Comparación entre técnicas inmunológicas y moleculares para el diagnóstico de la enfermedad de Chagas

Ferrer, Elizabeth; Lares, María; Viettri, Mercedes; Medina, Mehudy

doi:10.1016/j.eimc.2012.09.007

Cited by 32 publications

(28 citation statements)

References 27 publications

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“…Similarmente, la PCR constituye una herramienta muy útil para el diagnóstico de la leishmaniasis canina y la implicación de los caninos como reservorios en las distintas zonas endémicas para la LV, siendo de gran importancia para la detección de casos asintomáticos. A pesar de esta afirmación, se debe enfatizar que no siempre la técnica de PCR muestra mejores índices diagnósticos que las técnicas convencionales (35), por lo que es recomendable su uso en conjunto con otras técnicas para lograr un diagnóstico certero de leishmaniasis y así, basados en esto, fortalecer los programas de control vigente en nuestro país (36).…”

Section: Discussionunclassified

“…Este trabajo sugiere continuar con las pruebas realizando algunas modificaciones adecuadas a las condiciones de laboratorio, considerando que, un principio fundamental para el uso de la PCR continúa siendo su optimización, con el fin de garantizar una amplificación exitosa (35). Finalmente, esta técnica estandarizada será muy valiosa en el diseño e implementación de sistemas de control y vigilancia de las leishmaniasis.…”

Section: Discussionunclassified

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Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

Chaves¹,

Portillo²,

Thomaz-Soccol

et al. 2016

Mem. Inst. Investig. Cienc. Salud

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Cómo referenciar este artículo/ How to reference this article:Céspedes Chaves E, Portillo N, Thomaz-Soccol V, Gonçalves AL, González Brítez N. Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L.) infantum, en muestras caninas. Mem. Inst. Investig. Cienc. Salud. 2016;14(3):24-33 R E S U M E NLa leishmaniasis es una enfermedad producida por protozoarios parásitos del género Leishmania. Estos parásitos infectan a hospedadores mamíferos, entre los cuales los perros han sido implicados como reservorios del parásito. Este trabajo planteó estandarizar la técnica de la PCR-RFLP luego de la amplificación de la región ITS1 de Leishmania spp, como herramienta útil para la detección y caracterización molecular. Se utilizaron promastigotes de cultivo y muestras de biopsias procedentes de perros con leishmaniasis visceral previamente diagnosticados en el Centro Antirrábico Nacional. La región ITS1 del ADN genómico nuclear de Leishmania spp. fue amplificada utilizando los cebadores LITSR y L5,8S. La técnica ITS1 PCR-RFLP aplicada, permitió la detección de Leishmania (Leishmania) infantum en 10/10 aislados de parásitos mantenidos en medio NNN, en 10/18 muestras de bazo y 10/18 muestras de ganglio linfático poplíteo. Las condiciones óptimas de reacción fueron 0,2 mM de dNTPs, 0,1 pmol de cada cebador y 1U de Taq polimerasa. La sensibilidad de la PCR fue de 3 ng/µL de ADN en aislados de cultivo NNN y 60 ng/µL de ADN en muestras de biopsias, mientras que la especificidad fue de 100% para la detección de Leishmania sp. La enzima de restricción Hae III, determinó fragmentos de 184, 72 y 55 pb., que resultaron específicos para la especie Leishmania (L.) infantum. El marcador utilizado resultó confiable para la detección y caracterización de Leishmania sp. en perros procedentes de zonas endémicas, lo cual podría ser útil para verificar las especies de parásitos circulantes entre los perros.Palabras claves: Leishmania infantum, perros, ITS -1 (Espaciador transcripto interno 1). Standardization of PCR-RFLP technique of the ITS-1 region for Leishmania (L.) infantum molecular characterization in canine samples A B S T R A C TLeishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. These parasites infect to mammalian hosts, including canines that have been implicated as reservoirs of the parasite. The aim of this research was to standardize the technique of PCR-RFLP after amplification of the ITS1 region of Leishmania (Leishmania) infantum, as a useful tool for detection and molecular characterization. Promastigotes from culture and biopsies from dogs with visceral Leishmaniasis previously diagnosed by the Centro Antirrábico Nacional. The ITS1 region of the genomic DNA of Leishmania sp. was amplified using LITSR and L5,8S primers. The technique ITS1 PCR-RFLP applied, allowed the detection of

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Section: Discussionunclassified

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

Chaves¹,

Portillo²,

Thomaz-Soccol

et al. 2016

Mem. Inst. Investig. Cienc. Salud

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“…To this end, sera from patients infected with TcII and TcVI T. cruzi DTUs, sera from patients with different clinical forms of Chagas disease and sera from patients infected with non-typed T. cruzi were used. This sera panel was used because patients that are infected with different DTUs or present different clinical forms have been reported to exhibit discordant results when evaluated with conventional Chagas serologic tests [14], [15], [26]. Additionally, cross-reactivity between sera from patients infected with T. cruzi and Leishmania spp.…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Screening and Identification of New Trypanosoma cruzi Antigens with Potential Application for Chronic Chagas Disease Diagnosis

et al. 2014

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The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.

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“…The following epidemiological variables were considered in this study: presence of the vectors Triatoma mexicana (94%), Triatoma dimidiate (3%), Triatoma gerstaeckeri (2%) and Triatoma pallidipennis (1%) ( Villagrán et al 2009) , gender, age, housing type, co-habitation with animals, trips outside the community and reported symptomatology that was compatible with known signs and symptoms of the acute or chronic phases of Chagas disease. Comparisons between epidemiologic parameters and survival patterns were established in every studied population ( Ferrer et al 2013) . Statistical analyses were performed using SPSS v.11.5 to measure confidence values and statistical significance between parameters.…”

Section: Subjects Materials and Methodsmentioning

confidence: 99%

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

Villagrán-Herrera

Sánchez‐Moreno

Rodríguez-Méndez

et al. 2014

Mem. Inst. Oswaldo Cruz

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Immunological diagnostic methods for Trypanosoma cruzi depend specifically on the presence of antibodies and parasitological methods lack sensitivity during the chronic and “indeterminate” stages of the disease. This study performed a serological survey of 1,033 subjects from 52 rural communities in 12 of the 18 municipalities in the state of Querétaro, Mexico. We detected anti-T. cruzi antibodies using the following tests: indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IFA), ELISA and recombinant ELISA (rELISA). We also performed Western blot (WB) analysis using iron superoxide dismutase (FeSOD), a detoxifying enzyme excreted by the parasite, as the antigen. Positive test results were distributed as follows: ELISA 8%, rELISA 6.2%, IFA and IHA 5.4% in both cases and FeSOD 8%. A comparative study of the five tests was undertaken. Sensitivity levels, specificity, positive and negative predictive values, concordance percentage and kappa index were considered. Living with animals, trips to other communities, gender, age, type of housing and symptomatology at the time of the survey were statistically analysed using SPSS software v.11.5. Detection of the FeSOD enzyme that was secreted by the parasite and used as an antigenic fraction in WBs showed a 100% correlation with traditional ELISA tests.

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Comparación entre técnicas inmunológicas y moleculares para el diagnóstico de la enfermedad de Chagas

Cited by 32 publications

References 27 publications

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

Genome-Wide Screening and Identification of New Trypanosoma cruzi Antigens with Potential Application for Chronic Chagas Disease Diagnosis

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

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