Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, remains a significant public health challenge, primarily in Latin America but increasingly recognized worldwide due to migration. The disease progresses from an acute phase to a chronic phase, often remaining asymptomatic for decades before causing severe cardiac, digestive, or neurological complications (WHO, 2020).

Early and accurate diagnosis is crucial for effective management, epidemiological surveillance, and transmission prevention, particularly in endemic regions. The chronic phase of Chagas disease (CCD) poses significant diagnostic challenges due to low parasitemia levels, demanding the use of serological methods to detect specific antibodies against T. cruzi. Commonly employed assays include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and indirect hemagglutination (IHA) (Schijman et al., 2024). However, these methods are prone to false positives and inter-assay variability. To mitigate these issues, the World Health Organization (WHO) recommends using two concordant serological tests, with a third test performed in case of discordant results, to improve diagnostic accuracy.

The lack of a single assay with 100% sensitivity and specificity demands resources, infrastructure, and trained personnel that are often unavailable in regions where the disease is endemic. Beyond these logistical constraints, biological aspects such as cross-reactivity with antibodies from other parasitic infections and the genetic diversity of T. cruzi further complicate the diagnosis. The parasite’s extensive genetic variability is classified into six discrete typing units (DTUs), TcI–TcVI, and an additional genotype, Tcbat (Zingales et al., 2009, 2012). This diversity correlates with geographical distribution, eco-epidemiological traits, and disease progression (Silvestrini et al., 2024). For instance, while TcI is widely dispersed across the Americas, TcV and TcIV are predominantly found in southern and central South America. Consequently, serological tests developed with antigens from specific regions may underperform in detecting infections caused by strains from other areas (Truyens et al., 2021). Furthermore, the host’s antibody response is influenced by the antigenic variability of T. cruzi strains and the host’s genetic background (Silvestrini et al., 2024), directly impacting diagnostic sensitivity and specificity.

In recent work, we demonstrated the diagnostic accuracy of two immunogenic domains derived from the hypothetical T. cruzi protein Tc323, evaluated using an in-house ELISA. A panel of 333 CCD-positive and 141 CCD-negative plasma/serum samples from seven endemic countries (Argentina, Bolivia, Colombia, Mexico, Paraguay, El Salvador, and the USA) was tested. The recombinant domain rTcD3 achieved a sensitivity of 90.7% and specificity of 92.2%, while rTcD6 exhibited a sensitivity and specificity of 93.1 and 93.6%, respectively. Additionally, cross-reactivity was assessed using samples from individuals with related infectious (cutaneous leishmaniasis, strongyloidiasis, and toxoplasmosis), yielding false-positive results in 5/49 and 4/49 samples for rTcD3 and rTcD6, respectively (Ossowski et al., 2024).

In this study, we expanded our investigation of the diagnostic performance of the recombinant proteins rTcD3 and rTcD6 in distinguishing T. cruzi-positive and -negative individuals with CCD. To do this, we analyzed a panel of serum samples collected in Brazil, which includes both endemic regions (Bahia, Goiás, Minas Gerais, and Pernambuco) and a non-endemic region (Paraná). Notably, these regions exhibit a distinct distribution of T. cruzi discrete typing units (DTUs) compared to those evaluated in our previous study (Zingales and Macedo, 2023). Furthermore, we aimed to include not only a larger panel of sera from individuals with other infectious diseases, but also to broaden the range of pathogens represented—such as viruses and bacteria—in order to more comprehensively evaluate the potential cross-reactivity of the recombinant antigens.

