Usefulness of four different Echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (UHD) and postsurgical follow-up of patients treated for UHD

Abstract

Four different recombinant antigens derived from Echinococcus granulosus, designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories.

FIG. 1.

Recombinant proteins B1t and B2t. (a) The coding regions of these proteins were obtained by RT-PCR of E. granulosus cDNA. Primers were based on the nucleotide sequences from antigens B1 and B2 available in GenBank (accession no. AF143813 and U15001, respectively) to amplify the sequence encoding the C-terminal portion of antigens B1 and B2 (white boxes) but not the coding region for the putative signal peptides from both sequences (black boxes). Numbers indicate amino acid positions. (b) The resulting RT-PCR products encoding the proteins B1t and B2t were subcloned in the pGEX-4T1 vector and expressed in E. coli BL21 CodonPlus RIL cells. (c) The purified proteins after thrombin cleavage are shown in a 15% acrylamide Coomassie blue-stained gel. Lane 1, molecular mass markers; lane 2, B1t protein; and lane 3, B2t protein. Relative molecular masses are shown to the left of the gel.

FIG. 2.

Comparative analysis between the diagnostic performances of B1t and B2t recombinant proteins in IgG ELISA with a panel of defined sera. (a) The number of positive and negative (+/−) sera against B1t or B2t antigen. (b) ROC curves used to determine AUC and cutoff values for the B1t and B2t antigens. (c) The calculated AUCs ± standard errors (se), confidence intervals (CI), cutoffs, sensitivity, and specificity for each antigen are shown.

FIG. 3.

Applicability of B2t, E14t, and C317 recombinant proteins for the diagnosis of UHD by IgG ELISA compared to that of HF with a panel of defined sera. (a) Number of positive and negative (+/−) sera against HF, B2t, E14t, and C317 antigens. (b) ROC curves used to determine AUC and cutoff values for the HF, B2t, E14t, and C317 antigens. (c) The calculated AUCs ± standard errors (se), confidence intervals (CI), cutoffs, sensitivity, and specificity for each antigen are shown. Positive and negative predictive values (PPV and NPV, respectively) also are shown.

FIG. 4.

Influence of cyst location on the diagnostic performance of HF, B2t, E14t, and C317 antigens by IgG ELISA with a panel of defined sera. (a) Number of positive and negative (+/−) sera against HF, B2t, E14t, and C317 antigens. (b) ROC curves used to determine AUC and cutoff values for the HF, B2t, E14t, and C317 antigens in the detection of liver (dashed lines) or lung (black lines) cysts. (c) The calculated AUCs ± standard errors (se), confidence intervals (CI), and statistical significances (P) are shown. Differences are considered statistically significant for P values of <0.05.

FIG. 5.

Comparative performance of HF and B2t antigens in IgG ELISA for the follow-up of the treatment of UHD and for determining whether the patients are cured of UHD. (a) Time intervals after surgery, number of sera for each time interval, and number of positive and negative (+/−) sera against the HF and B2t antigens at each time interval. (b) Percentage of positive sera against HF (dotted line) and B2t (black line) antigens at each time interval after surgery. m, months; y, years.