International ring trial to detect anti-Trichinella IgG by ELISA on pig sera

To determine the reproducibility and robustness of an ELISA to detect anti-Trichinella IgG in pig sera which was previously validated at the Community Reference Laboratory for Parasites (CRLP), a ring trial was organized involving European and extra-European reference laboratories for Trichinella. The sensitivity and specificity of the assay determined by the CRLP validation resulted to be 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% and 96.9%, respectively. The analysis of the differences in optical density (OD) between duplicates indicated a high repeatability of the ELISA with about 95% of the differences between −0.16 and 0.17 absorbance units. The accuracy of the test was determined by calculating the area under the ROC curve (AUC). Overall, the ELISA index (I E) showed a very high accuracy (AUC=0.9965) and it performed significantly better than the mean of the duplicated ODs (AUC=0.9387). Of the 21 participating laboratories, nine performed the test without any modification of the original protocol, and 14 with some modifications. Of the laboratories that followed the protocol exactly, three produced false-negatives; whereas of the laboratories that modified the protocol, five produced false-negatives (differences between these two groups of laboratories were not significant, p =0.18). When comparing these two groups of laboratories, the AUCs were very similar (0.9988 and 0.9955, respectively). Finally, a normal mixed multiple model effect was used to evaluate if the I E obtained was only related to the serum or to other parameters such as the laboratory, dilution of the serum tested and application of the proposed protocol. The variability found in the test results was mainly due to the serum samples. The assay proposed is robust and reproducible and can be used for monitoring the lack of Trichinella infection in domestic pigs.