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

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.

Before: "Until 2017, TESA-blot was routinely used for confirmatory testing [30]; however, commercial production has since been discontinued, leaving a gap in the performance of confirmatory CD diagnosis." After (line 138-143): Until 2015 and 2017, respectively, HBK 740 Immunoblot Linhas anti-T. cruzi and TESA-blot were routinely used for confirmatory testing [32]; however, commercial productions have since been discontinued, leaving a gap in the performance of confirmatory chronic CD diagnosis. However, two other WB are commercially available worldwide. The Chagas blot and Abbott ESA Chagas, whose use is limited to Europe and the USA, respectively.

Discussion
Before: No text. After (line 368-376): Similar to the TESAcruzi, the Chagas Western blot IgG assay (Chagas blot) uses a complex mixture of antigenic molecules to coat a nylon strip. This assay is manufactured by a French company (LDBio Diagnostics) and uses native trypomastigote and amastigote antigens from the CL Brener T. cruzi strain (TcIV). In 2021, a study using samples from T. cruzi-positive individuals living in endemic areas of Argentina [49] showed that this kit has great potential for use as a confirmatory test (sensitivity and specificity of 100%). However, the authors note that these results must be confirmed in large tests with many sera from different regions of South America before this immunoblot can be considered a universal confirmatory test.
Before: "However, to date no studies have evaluated the use of chimeric proteins as an antigenic matrix in a WB-based diagnostic immunoassay platform. The diagnostic performance obtained here using WB corroborates our previous findings obtained using a variety of diagnostic platforms [16,17,54,[18][19][20][21][22][51][52][53]. Indeed, all four chimeric proteins achieved 100% sensitivity and specificity, with the exception of IBMP-8.3 (95% sensitivity)." After (line 385-397): However, to date few studies have evaluated the use of chimeric proteins as an antigenic matrix in a WB-based diagnostic immunoassay platform. In 2010, the Abbott ESA Chagas, an immunoblot based on 4 chimeric recombinant T. cruzi antigens (FP10, FP6, FP3, and TcF) [52], was evaluated using samples from different groups of T. cruzi infected and uninfected individuals. The high clinical and analytical sensitivity values as well as the simplicity of the method led the authors to conclude that the Abbott ESA Chagas could replace RIPA as the confirmatory test of choice for the detection of antibodies to T. cruzi [54]. In November 2011, the Abbott ESA Chagas was approved by the US FDA for the confirmation of blood donors who are repeatedly reactive in Chagas screening tests. The diagnostic performance obtained here using WB corroborates our previous findings obtained using a variety of diagnostic platforms [16][17][18][19][20][21][22][55][56][57][58]. Indeed, all four chimeric proteins achieved 100% sensitivity and specificity, with the exception of IBMP-8.3 (95% sensitivity).
To accommodate the editor's suggestions, we have added a new reference to the manuscript: Introduction Before: "Following WHO recommendations, around 5% of samples submitted to dual-serological assay testing return discordant or doubtful results (values falling within the cutoff range or indeterminate zone). While initial testing can be repeated, in some cases a third, preferably confirmatory test has also been used…" After (line 120-124): Following WHO recommendations, around 5% of samples submitted to dualserological assay testing return discordant or doubtful results (values falling within the cutoff range or indeterminate zone). Some studies have reported similar values ranging from 2.9% to 3.3% [23,24]. While initial testing can be repeated, in some cases a third, preferably confirmatory test has also been used…

Discussion
Before: "Several researchers have investigated the usefulness of WB in the confirmatory diagnosis of CD when conventional tests returned inconclusive results, reporting high values of sensitivity and specificity [40][41][42][43]. In 1986, a study used WB to assess the performance of epimastigote antigens in diagnosing CD; however, the low purity of the antigens employed resulted in cross reactions against anti-Leishmania braziliensis and anti-Leishmania donovani [44]." After (line 339-347): Several researchers have investigated the usefulness of WB in the confirmatory diagnosis of chronic CD when conventional tests returned inconclusive results, reporting high values of sensitivity and specificity [42][43][44][45]. The development a methodology to clarify results that are inconclusive after using conventional assays is extremely necessary because at least 50% of discordant results are from individuals affected by Chagas disease [23]. In 1986, a study used WB to assess the performance of epimastigote antigens in diagnosing chronic CD; however, the low purity of the antigens employed resulted in cross reactions against anti-Leishmania braziliensis and anti- Before: "Until 2017, TESA-blot was routinely used for confirmatory testing [30]; however, commercial production has since been discontinued, leaving a gap in the performance of confirmatory CD diagnosis." After (line 138-143): Until 2015 and 2017, respectively, HBK 740 Immunoblot Linhas anti-T. cruzi and TESA-blot were routinely used for confirmatory testing [32]; however, commercial productions have since been discontinued, leaving a gap in the performance of confirmatory chronic CD diagnosis. However, two other WB are commercially available worldwide. The Chagas blot and Abbott ESA Chagas, whose use is limited to Europe and the USA, respectively.

Discussion
Before: No text. After (line 368-376): Similar to the TESAcruzi, the Chagas Western blot IgG assay (Chagas blot) uses a complex mixture of antigenic molecules to coat a nylon strip. This assay is manufactured by a French company (LDBio Diagnostics) and uses native trypomastigote and amastigote antigens from the CL Brener T. cruzi strain (TcIV). In 2021, a study using samples from T. cruzi-positive individuals living in endemic areas of Argentina [49] showed that this kit has great potential for use as a confirmatory test (sensitivity and specificity of 100%). However, the authors note that these results must be confirmed in large tests with many sera from different regions of South America before this immunoblot can be considered a universal confirmatory test.

Reply:
We thank the reviewer for this comment. We used a miniblotter system with a constant width of the strips: 4 mm. For more details (if needed), we kindly ask the reviewer to read the data sheet available at https://www.interchim.fr/ft/B/BA369c.pdf. To clarify the width of the strips, we have added a new explanation to the Western Blot Assay subsection in the Materials and Methods section, which reads as follows.

Discussion
Before: "…a smaller amount of antigen was used: 12.5 ng for each of the four…" After (line 416): …a smaller amount of antigen was used: 12.5 ng/4 mm for each of the four…

Conclusion
The study was carried out nicely, with no major concerns. The main concern about this study is that the authors wrote the study results as a phase I study while the authors in 2019 (lines 402-404) evaluated the four IBMP antigens using 600 cutaneous and 229 visceral leishmaniasis samples. These IBMP antigens have been evaluated extensively by authors in ELISA format, so using these antigens in western blot format should be reported as a full report and not as a proof-of-concept or phase 1 study. In a different issue, the study used the best platform of Western Blot using chemiluminescent substrate and using the best reader. It is interesting to test the performance of the assay in different laboratories using the most available substrate for the Western Blot as the goal of this study is to fill the gap on the lack of confirmatory commercial chronic CD diagnosis.