Composition Vendor Newgen KnowledgeWorks (P) Ltd. PLoS Negl Trop Dis plos plosntds PLOS Neglected Tropical Diseases 1935-2735 Public Library of Science San Francisco, CA USA 10.1371/journal.pntd.0013892 PNTD-D-25-00569 Research Article Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoansTrypanosomaTrypanosoma cruzi Medicine and health sciencesEpidemiologyMedical risk factors Research and analysis methodsBioassays and physiological analysisElectrophysiological techniquesCardiac electrophysiologyElectrocardiography Medicine and health sciencesPathology and laboratory medicineSerology Medicine and health sciencesMedical conditionsTropical diseasesNeglected tropical diseasesChagas disease Medicine and health sciencesMedical conditionsParasitic diseasesProtozoan infectionsChagas disease People and placesGeographical locationsSouth AmericaBrazil Medicine and health sciencesPublic and occupational health Medicine and health sciencesMedical conditionsInfectious diseasesVector-borne diseases Seroepidemiological survey and seropositivity rate for Trypanosoma cruzi infection in a community-based cardiac screening initiative in Feira de Santana, Bahia, Brazil Seropositivity rate of T. cruzi infection in cardiac screening in Bahia Jesus Felipe Silva Santos de Formal analysis Methodology Writing – original draft Writing – review & editing 1 2 Fonseca Isabella Moreira Gonzalez Conceptualization Formal analysis Investigation Methodology Software Writing – original draft Writing – review & editing 3 Silva Ângelo Antônio Oliveira Formal analysis Methodology Writing – original draft Writing – review & editing 1 2 Gonçalves Noilson Lázaro Sousa Methodology Writing – review & editing 1 Sampaio Daniel Dias Formal analysis Writing – review & editing 2 Mothé Deborah Bittencourt Formal analysis Writing – review & editing 4 Celedon Paola Alejandra Fiorani Formal analysis Investigation Methodology Writing – review & editing 5 Zanette Dalila Luciola Methodology Writing – review & editing 5 Zanchin Nilson Ivo Tonin Data curation Formal analysis Funding acquisition Investigation Methodology Supervision Validation 6 Nunes Maria Carmo Pereira Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Supervision Validation Writing – original draft Writing – review & editing 3 Rocha Manoel Otávio da Costa Conceptualization Funding acquisition Project administration Supervision Writing – review & editing 3 Sable Craig Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Writing – review & editing 7 Ribeiro Antonio Luiz Pinho Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Supervision Writing – review & editing 3 https://orcid.org/0000-0002-3944-0818 Santos Fred Luciano Neves Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Writing – original draft Writing – review & editing 1 2 8 * Advanced Health Public Laboratory, Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil Interdisciplinary Research Group in Biotechnology and Epidemiology of Infectious Diseases (GRUPIBE), Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil Medical School and University Hospital, Federal University from Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Laboratory of Interaction Parasite Host and Epidemiology (LAIPHE), Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil Laboratory for Applied Science and Technology in Health, Carlos Chagas Institute, Oswaldo Cruz Foundation, Curitiba, Paraná, Brazil Structural Biology and Protein Engineering Laboratory, Carlos Chagas Institute, Oswaldo Cruz Foundation, Curitiba, Paraná, Brazil Ochsner Children’s Hospital, New Orleans, Louisiana, United States of America Integrated Translational Program in Chagas disease from Fiocruz – Fio-Chagas, Rio de Janeiro, Rio de Janeiro, Brazil Werneck Guilherme L Editor Universidade do Estado do Rio de Janeiro, BRAZIL * E-mail: fred.santos@fiocruz.br

The authors have declared that no competing interests exist.

☯ These authors contributed equally to this work.

 212026 12026 20 1 e0013892 1242025 22122025 2026 Jesus et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Chagas disease (CD), caused by Trypanosoma cruzi, is a significant public health issue in Latin America, particularly in endemic regions. This study integrates a seroepidemiological survey with large-scale echocardiographic screening conducted in Feira de Santana, a highly endemic city in Bahia, Brazil, to estimate the seropositivity rate of T. cruzi infection and identify associated risk factors. Peripheral blood samples were analyzed using in-house ELISA based on IBMP chimeric antigens and an indirect hemagglutination assay. Among 1,115 participants enrolled in the cardiac screening initiative, 140 underwent serological testing comprising individuals who screened positive based on clinical data, conventional ECG, and ECG-AI, and controls matched in a 2:1 ratio. Of these, 8.5% tested seropositive, with household exposure to triatomines identified as the strongest risk factor (prevalence ratio = 4.38, p = 0.004). Most seropositive individuals were migrants from other endemic areas, underscoring the influence of population mobility on CD epidemiology. This study highlights the importance of integrating diagnostic tools and vector control strategies into community-based health initiatives to improve early detection, reduce disease burden, and inform public health interventions in underserved regions.

 Author summary

Chagas disease, caused by the parasite Trypanosoma cruzi, remains a major health concern in Latin America, especially in areas with limited access to healthcare. In this study, we combined artificial intelligence-based electrocardiogram analysis and cardiac screening with serological testing to detect undiagnosed T. cruzi infections in Feira de Santana, a city in the state of Bahia, Brazil. By analyzing electrocardiogram data, epidemiological information, and laboratory assays, we identified individuals at increased risk and confirmed infection using validated serological methods. Our results showed that people who reported the presence of triatomine bugs inside their homes were significantly more likely to be infected, emphasizing the role of vector exposure in T. cruzi transmission. In addition, most seropositive individuals were unaware of their condition and had migrated from other endemic regions. These findings highlight the importance of combining innovative technologies with community-based screening initiatives can improve early detection of T. cruzi infection and guide public health strategies in vulnerable populations.