Developing a reliable reference test for diagnosing CCD remains a priority on the WHO’s agenda. The current necessity for performing multiple tests—often two or three in cases of discordant results—using diverse assay formats and antigen sources imposes substantial financial and logistical burdens. These challenges are particularly significant in resource-limited endemic regions or emergency room settings, where infected individuals may assist without prior diagnosis. Numerous recombinant protein-based ELISA assays for the diagnosis of CCD have been developed and documented, exhibiting specificity ranging from 93.8 to 100.0%, and sensitivity spanning 65.0 to 100.0%, contingent on the specific antigen employed and the testing conditions (Resende et al., 2024). Among the most widely studied antigens, proteins such as B13, 1F8, JL7, KMP11, FRA, and CRA have demonstrated promising diagnostic performance and have consequently been integrated into several commercial diagnostic kits. Despite these developments, a universal gold-standard antigen for Chagas disease diagnosis has not yet been established. A major limitation is the high variability in serological panels across studies, the diverse geographic origins of the tested populations, and the use of different laboratory methodologies, which complicate direct comparisons among assays. Furthermore, cross-reactivity with other infectious diseases remains a critical concern, particularly in regions where T. cruzi and Leishmania spp. co-exist (Resende et al., 2024). For example, a comparative study evaluating four commercial IgG T. cruzi enzyme immunoassays using 1,433 serum samples from Brazil reported for Gold ELISA Chagas (Rem) 100% sensitivity and 100% specificity; for Imuno-ELISA Chagas (Wama Diagnóstica) 99.5% sensitivity with 99.2% specificity; for ELISA Chagas III (BIOSChile) 97.3% sensitivity with 100% specificity, and for Pathozyme® Chagas (Omega) 99.5% sensitivity with 97.0% specificity (Santos et al., 2016). However, these assays exhibited cross-reactivity with pathogens such as rubella virus, measles virus, syphilis, hepatitis C virus, and, most notably, Leishmania spp., with values ranging from 8.6 to 42.9%. A previous study showed that Pathozyme® Chagas (Omega) yields 75% sensitivity and 100% specificity while Chagatest Rec v3.0 (Wiener) yields 81.5% sensitivity and 100% specificity when tested against serum samples from Brazilian individuals infected with T. cruzi and Leishmania sp. (Caballero et al., 2007). Such findings highlight the importance of evaluating diagnostic tools not only based on their analytical performance but also on their ability to distinguish T. cruzi infection from other common diseases in endemic regions. In addition to these concerns, it is important to note that many existing studies do not include sera from countries such as Mexico, where a high prevalence of false-negative results has been reported. This is likely due to differences in the circulating discrete typing units (DTUs) of T. cruzi and variations in host immune responses (Whitman et al., 2019; Kelly et al., 2021; Truyens et al., 2021). Consequently, despite decades of research, no single serological assay has achieved perfect sensitivity and specificity, largely due to the genetic diversity of the parasite and the heterogeneity of host antibody repertoires across endemic regions.

In this context, we recently proposed two recombinant proteins, rTcD3 and rTcD6, as potential diagnostic candidates in ELISA format. The proteins are derived from distinct regions of the hypothetical T. cruzi protein Tc323, with rTcD3 corresponding to an internal domain and rTcD6 to its C-terminal region. In a prior study using 333 CCD-positive and 141 CCD-negative samples from seven endemic countries across Latin America, rTcD3 exhibited sensitivity and specificity values of 90.7 and 92.2%, respectively, while rTcD6 demonstrated values of 93.1 and 93.6% (Ossowski et al., 2024). These results highlight the potential of both antigens to reliably distinguish T. cruzi-infected individuals from non-infected individuals, irrespective of parasite strain or host antibody variability.

This robust performance can be attributed to the conserved nature of Tc323 across parasite DTUs, with limited cross-reactivity restricted to non-pathogenic trypanosomatids (Trypanosoma rangeli and Paratrypanosoma) and absent in other eukaryotes outside the kinetoplastids, including mammalian hosts parasitized by T. cruzi. Sequence alignment using the MUSCLE algorithm confirmed that TcD3 and TcD6 exhibit >97% sequence conservation across different parasite lineages. Additionally, both recombinant proteins were effective in diagnosing CCD with and without cardiac manifestations, underscoring their diagnostic utility, though they do not provide prognostic information on disease severity (Ossowski et al., 2024).

