Dopico et al. BMC Infectious Diseases (2019) 19:251 https://doi.org/10.1186/s12879-019-3872-z RESEARCH ARTICLE Open Access Immune reactivity to Trypanosoma cruzi chimeric proteins for Chagas disease diagnosis in immigrants living in a non- endemic setting Eva Dopico1, Rodrigo Pimenta Del-Rei2, Bertha Espinoza3, Itziar Ubillos1, Nilson Ivo Tonin Zanchin4, Elena Sulleiro5, Zaira Moure5, Paola Alejandra Fiorani Celedon6, Wayner Vieira Souza7, Edimilson Domingos da Silva8, Yara Miranda Gomes7 and Fred Luciano Neves Santos9* Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Chagas disease, Trypanosoma cruzi, Chimeric antigens, Immunoassay, Accuracy * Correspondence: fred.santos@bahia.fiocruz.br Preliminary results were presented at XXIV Latin American Congress of Parasitology (FLAP XXIV); 10 December 2017; School of Medicine; Santiago, Chile. 9 Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dopico et al. BMC Infectious Diseases (2019) 19:251 Page 2 of 7 Background Study samples Chagas disease (CD) is a life-threatening infection caused by We employed anonymized human sera from individuals hemoflagellate protozoa Trypanosoma cruzi, generating an diagnosed at the Laboratory Clínic l’Hospitalet-Labora- estimated of 14,000 deaths every year [1] and morbidity in tori Clínic Territorial Metropolitana Sur, Catalan Insti- 5.7 to 9.4 million people in the continental Western Hemi- tute of Health (Barcelona-Spain). The minimum sample sphere [2, 3]. The epidemiological pattern of CD has under- with a 95% confidence interval, an absolute expected gone substantial changes in last decades as a consequence of error of 1.1% and sensitivity of 99% was 315 sera from control campaigns in endemic countries, which have re- non-infected and 315 from T. cruzi-infected individuals. duced vectorial and transfusional transmission [4]. Increas- We included sera from 331 non-infected and 347 T. cru- ing international migration flows and more affordable zi-infected Latin American individuals living in Barce- traveling conditions from Latin America to non-endemic lona (Fig. 1). The sample selection was based on areas have contributed to epidemiology changes [5, 6]. CD non-reactivity and reactivity by two serological assays: is no longer limited exclusively to the impoverished rural re- ORTHO® T. cruzi ELISA Test System (Ortho Clinical gions of Latin America; it is transformed into a global health Diagnostics Inc., Raritan, USA), which employs T. cruzi concern affecting people worldwide in both endemic and whole cell lysate antigen; and Bioelisa CHAGAS (Biokit non-endemic countries and placing 100 million people at S.A., Barcelona, Spain) or BIO-FLASH® Chagas (auto- risk for acquiring the infection [7]. mated chemiluminescent assay; Biokit S.A., Barcelona, In Spain, there are more than 6 million immigrants, Spain), the two latter composed by recombinant T. cruzi and more than 2 million (38%) are coming from CD en- antigens. Samples with repeatedly discrepant results be- demic Latin America countries [8], posing CD as a pub- tween both tests or inconclusive in one of them (or in gray lic health challenge [9]. Indeed, Ecuador, Bolivia, and zone) were defined as serodiscordant. Each sample was Argentina are the predominant areas of origin [7]. The given an identifier code in the laboratory to ensure a diversity of the geographic areas leads to another chal- blinded analysis. Antigenic cross-reactivity was assessed lenge: the need for an accurate diagnostic test capable of with sera samples from Latin American pregnant women identifying individuals infected with different T. cruzi with Toxoplasma gondii (n = 98) and Zika virus (ZIKV) strains. The high genetic variability of T. cruzi can be re- antibodies (n = 75). Pregnant women sera samples sponsible for false negative results [10]. These false nega- were employed in this study due to the availability of tive results