 National Council for Scientific and Technological Development (CNPQ) 304894/2023-0 ZanchinNilson Ivo Tonin National Council for Scientific and Technological Development (CNPQ) 306448/2023-8 https://orcid.org/0000-0002-3944-0818 SantosFred Luciano Neves http://dx.doi.org/10.13039/100006520 Edwards Lifesciences Coordination for the Improvement of Higher Education Personnel (CAPES) Finance Code 001 This study was supported by the Edwards Lifesciences Foundation and the Coordination for the Improvement of Higher Education Personnel in Brazil (CAPES; Finance Code 001). NITZ and FLNS are research fellows of the National Council for Scientific and Technological Development-Brazil (CNPq; processes 304894/2023-0 and 306448/2023-8, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are in the manuscript and its supporting information files. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2026-01-08 Introduction

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains a significant public health issue in Latin America, affecting millions and contributing to considerable morbidity and mortality [1,2]. Despite advances in control measures, gaps in early detection and treatment persist, particularly in underserved regions [3]. Chronic CD can lead to cardiac involvement, which represents the leading cause of mortality, highlighting the need for innovative strategies to improve healthcare delivery and patient outcomes.

The state of Bahia is recognized as one of the Brazilian states with persistent endemicity for T. cruzi infection, where both vectorial and oral transmission routes have been historically documented. Recent seroepidemiological surveys continue to report significant seroprevalence rates in rural and peri-urban communities [4–6]. Bahia also harbors the greatest diversity of triatomine species in Brazil, including several species that have adapted to human dwellings, thereby sustaining the risk of domestic transmission [7–10]. New triatomine species have been described or revalidated in the state, and changes in vector ecology and human–environment interactions have posed additional challenges to control strategies.

Within Bahia, Feira de Santana is one of the most populous municipalities and serves as an important regional commercial and transportation hub. The city has reported substantial morbidity and mortality associated with CD, reflecting the challenges in vector control, diagnosis, and clinical management [11]. Marked socioeconomic inequalities, population mobility, and limited access to specialized healthcare services further contribute to delays in the identification and follow-up of individuals infected with T. cruzi. In this context, innovative approaches that integrate diagnostic technologies into existing healthcare initiatives are essential to improve case detection and reduce disease burden. International initiatives such as the Every Heartbeat Matters Program have emerged as promising strategies to address cardiovascular health disparities in resource-limited settings [12]. This initiative incorporates telehealth-enabled electrocardiography (teleECG), artificial intelligence–based ECG analysis, and targeted echocardiographic screening to improve the detection and management of structural heart disease at the community level. Given the strong association between chronic T. cruzi infection and cardiac involvement, such platforms offer an opportunity to integrate seroepidemiological investigations into large-scale cardiac screening programs.

In this study, we leveraged data from a seroepidemiological survey conducted alongside the Every Heartbeat Matters initiative in Feira de Santana. The primary objectives were to estimate the seropositivity rate of T. cruzi infection, identify epidemiological and environmental factors associated with infection, and assess the feasibility of integrating T. cruzi screening into community-based echocardiographic programs in endemic settings. The findings aim to inform public health strategies and strengthen approaches for the early detection and management of T. cruzi infection in high-burden regions.

 Methods Ethics

This study adhered to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of the Gonçalo Moniz Institute, Oswaldo Cruz Foundation (IGM-FIOCRUZ), Salvador, Bahia, Brazil (protocol no. 67809417.0.0000.0040). Written informed consent was obtained from all participants prior to enrollment.

 Study population

This cross-sectional study was conducted in Feira de Santana, Bahia, Brazil, the second largest city in the state, located in the semi-arid region of Northeastern Brazil (11° 11′ S, 38° 58′ W) at an altitude of 234 m. According to the Brazilian Institute of Geography and Statistics (IBGE), the municipality has approximately 616,272 inhabitants and serves as an important commercial and transport hub [13]. The urban area is characterized by high population density and pronounced socioeconomic inequalities, particularly in peripheral neighborhoods with limited access to healthcare, sanitation, and adequate housing.

A total of 1,115 adults were recruited during a large-scale community-based cardiac screening initiative. All participants completed a structured questionnaire collecting sociodemographic data (age, sex, education, employment, household income), environmental and housing characteristics (construction materials, presence of domestic animals or chicken coops, and history of triatomine sightings), migration background, family history of Chagas disease, and previous medical diagnoses. Cardiovascular symptoms, including reduced exercise tolerance, chest pain, syncope, and palpitations, were also assessed. Questionnaires were administered by trained healthcare professionals or supervised medical students at the time of echocardiographic examination.

 Cardiac screening and risk stratification

All participants underwent a standard 12-lead electrocardiogram (ECG) acquired using a PC-based system (TEB, São Paulo, Brazil) and transmitted electronically to a telehealth platform for interpretation by cardiologists. ECG abnormalities were classified as major or minor according to the Minnesota Code criteria [14].

Transthoracic echocardiography was performed by a team of eight cardiologists using commercially available systems (Vivid Q, GE Healthcare; Affiniti 70, Philips, USA). Images were stored digitally and interpreted immediately after acquisition; uncertain findings were resolved by consensus reading. Abnormal echocardiograms were defined by the presence of major structural or functional abnormalities, as previously described [12].

An ECG-based artificial intelligence (AI) algorithm, implemented as a deep neural network trained on standard 12-lead digital ECG waveforms (time–voltage signals), was applied to estimate the probability of T. cruzi infection [15]. Risk stratification combined the AI-ECG output with responses to three epidemiological questions assessing whether participants had: (i) a family member diagnosed with CD, (ii) lived in areas with known triatomine (kissing bug) exposure, and (iii) resided in wooden houses, a recognized risk factor for vector transmission.