In the present study, we evaluated the diagnostic performance of these proteins in a larger number of individuals from Brazil, a country where TcII is prevalent (Zingales and Macedo, 2023). Specificity values for both antigens were consistently high (97.9%), but sensitivity compared to our previous work decreased to 79.5 and 81.5% for rTcD3 and rTcD6, respectively. The increase in specificity is particularly noteworthy, as a higher number of T. cruzi-negative samples were included in this analysis. Conversely, the decrease in sensitivity can primarily be attributed to a difference in false-negative results, which rose from 31 to 83 and 19 to 75 samples to rTcD3 and rTcD6, respectively (Supplementary Figure S1). In our opinion, as Tc323 domains are conserved across all parasite strains available in the database, the sensitivity reduction may reflect variations in antibody response mounted by individuals in the area. It is widely documented, particularly in response to vaccines, that host genetic variations—such as HLA class I and II alleles, and single-nucleotide polymorphisms in coding and noncoding regions of cytokines and their receptors—can contribute to differences in antibody response among countries. Furthermore, intrinsic factors such as age and sex, as well as non-genetic factors like smoking, diet, medical and vaccination history also influence the human antibody repertoire against pathogens (Meyer et al., 2021; Truyens et al., 2021; Olin et al., 2023). To improve diagnostic performance, a chimeric protein combining TcD3 and TcD6 could present additional antigenic determinants or a more favorable tertiary structure, enhancing antibody binding following plate immobilization.

Cross-reactivity testing with 764 samples from individuals infected with other parasites, viruses, and bacteria demonstrated minimal interference. Only one leptospirosis-positive sample showed reactivity with both proteins. Additionally, two visceral leishmaniasis-positive samples yielded false positives for rTcD3, while three of these, along with one HIV-positive sample, tested positive for rTcD6. These findings align with prior observations of minimal cross-reactivity with toxoplasmosis-positive sera, indicating that IgG-ELISAs exhibit excellent specificity (<0.9% cross-reactivity).

Although the specificity of the rTcD3 and rTcD6 ELISA assays is comparable to that of currently available commercial tests, their individual sensitivities remain slightly lower than those reported for the highest-performing assays (Resende et al., 2024). In the context of serological diagnosis, the World Health Organization (WHO) currently recommends the use of two concordant serological tests, followed by a third assay in cases of discordant results, to enhance diagnostic accuracy. In our study, the performance of rTcD3 and rTcD6 when used individually suggests that neither assay alone would be sufficient to replace the current diagnostic algorithm, primarily due to their marginally reduced sensitivity. However, when applied in a parallel testing strategy, the combination of both assays resulted in a marked improvement in diagnostic performance, yielding a sensitivity exceeding 96.0% and a high true negative rate of 95.9%. These findings underscore the potential of combining complementary antigen targets to enhance diagnostic yield. Building upon this observation, we are currently developing a chimeric protein that incorporates immunodominant regions of both TcD3 and TcD6, aiming to replicate the advantages of parallel testing within a single, streamlined assay. We anticipate that further optimization and validation of this approach may offer a more practical, cost-effective, and accurate serological tool for the diagnosis of Chagas disease, particularly in resource-limited settings.

In sum, this study demonstrates that rTcD3 and rTcD6-based IgG-ELISAs are valuable and sensitive diagnostic tools for detecting chronic T. cruzi infection across a broad spectrum of both endemic and non-endemic regions of Brazil. These assays exhibit specificity against antibodies generated in response to other pathogens, including closely related Leishmania species. Moreover, the results emphasize the promising potential of utilizing hypothetical proteins for high-specificity diagnostics, thus providing a solid foundation for further exploration of alternative antigens for CCD diagnosis. Future research should prioritize larger sample sizes, validation within natural populations, and an assessment of these assays’ applicability for post-treatment monitoring and the differentiation between acute and chronic infections.

This study is not without its limitations. Notably, the exclusion of samples with discordant results from commercial assays, which were retested, may have led to an overestimation of specificity. Additionally, the study’s geographic scope was limited to specific regions of Brazil, which may impact the generalizability of the findings. Furthermore, a comparative analysis with existing diagnostic methodologies, particularly regarding factors such as cost-effectiveness, time efficiency, and accessibility, was not performed.

Another limitation is the lack of information on the specific T. cruzi DTUs infecting each individual, and the absence of data characterizing the reactivity of the positive sera against specific DTUs. While our results suggest that the rTcD3 and rTcD6 antigens are capable of eliciting a consistent antibody response across a diverse population, further studies are needed to confirm this finding and to investigate the potential impact of DTU-specific variations on the diagnostic performance of our assay.