Original article

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Diagnostic accuracy of PCR for Jaagsiekte sheep retrovirus using field data from 125
Scottish sheep flocks
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F.I. Lewis a,*, F. Brülisauer a, C. Cousens c, I.J. McKendrick b, G.J. Gunn a
a
Epidemiology Research Unit, SAC (Scottish Agricultural College), King’s Buildings, West
Mains Road, Edinburgh, EH9 3JG, UK
b
Biomathematics and Statistics Scotland, King’s Buildings, West Mains Road, Edinburgh,
EH9 3JZ, UK
c
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK
* Corresponding author. Tel.: +44 1463 243030
Email address: fraser.lewis@sac.ac.uk (F.I. Lewis).
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Abstract
Using a representative sample of Scottish sheep comprising 125 flocks, the sensitivity
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and specificity of PCR for Jaagsiekte sheep retrovirus (JSRV) was estimated. By combining
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and adapting existing methods, the characteristics of the diagnostic test were estimated (in the
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absence of a gold standard reference) using repeated laboratory replicates. As the results of
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replicates within the same animal cannot be considered to be independent, the performance of
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the PCR was calculated at individual replicate level.
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The median diagnostic specificity of the PCR when applied to individual animals drawn
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from the Scottish flock was estimated to be 0.997 (95% confidence interval [CI] 0.996-0.999),
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whereas the median sensitivity was 0.107 (95% CI 0.077-0.152). Considering the diagnostic
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test as three replicates where a positive result on any one or more replicates results in a
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positive test, the median sensitivity increased to 0.279. Reasons for the low observed
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sensitivity were explored by comparing the performance of the test as a function of the
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concentration of target DNA using spiked positive controls with known concentrations of
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target DNA. The median sensitivity of the test when used with positive samples with a mean
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concentration of 1.0 target DNA sequence per 25 μL was estimated to be 0.160, which
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suggests that the PCR had a high true (analytical) sensitivity and that the low observed
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(diagnostic) sensitivity in individual samples was due to low concentrations of target DNA in
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the blood of clinically healthy animals.
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Keywords: Jaagsiekte sheep retrovirus; Diagnostic test validation; PCR; Modelling
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Introduction
Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary
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adenocarcinoma (OPA), an infectious lung tumour of sheep occurring in almost all countries
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but absent from Australia, New Zealand and Iceland. Currently, there is no treatment or
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vaccination for JSRV infection and clinical OPA is inevitably fatal. OPA can cause
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substantial losses in affected flocks and, in order to prevent spread of JSRV infection, a
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reliable diagnostic test for detection of infected sheep is needed.
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No cost effective serological assays are available for JSRV, since the virus does not
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induce a specific antibody response in infected animals (Sharp and Herring, 1983; Ortin et al.,
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1998). Current JSRV diagnostic tests are based on virus detection, e.g. from blood or
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bronchoalveolar lavage samples, observation of clinical signs of OPA in advanced clinical
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cases, and identification of OPA lesions at post mortem examination. However, no routine
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assays for pre-clinical diagnosis of JSRV infection are available.
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PCR for JSRV is not used routinely since there are reservations regarding its suitability
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for diagnosis of JSRV infection outside the research environment (De las Heras et al., 2005;
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Voigt et al., 2007). Viable implementation of any assay into routine diagnostics is dependent
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upon the accuracy of the diagnostic test. Hence, thorough validation of the test against the
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target population is essential.
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In this study, we used blood samples from a national survey commissioned by the
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Scottish Government for validation of a JSRV PCR assay. The diagnostic test used is similar
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to the hemi-nested PCR described by De las Heras et al. (2005). Previous work suggested that
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the diagnostic accuracy of this test is highly dependent upon (1) the specimen tested and (2)
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the stage of disease in the animal being sampled. De las Heras et al. (2005) noted that the
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sensitivity of the test when based on blood samples from infected but clinically healthy
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animals was too low to provide a reliable result at the individual animal level, and these
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authors recommended flock level testing. This conclusion was based on sampling from six
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animals infected with JSRV, but with no clinical evidence of disease.