Participants classified as at increased risk were invited to undergo serological testing. For comparison, control individuals classified as low risk were randomly selected using a computer-generated algorithm at a 2:1 ratio (controls to cases) and also underwent serological testing.

 Specimen collection and storage

Peripheral blood samples (9 mL) were collected into clot-activator tubes with gel separators (Vacuplast, Brazil). After clotting at room temperature for 30 minutes, samples were centrifuged at 3,000 × g for 10 minutes. Serum aliquots were transported to the Gonçalo Moniz Institute (Oswaldo Cruz Foundation, Bahia) and stored at −20 °C until analysis.

 Serological testing

Anti-T. cruzi IgG antibodies were initially detected using in-house ELISA assays based on four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4), following established protocols [16]. Recombinant antigens were expressed in Escherichia coli BL21-Star (DE3), purified by affinity and ion-exchange chromatography, and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). ELISAs were performed in 96-well microplates (Greiner Bio-One, Austria). Plates were sensitized with the antigens and blocked with a solution of Well-Champion (Ken-Em-Tec Diagnostics A/S, Denmark). Serum samples were diluted 1:100 in PBS-T (PBS containing 0.05% Tween-20; pH 7.4) and incubated at 37 °C for 30 minutes. After washing with PBS-T, a goat anti-human IgG-HRP conjugate (Bio-Manguinhos, FIOCRUZ, Brazil) was added and incubated at 37 °C for 30 minutes. Following additional washing, immune complexes were identified using TBM substrate (Kem-En-Tec, Denmark) with a 10-minute incubation in the dark at room temperature. The reaction was stopped with 0.3 M H₂SO₄, and optical density (OD) was measured at 450 nm using a SPECTRAmax 340PC microplate reader (Molecular Devices, USA).

All samples were additionally tested using a commercial indirect hemagglutination assay (Imuno-HAI Chagas, WAMA Diagnóstica, Brazil), performed according to the manufacturer’s instructions. Sera were diluted 1:32 in assay diluent supplemented with 2-mercaptoethanol to reduce nonspecific reactivity. Qualitative results were interpreted visually, with reactive samples formed a compact erythrocyte button at the bottom of the well, whereas reactive samples producing diffuse erythrocyte mats and non-reactive samples forming compact buttons. Reactive samples were further titrated by serial two-fold dilutions, with titers ≥1:32 considered positive.

 Reference testing

In the absence of a single gold standard for detecting anti-T. cruzi antibodies, infection status was determined using a combined approach based on indirect hemagglutination results and latent class analysis (LCA) [17–19]. Within the LCA framework, seropositivity was defined as reactivity in at least two of the four IBMP ELISA, yielding posterior probabilities between 87.9% and 100%; while seronegativity required non-reactivity in at least three assays, with posterior probabilities ≤0.8%. Concordance between LCA and indirect hemagglutination was assessed for all samples. All samples classified as seropositive or presenting discordant results were further evaluated using two commercial ELISA assays, Gold ELISA Chagas (REM Indústria e Comércio Ltda, Brazil) and Bioelisa Chagas Recombinante (Bioclin Quibasa Química Básica, Brazil), to provide additional confirmation.

 Data analysis

Descriptive statistics summarized sociodemographic and epidemiological characteristics. Age was expressed as median and interquartile range (IQR), and categorical variables (e.g., sex, serological test results) as relative frequencies. Associations between T. cruzi seropositivity and potential risk factors, including prior exposure to triatomines, contact with kissing bugs, presence of a chicken coop, and familial history of CD, were evaluated using contingency tables. Seropositivity rates were calculated as the proportion of seropositive cases among exposed individuals relative to non-exposed individuals, with 95% confidence intervals (CI) and p-values derived from Fisher’s exact test. Statistical significance was set at p-value < 0.05. Additionally, prevalence ratio (PR) was performed to assess the association of sociodemographic and environmental factors with positivity. Multivariable logistic regression models integrating ECG findings and epidemiological variables were used to estimate the probability of T. cruzi infection.

 Results

Between August 24 and 27, 2024, a total of 1,115 individuals participated in the Every Heartbeat Matters cardiac screening initiative conducted at the Holy House of Mercy in Feira da Santana (Fig 1). In the overall screened population, the median age was 57 years (IQR: 44–64 years), and women accounted for 78.4% of participants, corresponding to a female-to-male ratio of 4.9:1. According to the Minnesota Code criteria [14], 284 individuals (27.5%) presented minor ECG abnormalities, while 118 (11.4%) showed major abnormalities.

10.1371/journal.pntd.0013892.g001Study design of a seroepidemiological survey for Chagas disease in Feira de Santana, Bahia, Brazil.

The figure summarizes participant selection within the Every Heartbeat Matters cardiac screening initiative, with ECG and echocardiographic evaluation, risk stratification, and subsequent serological testing using IBMP chimeric antigen–based ELISA and commercial assays. The design follows the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines.

Based on the combined risk stratification approach integrating epidemiological information, conventional ECG findings, and ECG-AI output, 121 individuals were selected for serological evaluation. Of these, 80 were classified as being at increased risk for T. cruzi infection, and 41 low-risk individuals were randomly selected as controls at a 2:1 ratio. Blood samples were obtained from 112 participants, while the remaining screened individuals (n = 994) were not included in serological testing (S1 Table).

Among the 73 participants with ECG abnormalities who underwent serological testing, 13 (17.8%) were classified as seropositive for T. cruzi. In contrast, none of the 39 control individuals tested positive, resulting in a seropositivity rate of 0% in this group. Overall, the seropositivity rate among all tested participants was 11.6% (13/112) (Fig 1).

