feasible (see Terrestrial Manual Chapter 3.6.5
[footnote 14] for further details).

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Table 2.2. Diagnostic sensitivity and specificity estimates calculated from hypothetical set of results for samples tested
from known infected and non-infected populations.

Number of reference samples required*

Known positive (279) Known negative (95)

Test results Positive 270 7

TP FP

Negative FN TN

9 88

Diagnostic sensitivity* Diagnostic specificity*

TP/(TP+FN) TN/(TN+FP)
96.8% (94.0-98.5%)** 92.6% (85.4-97.0%)**

*Based on Table 2.1 for an assay with the following parameters:

1) Prior to testing, estimated DSe of 97% and DSp of 99%

2) 95% = required confidence in DSe and DSp estimates

3) 2% = Error margin in the estimates of DSe and DSp

TP and FP = True Positive & False Positive, respectively

TN and FN = True Negative and False Negative, respectively

**95% exact binomial confidence limits for DSe and DSp calculated values

(see Terrestrial ManualChapter 3.6.5 [footnote 14] for information on confidence limits)

2.2.6. Provisional assay recognition15

There are situations where it is not possible or desirable to fulfil Stage 2 of the Validation Pathway because
appropriate samples from the target population are scarce and animals are difficult to access (such as for
transboundary infectious diseases or wildlife diseases).

Experience has shown that the greatest obstacle for continuing through Stage 2 of the Validation Pathway is
the number of defined samples required to calculate DSe and DSp. The formula is well known and tables are
available for determining the number of samples required to estimate various levels of DSe and DSp,
depending on the desired error margin and the level of confidence in the estimates (Table 2.1.and Jacobson,
1998). The formula assumes that the myriad of host/organism factors that may affect the test outcome are all
accounted for. As that assumption may be questionable, the estimated sample sizes are at best minimal. For
a disease that is not endemic or widespread, it may be impossible, initially, to obtain the number of samples
required, but over time, accrual of additional data will allow adjustment of the cut-off (threshold) or if no
adjustment is needed, enhance confidence in the estimates.

Provisional recognition defines an assay that has been assessed through Stage 1 for critical assay
benchmark parameters (ASe, ASp and repeatability) with, in addition, a preliminary estimate of DSp and DSe
based on a small select panel of well-characterised samples containing the targeted analyte and a preliminary
estimate of reproducibility. This represents partial completion of Stage 2. Preliminary reproducibility estimates
of the candidate assay could be done using the select panel of well-characterised samples to enhance
provisional acceptance status for the assay. The candidate test method is then duplicated in laboratories in
at least two different institutes, and the panel of samples is evaluated using the candidate assay in each of
these laboratories, using the same protocol, same reagents as specified in the protocol, and comparable
equipment. This is a scaled-down version of the reproducibility study in Stage 3 of assay validation. In
following this procedure of provisional recognition the test protocol must not be varied.

15 Provisional recognition does not imply acceptance by the OIE. It does, however, recognise an informed decision of authorities at
local, state, national or international levels of their conditional approval of a partially validated assay.

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Provisional recognition of an assay by state or national authorities means that the assay has not been
evaluated for diagnostic performance characteristics. As such, the laboratory should develop and follow a
protocol for adding and evaluating samples, as they become available, to fulfil this requirement. Ideally, this
process should be limited to a specific timeframe in which such an accrual would be directed toward fulfilling
Stages 2 and 3 of the validation pathway, and to particular situations (emergencies, minor species, no other
test available, etc.)

2.3. Stage 3 - Reproducibility and augmented repeatability estimates

2.3.1. Reproducibility

Reproducibility is the ability of a test method to provide consistent results, as determined by estimates of
precision, when applied to aliquots of the same samples tested in different laboratories, preferably located in
distinct or different regions or countries using the identical assay (protocol, reagents and controls). To assess
the reproducibility of an assay, each of at least three laboratories should test the same panel of samples
(blinded) containing a suggested minimum of 20 samples, with identical aliquots going to each laboratory (see
Terrestrial ManualChapter 3.6.6 [footnote 7]). This exercise also generates preliminary data on non-random
effects attributable to deployment of the assay to other laboratories. In addition, within-laboratory repeatability
estimates are augmented by the replicates used in the reproducibility studies. Measurements of precision can
be estimated for both the reproducibility and repeatability data (see Terrestrial Manual Chapter 3.6.4
[footnote 13] for further explanation of the topic and its application)

For field tests, reproducibility should be evaluated under the conditions of intended use.