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Voigt et al. (2007) suggested that the sensitivity of a similar JSRV PCR used with blood
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samples may be as low as 10% at the individual animal level; this estimate was based on a
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study population of 47 Grey Heath sheep with histologically confirmed OPA lesions. These
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experimental studies used small sample sizes with repeated sampling of individual animals
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and confirmatory tests in live and dead animals.
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Although certain findings from these two experimental studies may not be applicable for
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diagnosis of JSRV infection under field conditions, the observed association between disease
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status and diagnostic accuracy is of relevance because in prevalence surveys it is expected
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that the majority of animals tested will be clinically healthy, i.e. the likelihood of detecting an
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individual infected sheep will be low. Diagnostic sensitivity and specificity are population
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parameters that describe the test performance for a given reference population (Greiner and
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Gardner, 2000). So it is important to question the accuracy of the JSRV PCR assay under
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given circumstances, in our case when applied to the Scottish sheep flock. The answer to this
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question has implications for future disease monitoring and control in the target population.
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Hughes and Totten (2003) proposed that the sensitivity of PCR assays should be
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specified as a function of the number of target DNA molecules present. However, in field
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samples the concentration of target DNA is unknown and estimates of sensitivity and
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specificity of the test used are not functions of concentration but rather averages over the
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range of possible concentrations which occur biologically within the subjects being sampled.
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An extensive body of literature exists on methods of validation for diagnostic tests in the
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absence of a gold standard reference test. Hui and Walter (1980) defined the necessary
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conditions for test sensitivities and specificities to be estimated using maximum likelihood
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methods. Later additions include Bayesian approaches (Joseph et al., 1995) and allowance for
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covariance between tests (Dendukuri and Joseph, 2001). The use of non-gold standard
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methods, particularly Bayesian methods, in diagnostic testing features heavily in modern
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veterinary epidemiology (Enoe et al., 2000).
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In this study, our primary objective was to estimate the accuracy of the JSRV PCR when
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applied to the Scottish sheep flock. A secondary objective was to present a novel statistical
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approach for estimating sensitivity and specificity of a diagnostic test in the absence of a gold
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standard reference test, using laboratory replicates to increase the amount of data available for
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analysis.
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Materials and methods
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Data
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Data were collected from a representative random sample of 125 Scottish sheep flocks.
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Study farms were stratified by the Scottish Government Animal Health Division to take
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account of the distribution of sheep flocks in different Scottish regions (see Supplementary
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material, Appendix A). Only flocks with at least 50 breeding ewes were eligible to take part in
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the study. In each flock, blood was collected from a random sample of animals, typically 27
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sheep, and each blood sample was subsequently tested for the presence of JSRV proviral
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DNA using a hemi-nested PCR (Palmarini et al., 1996), except that 800 ng DNA were used
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per replicate and the second round was a Taqman PCR using the carboxyfluorescein (FAM)
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labelled probe 5’-AGCAAACATCCGAGCCTTAAGAGCTTTC-3’ using an Applied
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Biosystems SDS7000.
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Samples from each flock were tested separately and comprised three replicate aliquots
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from each blood sample, along with a set of three positive controls of varying JSRV DNA
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concentrations (each with one aliquot) and typically four negative control samples (each with
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three replicate aliquots). The negative controls were from differing sources, namely, cow
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blood, Icelandic sheep blood, distilled water and a buffer solution. A total of 499 negative
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control samples were available, each with three replicate aliquots; samples from one flock
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were tested with three rather than four negative controls.
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Table 1 summarises the test results of the negative controls and field samples. We
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ignored the source of the negative control samples, as there was no evidence to suggest any
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differences associated with source in the mean proportion of replicates falsely testing positive.
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A total of 121 positive control samples was included in the analysis. Table 2 provides a
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summary of the positive control data.
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Statistical method
In the analysis of field samples three issues were relevant to statistical estimation of the
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sensitivity and specificity of the JSRV PCR. Firstly, test results from individual blood
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samples were not validated against a gold standard reference test to determine the true status
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of each sample. Secondly, replicate aliquots were available from each blood sample, which
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increased the amount of data available; however, these results could not be assumed to be
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independent and therefore an appropriate adjustment was needed to correct for correlations
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among replicates. Finally, the probability of a flock being free from the infectious agent (i.e.