False-positive alerts generated by the ECG-AI algorithm were observed in 22 individuals. Among these, six presented major ECG abnormalities and 16 presented minor abnormalities; however, all were classified as seronegative by both indirect hemagglutination and latent class analysis. Echocardiographic screening among individuals with confirmed T. cruzi infection revealed left or right ventricular involvement in only two participants, while the remaining seropositive individuals showed no major structural abnormalities at the time of screening.

Serological classification and assay concordance

The indirect hemagglutination assay classified 99 samples (88.4%) as non-reactive and 13 samples (11.6%) as reactive for anti-T. cruzi antibodies. Latent class analysis yielded an identical classification, with all 99 seronegative samples showing no reactivity to any of the four IBMP antigens and posterior probabilities of zero for positivity (S1 Table). Among the 13 seropositive samples, eight (61.5%) reacted with all four IBMP antigens, two (15.4%) reacted with three antigens, and three (23.1%) reacted with two antigens. Posterior probabilities for positivity ranged from 96.9% to 100%, confirming the classification of T. cruzi infection. No discordant results between indirect hemagglutination and LCA were observed. In addition, both commercial ELISA assays (Gold ELISA Chagas and Bioelisa Chagas Recombinante) detected seropositivity in 100% (13/13) of the confirmed positive samples (Table 1).

10.1371/journal.pntd.0013892.t001Reactivity patterns of serum samples from participants screened for Chagas disease in Feira de Santana, Bahia, Brazil.
Participant number In-house assays with IBMP antigens Commercial kits
-8.1 -8.2 -8.3 -8.4 LCA P (%) * IHA1 GEC2 BIO3
1 2.16 0.55 1.07 0.41 POS 96.9 1:64 2.64 3.96
2 2.00 0.59 1.03 0.58 POS 96.9 1:64 2.54 3.53
3 2.04 1.58 1.83 2.17 POS 100 1:128 12.41 3.03
4 0.75 1.47 1.53 1.54 POS 100 1:256 7.35 3.22
5 2.75 1.84 1.72 1.85 POS 100 1:256 9.54 2.57
6 2.95 1.83 1.69 2.47 POS 100 1:64 7.89 3.15
7 2.41 1.54 1.85 1.49 POS 100 1:256 11.01 2.99
8 1.44 0.62 0.58 1.01 POS 99 1:64 2.47 2.68
9 1.95 1.17 0.76 1.23 POS 100 1:128 5.27 2.29
10 1.89 1.77 1.82 2.48 POS 100 1:512 12.71 3.57
11 2.84 1.08 1.90 2.03 POS 100 1:256 10.56 3.26
12 2.56 1.52 1.81 1.73 POS 100 1:64 10.66 2.33
13 2.24 1.16 1.44 1.75 POS 100 1:512 9.01 2.61

1IHA (Imuno-HAI Chagas); 2GEC (Gold ELISA Chagas); 3BIO (Bioelisa Chagas Recombinante); LCA (Latent Class Analysis); POS (Positive). *Probability of being T. cruzi-positive by LCA.

 Demographic and epidemiological characteristics of seropositive individuals

Among individuals classified as seropositive for T. cruzi, the median age was 60 years (IQR: 39–69), and the female-to-male ratio was 5.5:1. Seronegative individuals had a median age of 56 years (IQR: 44–64) and a female-to-male ratio of 4.8:1 (S1 Table). Notably, 69.2% of seropositive participants were not born in Feira de Santana but had migrated from other endemic municipalities within the state of Bahia, including Conceição do Jacuípe (n = 1), Coração de Maria (n = 1), Ipirá (n = 1), Mundo Novo (n = 1), Muniz Ferreira (n = 1), Santa Maria da Vitória (n = 1), Utinga (n = 1), and Santo Estevão (n = 2) (Fig 2). This suggests migration as a potential factor influencing local epidemiological trends.

10.1371/journal.pntd.0013892.g002Geographic origins of individuals seropositive for <italic>Trypanosoma cruzi</italic> in Feira de Santana, Bahia, Brazil.

The map shows the municipalities of birth of participants who tested positive for Chagas disease during the Every Heartbeat Matters screening initiative. Public domain digital maps were obtained from the Brazilian Institute of Geography and Statistics (IBGE) and analyzed using QGIS version 3.10 (Geographic Information System, Open-Source Geospatial Foundation Project. http://qgis.osgeo.org).

Although most seropositive individuals reported awareness of their clinical condition, 23% were unaware of their T. cruzi infection status prior to participation in the screening initiative (Fig 2).

All seropositive individuals were older than 46 years and were either retired or beneficiaries of government assistance programs (Fig 2), except for one family cluster. This family consisted of two adults and three children and epidemiologically likely to a suspected episode of oral transmission after consumption of açaí juice in February 2024 near Feira de Santana. Two children (aged 9 and 13 years) and both parents (aged 40 and 38 years) tested seropositive, while one child who did not consume the juice tested seronegative. All infected family members received etiological treatment and remained under clinical follow-up.

 Risk factor analysis

In univariate analysis, self-reported observation of triatomine bugs inside the household was associated with a higher prevalence of T. cruzi seropositivity (PR = 4.38; 95% CI: 1.44–13.3, p = 0.004). Because triatomine exposure was not systematically compared between seropositive and seronegative individuals in the entire screened population, this association should be interpreted cautiously, as it may reflect broader characteristic of the surveyed community rather than a causal relationship. Other evaluated variables, including reported knowledge of the vector, presence of a chicken coop near the household, and family history of CD, were not significantly associated with seropositivity. This suggests that while reporting triatomine presence indoors may indicate increased risk, the association requires confirmation in broader community-based analyses (Table 2).