could be avoided by using synthetic chimeric the biological material in Laboratory Clínic l’Hospita- antigens with repetitive fragments of antigenic T. cruzi let-Laboratori Clínic Territorial Metropolitana Sur proteins for the detection of specific antibodies [11–14]. serum bank. Study participants were mostly from We performed ELISA [15, 16] and liquid microarray Bolivia (97.3%), but there were individuals from other [17] to assess the potential diagnostic of four chimeric CD endemic countries (Fig. 1). proteins, IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4, to identify T. cruzi-infected individuals from several Bra- Recombinant antigens zilian endemic (Bahia, Goiás, Minas Gerais, and Pernam- Proteins were expressed in Escherichia coli Bl21 star DE3 buco states; Brazil) and non-endemic settings (Paraná and purified from the soluble fraction of the total extract state; Brazil). These chimeric antigens were composed of of bacterial lysate. Both IBMP-8.1 and IBMP-8.4 antigens immunodominant and conserved sequences, as de- were purified by IMAC resin first, and the best fractions scribed previously [15]. We obtained high-performance dialyzed for buffer exchange and salt reduction before fol- values and low cross-reactivity to Leishmania spp., a lowing a second liquid chromatography step. The second pathogen showing relatively high antigenic similarity to purification was conducted on ionic exchange and heparin T. cruzi [15, 17]. Imprecision analyses showed that IBMP columns, respectively. Plasmidial construct has already chimeric antigens are highly reproducible and IBMP-8.1 been described in Santos et al. [18]. and IBMP-8.4 presented the highest performance values among the evaluated antigens. In this study, we endeav- Immunoassays ored to conduct an evaluation of the diagnostic perform- Assays were conducted according to previous reports ance of IBMP-8.1 and IBMP-8.4 chimeric antigens [14, 16]. Briefly, polystyrene “Maxisorp” 96-well micro- employing ELISA to diagnose T. cruzi-infected and plates (Nunc, Roskilde, Denmark) were coated with 25.0 non-infected immigrants living in Barcelona (Spain), a ng of IBMP-8.1 and IBMP-8.4 per well diluted in coating non-endemic setting for the CD. buffer (0.05 M carbonate-bicarbonate, pH 9.6). Micro- plates were blocked with Well Champion reagent Methods (Kem-En-Tec, Taastrup, Denmark) according to the man- We used the methodology previously described by San- ufacturer’s instructions. Serum samples were diluted in tos et al. [18] and Brito et al. [19]. 0.05 M phosphate-buffered saline (pH 7.2)-0.5% Tween 20 Dopico et al. BMC Infectious Diseases (2019) 19:251 Page 3 of 7 Fig. 1 T. cruzi-positive and negative samples selected from Latin American immigrants living in Barcelona-Spain (PBS-T), and 100 μl was added to each well. After 60 min set to 95% and p < 0.05 was considered as statistically sig- of incubation at 37 °C, microplates were washed in PBS-T nificant. A study workflow (Fig. 2) is provided according to remove unbound antibodies. HRP conjugated goat to the STARD guidelines [21]. Digital map (Fig. 1) was ac- anti-human IgG (Bio-Manguinhos, FIOCRUZ, Rio de quired from the Brazilian Institute of Geography and Sta- Janeiro, Brazil) was diluted 1:40,000 in PBS-T, and 100 μl tistics (IBGE) cartographic database in shapefile (.shp), were then added to each well, and the microplates were which were formatted and analyzed using TerraView ver- incubated for 30 min at 37 °C. Wells were washed five sion 4.2, public-access software provided by the National times and the immune complexes were revealed by the Institute for Space Research from Brazil (www.dpi.inpe.br/ addition of 100 μl TBM substrate (tetramethyl-benzidine; terraview). Kem-En-Tec, Taastrup, Denmark). After a new cycle of in- cubation (10 min at RT in the dark), the reaction was stopped by adding 50 μl 5 N H2SO4, and the absorbance