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.

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.

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.

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.

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.

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.

Proteínas recombinantes quiméricas do trypanosoma cruzi como ferramenta diagnóstica para identificar a doença de chagas crônica através do western blot

INTRODUÇÃO: A doença de Chagas (DC) é causada pelo Trypanosoma cruzi. Sua forma de infecção pode ser vetorial, oral ou congênita, além da transfusão sanguínea. No Brasil, o número de indivíduos infectados varia de 1,9 a 4,6 milhões e, na América Latina, de 5 a 7 milhões. A fase crônica da doença é caracterizada pela presença de anticorpos IgG anti-T. cruzi e o seu diagnóstico é realizado por métodos sorológicos, principalmente o ELISA indireto. A OMS preconiza a utilização de dois testes distintos para o diagnóstico da DC e a combinação dos resultados define os indivíduos como positivos, negativos ou, no caso de discordância entre eles, inconclusivos. Neste último caso, um teste confirmatório deve ser empregado, como o Western blot (WB) ou a PCR; no entanto, esta última apresenta baixa sensibilidade na fase crônica da doença. Quatro antígenos recombinantes quiméricos, denominados IBMP-8.1, IBMP-8.2, IBMP-8.3 e IBMP-8.4 foram expressos pelo nosso grupo e avaliados em estudos de fase I, II e III, utilizando o ELISA, o microarranjo líquido e a imunocromatografia, obtendo acurácia de 95 a 100%. Estes antígenos também foram submetidos à avaliação para detecção da DC em cães. Em 2016, o único teste de Western blot produzido no Brasil foi descontinuado e, por conta disso e devido ao elevado desempenho diagnóstico conferido aos antígenos IBMP, investigamos a utilização destas moléculas em diagnosticar a DC utilizando ensaios de WB. OBJETIVO: Avaliar e validar o desempenho diagnóstico dos antígenos recombinantes quiméricos do T. cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 e IBMP-8.4) como matrizes antigênicas utilizando plataforma de WB no diagnóstico da DC crônica. MATERIAL E MÉTODOS: Foram avaliadas 22 amostras positivas, 24 negativas e 3 positivas para leishmaniose visceral. A padronização dos ensaios foi realizada através de diferentes diluições dos antígenos, dos anticorpos conjugados e das amostras séricas. A validação do potencial diagnóstico foi realizada através de curvas ROC. A reatividade cruzada também foi avaliada para leishmaniose visceral. RESULTADOS: Todos os antígenos IBMP apresentaram 100% de sensibilidade, especificidade e acurácia, com exceção do antígeno IBMP-8.3, que obteve 90,9% de sensibilidade, 100% de especificidade e 95,7% de acurácia. Todavia, não houve diferença significativa nos referidos valores. CONCLUSÕES: No presente estudo (fase I ou prova de conceito), os antígenos IBMP apresentaram elevada capacidade em diferenciar amostras positivas das negativas para a DC, sendo, portanto, elegíveis para o estudo de fase II.

Proteínas recombinantes quiméricas do Trypanosoma cruzi como ferramenta diagnóstica para identificar a doença de Chagas crônica através do Western blot

INTRODUÇÃO: A doença de Chagas (DC) é causada pelo Trypanosoma cruzi. Sua forma de infecção pode ser vetorial, oral ou congênita, além da transfusão sanguínea. No Brasil, o número de indivíduos infectados varia de 1,9 a 4,6 milhões e, na América Latina, de 5 a 7 milhões. A fase crônica da doença é caracterizada pela presença de anticorpos IgG anti-T. cruzi e o seu diagnóstico é realizado por métodos sorológicos, principalmente o ELISA indireto. A OMS preconiza a utilização de dois testes distintos para o diagnóstico da DC e a combinação dos resultados define os indivíduos como positivos, negativos ou, no caso de discordância entre eles, inconclusivos. Neste último caso, um teste confirmatório deve ser empregado, como o Western blot (WB) ou a PCR; no entanto, esta última apresenta baixa sensibilidade na fase crônica da doença. Quatro antígenos recombinantes quiméricos, denominados IBMP-8.1, IBMP-8.2, IBMP-8.3 e IBMP-8.4 foram expressos pelo nosso grupo e avaliados em estudos de fase I, II e III, utilizando o ELISA, o microarranjo líquido e a imunocromatografia, obtendo acurácia de 95 a 100%. Estes antígenos também foram submetidos à avaliação para detecção da DC em cães. Em 2016, o único teste de Western blot produzido no Brasil foi descontinuado e, por conta disso e devido ao elevado desempenho diagnóstico conferido aos antígenos IBMP, investigamos a utilização destas moléculas em diagnosticar a DC utilizando ensaios de WB. OBJETIVO: Avaliar e validar o desempenho diagnóstico dos antígenos recombinantes quiméricos do T. cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 e IBMP-8.4) como matrizes antigênicas utilizando plataforma de WB no diagnóstico da DC crônica. MATERIAL E MÉTODOS: Foram avaliadas 22 amostras positivas, 24 negativas e 3 positivas para leishmaniose visceral. A padronização dos ensaios foi realizada através de diferentes diluições dos antígenos, dos anticorpos conjugados e das amostras séricas. A validação do potencial diagnóstico foi realizada através de curvas ROC. A reatividade cruzada também foi avaliada para leishmaniose visceral. RESULTADOS: Todos os antígenos IBMP apresentaram 100% de sensibilidade, especificidade e acurácia, com exceção do antígeno IBMP-8.3, que obteve 90,9% de sensibilidade, 100% de especificidade e 95,7% de acurácia. Todavia, não houve diferença significativa nos referidos valores. CONCLUSÕES: No presente estudo (fase I ou prova de conceito), os antígenos IBMP apresentaram elevada capacidade em diferenciar amostras positivas das negativas para a DC, sendo, portanto, elegíveis para o estudo de fase II.

Sífilis congênita e em gestantes no Brasil: análise da distribuição espaço-temporal de 2001 a 2017

Introdução: Apesar da existência de testes diagnósticos e tratamento eficaz, o aumento do número de casos de sífilis registrados no Brasil evidenciou a fragilidade do sistema público de saúde. A associação da doença a grupos específicos já está bem estabelecida, entretanto áreas vulneráveis necessitam ser mapeadas para o combate à infecção.