Performance Assessment of Treponemal and Nontreponemal Tests for the Diagnosis of Acquired Syphilis.

There are a variety of nontreponemal test (NTT) and treponemal test (TT) kits for the serologic diagnosis of syphilis. Because of the complexity of the infection (multiple clinical stages) and the different antigens used in these kits, a systematic evaluation of the accuracy of the currently available commercial tests is warranted. Our objective was to evaluate the performance of commercially available tests for the diagnosis of syphilis infection. In this study, we analyzed one NTT (Venereal Disease Research Laboratory [VDRL] test, Wiener Laboratories, Rosario, Argentina) and two TTs (fluorescent treponemal antibody absorption [FTA-ABS] test, Euroimmun, Lübeck, Germany, and syphilis recombinant ELISA v. 4.0 test [ELISA], Wiener Laboratories, Rosario, Argentina) using a panel of 187 samples, including serum samples from 31 individuals with primary syphilis, 77 with secondary syphilis, and 79 with latent syphilis. An additional 192 samples from uninfected individuals and 323 serum samples from individuals with other diseases were included. The sensitivities of the VDRL, ELISA, and FTA-ABS tests were 97.9%, 100%, and 96.3%, respectively. The VDRL and ELISA tests showed a specificity of 100%, and the FTA-ABS test showed a specificity of 99.5%. Accuracy was 98.9% for the VDRL test, 100% for the ELISA, and 97.9% for the FTA-ABS test. For primary, secondary, and latent syphilis, the ELISA achieved a diagnostic performance of 100%, whereas the sensitivity for the VDRL and FTA-ABS tests ranged from 96.8% to 98.7% and 93.7% to 98.7%, respectively. No difference was observed when the tests were used as traditional or reverse algorithms. In general, all three tests are able to discriminate positive and negative samples for syphilis, regardless of the diagnostic algorithm.

Post-therapeutic cure criterion in chronic Chagas disease using Trypanosoma cruzi chimeric proteins.

Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi Chagas, 1909. Causative treatment can be achieved with two drugs: benznidazole or Nifurtimox. There are some gaps that hinder progress in eradicating the disease. There is no test that can efficiently assess cure control after treatment. Currently, the decline in anti-T. cruzi antibody titres is assessed with conventional serological tests, which can take years. However, the search for new markers of cure must continue to fill this gap. The present study aimed to evaluate the decline in serological titres using chimeric proteins after treatment with benznidazole in chronic patients diagnosed with CD. It was a prospective cross-sectional cohort study between 2000 and 2004 of T. cruzi-positive participants from the Añatuya region (Argentina) treated with benznidazole. Serum samples from ten patients were collected before treatment (day zero) and after the end of treatment (2, 3, 6, 12, 24 and 36 months). For the detection of anti-T. cruzi antibodies, an indirect ELISA was performed using two chimeric recombinant proteins (IBMP-8.1 and IBMP-8.4) as antigens. The changes in reactivity index within the groups before and after treatment were evaluated using the Friedman test. All participants experienced a decrease in serological titres after treatment with benznidazole, especially IBMP-8.1. However, due to the small number of samples and the short follow-up period, it is premature to conclude that this molecule serves as a criterion for sustained cure. Further studies are needed to validate tests based on these or other biomarkers to demonstrate parasitological cure.

Evaluating the diagnostic accuracy of TpN17 and TmpA recombinant proteins in syphilis detection: a phase II study.

Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi. The chronic phase of CD is characterized by the presence of IgG anti-T. cruzi antibodies; and diagnosis is performed by serological methods. Because there is no reliable test that can be used as a reference test, WHO recommends the parallel use of two different tests for CD serodiagnosis. If results are inconclusive, samples should be subjected to a confirmatory test, e.g., Western blot (WB) or PCR. PCR offers low sensitivity in the chronic phase, whereas few confirmatory tests based on the WB method are commercially available worldwide. Therefore, new diagnostic tools should be evaluated to fill the gap in CD confirmatory tests. In recent years, four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) have been evaluated in phase I, II and III studies using ELISA, liquid microarray and immunochromatography with 95-100% accuracy. Given the high diagnostic performance of these antigens, the present study investigated the ability of these molecules to diagnose chronic CD using a WB testing platform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis (i.e., potentially cross-reactive) using WB as the diagnostic platform. Checkerboard titration with different dilutions of antigens, conjugated antigens, and serum samples was performed to standardize all assays. All IBMP antigens achieved 100% sensitivity, specificity, and accuracy, with the exception of IBMP-8.3, which had 100% specificity despite lack of significance, but lower sensitivity (95%) and accuracy (96.9%). No cross-reactivity was observed in samples positive for leishmaniasis. CONCLUSIONS/SIGNIFICANCE: The present phase I (proof-of-concept) study demonstrated the high diagnostic potential of these four IBMP antigens to discriminate between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with WB.

Spatiotemporal distribution analysis of syphilis in Brazil: Cases of congenital and syphilis in pregnant women from 2001-2017.

In Brazil, the notification of congenital (CS) and syphilis in pregnant women (SiP) is compulsory. Notification data provided by the Ministry of Health in combination with the mapping of vulnerable geographic areas is essential to forecasting possible outbreaks and more effectively combating infection through monitoring. We aim to evaluate the spatiotemporal distribution and epidemiological aspects of reported cases of CS and SiP in Brazil. A retrospective ecological study was carried out using secondary surveillance data obtained from the Brazilian National Notifiable Diseases Information System (SINAN) database, considering all reported cases of CS and SiP between 2001 to 2017. Epidemiological characteristics and time trends were analyzed using joinpoint regression models and spatial distribution, considering microregions or states/macroregions as units of analysis. A total of 188,630 (359/100,000 birth lives) CS and 235,895 of SiP (6.3/100,000 inhabitants) were reported during the period studied. In general, the epidemiologic profile of Brazil indicates most reported CS cases occurred in "mixed-race" newborns who were diagnosed within seven days of birth and whose mothers had received prenatal care, but the epidemiologic profile varies by Brazilian macroregion. Regarding SiP, most cases were among women who self-reported 'mixed-race', were aged 20-39 years, had up to eight years of formal education and were diagnosed with primary or latent syphilis. Approximately 549 (98.4%) and 558 (100%) microregions reported at least one case of CS and SiP, respectively. From 2012 to 2016, CS cases increased significantly in almost all Brazilian states, most notably in the South, Southeast, and Central-West macroregions, from 2001-2017 and the relative risk (RR) of SiP increased around 400% (RR: 1,00 to 445,50). Considering the epidemiological scenario of the infection in Brazil, it is necessary to enhance preventive, control and eradication measures.

Technological advances in the serological diagnosis of Chagas disease in dogs and cats: a systematic review.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi, which is transmitted mainly through the feces/urine of infected triatomine bugs. The acute phase lasts 2-3 months and is characterized by high parasitemia and nonspecific symptoms, whereas the lifelong chronic phase features symptoms affecting the heart and/or digestive tract occurring in 30-40% of infected individuals. As in humans, cardiac abnormalities are observed in T. cruzi-infected dogs and cats. We reviewed the technological advances in the serological diagnosis of CD in dogs and cats. METHODS: A review of the published literature during the last 54 years (1968-2022) on the epidemiology, clinical features, diagnosis, treatment and prevention of CD in dogs and cats was conducted. RESULTS: Using predefined eligibility criteria for a search of the published literature, we retrieved and screened 436 publications. Of these, 84 original studies were considered for inclusion in this review. Dogs and cats are considered as sentinels, potentially indicating an active T. cruzi transmission and thus the risk for human infection. Although dogs and cats are reputed to be important for maintaining the T. cruzi domestic transmission cycle, there are no commercial tests to detect past or active infections in these animals. Most published research on CD in dogs and cats have used in-house serological tests prepared with native and/or full-length recombinant antigens, resulting in variable diagnostic performance. In recent years, chimeric antigens have been used to improve the diagnosis of chronic CD in humans with encouraging results. Some of them have high performance values (> 95%) and extremely low cross-reactivity rates for Leishmania spp., especially the antigens IBMP-8.1 to IBMP-8.4. The diagnostic performance of IBMP antigens was also investigated in dogs, showing high diagnostic performance with negligible cross-reactivity with anti-Leishmania infantum antibodies. CONCLUSIONS: The development of a commercial immunodiagnostic tool to identify past or active T. cruzi infections in dogs and cats is urgently needed. The use of chimeric recombinant T. cruzi antigens may help to fill this gap and is discussed in this review.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform. METHODOLOGY/PRINCIPAL FINDINGS: DAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV. CONCLUSIONS/SIGNIFICANCE: DAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