Results was measured at 450 nm. The protocols were automatized Diagnostic performance and the runs carried out in an automated microplate The reactivity index and assay performance parameters immunoanalyser (BEST 2000®, Biokit, Werfen Group Bar- found for the IBMP-8.1 and IBMP-8.4 chimeric antigens celona, Spain). The blank readings (buffer dilution) was are illustrated in Fig. 3. Based on 678 samples from subtracted from all other values. non-infected and T. cruzi-infected individuals, the AUC (area under the curve) values were 1.000 and 0.9998 for Statistical analysis IBMP-8.1 and IBMP-8.4 proteins, respectively, demon- Data were coded and entered using computer graphic strating excellent overall diagnostic accuracy. The software (GraphPad Software Inc., La Jolla, CA, USA). IBMP-8.1 chimera yielded the highest diagnostic accur- Descriptive data were presented in the form of geomet- acy among T. cruzi-infected individuals, and no signifi- ric means ± standard deviation. Shapiro-Wilk test, cant difference was observed between chimeric antigens followed by Student’s t-test, was used to test data nor- in non-infected individuals. Sensitivity was 99.4% for mality. When assumed homogeneity was not confirmed, IBMP-8.1 and 99.1% for IBMP-8.4. Only one positive Wilcoxon’s signed rank test was adopted. Cut-off values sample was simultaneously false negative when assayed were determined under the receiver operating character- by IBMP-8.1 and IBMP-8.4 chimeric antigens. Specificity istic curve (ROC) analyzing the whole serum panel. For of IBMP-8.1 achieved 100 and 99.7% for IBMP-8.4. Both data normalization, all results were expressed by plotting proteins showed an accuracy of nearly 100%. No statisti- values in an indexed format, calculated as the ratio be- cally significant differences in sensitivity or specificity tween a given sample’s optical density (OD) and the scores were found between the chimeric antigens. Using cut-off OD values respective to each assay. Under this RI values of 1.0 ± 0.10 as the grey zone inconclusive index, referred to as a reactivity index (RI), all results interval, we observed two positive and one negative sam- ≥1.00 were considered positive. When a sample’s RI value ple fell in the inconclusive space using IBMP-8.1 while was 1.0 ± 10%, the result was considered as indeterminate two positive samples to IBMP-8.4 presented similar be- (i.e., in the grey zone), and these samples were deemed in- havior. Overall, the number of inconclusive results was conclusive. The test performances were computed using a 0.44% for IBMP-8.1 and 0.29% for IBMP-8.4 (Fig. 3). No dichotomous approach and compared regarding sensitiv- sample fell concomitantly inside the grey zone for ity, specificity, and accuracy [20]. Confidence interval was IBMP-8.1 and IBMP8.4 chimeric antigens. Dopico et al. BMC Infectious Diseases (2019) 19:251 Page 4 of 7 Fig. 2 Study workflow for testing IBMP T. cruzi chimeras. RI, reactivity index; ZIKV, Zika virus Fig. 3 Reactivity index (RI) was obtained with serum from non-infected and T. cruzi-infected immigrants living in Barcelona-Spain. The cut-off value is RI = 1.0 and the grey zone is RI = 1.0 ± 0.10. Lines and whiskers represent geometric means (± 95% CI). AUC, area under curve; GZ, grey zone; Sen, sensitivity; Spe, specificity; Acc; accuracy Dopico et al. BMC Infectious Diseases (2019) 19:251 Page 5 of 7 Antigenic cross-reactivity are able to diagnose chronic Chagas disease in areas with The RI values for sampled with T. gondii and ZIKV anti- predominance of TcII and TcV genetic groups. bodies were identical to that found for non-infected in- The assays exhibited high diagnostic accuracy values dividuals, indicating extremely low reactivity. No as demonstrated by AUC (nearly 100%), indicating a false-positive or inconclusive samples were found with substantial discriminative power between negative and