Performance of recombinant chimeric proteins in the serological diagnosis of Trypanosoma cruzi infection in dogs.

Background: Dogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera. Methodology/Principal findings: The IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7?100%), IBMP-8.2 (73.3?87.5%), and IBMP-8.1 (50?100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7?97.5%) and IBMP 8.1 (89.1?100%). Conclusions/Significance: The use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases

Identification of proteins from the cambial region of Eucalyptus grandis by bidimensional electrophoresis and mass spectrometry

A proteômica expandiu-se dentro da área biológica a partir da década de 90 com o desenvolvimento de técnicas de ionização branda que permitem analisar macromoléculas por espectrometria de massas (MS) e é uma nova estratégia na busca de genes de interesse e de informações sobre o controle da expressão gênica para a manipulação genética de plantas. Na busca por genes diferencialmente expressos durante o desenvolvimento da madeira, foi estabelecida uma plataforma 2D-LC-MS/MS (eletroforese bidimensional seguida de cromatografia líquida e espectrometria de massas em tandem) e identificadas 72 proteínas da região cambial de Eucalyptus grandis de árvores de 2 anos e 8 meses. A identificação foi feita por homologia com o banco de proteínas SwissProt, com um banco de ESTs específico de Eucalyptus spp. (GENOLYPTUS) e com o NCBI. Os dados de proteínas também foram confrontados com a expressão dos transcritos correspondentes obtidos a partir do mesmo tecido por SAGE (Serial Analysis of Gene Expression). Dentre as proteínas identificadas, a maior parcela (24,1 %) tem função relacionada com resposta a estresse, 20,5 % pertence a metabolismo de energia, 17,8 % são componentes estruturais, 14,3 % participam em metabolismo de macromoléculas e 15,2 % em metabolismos básicos como de nitrogênio, aminoácidos, nucleotídeos, lipídeos e metabolismo secundário.Proteomics impacted the biological sciences since the 90´s after the development of soft ionization techniques that allow to analyse macromolecules by mass spectrometry (MS). It became a new strategy to identify genes and obtain information about the molecular control of gene expression, of importance to genetic manipulation of plants. In order to search for genes involved in wood quality and to understand gene expression during wood development, a 2D-LC-MS/MS system (bidimensional electrophoresis followed by liquid cromatography coupled to mass spectrometry in tandem) was stablished. 72 proteins from the cambial region of Eucalyptus grandis trees at the age of 2 years and 8 months were identified. The MS/MS spectra were processed using the SwissProt databank, an EST (Expressed Sequence Tags) bank of Eucalyptus spp. (GENOLYPTUS) and the NCBI. A comparation of gene and protein expression was carried out using a databank constructed in our laboratory using SAGE (Serial Analysis of Gene Expression) obtained from the same cambial tissue. Most identified proteins are related to stress response (24.1 %), 20.5 % participate in energy metabolism, 17.8 % are important to the cell as structural components, 14,3 % are related to macromolecular metabolism and 15,2 % to basic metabolism of nitrogen, amino acids, nucleotides, lipids and secondary metabolism

Performance assessment of a Trypanosoma cruzi chimeric antigen in multiplex liquid microarray assays

Submitted by Manoel Barata ([email protected]) on 2017-09-04T15:40:42Z No. of bitstreams: 1 Santosperformanc.pdf: 1621790 bytes, checksum: e95251c68d55c20759b5a522c3f9599a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-09-06T19:44:57Z (GMT) No. of bitstreams: 1 Santosperformanc.pdf: 1621790 bytes, checksum: e95251c68d55c20759b5a522c3f9599a (MD5)Made available in DSpace on 2017-09-06T19:44:57Z (GMT). No. of bitstreams: 1 Santosperformanc.pdf: 1621790 bytes, checksum: e95251c68d55c20759b5a522c3f9599a (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Programa Integrado de Doença de Chagas. Rio de Janeiro, RJ, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil. / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Programa Integrado de Doença de Chagas. Rio de Janeiro, RJ, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Programa Integrado de Doença de Chagas. Rio de Janeiro, RJ, Brasil.Diagnosing chronic Chagas disease (CD) requires antibody--antigen detection methods, traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from Instituto de Biologia Molecular do Paraná (IBMP-8.1 to -8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1--99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania spp., a pathogen with high genome sequence similar to T. cruzi, showed cross-reactivity rates ranging from 0--2.17%. Inconclusive results were negligible (0--0.71%). Bland--Altman and Deming regression analysis based on 200 randomly selected CD-positive and -negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of a multiplex platform, like LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD

