The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

Objectives: To obtain and assess stable cage-like particles with low surface charge density, which can be prepared using a standardized, economic and scalable method. Methods: To form these nanoparticles, the lipid composition and proportion as well the method were modified in relation to cage-like particles previously described elsewhere. Bovine albumin was used to compare ISPA performance with that of other adjuvants in mice and to assess stability. Adjuvant efficacy was analysed using a mouse model of Trypanosoma cruzi infection, which shows protection against an intracellular infection that needs a strong cellular response. Key findings: The new particles were better in terms of level, kinetics and profile of humoral responses than Freund Adjuvant, aluminium hydroxide and Montanide TM ISA 206; they also tended to improve ISCOMATRIX™ performance. Particle size and adjuvant performance were conserved during the 6-month period assessed after preparation. In the model of Trypanosoma cruzi infection, mice immunized with ISPA and trans-sialidase developed high protection. Conclusions: The obtained nanoparticles were stable and outperformed the other assessed adjuvants in joining together the capacity of most adjuvants to enhance the immune response against specific antigen, to reduce the number of doses, to homogenize the response between individuals and to reach a balanced TH1/TH2 response.

We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe.

Chagas' disease is routinely diagnosed by detecting specific antibodies (Abs) using serological methods. The methodology has the drawback of potential cross-reactions with Abs raised during other infectious and autoimmune diseases (AID). Fusion of DNA sequences encoding antigenic proteins is a versatile tool to engineer proteins to be used as sensitizing elements in serological tests. A synthetic gene encoding a chimeric protein containing the C-terminal region of C29 and the N-terminal region of TcP2β was constructed. A 236-serum panel, composed of 104 reactive and 132 nonreactive sera to Chagas' disease, was used to evaluate the performance of the chimera. Among the nonreactive sera, 65 were from patients with AID (systemic lupus erythematosus and rheumatoid arthritis) or patients infected withLeishmania brasiliensis,Brucella abortus,Streptococcus pyogenes, orToxoplasma gondii. The diagnostic performances of the complete TcP2β (TcP2β_FL) and its N-terminal region (TcP2β_N) were evaluated. TcP2β_FLshowed unspecific recognition toward leishmaniasis (40%) and AID Abs (58%), while TcP2β_Nshowed no unspecific recognition. The diagnostic utility of the chimera was evaluated by analyzing reactivity and comparing the results with those obtained with TcP2β_N. The chimera reactivity was higher than that of the peptide fractions (0.874 versus 0.564 optical density,P= 0.0017). The detectability and specificity were both 100% for the whole serum panel tested. We conclude that the obtained chimera shows an improved selectivity and sensitivity compared with other ones previously reported, therefore displaying an optimized performance forTrypanosoma cruziinfection diagnosis.

This work deals with the use of predictors to identify useful B-cell linear epitopes to develop immunoassays. Experimental techniques to meet this goal are quite expensive and time consuming. Therefore, we tested 5 free, online prediction methods (AAPPred, ABCpred, BcePred, BepiPred and Antigenic) widely used for predicting linear epitopes, using the primary structure of the protein as the only input. We chose a set of 65 experimentally well documented epitopes obtained by the most reliable experimental techniques as our true positive set. To compare the quality of the predictor methods we used their positive predictive value (PPV), i.e. the proportion of the predicted epitopes that are true, experimentally confirmed epitopes, in relation to all the epitopes predicted. We conclude that AAPPred and ABCpred yield the best results as compared with the other programs and with a random prediction procedure. Our results also indicate that considering the consensual epitopes predicted by several programs does not improve the PPV.