Immune reactivity to Trypanosoma cruzi chimeric proteins for Chagas disease diagnosis in immigrants living in a non-endemic setting
Chronic Chagas Disease (CD) diagnosis is based on serological methods employing crude, semipurified or recombinant antigens, which may result in low sensitivity or cross-reactivity. To reduce these restrictions, we developed a strategy involving use of molecules containing repetitive fragments of Trypanosoma cruzi conserved proteins. Diagnostic performance of IBMP-8.1 and IBMP-8.4 chimeric antigens (Molecular Biology Institute of Paraná - IBMP in Portuguese acronym) was assessed to diagnose T. cruzi-infected and non-infected immigrants living in Barcelona (Spain), a non-endemic setting for Chagas disease.
Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house automated ELISA with 347 positive and 331 negative individuals to Chagas disease. Antigenic cross-reactivity was measured with sera samples from pregnant women with Toxoplasma gondii (n = 98) and Zika virus (n = 75) antibodies.
The area under the curve values was 1 and 0.99 for the IBMP-8.1 and IBMP-8.4 proteins, respectively, demonstrating excellent diagnostic accuracy. The reactivity index was higher for IBMP-8.1 than IBMP-8.4 in positive samples and no significant difference in reactivity index was observed in negative samples. Sensitivity ranged from 99.4% for IBMP-8.1 to 99.1% for IBMP-8.4 and was not statistically different. Specificity for IBMP-8.1 reached 100 and 99.7% for IBMP-8.4, both nearly 100% accurate. No antigenic cross-reactivity was observed and reactivity index was similar to that for negative Chagas disease individuals.
Our results showed an outstanding performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA and suggest both chimeric antigens could also be used for Chagas disease diagnosis in immigrants living in non-endemic settings.
KeywordsChagas disease Trypanosoma cruzi Chimeric antigens Immunoassay Accuracy
Area under the curve
Discrete typing unit
Molecular Biology Institute of Paraná (IBMP in Portuguese acronym)
Phosphate-buffered saline-0.5% Tween 20
Receiving operator curve
Standards for reporting of diagnostic accuracy studies
- T. cruzi
- T. gondii
Chagas disease (CD) is a life-threatening infection caused by hemoflagellate protozoa Trypanosoma cruzi, generating an estimated of 14,000 deaths every year  and morbidity in 5.7 to 9.4 million people in the continental Western Hemisphere [2, 3]. The epidemiological pattern of CD has undergone substantial changes in last decades as a consequence of control campaigns in endemic countries, which have reduced vectorial and transfusional transmission . Increasing international migration flows and more affordable traveling conditions from Latin America to non-endemic areas have contributed to epidemiology changes [5, 6]. CD is no longer limited exclusively to the impoverished rural regions of Latin America; it is transformed into a global health concern affecting people worldwide in both endemic and non-endemic countries and placing 100 million people at risk for acquiring the infection .
In Spain, there are more than 6 million immigrants, and more than 2 million (38%) are coming from CD endemic Latin America countries , posing CD as a public health challenge . Indeed, Ecuador, Bolivia, and Argentina are the predominant areas of origin . The diversity of the geographic areas leads to another challenge: the need for an accurate diagnostic test capable of identifying individuals infected with different T. cruzi strains. The high genetic variability of T. cruzi can be responsible for false negative results . These false negative results could be avoided by using synthetic chimeric antigens with repetitive fragments of antigenic T. cruzi proteins for the detection of specific antibodies [11, 12, 13, 14].
We performed ELISA [15, 16] and liquid microarray  to assess the potential diagnostic of four chimeric proteins, IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4, to identify T. cruzi-infected individuals from several Brazilian endemic (Bahia, Goiás, Minas Gerais, and Pernambuco states; Brazil) and non-endemic settings (Paraná state; Brazil). These chimeric antigens were composed of immunodominant and conserved sequences, as described previously . We obtained high-performance values and low cross-reactivity to Leishmania spp., a pathogen showing relatively high antigenic similarity to T. cruzi [15, 17]. Imprecision analyses showed that IBMP chimeric antigens are highly reproducible and IBMP-8.1 and IBMP-8.4 presented the highest performance values among the evaluated antigens. In this study, we endeavored to conduct an evaluation of the diagnostic performance of IBMP-8.1 and IBMP-8.4 chimeric antigens employing ELISA to diagnose T. cruzi-infected and non-infected immigrants living in Barcelona (Spain), a non-endemic setting for the CD.