Accuracy of chimeric proteins in the serological diagnosis of chronic chagas disease - a Phase II study

Submitted by Adagilson Silva ([email protected]) on 2017-06-22T12:51:41Z No. of bitstreams: 1 28273127 2017 san-acc.oa.pdf: 5179215 bytes, checksum: 12005c4a0c8fd5d5f9e1ed44052231f9 (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-06-22T12:55:35Z (GMT) No. of bitstreams: 1 28273127 2017 san-acc.oa.pdf: 5179215 bytes, checksum: 12005c4a0c8fd5d5f9e1ed44052231f9 (MD5)Made available in DSpace on 2017-06-22T12:55:35Z (GMT). No. of bitstreams: 1 28273127 2017 san-acc.oa.pdf: 5179215 bytes, checksum: 12005c4a0c8fd5d5f9e1ed44052231f9 (MD5) Previous issue date: 2017-03Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil / Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Chagas Disease Integrated Program. Rio de Janeiro, RJ, Brazil.Molecular Biology Institute of Paraná. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Chagas Disease Integrated Program. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Bio-Manguinhos. Rio de Janeiro, RJ, Brazil.Molecular Biology Institute of Paraná. Curitiba, PR, Brazil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Chagas Disease Integrated Program. Rio de Janeiro, RJ, Brazil / Molecular Biology Institute of Paraná. Curitiba, PR, Brazil / Fundação Oswaldo Cruz. Instituto Carlos Chagas Institute. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Chagas Disease Integrated Program. Rio de Janeiro, RJ, Brazil.The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results

Detection of anti-Trypanosoma cruzi antibodies by chimeric antigens in chronic Chagas disease-individuals from endemic South American countries

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-05-07T14:26:01Z No. of bitstreams: 1 Del-Rei et al., Detection ...2019.pdf: 2364416 bytes, checksum: 1fb04e34df00f90597065a5a189a69b6 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-05-07T16:39:03Z (GMT) No. of bitstreams: 1 Del-Rei et al., Detection ...2019.pdf: 2364416 bytes, checksum: 1fb04e34df00f90597065a5a189a69b6 (MD5)Made available in DSpace on 2019-05-07T16:39:03Z (GMT). No. of bitstreams: 1 Del-Rei et al., Detection ...2019.pdf: 2364416 bytes, checksum: 1fb04e34df00f90597065a5a189a69b6 (MD5) Previous issue date: 2019Fundación Bunge y Born 2012 Dr. Silvia Andrea Longhi; Consejo Nacional de Investigaciones Científicas y Tecnológicas PIP/0974-2011 Dr. Alejandro Gabriel Schijman; Gonçalo Moniz Institute PROEP/IGM 400904/2013-6 Dr. Mitermayer Galvão dos Reis;Coordination of Superior Level Staff Improvement CAPES - PROEX 0720/2018 Dr. Fred Luciano Neves Santos; National Council for Scientific and Technological Development CNPq Proc. No. 312195/2015-0 Dr. Nilson Ivo Tonin Zanchin; and National Council for Scientific and Technological Development CNPq Proc. No. 307319/2016-4 Dr. Mitermayer Galvão dos Reis. The funders were not involved in the design of the study, in the collection, analysis and interpretation of the data, or in writing the manuscript.Faculty of Technology and Sciences of Bahia. Salvador, BA, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Molecular Biology Institute of Paraná. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Federal University of Bahia. Department of Pathology and Legal Medicine. Salvador, BA, Brazil / Yale University. School of Public Health. Department of Epidemiology of Microbial Diseases. New Haven, Connecticut, United States of America.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Institute for Research on Genetic Engineering and Molecular Biology “Dr Héctor Torres”. Laboratory of Molecular Biology of Chagas Disease. Buenos Aires, Argentina.Institute for Research on Genetic Engineering and Molecular Biology “Dr Héctor Torres”. Laboratory of Molecular Biology of Chagas Disease. Buenos Aires, Argentina.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees

Highly Accurate Chimeric Proteins for the Serological Diagnosis of Chronic Chagas Disease: A Latent Class Analysis