10.1371/journal.pntd.0013892.t002Univariate analysis of epidemiological factors associated with <italic>Trypanosoma cruzi</italic> seropositivity in participants from Feira de Santana, Bahia, Brazil.
Variables Univariate analysis
PR 95%CI p-value
Prior exposure to triatomines 4.38 1.44 to 13.3 0.004
Presence of a chicken coop 0.82 0.27 to 2.48 0.730
Familial history of CD 1.81 0.59 to 5.55 0.285

PR (Prevalence ratio), β (Log-odds coefficient), CD (Chagas disease) CI (Confidence interval).

 Discussion

This study leverages a community-based cardiac screening initiative to conduct a seroepidemiological survey, integrating large-scale ECG and echocardiographic screening for the identification of CD. A significant association was identified between self-reported household observation of triatomine bugs and T. cruzi seropositivity, underscoring vector exposure as a key factor associated with CD occurrence in endemic regions. While other environmental factors, such as the presence of chicken coops near households and family history of CD, were not significantly associated, direct exposure to the vector emerged as the strongest factor linked to a higher prevalence of infection. These findings are consistent with previous studies emphasizing the critical role of domestic vector infestation in sustaining transmission cycles [20,21].

The overall seropositivity rate of T. cruzi infection observed in this study (8.5%) is comparable to reports from other endemic areas of Bahia [4–6], reinforcing the persistent public health challenge posed by CD. The high seropositivity rate among individuals reporting the presence of triatomines inside their households highlights the need for effective vector control programs. In addition to reducing domestic infestation, community education focused on vector recognition and preventive measures remains essential to disrupt transmission dynamics.

A notable observation was that most T. cruzi-positive individuals currently residing in Feira de Santana were born in other endemic areas. This finding suggests that many infections likely occurred prior to migration, highlighting the influence of population mobility on CD epidemiology. However, it remains unclear whether migration itself represents an independent risk factor for T. cruzi transmission or whether expose occurred in the individuals’ places of origin before relocation. The absence of longitudinal data on the timing of infection limits causal inferences related to migration, as transmission may have occurred either before or after resettlement. Furthermore, differences in vector control programs and access to healthcare across endemic regions may differentially influence infection risks among migrant populations. These findings support the need for targeted interventions and emphasize the importance of integrating migrant populations into local healthcare systems. Tailored strategies to improve access to diagnostic and treatment services for migrant communities are essential to reduce the overall burden of CD in endemic regions.

Further analysis revealed that individuals who reported finding triatomines in their households presented fourfold higher prevalence of T. cruzi infection (PR = 4.38, p = 0.004) compared with those who did not report vector presence indoors. This result further underscores the central role of domestic vector infestation in maintaining transmission cycles and corroborates previous research identifying in-home triatomine presence as a critical determinant of infection risk [20,21]. The lack of significant associations for other variables, such as general knowledge about the vector or the presence of chicken coops, reinforces the primacy of direct vector contact in transmission dynamics.

From a methodological perspective, the use of IBMP chimeric antigens for serological screening, complemented by commercial assays for case validation, resulted in high diagnostic accuracy. The application of latent class analysis to classify seropositive cases minimized misclassification bias and enhanced the reliability of the findings. These methodological strengths demonstrate the value of integrating innovative diagnostic tools into community-based health initiatives to effectively address CD in endemic regions.

Although most participants were aware of their clinical conditions, it is noteworthy that 23% of individuals identified as T. cruzi-positive were unaware of their infection status. This finding highlights the insidious nature of chronic CD, which may remain asymptomatic or manifest with nonspecific symptoms for prolonged periods, thereby complicating timely diagnosis. Limited access to healthcare services, suboptimal screening practices, and low disease awareness in at-risk communities likely contribute to this diagnostic gap. The fact that nearly one quarter of seropositive individuals were previously undiagnosed underscores the need for enhanced community-based screening programs, particularly in endemic settings. By integrating serological testing with cardiac screening initiatives, as demonstrated in this study, early detection and intervention become feasible, potentially reducing disease progression and long-term morbidity. This observation aligns with previous studies reporting substantial proportions of undiagnosed infections in similar contexts [22–24].

However, the cross-sectional design of this study limits causal inferences, and the relatively small number of seropositive cases may affect the generalizability of the findings. In addition, recruitment through a cardiac screening initiative introduces potential selection bias, as individuals with underlying cardiovascular abnormalities may not be representative of the broader population at risk for T. cruzi infection. This could lead to an overestimation of seropositivity in this subgroup while underrepresenting asymptomatic or early-stage infections in the general community. To mitigate sampling bias in serologic testing, a computer-generated random selection of low-risk controls based on ECG-AI scores was applied, improving the representativeness of the tested sample. Future longitudinal studies with larger, community-based populations, such as those planned within the Oxente Chagas Project [25], are needed to further explore the temporal dynamics of infection risk and validate these findings.

In conclusion, this study provides important insights into the epidemiology of T. cruzi infection in Feira de Santana, Bahia, emphasizing the central role of vector exposure in CD transmission. The integration of seroepidemiological surveys with cardiac screening initiatives represents a valuable model for identifying at-risk populations and improving early detection of CD. Continued investment in vector control, diagnostic innovation, and public health education remains essential to reduce the burden of CD in endemic regions [20,26]. These findings underscore the importance of community-based health initiatives in advancing CD control and improving outcomes in underserved and vulnerable populations.

 Supporting information Sociodemographic, epidemiological, and serological characteristics of the study samples.

(XLSX)

We are grateful to the Holy House of Mercy team in Feira da Santana for their invaluable assistance in performing this study.

