Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans

Paiva-Cavalcanti, Milena de; Dantas‐Torres, Filipe; Albuquerque, Suênia da Cunha Gonçalves de; Morais, Rayana Carla Silva de; Brito, Maria Edileuza Felinto de; Otranto, Domenico; Brandão-Filho, Sinval Pinto

doi:10.1016/j.mcp.2013.01.003

Cited by 38 publications

(29 citation statements)

References 29 publications

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“…However, traditional methods have multiple disadvantages including long procedures that entail an increased risk of DNA contamination, interpretation and processing of complex data, low sensitivity, high cost and cross-reactivity [21]. To overcome these disadvantages, Real-Time PCR methods, particularly those that use SYBR Green, TaqMan probes or FRET [11][12][13]17,22], have become available in the last few years to ensure better reproducibility, specificity, sensitivity, and velocity during diagnosing and genotyping [23,24].…”

Section: Resultsmentioning

confidence: 99%

“…Currently, the methods employed for the discrimination of New World Leishmania species are laborious, subjective and often lacking of specificity [21]. Therefore, it is crucial to count on a low-cost, effective and rapid tool that allows an accurate detection and identification of the Leishmania species present in the New World or at least the most prevalent.…”

Section: Resultsmentioning

confidence: 99%

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Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Hernández

Álvarez

González

et al. 2014

Parasites & Vectors

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Background: Leishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species. Findings: Herein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL. Conclusions: This is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Hernández

Álvarez

González

et al. 2014

Parasites & Vectors

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“…B. Gel de agarosa al 2 % con los productos de PCR de cada una de las diluciones se han utilizado varios blancos moleculares como el gen ssu rRNA, el gen hsp70, microsatélites o ADN mitocondrial (kDNA) (15,27,28), algunos de los cuales son menos sensibles que otros debido a su bajo número de copias. Se han descrito varios ensayos para la detección de parásitos y la discriminación de especies de Leishmania en el Nuevo Mundo (29,30), sin embargo, ninguno de estos métodos cumple con los dos objetivos de manera simultánea y, en ocasiones, pueden carecer de especificidad (31). Un diagnóstico molecular rápido específico de especie permite elegir el tratamiento más adecuado y hacer un seguimiento del parásito para fines epidemiológicos (investigación de brotes o seguimiento de cepas de parásitos resistentes a los medicamentos) (2).…”

Section: Fluorescencia Ciclosunclassified

Curvas de fusión de regiones genómicas específicas: una herramienta prometedora para el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia

et al. 2017

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Introducción. La leishmaniasis cutánea es una enfermedad causada por parásitos del género Leishmania que tiene gran incidencia en Colombia. El diagnóstico y la identificación de la especie infecciosa son factores críticos en el momento de escoger e iniciar el tratamiento. Actualmente, los métodos de diagnóstico y tipificación requieren procedimientos complejos, por lo que es necesario validar nuevos marcadores moleculares y métodos que simplifiquen el proceso.Objetivo. Desarrollar una herramienta basada en la reacción en cadena de la polimerasa (PCR) con curvas de fusión (High Resolution Melting; PCR-HRM) para el diagnóstico y tipificación de las tres especies de Leishmania de importancia epidemiológica en casos de leishmaniasis cutánea en Colombia.Materiales y métodos. Los genomas de Leishmania panamensis, L. braziliensis y L. guyanensis se compararon mediante métodos bioinformáticos. Las regiones específicas de especie identificadas se validaron mediante PCR. Para los marcadores seleccionados se diseñó una PCR-HRM y se estimaron algunos parámetros de validez y seguridad usando aislamientos de pacientes colombianos caracterizados previamente mediante PCR y análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism – RFLP; PCR-RFLP) del gen hsp70.Resultados. El análisis genómico comparativo mostró 24 regiones específicas de especie. Sin embargo, la validación mediante PCR solo identificó un marcador específico para cada especie de Leishmania. Los otros marcadores mostraron amplificación cruzada. El límite de detección para los tres marcadores seleccionados fue de un parásito, mientras que la sensibilidad, la especificidad, el valor predictivo positivo y el negativo fueron de 91,4, 100, 100 y 75 %, respectivamente.Conclusiones. Las tres regiones seleccionadas pueden emplearse como marcadores moleculares en el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia.

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“…Samples from dogs and humans living in a non-endemic area, apparently healthy and negative for VL and CL using conventional PCR and qPCR [8,10] were used as negative controls in the optimization (human samples) and validation (dogs samples) steps [15]. Samples known to be positive were obtained by the addition of genomic DNA from Leishmania infantum (MHOM/TN/1980/IPT1) or Leishmania braziliensis (MHOM/BR/1975/M2903) to DNA extracted from healthy dogs and humans.…”

Section: Samplesmentioning

confidence: 99%

“…The limitations inherent to the microscopy and immunologybased assays have spurred parasitologists toward the use of more refined molecular tools [7]. Besides the conventional PCR, other PCR-based methods have been recently implemented for the detection of several parasitic infections [8][9][10]. The use of molecular diagnostic techniques has shown that PCR can achieve ''gold standard'' criteria, in some instances [11].…”

Section: Introductionmentioning

confidence: 99%

Inclusion of Quality Controls on Leishmaniases Molecular Tests to Increase Diagnostic Accuracy in Research and Reference Laboratories

Gonçalves-de-Albuquerque

Silva

Trajano-Silva

et al. 2014

Mol Biotechnol

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Early detection of leishmaniases and prompt institution of treatment are paramount for individuals and communities affected by these diseases. To overcome the remaining limitations inherent to molecular methods currently used and to ensure the accuracy of results in leishmaniases diagnosis, two triplex polymerase chain reaction (PCR) assays with quality controls for the reactions were developed. Validity indicators were assessed in 186 dog blood samples from endemic areas in Brazil. The level of agreement between the new tools and their singleplex protocols was assessed by kappa analysis. The triplex PCR for visceral leishmaniasis showed sensitivity (S) = 78.68 %, specificity (E) = 85.29 %, and efficiency (e) = 81.05 %. The cutaneous leishmaniasis protocol showed S = 97.29 %, E = 79.16 %, and e = 90.16 %. Both protocols showed good agreement with gold standards. These new tools enable, in a single reaction, the diagnosis of the diseases and the evaluation of the sample quality and DNA extraction process, thus reducing the cost of reagents and avoiding the eventual need for collecting a second sample.

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Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans

Cited by 38 publications

References 29 publications

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Curvas de fusión de regiones genómicas específicas: una herramienta prometedora para el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia

Inclusion of Quality Controls on Leishmaniases Molecular Tests to Increase Diagnostic Accuracy in Research and Reference Laboratories

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