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-10-23T12:17:11Z No. of bitstreams: 1 Santos FL Highly Accurate Chimeric...2018.pdf: 553405 bytes, checksum: a45acff4b655375c0b330fd036a1f045 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-10-23T12:34:46Z (GMT) No. of bitstreams: 1 Santos FL Highly Accurate Chimeric...2018.pdf: 553405 bytes, checksum: a45acff4b655375c0b330fd036a1f045 (MD5)Made available in DSpace on 2018-10-23T12:34:46Z (GMT). No. of bitstreams: 1 Santos FL Highly Accurate Chimeric...2018.pdf: 553405 bytes, checksum: a45acff4b655375c0b330fd036a1f045 (MD5) Previous issue date: 2018Gonçalo Moniz Institute, Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (Proc. 404242/2012-0) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco-FACEPE (Proc. APQ-1257-2.11/12). W. V. Sousa and M. A. Krieger are research fellows of CNPq Proc. No. 306222/2013-2 and 590032/2011-9, respectively.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Fio-Chagas. Rio de Janeiro, RJ, Brasil.Universidade Federal da Bahia. Instituto de Matemática. Departamento de Estatística. Salvador, BA, Brasil.Universidade Federal da Bahia. Instituto de Matemática. Departamento de Estatística. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Molecular Biology Institute of Paraná. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Fio-Chagas. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Molecular Biology Institute of Paraná. Curitiba, PR, Brazil.Fundação Oswaldo Cruz. Fio-Chagas. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.The existence of an imperfect reference standard presents complications when evaluating the unbiased performance of novel diagnostic techniques. This is especially true in the absence of a gold standard, as is the case in chronic Chagas disease (CD) diagnosis. To circumvent this constraint, we elected to use latent class analysis (LCA). Previously, our group demonstrated the high performance of four Trypanosoma cruzi–chimeric proteins (Molecular Biology Institute of Parana´ [IBMP]-8.1, -8.2, -8.3, and -8.4) for CD diagnosis using several distinct immunoassays. Although commercial tests had previously been established as a reference standard, the diagnostic performance of these chimeric antigens could present bias because these tests fail to produce 100% accurate results. Thus, we used LCA to assess the performance of these IBMP chimeric antigens in chronic CD diagnosis. Using the LCA model as a gold standard, sensitivity and specificity values ranged from 93.5% to 99.4% and 99.6% to 100%, respectively. The accuracy values were 96.2% for IBMP-8.2, approximately 98% for IBMP-8.1 and IBMP-8.3, and nearly 100% for IBMP-8.4. For IBMP-8.1 and IBMP-8.2, higher positive predictive values were associated with increases in hypothetical prevalence. Similarly, higher hypothetical prevalence resulted in lower negative predictive values for IBMP-8.1, IBMP-8.2, and IBMP- 8.3. In addition, samples with serodiscordant results from commercial serological tests were analyzed using LCA. Molecular Biology Institute of Parana´ -8.1 demonstrated potential for use in confirmatory testing with regard to samples with inconsistent results. Moreover, our findings further confirmed the remarkable performance of the IBMP-8.4 antigen to diagnose chronic CD in both endemic and non-endemic areas

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

Antibodies response induced by recombinant virus-like particles from Triatoma virus and chimeric antigens from Trypanosoma cruzi

The infection caused by the protozoan Trypanosoma cruzi affects humans and is called as Chagas disease. Currently, the main measures available to reduce the incidence of this disease are drug treatment and vector control. Traditionally, the development of vaccines occurs mainly through the use of antigenic candidates of the etiologic agent in the form of a vaccine preparation. Virus-like particles (VLPs) are structures analogous to viral capsids composed essentially of structural proteins and are widely used in vaccination protocols because of their immunostimulatory properties. In this context, the objective of this study was to use strategies in a murine immunization model to characterize the immunostimulatory capacity of VLPs from Triatoma virus (TrV-VLPs), analysed in the presence or absence of the aluminium vaccine adjuvant. In parallel, to characterize the immunogenic behaviour of four T. cruzi chimeric recombinant proteins (mix-IBMP) associated with TrV-VLPs or aluminium vaccine adjuvant.This research was funded by Consejo Superior de Investigaciones Científicas (grant number COOPB20503 - CSIC, Spain) and Global Health and Tropical Medicine [grant number GHTM-UID/multi/04413/2013] and partially supported by grants to DMAG, from the Ministerio de Ciencia e Innovación [BFU2012-36241], Grupos Investigación UPV/EHU 2018 [GIU18/172], and Gobierno Vasco [Elkartek KK-2017/00008], Programa de empresas Innovadoras A1 2020 EKINZAILE, The Basque Country, Spain. This study also was supported by Coordenação de Aperfeiçaomento de Pessoal de Nível Superior (CAPES, Brazil).Peer reviewe