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Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021. Mem Inst Oswaldo Cruz. 2021;116:e210015. doi: 10.1590/0074-02760210015 34076075 Gurgel-Gonçalves R, Galvão C, Costa J, Peterson AT. Geographic distribution of chagas disease vectors in Brazil based on ecological niche modeling. J Trop Med. 2012;2012:705326. doi: 10.1155/2012/705326 22523500 Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC, et al. The epidemiologic importance of Triatoma brasiliensis as a chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem Inst Oswaldo Cruz. 2003;98(4):443–9. doi: 10.1590/s0074-02762003000400002 12937751 Carvalho CMM de, Ribeiro-Jr G, Gurgel-Gonçalves R, Andrade LS, Moraes CA, Figueiredo MAA. Spatio-temporal trends in mortality due to Chagas disease in the State of Bahia, Brazil, from 2008 to 2018. Rev Soc Bras Med Trop. 2024;57:e004172024. doi: 10.1590/0037-8682-0058-2024 39476074 Diamantino AC, Nascimento BR, Nunes MCP, Sable CA, Oliveira KKB, Rabelo LC, et al. Impact of incorporating echocardiographic screening into a clinical prediction model to optimise utilisation of echocardiography in primary care. Int J Clin Pract. 2021;75(3):e13686. doi: 10.1111/ijcp.13686 32852108 Instituto Brasileiro de Geografia e Estatística IBGE. Feira de Santana. 2023. Accessed 2025 September 25. https://cidades.ibge.gov.br/brasil/ba/feira-de-santana/panorama Ribeiro ALP, Marcolino MS, Prineas RJ, Lima-Costa MF. Electrocardiographic abnormalities in elderly Chagas disease patients: 10-year follow-up of the Bambui Cohort Study of Aging. J Am Heart Assoc. 2014;3(1):e000632. doi: 10.1161/JAHA.113.000632 24510116 Jidling C, Gedon D, Schön TB, Oliveira CDL, Cardoso CS, Ferreira AM, et al. Screening for Chagas disease from the electrocardiogram using a deep neural network. PLoS Negl Trop Dis. 2023;17(7):e0011118. doi: 10.1371/journal.pntd.0011118 37399207 Santos FLN, Celedon PAF, Zanchin NIT, Brasil T de AC, Foti L, Souza WV de, et al. Performance assessment of four chimeric Trypanosoma cruzi antigens based on antigen-antibody detection for diagnosis of chronic Chagas disease. PLoS One. 2016;11(8):e0161100. doi: 10.1371/journal.pone.0161100 27517281 Santos FLN, Campos ACP, Amorim LDAF, Silva ED, Zanchin NIT, Celedon PAF, et al. Highly accurate chimeric proteins for the serological diagnosis of chronic chagas disease: a latent class analysis. Am J Trop Med Hyg. 2018;99(5):1174–9. doi: 10.4269/ajtmh.17-0727 30226130 Santos EF, Leony LM, Silva ÂAO, Daltro RT, Freitas NEM, Vasconcelos LCM, et al. Assessment of Liaison XL murex chagas diagnostic performance in blood screening for Chagas disease using a reference array of chimeric antigens. Transfusion. 2021;61(9):2701–9. doi: 10.1111/trf.16583 34240750 Dos Santos EF, Silva ÂAO, Freitas NEM, Leony LM, Daltro RT, Santos CA de ST, et al. Performance of chimeric Trypanosoma cruzi antigens in serological screening for chagas disease in blood banks. Front Med (Lausanne). 2022;9:852864. doi: 10.3389/fmed.2022.852864 35330587 Dias JCP, Ramos AN Jr, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. 2 nd Brazilian consensus on Chagas disease, 2015. Rev Soc Bras Med Trop. 2016;49Suppl 1(Suppl 1):3–60. doi: 10.1590/0037-8682-0505-2016 27982292 Pan American Health Organization PAHO. Updated estimate of Chagas disease in the endemic countries of the Americas 2018. Washington, D.C. 2025. https://iris.paho.org/items/cd0ec286-92c9-4a2d-9b11-c1d2c08249b4 Fidalgo ASO de BV, Costa AC da, Ramos Júnior AN, Leal LKAM, Martins AMC, Silva Filho JD da, et al. Seroprevalence and risk factors of Chagas disease in a rural population of the Quixeré municipality, Ceará, Brazil. Rev Soc Bras Med Trop. 2021;54:e0247-2020. doi: 10.1590/0037-8682-0247-2020 33681912 Freitas EC, Oliveira M de F, Vasconcelos ASO de B, Silva JD da Filho, Viana CEM, Gomes KCMS, et al. Analysis of the seroprevalence of and factors associated with Chagas disease in an endemic area in Northeastern Brazil. Rev Soc Bras Med Trop. 2017;50(1):44–51. doi: 10.1590/0037-8682-0242-2016 28327801 de Aquino Santana M, da Silva Ferreira AL, Dos Santos LVB, Furtado Campos JH, de Sena LLJ, Mendonça VJ. Seroprevalence of Chagas disease in rural communities at Campinas do Piauí city, Brazil. Trop Med Int Health. 2021;26(3):281–9. doi: 10.1111/tmi.13516 33159709 Santos FLN, Pavan TBS, Valle CS, Sampaio DD, Vasconcelos LCM, Cristóbal MH, et al. The Oxente Chagas Bahia Project: evaluating the efficacy of a rapid diagnostic test and treatments for Chagas disease. Mem Inst Oswaldo Cruz. 2024;119:e240140. doi: 10.1590/0074-02760240140 39476029 Santos FLN, Costa VM da, Silva RAE. Chagas disease in Brazil: new challenges and perspectives for old problems. Mem Inst Oswaldo Cruz. 2025;120:e240279. doi: 10.1590/0074-02760240279 40699036 10.1371/journal.pntd.0013892.r001 Decision Letter 0 Werneck Guilherme Section Editor 2026 Guilherme Werneck This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

31 Jul 2025

Seroepidemiological survey and seropositivity rate for Trypanosoma cruzi infection in a community-based cardiac screening initiative in Feira de Santana, Bahia, Brazil

PLOS Neglected Tropical Diseases

Dear Dr. Santos,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 60 days Sep 29 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

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If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Marilia Sá Carvalho

Academic Editor

PLOS Neglected Tropical Diseases

Guilherme Werneck

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Journal Requirements:

- We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- ® on pages: 7, 9, and 10.

Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods:

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Once Bahia State presents one of the highest triatomine infestation rates in Brazil, the hypothesis of the study should be presented in a testable way.

The design is appropriate and in accordance with the objectives and the obtained results; the sample size is appropriate and the ethical issues are well addressed.

Reviewer #2: The objectives are clear and consistent with the cross-sectional study design. I believe that the sample size is sufficient and appropriate for the hypothesis tested using algorithm-based artificial intelligence as a technological tool, and the statistical analysis supports the conclusions. There are no ethical concerns.

Reviewer #3: In the absence of a parasitological test, the WHO recommends and is internationally accepted that a definitive serological diagnosis must be based on two distinct serological tests. This is especially important in trypanosomatids, where cross-reactivity with other Trypanosoma species and even Leishmania is reported. The inclusion of a second test should not be presented as “Reference Testing”, but as second confirmatory for the ELISA. Even using four chimeric proteins, it remains the same test (ELISA), and the high chance of cross-reactivity (Leishmaniasis is a reality throughout Brazil) increases the risk of false positives. Morevoer, The echocardiographic test cannot be considered a screening test, given that several other diseases present with cardiac dysfunction, and a high percentage of Chagas seropositive individuals do not develop symptoms.

The “structured questionnaire” (line 100) wasn't presented, and I imagine it's not just the three questions that appear in the following paragraph.

Electrocardiogram (ECG) and echocardiography: Which equipment? How was it performed? By the same professional? Who interpreted the results? The methodological approach is lacking in detail.

Artificial intelligence (AI)-based algorithm: How was it done? What was the methodology?

“Clinical data” (line 110): What clinical symptoms formed the basis for the clinical data? Who collected it? What aspects were assessed?

Results:

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are well presented and ilustrated in the tables and figs

The legends must be revised

Reviewer #2: The results are presented as planned, in a clear and concise manner with self-explanatory figures and tables. Positive highlights of the study include the integration of new diagnostic tools into health initiatives and the efficient approach to detecting Chagas disease in endemic regions.

Reviewer #3: The association with presence of triatomine bugs cannot be done because authors didn’t compare this trait to the negatives. It could just reflect the overall population characteristic.

Lines 177-178: These results need to be better described. If they are part of another study, you need to reference the other study that contains these data.

The “AI algorithm” (line 179): There is also a lack of information on how it was produced and tested.

“…based on predefined criteria…” (line 181): criteria not presented in the article

Line 187: Why false positives, since several other diseases besides Chagas' disease cause heart problems? Several other diseases present with cardiac dysfunction, and a high percentage of Chagas seropositive individuals do not develop symptoms.

Lines 202-203: This affirmation cannot be done because negative samples, in one and/or the commercial tests, were evaluated.

Conclusions:

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions are in accordance with the obtained results which are well discussed

The manuscript presents a relevant analysis on Chagas disease, which represents an important public health concern

Reviewer #2: The conclusions are appropriate to the data and highlight the limitations of the study. There is good discussion of the data in light of the epidemiologic indicators related to Chagas disease. It also addresses the importance of reporting suspected cases in primary care and public health services, early diagnosis, and gaps in the availability of etiologic treatment as relevant factors in controlling parasite transmission. It also highlights the importance of integrating seroepidemiologic surveys with cardiac screening, which I believe is a valuable model for identifying at-risk populations and improving early detection of T. cruzi infection. The data also show that it is necessary and essential to invest in diagnostic innovation to fill the gaps that still exist in the availability of etiologic treatment and relevant factors in the control of parasite transmission and ongoing epidemiologic surveillance.

Reviewer #3: It could not be evaluated because of the appointments above described.

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Scientific names should be written in italic letters.

Reviewing the list of references.

Corrections are highlighted in yellow in the text. See attached manuscript.

Reviewer #3: Reject

Summary and General Comments:

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Review PNTD-D-25-00569

General Comments: This is a very relevant manuscript, which updates and integrates a seroepidemiological survey with large-scale echocardiographic screening to estimate the prevalence of T. cruzi infection and identify associated risk factors in Feira de Santana, Bahia, Brasil. The manuscript is well written, presented, the methodology is in accordance with the objectives, and results. The main concern is the short introduction. A few other issues were also detected therefore, changes are recommended before the final acceptance of the manuscript in the PNTD.

Specific comments

Abstract please provide brief information on the place the study was carried out.

Introduction

Please provide a brief background on Bahia State related to epidemiological survey on Chagas disease as well as for Feira de Santana. The authors cite reference n. 4 however, we recommend a few words on the CD in Bahia and specially in Feira de Santana Municipality.

Also, it is recommended some words on the triatomine fauna in Bahia State, which presents the richest biodiversity of CD vector species, being one of the most infested states in Brasil. In addition to that new species have been described, revalidated in that state and also new triatomine species have been getting adapted to the human dwellings enhancing the risk of transmission of Chagas disease.

The authors mention the vectors as a risk factor for CD therefore, it is recommended including some references mentioning the vector species in Bahia.

Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC, et al. The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem Inst Oswaldo Cruz. 2003; 98(4): 443-9.

Gurgel-Gonçalves R, Galvão C, Costa J, Peterson AT. Geographic distribution of Chagas disease vectors in Brazil based on ecological niche modeling. J Trop Med. 2012; 705326.

