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Lourena E Costa - One of the best experts on this subject based on the ideXlab platform.
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a conserved leishmania hypothetical protein evaluated for the Serodiagnosis of canine and human visceral and tegumentary leishmaniasis as well as a serological marker for the posttreatment patient follow up
Diagnostic Microbiology and Infectious Disease, 2018Co-Authors: Patricia A F Ribeiro, Daniela P Lage, Fernanda F Ramos, Daniel Dias, Lourena E Costa, Thais T O Santos, Bethina T Steiner, Beatriz C S Salles, Mariana P Lima, Ana Thereza ChavesAbstract:Abstract In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the Serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the Serodiagnosis of canine and human leishmaniasis.
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Performance of Leishmania braziliensis enolase protein for the Serodiagnosis of canine and human visceral leishmaniosis
Veterinary parasitology, 2017Co-Authors: Mariana C Duarte, Daniela P Lage, Daniel Dias, Patricia A F Ribeiro, Lourena E Costa, Thais T O Santos, Vivian T Martins, Beatriz C S Salles, Ana Maria Ravena Severino Carvalho, Miguel A. Chávez-fumagalliAbstract:In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the Serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec®-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL Serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the Serodiagnosis of human and canine visceral leishmaniosis.
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leishmania infantum mimotopes and a phage elisa assay as tools for a sensitive and specific Serodiagnosis of human visceral leishmaniasis
Diagnostic Microbiology and Infectious Disease, 2017Co-Authors: Beatriz C S Salles, Fernanda F Ramos, Lourena E Costa, Mariana C Duarte, Daniel Menezessouza, Patricia Terra Alves, Ana C S Dias, Emilia Rezende Vaz, Bruno Mendes Roatt, Miguel A ChavezfumagalliAbstract:Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the Serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive Serodiagnosis of human VL.
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evaluation of a hypothetical protein for Serodiagnosis and as a potential marker for post treatment serological evaluation of tegumentary leishmaniasis patients
Parasitology Research, 2017Co-Authors: Mariana P Lima, Fernanda F Ramos, Lourena E Costa, Thais T O Santos, Miguel A Chavezfumagalli, Mariana C Duarte, Daniel Menezessouza, Beatriz C S Salles, Amanda Christine Da Silva Kursancew, Roberta Passamani AmbrosioAbstract:The Serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the Serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the Serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
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a new leishmania specific hypothetical protein and its non described specific b cell conformational epitope applied in the Serodiagnosis of canine visceral leishmaniasis
Parasitology Research, 2016Co-Authors: Daniela P Lage, Lourena E Costa, Vivian T Martins, Miguel A Chavezfumagalli, Mariana C Duarte, Esther Garde, Danielle F De Magalhaessoares, Amanda Christine Da Silva Kursancew, Laura M Dimer, Daniel MenezessouzaAbstract:The Serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL Serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL Serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for Serodiagnosis of CVL.
Miguel A Chavezfumagalli - One of the best experts on this subject based on the ideXlab platform.
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leishmania infantum mimotopes and a phage elisa assay as tools for a sensitive and specific Serodiagnosis of human visceral leishmaniasis
Diagnostic Microbiology and Infectious Disease, 2017Co-Authors: Beatriz C S Salles, Fernanda F Ramos, Lourena E Costa, Mariana C Duarte, Daniel Menezessouza, Patricia Terra Alves, Ana C S Dias, Emilia Rezende Vaz, Bruno Mendes Roatt, Miguel A ChavezfumagalliAbstract:Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the Serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive Serodiagnosis of human VL.
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evaluation of a hypothetical protein for Serodiagnosis and as a potential marker for post treatment serological evaluation of tegumentary leishmaniasis patients
Parasitology Research, 2017Co-Authors: Mariana P Lima, Fernanda F Ramos, Lourena E Costa, Thais T O Santos, Miguel A Chavezfumagalli, Mariana C Duarte, Daniel Menezessouza, Beatriz C S Salles, Amanda Christine Da Silva Kursancew, Roberta Passamani AmbrosioAbstract:The Serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the Serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the Serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
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a new leishmania specific hypothetical protein and its non described specific b cell conformational epitope applied in the Serodiagnosis of canine visceral leishmaniasis
Parasitology Research, 2016Co-Authors: Daniela P Lage, Lourena E Costa, Vivian T Martins, Miguel A Chavezfumagalli, Mariana C Duarte, Esther Garde, Danielle F De Magalhaessoares, Amanda Christine Da Silva Kursancew, Laura M Dimer, Daniel MenezessouzaAbstract:The Serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL Serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL Serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for Serodiagnosis of CVL.
