Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia

Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in...

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Autores:
Murillo Casas, Angie Tatiana
Castro Martinez, Paula Andrea
Vega Morales, Luz Angela
Borda Rojas, Fernando
Souza, Anna Cláudia Alves de
Fietto, Juliana
Hell Mor, Natalie
Tafur Gómez, Gabriel Andres
Tipo de recurso:
Article of investigation
Fecha de publicación:
2024
Institución:
Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
Repositorio:
Repositorio Institucional UDCA
Idioma:
eng
OAI Identifier:
oai:repository.udca.edu.co:11158/6682
Acceso en línea:
https://repository.udca.edu.co/handle/11158/6682
https://doi.org/10.1016/j.jim.2024.113765
https://repository.udca.edu.co/
Palabra clave:
630 - Agricultura y tecnologías relacionadas::636 - Producción animal
Perros
Ensayo de Inmunoadsorción Enzimática
Pruebas Serológicas
Vacunas Sintéticas
Leishmaniasis
Enfermedades de los perros
Rights
openAccess
License
https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.es
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network_acronym_str RepoUDCA2
network_name_str Repositorio Institucional UDCA
repository_id_str
dc.title.eng.fl_str_mv Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
title Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
spellingShingle Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
630 - Agricultura y tecnologías relacionadas::636 - Producción animal
Perros
Ensayo de Inmunoadsorción Enzimática
Pruebas Serológicas
Vacunas Sintéticas
Leishmaniasis
Enfermedades de los perros
title_short Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
title_full Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
title_fullStr Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
title_full_unstemmed Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
title_sort Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia
dc.creator.fl_str_mv Murillo Casas, Angie Tatiana
Castro Martinez, Paula Andrea
Vega Morales, Luz Angela
Borda Rojas, Fernando
Souza, Anna Cláudia Alves de
Fietto, Juliana
Hell Mor, Natalie
Tafur Gómez, Gabriel Andres
dc.contributor.author.none.fl_str_mv Murillo Casas, Angie Tatiana
Castro Martinez, Paula Andrea
Vega Morales, Luz Angela
Borda Rojas, Fernando
Souza, Anna Cláudia Alves de
Fietto, Juliana
Hell Mor, Natalie
Tafur Gómez, Gabriel Andres
dc.subject.ddc.none.fl_str_mv 630 - Agricultura y tecnologías relacionadas::636 - Producción animal
topic 630 - Agricultura y tecnologías relacionadas::636 - Producción animal
Perros
Ensayo de Inmunoadsorción Enzimática
Pruebas Serológicas
Vacunas Sintéticas
Leishmaniasis
Enfermedades de los perros
dc.subject.armarc.none.fl_str_mv Perros
dc.subject.decs.none.fl_str_mv Ensayo de Inmunoadsorción Enzimática
Pruebas Serológicas
Vacunas Sintéticas
Leishmaniasis
dc.subject.agrovoc.none.fl_str_mv Enfermedades de los perros
description Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.
publishDate 2024
dc.date.issued.none.fl_str_mv 2024
dc.date.accessioned.none.fl_str_mv 2025-10-01T19:59:43Z
dc.date.available.none.fl_str_mv 2025-10-01T19:59:43Z
dc.type.none.fl_str_mv Artículo de revista
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dc.identifier.citation.none.fl_str_mv Murillo Casas, A. T., Castro Martinez, P. A., Borda Rojas, F., Vega, L. A., de Sousa, A. C. A., Fietto, J. L. R., Hell-Mor, N., & Tafur-Gómez, G. A. (2024). Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia. Journal of Immunological Methods, 534(113765), 113765. https://doi.org/10.1016/j.jim.2024.113765
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dc.identifier.instname.none.fl_str_mv Universidad de Ciencias Aplicadas y Ambientales
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identifier_str_mv Murillo Casas, A. T., Castro Martinez, P. A., Borda Rojas, F., Vega, L. A., de Sousa, A. C. A., Fietto, J. L. R., Hell-Mor, N., & Tafur-Gómez, G. A. (2024). Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia. Journal of Immunological Methods, 534(113765), 113765. https://doi.org/10.1016/j.jim.2024.113765
00221759
Universidad de Ciencias Aplicadas y Ambientales
UDCA
url https://repository.udca.edu.co/handle/11158/6682
https://doi.org/10.1016/j.jim.2024.113765
https://repository.udca.edu.co/
dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.indexed.none.fl_str_mv LaReferencia
dc.relation.citationedition.none.fl_str_mv (Nov., 2024) Artículo número 113765
dc.relation.citationendpage.none.fl_str_mv 5
dc.relation.citationstartpage.none.fl_str_mv 1
dc.relation.citationvolume.none.fl_str_mv 534
dc.relation.ispartofjournal.none.fl_str_mv Journal of Immunological Methods
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spelling Murillo Casas, Angie TatianaCastro Martinez, Paula AndreaVega Morales, Luz Angelavirtual::915-1Borda Rojas, Fernandovirtual::916-1Souza, Anna Cláudia Alves devirtual::917-1Fietto, Julianavirtual::918-1Hell Mor, Natalievirtual::919-1Tafur Gómez, Gabriel Andresvirtual::920-12025-10-01T19:59:43Z2025-10-01T19:59:43Z2024Murillo Casas, A. T., Castro Martinez, P. A., Borda Rojas, F., Vega, L. A., de Sousa, A. C. A., Fietto, J. L. R., Hell-Mor, N., & Tafur-Gómez, G. A. (2024). Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia. Journal of Immunological Methods, 534(113765), 113765. https://doi.org/10.1016/j.jim.2024.113765https://repository.udca.edu.co/handle/11158/6682https://doi.org/10.1016/j.jim.2024.11376500221759Universidad de Ciencias Aplicadas y AmbientalesUDCAhttps://repository.udca.edu.co/Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.Incluye referencias bibliográficasapplication/pdfenghttps://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.eshttps://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2https://www.sciencedirect.com/science/article/pii/S0022175924001509?via%3Dihub630 - Agricultura y tecnologías relacionadas::636 - Producción animalPerrosEnsayo de Inmunoadsorción EnzimáticaPruebas SerológicasVacunas SintéticasLeishmaniasisEnfermedades de los perrosPreliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in ColombiaArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionTexthttp://purl.org/redcol/resource_type/ARThttp://purl.org/coar/version/c_970fb48d4fbd8a85LaReferencia(Nov., 2024) Artículo número 11376551534Journal of Immunological 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jecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.</li>
      <li>
        Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:
        <ol type="i">
          <li>Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.</li>
          <li>Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
        </ol>
      </li>
      <li>Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
    </ol>
  </li>
  <br/>
  <li>
    Representaciones, Garantías y Limitaciones de Responsabilidad.
    <p>A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Limitación de responsabilidad.
    <p>A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Término.
    <ol type="a">
      <li>Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.</li>
      <li>Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.</li>
    </ol>
  </li>
  <br/>
  <li>
    Varios.
    <ol type="a">
      <li>Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.</li>
      <li>Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.</li>
      <li>Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.</li>
      <li>Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.</li>
    </ol>
  </li>
  <br/>
</ol>
