Field evaluation of fluorescence polarization assay, and comparison with competitive ELISA for the detection of antibodies against Brucella melitensis in sheep in Sicily, Italy

The relative sensitivity of the fluorescence polarization assay (FPA) was estimated by testing blood sera from 1515 sheep, belonging to flocks infected with Brucella melitensis in Sicily, which were previously shown as positive to at least two of the following tests: rose bengal test (RBT), complement fixation test (CFT), competitive ELISA (c-ELISA). FPA specificity was estimated by testing sera from 719 sheep from B. melitensis-free flocks from Sicily and Sardinia. Based upon results of a receiver-operating-characteristic (ROC) curve, optimal FPA cut-off value in millipolarization units (mP) was chosen at the mean of negative controls tested in the same assay, plus 6, which corresponded to 96.0% sensitivity (95% confidence interval: 93.9, 97.3) and 100% (99.5, 100) specificity. Such a cut off value was the one yielding the maximum sum of sensitivity and specificity (196.0), and it was adopted for subsequent analyses. Maximum FPA sensitivity (98.8%; 91.7, 99.8) was obtained on sera which were positive to CFT and c-ELISA, but negative to RBT. Median FPA values (first, and third quartiles) (109.7, 53.7, 169.3) were greatest for sera which were positive to RBT, CFT, and c-ELISA. Sensitivity of c-ELISA, which was estimated as the proportion of positive results on RBT and CFT positive sera, was 99.8% (99.4, 1.0), whereas specificity was 100.0% (98.5, 100.0). Although FPA resulted as an accurate test, c-ELISA was characterized by a superior sensitivity and it is, therefore, proposed to be used, in parallel with RBT, to increase the testing sensitivity in regions and flocks which can be considered as at a great risk of infection.