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proteins selected in leishmania viannia braziliensis by an immunoproteomic approach with potential Serodiagnosis applications for tegumentary leishmaniasis
Clinical and Vaccine Immunology, 2015Co-Authors: Mariana C Duarte, Lourena E Costa, Miguel A Chavezfumagalli, Daniel Menezessouza, Paula S Lage, Daniel C Pimenta, Rubens D M Magalhaes, Joao P Diniz, Daniela C Bartholomeu, Maria Julia Manso AlvesAbstract:The Serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the Serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the Serodiagnosis of TL.
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sensitive and specific Serodiagnosis of leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach
Clinical and Vaccine Immunology, 2013Co-Authors: Miguel A Chavezfumagalli, Daniela P Lage, Lourena E Costa, Vivian T Martins, Mariana C Duarte, Miriam C S Testasicca, Paula S Lage, Henrique Gama Ker, Tatiana G Ribeiro, Fernando Aecio A CarvalhoAbstract:In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher thanfigures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the Serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the Serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the Serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study’sfindings suggest that these three peptides can constitute a potential tool for more sensitive and specific Serodiagnosis of L. infantum infection in dogs.
Mariana C Duarte - One of the best experts on this subject based on the ideXlab platform.
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Performance of Leishmania braziliensis enolase protein for the Serodiagnosis of canine and human visceral leishmaniosis
Veterinary parasitology, 2017Co-Authors: Mariana C Duarte, Daniela P Lage, Daniel Dias, Patricia A F Ribeiro, Lourena E Costa, Thais T O Santos, Vivian T Martins, Beatriz C S Salles, Ana Maria Ravena Severino Carvalho, Miguel A. Chávez-fumagalliAbstract:In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the Serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec®-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL Serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the Serodiagnosis of human and canine visceral leishmaniosis.
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leishmania infantum mimotopes and a phage elisa assay as tools for a sensitive and specific Serodiagnosis of human visceral leishmaniasis
Diagnostic Microbiology and Infectious Disease, 2017Co-Authors: Beatriz C S Salles, Fernanda F Ramos, Lourena E Costa, Mariana C Duarte, Daniel Menezessouza, Patricia Terra Alves, Ana C S Dias, Emilia Rezende Vaz, Bruno Mendes Roatt, Miguel A ChavezfumagalliAbstract:Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the Serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive Serodiagnosis of human VL.
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evaluation of a hypothetical protein for Serodiagnosis and as a potential marker for post treatment serological evaluation of tegumentary leishmaniasis patients
Parasitology Research, 2017Co-Authors: Mariana P Lima, Fernanda F Ramos, Lourena E Costa, Thais T O Santos, Miguel A Chavezfumagalli, Mariana C Duarte, Daniel Menezessouza, Beatriz C S Salles, Amanda Christine Da Silva Kursancew, Roberta Passamani AmbrosioAbstract:The Serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the Serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the Serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
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a new leishmania specific hypothetical protein and its non described specific b cell conformational epitope applied in the Serodiagnosis of canine visceral leishmaniasis
Parasitology Research, 2016Co-Authors: Daniela P Lage, Lourena E Costa, Vivian T Martins, Miguel A Chavezfumagalli, Mariana C Duarte, Esther Garde, Danielle F De Magalhaessoares, Amanda Christine Da Silva Kursancew, Laura M Dimer, Daniel MenezessouzaAbstract:The Serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL Serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL Serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for Serodiagnosis of CVL.
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proteins selected in leishmania viannia braziliensis by an immunoproteomic approach with potential Serodiagnosis applications for tegumentary leishmaniasis
Clinical and Vaccine Immunology, 2015Co-Authors: Mariana C Duarte, Lourena E Costa, Miguel A Chavezfumagalli, Daniel Menezessouza, Paula S Lage, Daniel C Pimenta, Rubens D M Magalhaes, Joao P Diniz, Daniela C Bartholomeu, Maria Julia Manso AlvesAbstract:The Serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the Serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the Serodiagnosis of TL.
Daniel Menezessouza - One of the best experts on this subject based on the ideXlab platform.
