The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

BackgroundThe performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results.Methodology/Principal findingsHere, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible.Conclusions/SignificanceOur findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis.

BackgroundInconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening.MethodsIn order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction.ResultsThe comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 105 to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes – Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples.ConclusionsTaken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.

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Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. , a pathogen with high similarity to, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.