Proteins were expressed in Escherichia coli Bl21 star DE3 and purified from the soluble fraction of the total extract of bacterial lysate. Both IBMP-8.1 and IBMP-8.4 antigens were purified by IMAC resin first, and the best fractions dialyzed for buffer exchange and salt reduction before following a second liquid chromatography step. The second purification was conducted on ionic exchange and heparin columns, respectively. Plasmidial construct has already been described in Santos et al. .
Assays were conducted according to previous reports [14, 16]. Briefly, polystyrene “Maxisorp” 96-well microplates (Nunc, Roskilde, Denmark) were coated with 25.0 ng of IBMP-8.1 and IBMP-8.4 per well diluted in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Microplates were blocked with Well Champion reagent (Kem-En-Tec, Taastrup, Denmark) according to the manufacturer’s instructions. Serum samples were diluted in 0.05 M phosphate-buffered saline (pH 7.2)-0.5% Tween 20 (PBS-T), and 100 μl was added to each well. After 60 min of incubation at 37 °C, microplates were washed in PBS-T to remove unbound antibodies. HRP conjugated goat anti-human IgG (Bio-Manguinhos, FIOCRUZ, Rio de Janeiro, Brazil) was diluted 1:40,000 in PBS-T, and 100 μl were then added to each well, and the microplates were incubated for 30 min at 37 °C. Wells were washed five times and the immune complexes were revealed by the addition of 100 μl TBM substrate (tetramethyl-benzidine; Kem-En-Tec, Taastrup, Denmark). After a new cycle of incubation (10 min at RT in the dark), the reaction was stopped by adding 50 μl 5 N H2SO4, and the absorbance was measured at 450 nm. The protocols were automatized and the runs carried out in an automated microplate immunoanalyser (BEST 2000®, Biokit, Werfen Group Barcelona, Spain). The blank readings (buffer dilution) was subtracted from all other values.
Diagnosis of chronic CD is not a simple task due to the high genetic diversity of T. cruzi, which might lead to misdiagnosis . In fact, T. cruzi parasite has remarkable genetic heterogeneity, it is classified into seven evolutionary genetic groups or discrete typing units (DTUs) termed TcI-TcVI and TcBat, with sub-classifications for regional strains [22, 23, 24]. Regional differences in sensitivity of serological tests had been reported, leading to negative CD diagnosis, mostly in non-endemic countries that receive immigrants from several endemic areas [25, 26, 27, 28]. Therefore, a serological test should be able to diagnose CD regardless of the T. cruzi antigenic heterogeneity. The main benefit in the use of chimeric antigens as antigenic matrix is the increased repertoire of epitopes in comparison to non-chimeric recombinant antigens, reducing the number of false negative results. In previous studies, our group analyzed the IBMP performance to diagnose T. cruzi-infected individuals in several endemic and non-endemic geographical areas from Brazil [14, 15], a country where TcII is predominant . Here, we used two chimeric antigens to capture specific anti-T. cruzi antibodies in the sera of Latin American immigrants living in Barcelona/Spain, a non-endemic setting for CD. The majority of CD-positive samples were collected from Bolivian immigrants, where TcV is the most common DTU found in Bolivia  and predominates in Bolivian immigrants living in Barcelona . Based on the result from previous studies, we suggest that IBMP-8.1 and IBMP-8.4 antigens are able to diagnose chronic Chagas disease in areas with predominance of TcII and TcV genetic groups.