2.3.2. Designation of a validated assay

On completion of Stage 3 validation, assuming the earlier stages have been fully and satisfactorily completed,
the assay may be designated as “validated for the original intended purpose”. Retention of this designation is
dependent on continual monitoring of the assay performance, as described in Section 2.5.1.

2.4. Stage 4 - Programme implementation

The successful deployment of an assay provides additional and valuable evidence for its performance according
to the expectations. Moreover, the (true) prevalence of the diagnostic trait in the target population is an important
factor that needs to be accounted for as described below

2.4.1. Fitness for use

While this chapter deals with validation and fitness for purpose from a scientific perspective, it should also be
noted that other practical factors might impact the utility of an assay with respect to its intended application.
These factors include not only the diagnostic suitability of the assay, but also its acceptability by scientific and
regulatory communities, acceptability to the client, and feasibility given available laboratory resources. For
some diseases, multiple assays might be available for use in combination in disease control and surveillance
programmes and hence, an assay’s utility might need to be assessed by evaluating incremental changes in
DSe, DSp and predictive values of the combined tests.

An inability to meet operational requirements of an assay also may make it unfit for its intended use. Such
requirements may include performance costs, equipment availability, level of technical sophistication and
interpretation skills, kit/reagent availability, shelf life, transport requirements, safety, biosecurity, sample
throughput, turn-around times for test results, aspects of quality control and quality assurance, and whether
the assay can practically be deployed to other laboratories. Test kits used in the field are highly desirable from
an ease-of-use viewpoint, but because they are performed outside the confines of a controlled laboratory
environment, they require added precautions to maintain fitness for purpose (Crowther et al., 2006).

2.4.2. Interpretation of test results

Predictive values of test results: The positive predictive value (PPV) is the probability that an animal that has
tested positive is in fact positive with regard to the true diagnostic status. The negative predictive value (NPV)
is the probability that an animal that has tested negative is in fact negative with regard to the true diagnostic
status.

Predictive values of test results are an application of Bayes’ theorem and are calculated as follows:

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Where:

PPV = Predictive value of a positive test result

NPV = Predictive value of a negative test result

P = Prevalence of infection

DSe = Diagnostic sensitivity

DSp = Diagnostic specificity

In contrast to DSe and DSp, predictive values are influenced by the true prevalence of the true diagnostic
status of the target population. In other words, predictive values are not inherent characteristics of a specific
diagnostic test, but are a function of its DSe and DSp and the local prevalence of infection in a defined
population at a given point in time.

Predictive values are of great importance to field veterinarians for the interpretation of results. For example,
a PPV of 0.9 means that an animal reacting positive to the test has 90% chance of being indeed infected and
10% probability of being a false positive.

The predictive value of a positive result also has great importance for the veterinary services in charge of the
management of control or eradication programmes. If we consider the inverse of the PPV (i.e. 1/PPV) it gives
the information on how much money is spent in the culling of true and false positives for each true positive
animal detected by the surveillance activity. In other words, if the PPV V is 0.67, it means that two positive
animals out of three are true positives and the remaining is a false positive. Since during the application of a
control programme, the prevalence of infection is continually changing, the monitoring of the PPV is a way of
evaluating the costs of the programme.

Furthermore, during the application of a control programme it is usually advisable to change the sensitivity of
the tests employed, based on the variation of prevalence of infection in the target population and on the
objective of the programme, the PPV may be used to make the changes in DSe and DSp based on economic
considerations. In other words, when the need for a change in DSe and DSp of the test arises, a number of
putative cut-offs may be set along the ROC curve of the test validation and the relevant values of DSe and
DSp for each cut-off may be used to evaluate the expected cost for the culling of each infected animal.

If the purpose is establishing evidence for freedom from disease, the NPV is the more important measure.
The NPV critically depends on DSe.