Eco-epidemiology of vectorial Trypanosoma cruzi transmission in a region of northeast Brazil.

Chagas disease (CD) is a parasitic zoonosis endemic in Brazil. Despite virtual control of Triatoma infestans, the main domesticated vector of Trypanosoma cruzi, vectorial transmission by other triatomine species persists in some rural communities. This study aims to characterize triatomines role in transmitting T. cruzi to dogs and humans in the district of Santo Inácio, located in the northwest region of the state of Bahia, Brazil. It also describes environmental factors in housings associated with insect occurrence and assesses the perception, knowledge, and preventive practices adopted by the population regarding CD. Blood samples of humans and dogs, and biological samples of triatomines, were collected between November 2018 and February 2019 and subjected to the detection of T. cruzi by serological and molecular biology tests. Also, we applied a questionnaire to research the perception, knowledge, and local practices of people related to CD. The capture of triatomines in households was associated with exploratory variables of the questionnaires using multivariate logistic regression (p < 0.05). The 155 triatomines captured in the wild and domestic environment were of the species Triatoma sherlocki (n = 151), Panstrongylus sherlocki (n = 1) and Triatoma sordida (n = 3), and had a natural infection rate for T. cruzi by PCR of 18.5%, 100% and 0%, respectively. District residents (n = 126) were seronegative for T. cruzi, while 17.5% (7/40) of the dogs were seropositive. The fact that residents are aware that triatomines can "cause" CD was configured as a protection factor for residents according to the fitted logistic regression model (p = 0.04). However, respondents have limited perception and knowledge about the CD, prevention and control practices for triatomines in a household. The results suggest the existence of a domestic cycle of transmission of T. cruzi between triatomines and dogs, configuring a latent risk of infection to the human population of Santo Inácio. Studies that clarify the potential for the establishing of intrusive triatomines in households, surveillance actions for triatomines, and health education in rural communities are indispensable to prevent the reemergence of CD in vulnerable regions of Brazil and other American countries with similar epidemiological characteristics.

Seroprevalence and detection of Trypanosoma cruzi in dogs living in a non-endemic area for Chagas disease in the legal Amazon region, Brazil.

Trypanosoma cruzi, the etiological agent for Chagas disease, is widely distributed in the Americas. Its hosts are humans and wild and domestic mammals, and its vectors are triatomine insects. Studies have indicated that domestic dogs are sentinel animals in the epidemiology of Chagas disease in endemic regions, including states in the Legal Amazon region of Brazil. In São Luís, the capital of Maranhão, a non-endemic state, the existence of a domestic cycle involving domestic rats has been proven, along with a wild cycle maintained by didelphids. However, no studies on T. cruzi infection in domestic animals in this locality have been conducted. The aim of this study was to investigate occurrence of T. cruzi in dogs living in the Itaqui Bacanga district of São Luís, Maranhão, by means of serological and molecular tests. Blood samples were obtained from 330 dogs and structured epidemiological questionnaires were applied to their keepers. These samples were used in the indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Fisher's exact test was used for statistical calculations with the aim of identifying risk factors. Out of the 330 animals, 105 (31.8%) were reactive in IFAT, 46 (13.0%) in ELISA and 20 (6.0%) in both serological tests. The results were not significant (p > 0.05) when submitted to statistical analysis for the studied variables. From PCR, 58 samples (17.5%) were found to be positive and, of these, one (0.3%) showed similarity to T. cruzi after sequencing. These data demonstrate that dogs were exposed to and infected by T. cruzi. Thus, they can be considered sentinel animals for Chagas disease in the locality studied, which signals that there is a need for epidemiological surveillance actions.