The assays exhibited high diagnostic accuracy values as demonstrated by AUC (nearly 100%), indicating a substantial discriminative power between negative and chronic CD-positive samples. Similar results were previously found in samples from several Brazilian settings both by ELISA (AUC > 99.7%)  and liquid microarray (AUC > 99.1%) . Moreover, the reactivity index from IBMP-8.1 and IBMP-8.4 chimeric antigens, achieved from immigrants living in a non-endemic setting with CD, was higher than those previously obtained for Brazilian samples . It is interesting to note that no differences were observed concerning negative samples. Further studies need to evaluate the performance of the chimeric antigens in settings where other DTUs are predominant, i.e., Argentina, Mexico and Costa Rica.
The present study showed high sensitivity and specificity for both IBMP-8.1 and IBMP-8.4, similar to those found previously using Brazilian samples [14, 15]. Also, the chimeric antigens were found to be nearly 100% accurate, suggesting that the number of misdiagnoses was negligible. In fact, only two and three CD-positive samples were misclassified when assayed with IBMP-8.1 and IBMP-8.4, respectively. In previous studies we evaluated the increase of sensitivity values and the RI signal using a multiplex methodology  or the equimolar mixture of the antigens , however no gains of were achieved. Hence, we believe that the lack of reactivity from these samples could be due to host immunological reasons or low levels of antibodies on the sera. In CD-negative samples, only one sample was classified as false-positive by IBMP-8.4 antigen. Although this sample presented low signal to IBMP-8.4 (1.28), it was classified as negative when assayed by commercial tests (RI < 0.25) and by the IBMP-8.1 chimera (0.22).
No cross-reactivity of IBMP chimeric antigens against antibodies of T. gondii and ZIKV was observed. Indeed, previous studies have shown extremely low reactivity of IBMP chimeric antigens for several infectious diseases, even for Leishmania spp. [15, 17], a pathogen phylogenetically similar to T. cruzi. T. gondii and ZIKV positive samples were used in this study due to serum bank availability and because these infectious diseases did not assay before using IBMP proteins.
Our results showed a remarkable performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA and suggest both antigens could also be used for CD diagnosis in immigrants living in non-endemic settings. The high accuracy of IBMP-8.1 and IBMP-8.4 chimeric antigens suggests that they are useful for CD diagnosis in individuals infected with other DTUs.
The authors would like to address a special thanks to Ignacio Martínez (Departmento de Immunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico) and Jesus Almeda (Unitat de Suport a la Recerca, Direcció d’Atenció Primària Costa de Ponent-IDIAP J Gol) for their critical review. We also thank the staff at the Laboratori Clínic Hospitalet (Laboratori Clínic Territorial Metropolitana Sud, Catalan Institute of Health, Barcelona/Spain), especially Elisabeth Castro, as well as Montse Graells and Cristina Fernández for help with sample collection and laboratory analysis.
Availability of the data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
This research project was funded by a Gonçalo Moniz Institute, Oswaldo Cruz Foundation (Fiocruz/BA). The funder was not involved in the design of the study, in the collection, analysis and interpretation of the data, or in writing the manuscript.
FLNS and ED were involved in the design and operational management of the study. FLNS and WVS were the lead study statistician. ED and RPDR were principal investigators in this study. All authors (ED, RPDR, BE, IU, NITZ, ES, ZM, PAFC, WVS, EDS, YMG, and FLNS) contributed to the interpretation of the data, contributed to this publication and approved the final manuscript for submission. All authors (ED, RPDR, BE, IU, NITZ, ES, ZM, PAFC, WVS, EDS, YMG, and FLNS) had access to the study data and are responsible for the veracity and completeness of the data reported.
FLNS is investigator in public health at Gonçalo Moniz Institute, Oswaldo Cruz Foundation (Fiocruz), Brazil.
Ethics approval and consent to participate
Ethical permission was sought and granted by Clinical Research Ethics Committee of Bellvitge University Hospital (Barcelona, Spain; IRB OHRP: IRB00005523), and was carried out in accordance with the guidelines of the Helsinki Declaration. With the purpose of protecting the patient’s private information, the Clinical Research Ethics Committee of Bellvitge University Hospital approved that the samples were anonymized so that the investigators do not have access to patient’s private information, therefore, avoiding the requirement of verbal or written consent. This manuscript has also been revised for its publication by the Clinical Research Ethics Committee of Bellvitge University Hospital.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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