IBMP chimeric antigens (Fig. 4). chronic CD-positive samples. Similar results were previ- ously found in samples from several Brazilian settings Discussion both by ELISA (AUC > 99.7%) [14] and liquid microarray Diagnosis of chronic CD is not a simple task due to the (AUC > 99.1%) [17]. Moreover, the reactivity index from high genetic diversity of T. cruzi, which might lead to mis- IBMP-8.1 and IBMP-8.4 chimeric antigens, achieved diagnosis [22]. In fact, T. cruzi parasite has remarkable from immigrants living in a non-endemic setting with genetic heterogeneity, it is classified into seven evolution- CD, was higher than those previously obtained for Bra- ary genetic groups or discrete typing units (DTUs) termed zilian samples [14]. It is interesting to note that no dif- TcI-TcVI and TcBat, with sub-classifications for regional ferences were observed concerning negative samples. strains [22–24]. Regional differences in sensitivity of sero- Further studies need to evaluate the performance of the logical tests had been reported, leading to negative CD chimeric antigens in settings where other DTUs are pre- diagnosis, mostly in non-endemic countries that receive dominant, i.e., Argentina, Mexico and Costa Rica. immigrants from several endemic areas [25–28]. There- The present study showed high sensitivity and specifi- fore, a serological test should be able to diagnose CD re- city for both IBMP-8.1 and IBMP-8.4, similar to those gardless of the T. cruzi antigenic heterogeneity. The main found previously using Brazilian samples [14, 15]. Also, benefit in the use of chimeric antigens as antigenic matrix the chimeric antigens were found to be nearly 100% ac- is the increased repertoire of epitopes in comparison to curate, suggesting that the number of misdiagnoses was non-chimeric recombinant antigens, reducing the number negligible. In fact, only two and three CD-positive sam- of false negative results. In previous studies, our group an- ples were misclassified when assayed with IBMP-8.1 and alyzed the IBMP performance to diagnose T. cruzi-in- IBMP-8.4, respectively. In previous studies we evaluated fected individuals in several endemic and non-endemic the increase of sensitivity values and the RI signal using geographical areas from Brazil [14, 15], a country where a multiplex methodology [17] or the equimolar mixture TcII is predominant [10]. Here, we used two chimeric an- of the antigens [15], however no gains of were achieved. tigens to capture specific anti-T. cruzi antibodies in the Hence, we believe that the lack of reactivity from these sera of Latin American immigrants living in Barcelona/ samples could be due to host immunological reasons or Spain, a non-endemic setting for CD. The majority of low levels of antibodies on the sera. In CD-negative sam- CD-positive samples were collected from Bolivian immi- ples, only one sample was classified as false-positive by grants, where TcV is the most common DTU found in IBMP-8.4 antigen. Although this sample presented low Bolivia [10] and predominates in Bolivian immigrants liv- signal to IBMP-8.4 (1.28), it was classified as negative ing in Barcelona [29]. Based on the result from previous when assayed by commercial tests (RI < 0.25) and by the studies, we suggest that IBMP-8.1 and IBMP-8.4 antigens IBMP-8.1 chimera (0.22). Fig. 4 Analysis of the cross-reactivity of the IBMP-8.1 and IBMP-8.4 chimeras to sera with Toxoplasma gondii antibodies (n = 98) and Zika virus antibodies (n = 75). The cut-off value is 1.0 and the grey zone is RI = 1.0 ± 0.10. Lines and whiskers represent geometric means (± 95% CI). GZ, grey zone; RI, reactivity index; ZIKV, Zika virus Dopico et al. BMC Infectious Diseases (2019) 19:251 Page 6 of 7 No cross-reactivity of IBMP chimeric antigens against IRB00005523), and was carried out in accordance with the guidelines of the antibodies of T. gondii and ZIKV was