Stability Assessment of Four Chimeric Proteins for Human Chagas Disease Immunodiagnosis.

The performance of an immunoassay relies on antigen-antibody interaction; hence, antigen chemical stability and structural integrity are paramount for an efficient assay. We conducted a functional, thermostability and long-term stability analysis of different chimeric antigens (IBMP), in order to assess effects of adverse conditions on four antigens employed in ELISA to diagnose Chagas disease. ELISA-based immunoassays have served as a model for biosensors development, as both assess molecular interactions. To evaluate thermostability, samples were heated and cooled to verify heat-induced denaturation reversibility. In relation to storage stability, the antigens were analyzed at 25 °C at different moments. Long-term stability tests were performed using eight sets of microplates sensitized. Antigens were structurally analyzed through circular dichroism (CD), dynamic light scattering, SDS-PAGE, and functionally evaluated by ELISA. Data suggest that IBMP antigens are stable, over adverse conditions and for over a year. Daily analysis revealed minor changes in the molecular structure. Functionally, IBMP-8.2 and IBMP-8.3 antigens showed reactivity towards anti-T. cruzi antibodies, even after 72 h at 25 °C. Long-term stability tests showed that all antigens were comparable to the control group and all antigens demonstrated stability for one year. Data suggest that the antigens maintained their function and structural characteristics even in adverse conditions, making them a sturdy and reliable candidate to be employed in future in vitro diagnostic tests applicable to different models of POC devices, such as modern biosensors in development.

Distribution of Human Immunodeficiency Virus and Human T-Leukemia Virus Co-infection in Bahia, Brazil.

Human Immunodeficiency Virus (HIV) and Human T-Leukemia Virus (HTLV) are retroviruses that share similar routes of transmission. In Brazil, the prevalence of HIV and HTLV varies according to geographic region. The state of Bahia, located in the Northeast region, is considered endemic for both retroviruses. The present study aimed to characterize the frequency of HIV/HTLV coinfection and evaluate the geographic distribution of coinfection throughout the state. This cross-sectional study was conducted at the state's Central Laboratory of Public Health (LACEN-BA) and included all samples from 2004 to 2013 submitted to serological testing for anti-HIV and anti-HTLV-1/2, screened by chemiluminescence/ELISA and confirmed by Western blot. Infection rates are expressed as the number of infected individuals per 100,000 inhabitants from each municipality. A total of 129,158 samples originating from 358/417 (85.8%) municipalities in Bahia were evaluated. HTLV was detected in 2.4% of the HIV-positive samples (n = 42) compared to 0.5% of those with negative HIV serology (n = 677) (OR: 4.65; CI: 3.39-6.37). HIV/HTLV coinfection was more frequent in women (69.0%); the median age of coinfected individuals was 47.2 years [interquartile range (IQR): 41.6-55.4 years]. In the 14/417 (3.4%) municipalities where at least one case of HIV/HTLV coinfection was detected, the overall HTLV coinfection rate in HIV-positive samples was 0.25 (range: 0.17-13.84) per 100,000 inhabitants. Most cases of HIV/HTLV-1 coinfection (21/37, 57%) were concentrated in the municipality of Salvador. Isolated instances (one or two cases) of HIV/HTLV-1 coinfection were distributed across municipalities known to be endemic for HTLV infection.

Correction: Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

[This corrects the article DOI: 10.1371/journal.pone.0234043.].

Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.

Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic.

Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.