2.4.3. International recognition

Traditionally, assays have been recognised internationally by the OIE when they are designated as
prescribed or alternate tests for trade purposes. This has often been based on evidence of their usefulness
on a national, regional or international basis. For commercial diagnostic kits that have gone through the OIE
procedure for validation and certification of diagnostic assays, the final step is listing of the test in the OIE
Register. Tests listed in the Register are certified as fit for a specific purpose if they have completed Validation
Stages 1, 2 and 3. The Register is intended to provide potential kit users with an informed and unbiased
source of information about the kit and its performance characteristics for an intended purpose. The Register
is available on the OIE website at:
http://www.oie.int/en/our-scientific-expertise/certification-of-diagnostic-tests/the-register-of-diagnostic-tests/

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2.4.4. Deployment of the assay

Ultimate evidence of the usefulness of an assay is its successful application(s) in other laboratories and
inclusion in national, regional and/or international control or surveillance programmes. Reference laboratories
play a critical role in this process. In the natural progression of diagnostic and/or technological improvements,
new assays will become the new standard method to which other assays will be compared. As such, they may
progressively achieve national, regional and international recognition. As a recognised standard, these
assays will also be used to develop reference reagents for quality control, proficiency and harmonisation
purposes. These reference reagents may also become international standards.

An assessment of the reproducibility should be repeated when the test is transferred from the development
laboratory to the field, whether for use in local laboratories or in field applications. Predictable changes, e.g.
extremes of temperature and levels of operator experience, should be assessed as additional sources of
variation in assay results that may affect estimates of reproducibility.

2.5. Monitoring assay performance after initial validation

2.5.1. Monitoring the assay

To retain the status of a validated assay it is necessary to assure that the assay as originally validated
consistently maintains the performance characteristics as defined during validation of the assay. This can be
determined in a quality assurance programme characterised by carefully monitoring the assay’s daily
performance, primarily through precision and accuracy estimates for internal controls, as well as outlier
tendencies. The performance can be monitored graphically by plotting measurements from assay controls in
control charts16. Deviations from the expected performance should be investigated so corrective action can
be taken if necessary. Such monitoring provides critical evidence that the assay retains its ‛validated’
designation during the implementation phase of the assay. Reproducibility is assessed through external
quality control programmes such as proficiency testing. Should the assay cease to produce results consistent
with the original validation data, the assay would be rendered unfit for its intended purpose. Thus, a validated
assay must be continuously assessed to assure it maintains its fitness for purpose.

2.5.2. Modifications and enhancements ? considerations for changes in the assay

Over time, modifications of the assay likely will be necessary to address changes in the intended purpose,
analytes targeted (i.e. modification of the assay to adjust diagnostic performance) or technical modifications
to improve assay efficiency or cost-effectiveness. For a change in intended purpose of the assay, then a
revised validation from Stage 2 onwards is obligatory.

If the assay is to be applied in another geographical region and/or population, revalidation of the assay under
the new conditions is recommended. Lineages or sub-lineages of an infectious agent, derived from animals
in different geographic locations, are known to vary requiring revalidation of the assay for the specified target
population. This is especially true for nucleic acid detection (NAD) systems as it is very common for point
mutations to occur in many infectious agents (especially RNA viruses). Mutations, which may occur within the
primer or probe sites can affect the efficiency of the assay and even invalidate the established performance
characteristics. It is also advisable to regularly confirm the target sequence at the selected genomic regions
for national or regional isolates of the infectious agents. This is especially true for the primer and probe sites,
to ensure that they remain stable and the DSe and DSp for the assay are not compromised. Similar issues
can arise with immunologically based assays for antigen or antibody.

A similar situation may occur with emergence of new subtypes of existing pathogens. In these circumstances,
existing assays may need to be modified.

2.5.2.1. Technical modifications and comparability assessments

Technical modifications to a validated assay such as changes in instrumentation, extraction protocols, and
conversion of an assay to a semi-automated or fully automated system using robotics will typically not
necessitate full revalidation of the assay. Rather, a methods comparison study is done to determine if the
relatively minor modification to the assay affected the previously documented performance characteristics
of the assay. Comparability can be established by running the modified procedure and original procedure
side-by-side, with the same panel of samples in both, over several runs. The panel chosen for this
comparison should represent the entire operating range of both assays. If the results from the modified

16 Control chart: A graphical representation of data from the repetitive measurement of a control sample(s) tested in different runs
of the assay over time.

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procedure and originally validated method are determined to be comparable in an experiment based on a
pre-specified criterion, the modified assay remains valid for its intended purpose. See Terrestrial Manual
Chapter 3.6.8 (footnote 8) for description of experiments that are appropriate for comparability testing and
Terrestrial Manual Chapter 3.6.6 (footnote 7) on reference sample panels.

2.5.2.2. Biological modifications and comparability assessments

There may be situations where changes to some of the biologicals used in the assay may be necessary or
warranted. This may include changes to the test specimen itself (e.g. a change in tissue to be tested or
perhaps testing of a different species altogether). It may include changes to reagents (e.g. the substitution
of a recombinant antigen for a cell culture derived antigen or one antibody conjugate for another of similar
immunological specificity in an ELISA). The difficulty in making any modification lies in determining whether
the change requires a complete revalidation of the assay at both bench and field levels. At the very least,
any modification requires that the appropriate Stage 1 ‘analytical requisites’ be assessed. The more difficult
decision relates to Stage 2 ‘diagnostic performance’. To assist here, the original (reference) assay should
initially be compared to the modified (candidate) assay in a controlled trial using a defined panel of positive
and negative diagnostic samples. See Terrestrial Manual Chapter 3.6.8 (footnote 8) for a description of
comparability assessment. If the comparability assessment does not suggest a change in diagnostic
performance, the modified assay may be phased into routine use. If, on the other hand, differences in DSp
and DSe are observed, the modified assay would require additional Stage 2 or field validation before being
adopted.

2.5.2.3. Replacement of depleted reagents

When a reagent such as a control sample or working standard is nearing depletion, it is essential to prepare
and repeatedly test a replacement before such a control is depleted. The prospective control sample should
be included in multiple runs of the assay in parallel with the original control to establish their proportional
relationship. It is important to change only one reagent at a time to avoid the compound problem of
evaluating more than one variable.

2.5.3. Enhancing confidence in validation criteria

Because many host variables have an impact on the diagnostic performance of assays, it is highly desirable
over time to increase the number of reference samples or samples suitable for latent class analysis. The
sampling design, collection, transportation, and testing environment for the new samples should be the same
as used for the original validation study. Increases in sample numbers improves the precision of the overall
estimates of DSe and DSp, and may allow calculations of DSe estimates by factors such as age, stage of
disease, and load of organisms. New data should be included annually in relevant test dossiers.

2.5.4. Verification of existing assays (in-house validation)

If a laboratory is considering the use of a validated commercial kit or a candidate assay based on published
literature with validation data, some form of verification will be required to determine whether the assay
complies with either the kit manufacturer’s or the author's assertions, with respect to Stage 1 validation
criteria, in the context of the intended application. This may require a limited verification of both ASp and ASe
using available reference materials, whether they be external and/or locally acquired from the target
population. Once the laboratory is confident that the assay is performing as described from an analytical
perspective, then proceeding to a limited Stage 2 validation should be considered in the context of the
intended application and target population before the assay is put into routine diagnostic use.

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FINDLAY J.W.A. & DILLARD R.F. (2007). Appropriate calibration curve fitting in ligand binding assays. AAPS J., 9 (2),
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HUI S.L. & WALTER S.D. (1980). Estimating the error rates of diagnostic tests. Biometrics, 36, 167–171.

JACOBSON R.H. (1998). Validation of serological assays for diagnosis of infectious diseases. Rev. sci. tech. Off. int.
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VESSMAN J., STEFAN R., VAN STADEN J., DANZER K., LINDNER W., BURNS D., FAJGELJ A. & MULLER H.
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WORLD ORGANISATION FOR ANIMAL HEALTH (OIE) (2008). OIE Standard for Management and Technical
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Chapter 1.1.1. - Principles and methods of validation of diagnostic assays for infectious diseases

18 2018 © OIE - Manual of Diagnostic Tests for Aquatic Animals - 22/06/2018