BackgroundThe objective was to evaluate the diagnostic accuracy of two serum ELISAs and one quantitative PCR on feces for the diagnosis of paratuberculosis in sub-clinically infected young-adult sheep. A cross-sectional study was performed to collect 1197 individual blood and fecal samples from 2- to 3-year-old sub-clinically infected ewes in 14 closed meat sheep flocks in France. Fecal excretion was determined using qPCR based on IS900 sequence detection, and serology was performed on serum samples using two commercial ELISAs. Data were analyzed in a 3-test multiple-population Bayesian latent class model accounting for potential dependence between the three tests fitted in OpenBUGS. Separate analyses were performed according to whether doubtful ELISA results were handled as positive or negative and based on two thresholds for fecal qPCR (Ct ≤ 42 or Ct ≤ 40).ResultsThe best fit to the data was provided by accounting for a pairwise dependence between the two ELISAs on sensitivity and pairwise dependence between the three tests on specificity. Under this model, the estimated ELISA sensitivities were 17.4% (95% PCI: 10.6 – 25.9) and 17.9% (95% PCI 11.4 – 25.6), with estimated specificities of 94.8% (95% PCI: 93.1 – 96.3) and 94.0% (95% PCI: 92.2 – 95.7). Fecal qPCR demonstrated significantly higher sensitivity (47.5%; 95% PCI: 29.3 – 69.9) and specificity (99.0%; 95% PCI: 97.9 – 99.9) than the ELISAs. Assumptions regarding doubtful ELISA results and qPCR thresholds had only a slight impact on test accuracy estimates. Models not accounting for pairwise dependence between ELISA and fecal qPCR results yielded higher sensitivity and specificity estimates but always provided a worse fit to the data.ConclusionsAlthough the overall sensitivity of serum ELISAs and fecal qPCR remains low, the higher diagnostic performances of fecal qPCR make it more suitable for paratuberculosis diagnosis in sub-clinically infected sheep. Our results also illustrate that all dependence structures should be investigated when evaluating diagnostic test accuracy and selection based on a rigorous statistical approach.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-017-1145-x) contains supplementary material, which is available to authorized users.
The aim of our study was to evaluate the flock sensitivity and specificity of fecal qPCR and serum ELISA using pooled samples for screening paratuberculosis in French sheep.Using individual feces with low or high qPCR Ct values from ewes sampled in 14 infected flocks, a total of 555 pools of size 5, 10 and 20 were created by diluting individual materials in negative feces and analysed using a commercial IS900 qPCR kit. The relative performances of pooled serum ELISA analysis were evaluated based on the analysis of 181 different pools of size 5 and 10, composed of individual serum samples of various individual S/P values. Results showed that for pools of size 5, 10 or 20, individual fecal samples with low Ct values were invariably detected. Conversely fecal samples with high Ct values were associated with a lower detection rate in both pools of size 5 (87.0% to 90.0%), 10 (63.0% to 70.7%) and 20 (46.7% to 60.0%). After lowering the decision threshold to 25% and 15% for serum pools of size 5 and 10 respectively, the pooled serum ELISA relative sensitivity ranged between 62.2% and 100.0% depending on the composition of the pools.Finally, a simulation study was carried out to evaluate the performances of 16 screening strategies at flock level, with varying pool size (5 to 20) and number (5 to 60). The use of pooled serum ELISA led to very false positive detection rate ranging between 37.6% and 91.8% in paratuberculosis free flocks and prevents its further use in that context. For infection prevalence ≤ 5%, the flock sensitivity based on pooled fecal qPCR ranged between 39.0% (5 pools of size 10) and 99.9% (300 sampled individuals, with pools of size 5,10 or20), and was always above 93% when the infection prevalence was greater or equal to 15%. We conclude that pooled-fecal qPCR but not pooled-serum ELISA could be a useful tool to detect sheep flocks infected with paratuberculosis.
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