observed. Indeed, Helsinki Declaration. With the purpose of protecting the patient’s private information, the Clinical Research Ethics Committee of Bellvitge University previous studies have shown extremely low reactivity of Hospital approved that the samples were anonymized so that the investigators IBMP chimeric antigens for several infectious diseases, do not have access to patient’s private information, therefore, avoiding the even for Leishmania spp. [15, 17], a pathogen phylogen- requirement of verbal or written consent. This manuscript has also been revised for its publication by the Clinical Research Ethics Committee of Bellvitge etically similar to T. cruzi. T. gondii and ZIKV positive University Hospital. samples were used in this study due to serum bank avail- ability and because these infectious diseases did not Consent for publication Not applicable. assay before using IBMP proteins. Competing interests Conclusion The authors declare that they have no competing interests. Our results showed a remarkable performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in and suggest both antigens could also be used for CD published maps and institutional affiliations. diagnosis in immigrants living in non-endemic settings. The high accuracy of IBMP-8.1 and IBMP-8.4 chimeric Author details 1 Laboratori Clínic Territorial Metropolitana Sud, Catalan Institute of Health, antigens suggests that they are useful for CD diagnosis Barcelona, Catalonia, Spain. 2Faculty of Technology and Sciences of Bahia, in individuals infected with other DTUs. Salvador, Bahia, Brazil. 3Instituto de Investigaciones Biomédicas, Departamento de Inmunología, Universidad Nacional Autónoma de México, Abbreviations Ciudad de México, Mexico. 4Carlos Chagas Institute, Oswaldo Cruz Acc: Accuracy; AUC: Area under the curve; CD: Chagas disease; DTU: Discrete Foundation, Curitiba, Paraná, Brazil. 5Microbiology Department, Vall d’Hebron typing unit; GZ: Grey zone; HRP: Horseradish peroxidase; IBMP: Molecular University Hospital, PROSICS, Barcelona, Catalonia, Spain. 6Molecular Biology Biology Institute of Paraná (IBMP in Portuguese acronym); OD: Optical Institute of Paraná, Curitiba, Paraná, Brazil. 7Aggeu Magalhães Institute, density; PBS: Phosphate-buffered saline; PBS-T: Phosphate-buffered saline- Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil. 8Immunobiological 0.5% Tween 20; RI: Reactivity index; ROC: Receiving operator curve; Technology Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. 9 Sen: Sensitivity; Spe: Specificity; STARD: Standards for reporting of diagnostic Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil. accuracy studies; T. cruzi: Trypanosoma cruzi; T. gondii: Toxoplasma gondii; TBM: Tetramethyl-benzidine; ZIKV: Zika virus Received: 24 September 2018 Accepted: 4 March 2019 Acknowledgements The authors would like to address a special thanks to Ignacio Martínez References (Departmento de Immunologia, Instituto de Investigaciones Biomedicas, 1. Hotez PJ, Dumonteil E, Woc-Colburn L, Serpa JA, Bezek S, Edwards MS, et al. Universidad Nacional Autonoma de Mexico) and Jesus Almeda (Unitat de Chagas disease: “the new HIV/AIDS of the Americas”. PLoS Negl Trop Dis. Suport a la Recerca, Direcció d’Atenció Primària Costa de Ponent-IDIAP J Gol) 2012;6(5):e1498. for their critical review. We also thank the staff at the Laboratori Clínic Hospitalet 2. World Health Organization. Chagas disease in Latin America: an (Laboratori Clínic Territorial Metropolitana Sud, Catalan Institute of Health, epidemiological update based on 2010 estimates. Wkly Epidemiol Rec. Barcelona/Spain), especially Elisabeth Castro, as well as Montse Graells 2015;90(6):33–43. and Cristina Fernández for help with sample collection and laboratory analysis. 3. Global Burden of Disease Study 2013 Collaborators, Collaborators GB of DS 2013. 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