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the within flock prevalence can equal zero with non-zero probability) needed to be accounted
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for. To accommodate each of these complications, we used a Bayesian non-gold standard
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latent variable model (Enoe et al., 2000), with conditional dependence between replicates
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from the same sample (Dendukuri and Joseph, 2001), where the latent variable denoting
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within flock JSRV prevalence has a mixture distribution (Branscum et al., 2004).
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Our study was based on a single diagnostic test with conditionally dependent replicates
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and was considered to be a special case based on the approach of Dendukuri and Joseph
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(2001), with the sensitivity and specificity being the same in each test. The observed data
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within a single flock were modelled using a multinomial distribution, which defines the
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probability of observing animals with zero, one, two or three positive replicates, given a fixed
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total number of animals sampled (see Supplementary file).
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The statistical model allowed the prevalence of JSRV to vary between flocks and we
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estimated sensitivity and specificity across all flocks. The likelihood function for a single
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flock is multinomial and the likelihood function for all flocks in our study is the product of
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the likelihood functions for individual flocks, where we allowed the prevalence of JSRV in
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each flock to vary independently (see Supplementary file). We used a Bayesian model with
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uninformative priors for all parameters and fitted the model using JAGS, an open source
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software package for running Markov chain Monte Carlo analyses similar to WinBUGS (see
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Supplementary file).
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In the analysis of control samples we estimated the sensitivity and specificity of the test
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when applied to the JSRV positive and JSRV negative control samples. These control samples
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were primarily used as quality assurance checks during the laboratory testing process;
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however, they also provided potential bounds on the accuracy of the test when applied to
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samples of unknown status. Analysis of the negative control samples followed the same
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method as for the field samples, since they were distinct samples with three replicates each,
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with the knowledge that the true status of the sample was negative.
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The positive control samples required a different approach, since we had no replicates
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but rather a single sample at three different dilutions. We adopted the parametric approach of
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Hughes and Totten (2003), which discriminated between ‘observed’ sensitivity and ‘true’
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sensitivity. The former includes test error due to (1) the aliquot under study contains no copies
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of the target DNA sequence; or (2) although target DNA is present, the PCR fails to amplify
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the DNA. It is argued that ‘true’ sensitivity only includes the error associated with (2) and that
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sensitivity should be a function of the number of target DNA molecules. The observed
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sensitivity may be estimated using standard methods, such as logistic regression with dilution
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as a covariate. In contrast, estimating true sensitivity requires certain probabilistic
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assumptions, e.g. the number of DNA molecules follows a Poisson distribution (see
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Supplementary file).
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Results
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Field samples
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Fitting our statistical model to the field data, we estimated that sensitivity (S) of the PCR
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had a posterior median of 0.107 and a 95% CI of 0.077-0.152. In contrast, we found that the
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test was highly specific, with a posterior median for specificity (C) of 0.997 (95% CI 0.996-
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0.999). Estimates of the posterior densities for S and C are illustrated in Fig. 1. The estimated
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covariance within sample replicates was low, with a median of 2.59 x 10-3 when JSRV was
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present (covs) and a median of 3.63 x 10-6 when JSRV was absent (covc).
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Control samples
Fitting our statistical model to the negative control samples (Table 1), the estimated 95%
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CI was 0.982-0.993 for S and 1.04 x 10-6 to 1.41 x 10-4 for covc. Using the method of Hughes
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and Totten (2003) for estimating the true S of the test on the positive control samples, median
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S estimates for mean concentrations of 1, 6, 12.5 and 25 target DNA molecules per 25 µL
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were 0.160, 0.648, 0.886 and 0.987, respectively. In contrast, the raw observed S of the test
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using the data in Table 2 were 0.793, 0.884 and 0.901 for mean concentrations of 6, 12.5 and
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25 target DNA molecules per 25 μL, respectively. The method also allows for explicit
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estimation of C; however, given that we had median estimates for C from both the field
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samples and negative control samples in excess of 0.99, we assumed that the probability of
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observing a false positive is zero.