Costa, J.; Dale, C.; Galvão, C.; Almeida, C.E.; Dujardin, J.P. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979–2021. Mem. Inst. Oswaldo Cruz 2021, 116, e210015.

M & M

Please provide relevant information about Feira de Santana, physio geographic information based for instance in IBGE data and more specifically about the place the analyzed people reside, age, sex, socio economic status etc.

Discussion

From lines 312 – 315 please, include a reference in the text

From lines 333-335 please, include a reference in the text

Figs and Legends

Legends should be self-explanatory, please revise the legend of fig 1 providing information about the locality, the disease and any other relevant additional information. The same is applied for Table 1, fig 2 and Table 2

Reviewer #2: This study demonstrates how we can improve diagnostic strategies for T. cruzi infection using large-scale digital health AI tools aimed at overcoming barriers to identifying seropositive patients with excellent results. The significant association between household observation and the presence of triatomine highlights the ongoing exposure to the vector and the occurrence of parasite transmission in endemic areas. Given the possibility of increased risk of infection, it is important to emphasize the importance of early diagnosis using innovative methods, prompt etiologic treatment of cases, and well-designed clinical follow-up.

Reviewer #3: There are structural issues and a lack of clarity in the text. The methodology is not presented in full, and as a result, there is a lack of information to evaluate the results and discussion presented. The primary objective “estimate the seropositivity rate of T. cruzi infection” (lines 89-90) cannot be achieved with the proposal methodology.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Reproducibility:

?>

Submitted filename: PNTD-D-25-00569_reviewer.pdf

 10.1371/journal.pntd.0013892.r002 Author response to Decision Letter 1 Submission Version 1

30 Sep 2025

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pntd.0013892.r003 Decision Letter 1 Werneck Guilherme Section Editor 2026 Guilherme Werneck This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

7 Dec 2025

Response to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments:--> -->-->The revised manuscript represents a significant improvement, but we need an explicit consideration regarding the concern about "the absence of a second serological test to confirm infection" raised by Reviewer #3. Could the lack of such a confirmatory test have biased the results of the study?-->--> -->--> -->-->?>Journal Requirements:

Reviewers' comments:

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The manuscript was greatly improved. The objectives are clear and the design is appropriate to address the objectives.

The manuscrilpt presents sufficient sample size to anwer the objectives. All concerns about ethical or regulatory requrements were clarified.

Reviewer #2: The objectives are clear and consistent with the cross-sectional study design. In this study, the sample population corresponds to the tested hypothesis, and the statistical analysis supports the conclusions. There are no ethical concerns.

Reviewer #3: The main point made by me in the previous version was not answered: the absence of a second serological test to confirm infection. This absence impacts the entire article because it lacks the necessary robustness to define those infected humans. Another unresolved point is associating the questionnaire on triatomine exposure only with serologically positive and not negative tests. The authors agreed and included this as a limitation, but simply including this limitation does not validate the analysis. Because I understand the first aspect impacts the accuracy of the diagnosis on which the entire article is based, I maintain my recommendation to reject the article.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are clear and matcher the analysis plan and presented in full. Figs and tables are sufficient to illustrate the obtained data.

Reviewer #2: The data analysis corresponds to the study plan, which involved investigating the prevalence of Trypanosoma cruzi infection and associated risk factors in an area endemic for Chagas disease in the state of Bahia, Brazil using a cardiac screening programme. The seropositivity rate in this area is 8.5%. The data are clear and complete, figures and tables are self-explanatory and of a high standard.

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusion is now addressing all relevant results obtained in the revised study encompassing a important topic on public heath once it updates the seroepidemiological and seropositivity data in Ferira de Santana, BA, Brazil. The obtained data was clearly discussed and contextualized in terms of advances and understanding Chagas disease in the studied area

Reviewer #2: The conclusions are appropriate to the data emphasizing the study limitations to study cross-sectional design. The study emphasises the significance of vector exposure in the transmission of Chagas disease, underscoring the necessity of targeted interventions. Further longitudinal studies involving larger community-based samples are required to investigate the temporal dynamics of infection risk by the parasite, and to validate these findings within broader populations.

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The manuscript can now be accepted for publication

Reviewer #2: I suggest reviewing the entire manuscript of the text and references.

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: All issues were already addressed

Reviewer #2: I suggest that the authors review the entire manuscript, including the references. I recommend stating that transmission is of the etiological agent, T. cruzi, rather than of the disease itself, since most individuals with positive serology remain asymptomatic.

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure resubmission:

Reproducibility:--> -->-->To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols-->?>

Submitted filename: Main document with tracks.docx

 10.1371/journal.pntd.0013892.r004 Author response to Decision Letter 2 Submission Version 2

20 Dec 2025

Submitted filename: Response to Reviewers.pdf

 10.1371/journal.pntd.0013892.r005 Decision Letter 2 Werneck Guilherme Section Editor 2026 Guilherme Werneck This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

22 Dec 2025

Dear Dr Santos,

We are pleased to inform you that your manuscript 'Seroepidemiological survey and seropositivity rate for Trypanosoma cruzi infection in a community-based cardiac screening initiative in Feira de Santana, Bahia, Brazil' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Guilherme L Werneck

Section Editor

PLOS Neglected Tropical Diseases

Guilherme Werneck

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

***********************************************************

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 10.1371/journal.pntd.0013892.r006 Acceptance letter Werneck Guilherme Section Editor 2026 Guilherme Werneck This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Dear Dr Santos,

We are delighted to inform you that your manuscript, " 

Seroepidemiological survey and seropositivity rate for Trypanosoma cruzi infection in a community-based cardiac screening initiative in Feira de Santana, Bahia, Brazil," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases