Research Article: Detection of anti-Trypanosoma cruzi antibodies by chimeric antigens in chronic Chagas disease-individuals from endemic South American countries

Date Published: April 18, 2019

Publisher: Public Library of Science

Author(s): Rodrigo Pimenta Del-Rei, Leonardo Maia Leony, Paola Alejandra Fiorani Celedon, Nilson Ivo Tonin Zanchin, Mitermayer Galvão dos Reis, Yara de Miranda Gomes, Alejandro Gabriel Schijman, Silvia Andrea Longhi, Fred Luciano Neves Santos, Walderez O. Dutra.


Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries.

IBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant.

Our findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur.

Partial Text

Chagas’ disease (CD) is a life-threating zoonosis caused by the hemoflagellate protozoan Trypanosoma cruzi. The parasite is transmitted by contact with dejections of infected blood-sucking triatomine bugs, by tissue and organ transplantation, consumption of parasite-contaminated food or beverages, blood transfusion, and from mother-to-child during pregnancy [1]. Although T. cruzi was discovered over a century ago, it is still posing a substantial public health threat, bearing in mind that the vast majority of the affected individuals lack access to treatment and diagnosis. As a matter of fact, CD is considered an essential neglected disease in the Americas [2].

The reactivity index (RI) distributions and assay performance parameters obtained for IBMP-8.1 and IBMP-8.4 chimeras are illustrated in Fig 3 (individual data points are available in the S2 Table). ROC curves were generated from 122 non-infected and 215 T. cruzi-infected individuals assayed by ELISA. Area under the curve (AUC) values were > 99.7%, demonstrating high overall diagnostic accuracy. IgG levels in T. cruzi-positive samples were variable, ranging from 1.64 for IBMP-8.1 to 1.84 for IBMP-8.4. For the panel of T. cruzi-positive samples, IBMP-8.4 chimera produced the highest sensitivity (100%). IBMP-8.1 showed 95.3% sensitivity with 10 cases classified as false-negatives. The differences between these values were statistically significant. Nonetheless, this difference was almost negligible, considering that the 95% CI values practically overlapped. Conversely, no false-positive results were obtained when T. cruzi-negative samples were assayed with IBMP-8.1 and IBMP-8.4, resulting in a specificity score of 100%.

The high genetic and phenotypic intraspecific diversity of T. cruzi is extensively recognized [13]; as it has been demonstrated using different biochemical, immunological and molecular markers [14,15]. Homologous pairs of chromosomes can vary in number and sizes between strains, as well as sequences and copy numbers of many genes, resulting in great genome plasticity [16]. Accordingly, the parasite has been grouped into seven evolutionary discrete typing units (DTUs) termed TcI–TcVI and Tcbat, with sub-classifications for regional strains in clonets and clones [4,17]. Regional parasite genetic variations have substantial implications in several features, such as epidemiological surveys [18], treatment response of T. cruzi-infected individuals [19], development of vaccines and drugs [20], prevalence of clinical forms and severity of manifestations [21,22], and even diagnosis [23]. Therefore, no single immunological test has sufficient performance to be used alone. In this way, we emphasize the need for the development of a diagnostic test able to identify chronic CD regardless of parasite genetic diversity. Here, two T. cruzi chimeric antigens were assayed with serum samples from patients residing in three endemic Latin America countries and returned accuracy values higher than 97%. The assays revealed a high diagnostic value. Indeed, the AUC values were greater than 99.7%, thereby showing an optimal discriminative power between chronic CD-positive and negative samples. These data are similar to previous results found by our group when samples from both Chagas disease endemic and non-endemic Brazilian settings were assayed either by ELISA [9] and liquid microarray tests [10].