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Fig. 2 shows estimates of the posterior density for S at the three observed concentrations,
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plus extrapolation when the mean concentration is one copy of target DNA sequence in 25
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μL. A key parameter in the mechanistic model used by Hughes and Totten (2003) is the
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probability that each of the target molecules in the sample being tested fails to escape
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amplification by PCR, where we estimate this to be 0.160. As we have assumed that the
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specificity is 1.0, then this is equivalent to the S when the mean concentration is 1.0 target
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DNA sequence per 25 μL.
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Summary probabilities
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Table 3 contains summary statistics for each of the eight independent conditional
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probabilities estimated from our model. These summarise the probabilities of observing zero
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or more positive replicates conditional on whether JSRV infection is truly present. The
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probability of observing one or more positive replicates is considerably larger when JSRV is
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truly present, as should be expected.
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Discussion
We estimated that the median sensitivity of the JSRV PCR was 0.107 per individual
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replicate, where this accounted for covariance between replicates from the same sample. We
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found that the covariance between replicates was low, which was unsurprising, given the
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observed data: out of a total 3,361 sets of triple replicates, only 106 had one positive replicate,
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14 had two positive replicates and four had three positive replicates. Hence, there is little
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obvious covariance between positive replicates, even from positive animals.
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Considering the performance of the PCR when applied to control samples it was
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interesting to note that estimates of C using the field samples were slightly higher than those
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derived from the negative controls. This could in part be explained by the fact that S and C in
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the field data were negatively correlated. This is to be expected, since S + C must be greater
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than 1 to have a ‘legitimate’ (better than random guessing) diagnostic test. In our modelling,
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we assumed uninformative independent priors for S and C. An alternative would be to
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explicitly model this correlation using a joint prior, as for Chu et al. (2006).
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For the positive control samples, we found that the estimates of observed S were lower
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than estimates of true S; this was to be expected, since the former also includes the error due
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to individual samples not containing any target DNA. Table 3 showed an alternative and
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potentially more informative assessment of test accuracy. The probability of observing no
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positive test replicates, given that JSRV is truly present within the blood sample, is in excess
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of 0.70. If we define a positive test result as where any one of the available three replicates is
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positive, then we can approximate the sensitivity of the test across all three replicates by
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summing the individual probabilities in rows 3, 5 and 7 in Table 3, giving a median observed
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sensitivity of 0.279. This result is in line with observations in previous studies, where PCR
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results based on blood samples were compared with other diagnostic procedures (Gonzalez et
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al., 2001; De las Heras et al., 2005; Voigt et al., 2007).
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The primary objective of our modelling was to estimate the diagnostic accuracy of the
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JSRV PCR when applied to the Scottish sheep flock. A necessary and interdependent part of
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this process is estimation of the prevalence of JSRV within each flock sampled. We explicitly
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allowed the prevalence of JSRV to vary independently within each of the study flocks. If,
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instead, the primary goal of our study was estimation of prevalence and, in particular, at
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regional or national flock level, then a natural extension to our model would be to incorporate
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it into the hierarchical framework of Branscum et al. (2004).
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In previous work, the study population consisted largely of animals which were likely to
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be in early and late stages of OPA; these studies concluded that the ‘observed’ sensitivity in
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preclinical animals is considerably lower than in animals with clinical OPA (De las Heras et
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al., 2005; Voigt et al., 2007). Samples for the current study were taken from a random
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selection of Scottish sheep; the vast majority of these animals were clinically healthy. The test
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characteristics assessed in this validation based on a representative field sample would
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therefore be applicable for prevalence studies or diagnostic screening of clinically healthy
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sheep.
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We estimated the ‘true’ sensitivity of the JSRV PCR as a function of the number of
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target DNA molecules present using the associated spiked positive control samples to assess
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the detection limit of the PCR. There were two key findings from this analysis. Firstly, we
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estimated a median sensitivity of 0.160 when the mean number of target DNA molecules per
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25 μL is 1.0; this observation is indicative of a high technical performance of the PCR assay
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and a good ‘true’ sensitivity. Secondly, comparing the estimate of 0.160 with the median
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sensitivity from field samples of 0.107 strongly suggests that the concentrations of target
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DNA in the latter are generally very low. The fact that only a few samples from infected
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animals tested positive in more than one replicate leads to the same conclusion and hence
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potentially explains the low ‘observed’ sensitivity.
