Accuracy of chimeric proteins in the serological diagnosis of chronic chagas disease – a Phase II study

Abstract

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.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays

“…Here, we expanded the sample size to 1,333 sera and found AUC values higher than 99% for all 4 proteins. These data are in accordance with results from a phase II study, where these same proteins were tested by ELISA (16), thereby indicating the high discriminative power these antigens potentially possess with respect to other diagnostic platforms. Most importantly, these IBMP chimeric proteins provided much better AUC values than did T. cruzi cell lysates, single recombinant proteins, or other recombinant chimeric proteins commonly used in diagnostic kits (17,18).…”

“…Nonetheless, this difference was almost negligible, considering that the 95% CI values practically overlapped. LMA assay performance was comparable to previously published data with ELISAs (16). With the exception of the IBMP-8.2 antigen, both testing methods offered similar performance.…”

“…In addition, differences greater than 4.40 were seen between the RI signals from the positive and negative samples for all proteins, providing further evidence of their high discriminatory capability. Moreover, the RI signals obtained from positive samples assayed by LMA were up to 56% stronger than those previously obtained by ELISA (16). Conversely, the average RI signals from negative samples were 32% lower by LMA.…”

“…When evaluated by LMA, the IBMP-8.2 protein showed 99.0% accuracy, while it showed 96.6% accuracy by ELISA. According to a previous study, the lower value obtained by ELISA was probably due to the amino acid sequence of this protein, which impaired its recognition by specific anti-T. cruzi antibodies from CD-positive samples collected in distinct geographical regions (16). However, this discrepancy in accuracy may also be the result of characteristics inherent to each diagnostic platform used.…”

“…In addition, no significant differences were observed with respect to the diagnostic performances of the ELISA and LMA test methods. Data from a subsequent phase II study confirmed the high performance of these proteins in ELISAs (16). In the present study, we aimed to assess the diagnostic performance of the IBMP chimeras to diagnose CD using LMA in singleplex and multiplex formats.…”