Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

Autores
Besuschio, Susana Alicia; Llano Murcia, Mónica; Benatar, Alejandro Francisco; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, Maria de Los Angeles; Kubota, Yutaka; Wehrendt, Diana Patricia; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M.; Schijman, Alejandro Gabriel
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after ?Boil & Spin? rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), and as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2parasite equivalents/mL in spiked EDTA blood and 1x10-1par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.
Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Llano Murcia, Mónica. Pontificia Universidad Javeriana; Colombia
Fil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Monnerat, Severine. Foundation for Innovative New Diagnostics; Suiza
Fil: Cruz, Israel. Foundation for Innovative New Diagnostics; Suiza
Fil: Picado, Albert. Foundation for Innovative New Diagnostics; Suiza
Fil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Kubota, Yutaka. Eiken Chemical Company; Japón
Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Pavia, Paula. Pontificia Universidad Javeriana; Colombia
Fil: Mori, Yasuyoshi. Eiken Chemical Company; Japón
Fil: Puerta, Concepción. Pontificia Universidad Javeriana; Colombia
Fil: Ndung'u, Joseph M.. Foundation for Innovative New Diagnostics; Suiza
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Materia
LAMP
TRYPANOSOMA CRUZI
DETECTION
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/40954

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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samplesBesuschio, Susana AliciaLlano Murcia, MónicaBenatar, Alejandro FranciscoMonnerat, SeverineCruz, IsraelPicado, AlbertCurto, Maria de Los AngelesKubota, YutakaWehrendt, Diana PatriciaPavia, PaulaMori, YasuyoshiPuerta, ConcepciónNdung'u, Joseph M.Schijman, Alejandro GabrielLAMPTRYPANOSOMA CRUZIDETECTIONhttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after ?Boil & Spin? rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), and as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2parasite equivalents/mL in spiked EDTA blood and 1x10-1par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Llano Murcia, Mónica. Pontificia Universidad Javeriana; ColombiaFil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Monnerat, Severine. Foundation for Innovative New Diagnostics; SuizaFil: Cruz, Israel. Foundation for Innovative New Diagnostics; SuizaFil: Picado, Albert. Foundation for Innovative New Diagnostics; SuizaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Kubota, Yutaka. Eiken Chemical Company; JapónFil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Pavia, Paula. Pontificia Universidad Javeriana; ColombiaFil: Mori, Yasuyoshi. Eiken Chemical Company; JapónFil: Puerta, Concepción. Pontificia Universidad Javeriana; ColombiaFil: Ndung'u, Joseph M.. Foundation for Innovative New Diagnostics; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaPublic Library of Science2017-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/40954Besuschio, Susana Alicia; Llano Murcia, Mónica; Benatar, Alejandro Francisco; Monnerat, Severine; Cruz, Israel; et al.; Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples; Public Library of Science; Neglected Tropical Diseases; 11; 7; 7-20171935-2735CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pntd.0005779info:eu-repo/semantics/altIdentifier/url/http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0005779info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:12:00Zoai:ri.conicet.gov.ar:11336/40954instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:12:00.34CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
title Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
spellingShingle Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
Besuschio, Susana Alicia
LAMP
TRYPANOSOMA CRUZI
DETECTION
title_short Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
title_full Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
title_fullStr Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
title_full_unstemmed Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
title_sort Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples
dc.creator.none.fl_str_mv Besuschio, Susana Alicia
Llano Murcia, Mónica
Benatar, Alejandro Francisco
Monnerat, Severine
Cruz, Israel
Picado, Albert
Curto, Maria de Los Angeles
Kubota, Yutaka
Wehrendt, Diana Patricia
Pavia, Paula
Mori, Yasuyoshi
Puerta, Concepción
Ndung'u, Joseph M.
Schijman, Alejandro Gabriel
author Besuschio, Susana Alicia
author_facet Besuschio, Susana Alicia
Llano Murcia, Mónica
Benatar, Alejandro Francisco
Monnerat, Severine
Cruz, Israel
Picado, Albert
Curto, Maria de Los Angeles
Kubota, Yutaka
Wehrendt, Diana Patricia
Pavia, Paula
Mori, Yasuyoshi
Puerta, Concepción
Ndung'u, Joseph M.
Schijman, Alejandro Gabriel
author_role author
author2 Llano Murcia, Mónica
Benatar, Alejandro Francisco
Monnerat, Severine
Cruz, Israel
Picado, Albert
Curto, Maria de Los Angeles
Kubota, Yutaka
Wehrendt, Diana Patricia
Pavia, Paula
Mori, Yasuyoshi
Puerta, Concepción
Ndung'u, Joseph M.
Schijman, Alejandro Gabriel
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv LAMP
TRYPANOSOMA CRUZI
DETECTION
topic LAMP
TRYPANOSOMA CRUZI
DETECTION
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after ?Boil & Spin? rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), and as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2parasite equivalents/mL in spiked EDTA blood and 1x10-1par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.
Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Llano Murcia, Mónica. Pontificia Universidad Javeriana; Colombia
Fil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Monnerat, Severine. Foundation for Innovative New Diagnostics; Suiza
Fil: Cruz, Israel. Foundation for Innovative New Diagnostics; Suiza
Fil: Picado, Albert. Foundation for Innovative New Diagnostics; Suiza
Fil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Kubota, Yutaka. Eiken Chemical Company; Japón
Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Pavia, Paula. Pontificia Universidad Javeriana; Colombia
Fil: Mori, Yasuyoshi. Eiken Chemical Company; Japón
Fil: Puerta, Concepción. Pontificia Universidad Javeriana; Colombia
Fil: Ndung'u, Joseph M.. Foundation for Innovative New Diagnostics; Suiza
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
description This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after ?Boil & Spin? rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), and as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2parasite equivalents/mL in spiked EDTA blood and 1x10-1par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.
publishDate 2017
dc.date.none.fl_str_mv 2017-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/40954
Besuschio, Susana Alicia; Llano Murcia, Mónica; Benatar, Alejandro Francisco; Monnerat, Severine; Cruz, Israel; et al.; Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples; Public Library of Science; Neglected Tropical Diseases; 11; 7; 7-2017
1935-2735
CONICET Digital
CONICET
url http://hdl.handle.net/11336/40954
identifier_str_mv Besuschio, Susana Alicia; Llano Murcia, Mónica; Benatar, Alejandro Francisco; Monnerat, Severine; Cruz, Israel; et al.; Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples; Public Library of Science; Neglected Tropical Diseases; 11; 7; 7-2017
1935-2735
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pntd.0005779
info:eu-repo/semantics/altIdentifier/url/http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0005779
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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