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We used a non-gold standard method to validate the diagnostic test. The use of non-gold
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standard methods in practice requires considerable care, e.g. requiring sufficient observations
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to enable robust estimation (Toft et al., 2005). In the work presented, we required data from at
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least four flocks, each with different levels of prevalence, in order to calculate test
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characteristics (see Supplementary file).
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The JSRV PCR assay was assessed to have a generally low ‘observed’ sensitivity when
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used with blood samples from clinically healthy sheep. Therefore, improvements and
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adjustments would be necessary should the test become part of routine diagnostic
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investigations. Theoretically, the assay could be further enhanced, but, given that various
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technical refinements have been implemented in the past, the PCR seems to have reached
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what is currently possible with state of the art technology. On an individual animal level, the
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‘observed’ sensitivity could be improved by testing specimens which have a higher
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concentration of JSRV proviral DNA in infected animals, e.g. bronchoalveolar lavage
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samples. An obvious next step is estimation of the flock level sensitivity of the test when
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applied to the Scottish sheep flock. This is, however, a considerably more complex task, since
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flock level sensitivity depends jointly on the accuracy of the JSRV PCR assay at individual
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animal/replicate level, the number of animals sampled from within each flock and, crucially,
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the distribution of within flock prevalence of JSRV in the population under study.
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Conclusions
Using non-gold standard methods, which make use of laboratory replicates to maximise
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available data, the JSRV PCR was assessed to have a high ‘true’ sensitivity and low
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‘observed’ sensitivity, where the latter can be explained by the low concentration of JSRV
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proviral DNA in the blood of infected sheep. The analytical method presented is generic and
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applicable to diagnostic test validation when repeated measurements are available.
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Conflict of interest statement
None of the authors has any financial or personal relationships that could
inappropriately influence or bias the content of the paper.
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Acknowledgements
Fieldwork and data collection were funded as part of RERAD grant MRI843/04 awarded
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to MRI, BioSS and SAC. Statistical analyses were undertaken by the Scottish Government
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Centre of Excellence in epidemiology, population health and infectious disease control
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(EPIC). Leenadevi Thonur and Joanne Crawford performed all PCR assays. Biomathematics
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and Statistics Scotland, Moredun Research Institute and the Scottish Agricultural College all
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receive financial support from the Scottish Government (RERAD).
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found in the online version, at
doi:….
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Table 1
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Observed test results from field and negative control samples
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Number of positive
Number of field
Number of negative
replicates
samples
control samples
0
3237
486
1
106
11
2
14
2
3
4
0
Total
3361
499
384
385
Each observation is the number of replicates out of a total of three which tested positive for
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JSRV.
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Table 2
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Observed test results from positive control samples
389
390
Mean number of
Number of
Number of samples
DNA plasmids
samples
positive for JSRV
6
121
96
12.5
121
107
25
121
109
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Table 3
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Posterior estimates of test accuracy applied to field samples.
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Parameter
Median
95% Confidence interval
P (0; I+) a
7.21 x 10-1
6.24 x 10-1;7.92 x 10-1
P (0; I-)
9.92 x 10-1
9.87 x 10-1;9.96 x 10-1
P (1; I+)
2.48 x 10-1
1.92 x 10-1;3.14 x 10-1
P (1; I-)
7.61 x 10-3
3.82 x 10-3;1.25 x 10-2
P (2; I+)
2.29 x 10-2
1.14 x 10-2;4.67 x 10-2
P (2; I-)
8.59 x 10-6
3.81 x 10-7;3.61 x 10-5
P (3; I+)
8.16 x 10-3
2.61 x 10-3;2.13 x 10-2
P (3; I-)
8.39 x 10-6
3.54 x 10-7;3.58 x 10-5
394
395
a
396
infection negative for JSRV, respectively.
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Number of positive replicates; Infection status. I+ and I- denote infection positive and
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Figure legends
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Fig. 1. Posterior density estimates for test sensitivity and specificity against field samples
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from 125 flocks.
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Fig. 2. Posterior density estimates for test sensitivity (S) using positive control samples at
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mean concentrations of 1.0, 6.0, 12.5 and 25 target DNA molecules per 25 μL.
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