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leishmania infantum mimotopes and a phage elisa assay as tools for a sensitive and specific Serodiagnosis of human visceral leishmaniasis
Diagnostic Microbiology and Infectious Disease, 2017Co-Authors: Beatriz C S Salles, Fernanda F Ramos, Lourena E Costa, Mariana C Duarte, Daniel Menezessouza, Patricia Terra Alves, Ana C S Dias, Emilia Rezende Vaz, Bruno Mendes Roatt, Miguel A ChavezfumagalliAbstract:Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the Serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive Serodiagnosis of human VL.
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evaluation of a hypothetical protein for Serodiagnosis and as a potential marker for post treatment serological evaluation of tegumentary leishmaniasis patients
Parasitology Research, 2017Co-Authors: Mariana P Lima, Fernanda F Ramos, Lourena E Costa, Thais T O Santos, Miguel A Chavezfumagalli, Mariana C Duarte, Daniel Menezessouza, Beatriz C S Salles, Amanda Christine Da Silva Kursancew, Roberta Passamani AmbrosioAbstract:The Serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the Serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the Serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
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a new leishmania specific hypothetical protein and its non described specific b cell conformational epitope applied in the Serodiagnosis of canine visceral leishmaniasis
Parasitology Research, 2016Co-Authors: Daniela P Lage, Lourena E Costa, Vivian T Martins, Miguel A Chavezfumagalli, Mariana C Duarte, Esther Garde, Danielle F De Magalhaessoares, Amanda Christine Da Silva Kursancew, Laura M Dimer, Daniel MenezessouzaAbstract:The Serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL Serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL Serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for Serodiagnosis of CVL.
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proteins selected in leishmania viannia braziliensis by an immunoproteomic approach with potential Serodiagnosis applications for tegumentary leishmaniasis
Clinical and Vaccine Immunology, 2015Co-Authors: Mariana C Duarte, Lourena E Costa, Miguel A Chavezfumagalli, Daniel Menezessouza, Paula S Lage, Daniel C Pimenta, Rubens D M Magalhaes, Joao P Diniz, Daniela C Bartholomeu, Maria Julia Manso AlvesAbstract:The Serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the Serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the Serodiagnosis of TL.
Beatriz C S Salles - One of the best experts on this subject based on the ideXlab platform.
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a conserved leishmania hypothetical protein evaluated for the Serodiagnosis of canine and human visceral and tegumentary leishmaniasis as well as a serological marker for the posttreatment patient follow up
Diagnostic Microbiology and Infectious Disease, 2018Co-Authors: Patricia A F Ribeiro, Daniela P Lage, Fernanda F Ramos, Daniel Dias, Lourena E Costa, Thais T O Santos, Bethina T Steiner, Beatriz C S Salles, Mariana P Lima, Ana Thereza ChavesAbstract:Abstract In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the Serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the Serodiagnosis of canine and human leishmaniasis.
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Performance of Leishmania braziliensis enolase protein for the Serodiagnosis of canine and human visceral leishmaniosis
Veterinary parasitology, 2017Co-Authors: Mariana C Duarte, Daniela P Lage, Daniel Dias, Patricia A F Ribeiro, Lourena E Costa, Thais T O Santos, Vivian T Martins, Beatriz C S Salles, Ana Maria Ravena Severino Carvalho, Miguel A. Chávez-fumagalliAbstract:In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the Serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec®-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL Serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the Serodiagnosis of human and canine visceral leishmaniosis.
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leishmania infantum mimotopes and a phage elisa assay as tools for a sensitive and specific Serodiagnosis of human visceral leishmaniasis
Diagnostic Microbiology and Infectious Disease, 2017Co-Authors: Beatriz C S Salles, Fernanda F Ramos, Lourena E Costa, Mariana C Duarte, Daniel Menezessouza, Patricia Terra Alves, Ana C S Dias, Emilia Rezende Vaz, Bruno Mendes Roatt, Miguel A ChavezfumagalliAbstract:Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the Serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive Serodiagnosis of human VL.
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evaluation of a hypothetical protein for Serodiagnosis and as a potential marker for post treatment serological evaluation of tegumentary leishmaniasis patients
Parasitology Research, 2017Co-Authors: Mariana P Lima, Fernanda F Ramos, Lourena E Costa, Thais T O Santos, Miguel A Chavezfumagalli, Mariana C Duarte, Daniel Menezessouza, Beatriz C S Salles, Amanda Christine Da Silva Kursancew, Roberta Passamani AmbrosioAbstract:The Serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the Serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the Serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
