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. 2022 Nov 28;16(11):e0010944.
doi: 10.1371/journal.pntd.0010944. eCollection 2022 Nov.

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study

Ramona Tavares Daltro  1 , Emily Ferreira Santos  1 , Ângelo Antônio Oliveira Silva  1 , Natália Erdens Maron Freitas  1 , Leonardo Maia Leony  1 , Larissa Carvalho Medrado Vasconcelos  1 , Alejandro Ostermayer Luquetti  2 , Paola Alejandra Fiorani Celedon  3 , Nilson Ivo Tonin Zanchin  4 , Carlos Gustavo Regis-Silva  1 , Fred Luciano Neves Santos  1   5
Affiliations

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study

Ramona Tavares Daltro et al. PLoS Negl Trop Dis. .

Abstract

Background: Chagas disease (CD) is caused by Trypanosoma cruzi. The chronic phase of CD is characterized by the presence of IgG anti-T. cruzi antibodies; and diagnosis is performed by serological methods. Because there is no reliable test that can be used as a reference test, WHO recommends the parallel use of two different tests for CD serodiagnosis. If results are inconclusive, samples should be subjected to a confirmatory test, e.g., Western blot (WB) or PCR. PCR offers low sensitivity in the chronic phase, whereas few confirmatory tests based on the WB method are commercially available worldwide. Therefore, new diagnostic tools should be evaluated to fill the gap in CD confirmatory tests. In recent years, four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) have been evaluated in phase I, II and III studies using ELISA, liquid microarray and immunochromatography with 95-100% accuracy. Given the high diagnostic performance of these antigens, the present study investigated the ability of these molecules to diagnose chronic CD using a WB testing platform.

Methodology/principal findings: In this study, we analyzed the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis (i.e., potentially cross-reactive) using WB as the diagnostic platform. Checkerboard titration with different dilutions of antigens, conjugated antigens, and serum samples was performed to standardize all assays. All IBMP antigens achieved 100% sensitivity, specificity, and accuracy, with the exception of IBMP-8.3, which had 100% specificity despite lack of significance, but lower sensitivity (95%) and accuracy (96.9%). No cross-reactivity was observed in samples positive for leishmaniasis.

Conclusions/significance: The present phase I (proof-of-concept) study demonstrated the high diagnostic potential of these four IBMP antigens to discriminate between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with WB.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flowchart illustrating study design in conformity with the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines.
Source base layer and credit base layer: https://data.humdata.org/ published under creative commons attribution for intergovernmental organizations: https://data.humdata.org/dataset/geoboundaries-admin-boundaries-for-brazil.
Fig 2
Fig 2. Relative band intensity (RBI) and diagnostic performance parameters obtained from Trypanosoma cruzi-positive (Tc-Pos) and T. cruzi-negative (Tc-Neg) serum samples.
The cut-off value was set at a reactivity index value of 1.0, with the shaded area representing the grey zone (i.e., an indeterminate result; RI = 1.0 ± 0.10). Horizontal lines and numbers for each result group represent geometric means (± 95%CI). GSD represents geometric standard deviation.
Fig 3
Fig 3. Photographic images of nitrocellulose test strips result from Trypanosoma cruzi-positive samples.
Relative band intensity (RBI) values respective to each IBMP antigen are shown below the images pertaining to each sample. Abbreviations: AL (Alagoas); AP (Amapá); BA (Bahia); GO (Goiás); MG (Minas Gerais); PE (Pernambuco).
Fig 4
Fig 4. Photographic images of nitrocellulose test strips result from Trypanosoma cruzi-negative samples.
Relative band intensity (RBI) values respective to each IBMP antigen are shown below the images pertaining to each sample. Abbreviations: AL (Alagoas); AP (Amapá); BA (Bahia); GO (Goiás); MG (Minas Gerais); PE (Pernambuco).
Fig 5
Fig 5. Analysis of cross-reactivity using chimeric Trypanosoma cruzi proteins to diagnose sera from visceral leishmaniasis patients.
Relative band intensity (RBI) values for each sample are shown below the images.
See this image and copyright information in PMC

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This work was supported by the Coordination of Superior Level Staff Improvement (CAPES; Finance Code 001 to FLNS, 88887.637869/2021-00 to LCMV, 88887.509223/2020-00 to LML, 88887.637641/2021-00 to RTD, and 88887.637758/2021-00 to NEMF), Research Support Foundation of the State of Bahia (FAPESB; BOL0543/2020 to ÂAOS and BOL0413/2021 to EFS), and Inova Fiocruz/VPPCB (FICORUZ; grant number VPPCB-008-FIO-18-2-20 to FLNS). NITZ and FLNS are research fellows at National Council for Scientific and Technological Development (CNPq; process no. 304167/2019-3 and 309263/2020-4, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.