Diagnosis of Leishmania Using the Polymerase Chain Reaction: a Simplified Procedure for Field Work

López, Martha; Inga, Richard Montañez; Cangalaya, M; Echevarría, Juan; Llanos-Cuentas, Alejandro; Orrego, Cristián; Arévalo, Jorge

doi:10.4269/ajtmh.1993.49.348

Cited by 180 publications

(165 citation statements)

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“…Since in the north and northwestern regions of Paraná the majority of Leishmania isolates (98.7%) has been identified as L. (V.) braziliensis (Silveira et al 1996(Silveira et al , 1999, it was decided to use the MP1L/MP3H primers described by Lopez et al (1993), that amplify a fragment of 70 base pairs of minicircle region present in the kDNA of members of Leishmania (Viannia) subgenus. However, it is necessary to remember that these primers are specific for Leishmania (Viannia), and this PCR can be negative if the patient was infected by L. (L.) amazonensis.…”

Section: Discussionmentioning

confidence: 99%

“…For DNA amplification it was used the MP1L (5' -TAC TCC CCG ACA TGC CTC TG -3') and MP3H (5' -GAA CGG GGT TTC TGT ATG C -3') primers described by Lopez et al (1993), that amplify a fragment of 70 base pairs of the mini-circles present in the kDNA of members of Leishmania (Viannia) subgenus. The PCR reaction mixture (25 µl) contained 1 µM of each one of the primers (Invitrogen ® ), 0.2 mM dNTP (Invitrogen ® ), 2U of Taq DNA Polymerase (Invitrogen ® ), 3 mM of MgCl 2 , 1 × enzyme buffer and 5 µl of the extracted DNA.…”

Section: Studied Populationmentioning

confidence: 99%

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Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

Venazzi

Roberto

Barbosa-Tessmann

et al. 2006

Mem. Inst. Oswaldo Cruz

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Section: Discussionmentioning

confidence: 99%

Section: Studied Populationmentioning

confidence: 99%

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

Venazzi

Roberto

Barbosa-Tessmann

et al. 2006

Mem. Inst. Oswaldo Cruz

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“…The polymerase chain reaction (PCR) represents a new option among them (2)(3)(4)(5). PCR has been shown to be highly sensitive for the diagnosis of ACL, with rapid detection of leishmania (6)(7)(8)(9)(10)(11). PCR can also be used for the typing of leishmania isolated from clinical material, insect vectors, reservoirs, or culture media (12,13).…”

Section: Introductionmentioning

confidence: 99%

Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

Medeiros

Rodrigues

Roselino

2002

Braz J Med Biol Res

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More precise and rapid diagnostic methods for American cutaneous leishmaniasis (ACL) are necessary because of the growing number of cases observed in Brazil, including the northeastern region of the State of São Paulo. We applied PCR to 54 skin or mucosal biopsies from patients with a clinical and/or laboratory diagnosis of ACL using primers 13A and 13B, with positive results being obtained for 82% of the samples. When the PCR results were compared to those of histopathological leishmania detection, PCR showed superior results with 81.5% sensitivity and 95% CI of 68.0-95.1%. The Montenegro skin test (MST) was positive in 88.7% of patients. Since MST cannot be used as a diagnostic tool in endemic areas, the present results strongly suggest the use of PCR for the etiological confirmation of ACL, with emphasis on the mucosal form. Correspondence

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“…Actualmente, la detección de parásitos del género Leishmania en los insectos incluye métodos que van desde la búsqueda directa de los flagelados en el tracto digestivo del insecto hasta técnicas moleculares que usan principalmente K ADN (12,(26)(27)(28)(29)(30). La técnica de reacción en cadena de la polimerasa (PCR), por su alta sensibilidad y especificidad, ha facilitado adelantar estudios ecoepidemiológicos en áreas de alta endemicidad, suministrando datos más precisos en periodos de tiempo más cortos (17,29).…”

Section: Discussionunclassified

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

Cabrera¹,

Munsterman

Cárdenas³

et al. 2002

biomedica

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Para estudios epidemiológicos y programas de control de las leishmaniasis es importante la determinación taxonómica del insecto vector y del agente etiológico causante de esta enfermedad. Por su eficacia y sensibilidad, la utilización de técnicas moleculares, como la reacción en cadena de la polimerasa (PCR) ha permitido avances en entomología médica. La metodología tradicional usada para la búsqueda de infección en los flebótomos es un método dispendioso que requiere mucho tiempo y capacitación para emitir un diagnóstico acertado. En el presente trabajo se evaluaron las condiciones y prácticas adecuadas para el almacenamiento de flebótomos con el propósito de aplicar la PCR. Hembras de Lutzomyia longipalpis de la colonia del Instituto Nacional de Salud se infectaron experimentalmente con una cepa de Leishmania chagasi del valle alto del río Magdalena (Quipile, Cundinamarca). Los insectos infectados se preservaron en tres soluciones: etanol al 100%, etanol al 70% y en buffer Tris-EDTA (TE); submuestras de cada una se almacenaron a -80 °C, -20 °C y a temperatura ambiente. Para determinar los porcentajes de infección, algunas de las hembras se disecaron para buscar las formas flageladas al microscopio. La extracción del K ADN se realizó con Chelex 100. Para la amplificación se utilizaron los iniciadores OL1 y OL2 de Leishmania con electroforesis en geles de agarosa al 1%. En cada una de las condiciones descritas se logró la amplificación de un fragmento de ≈120 pb, correspondiente a Leishmania spp. Estos resultados muestran la ventaja de incorporar como técnica de rutina la PCR para detectar flebótomos infectados de zonas endémicas de leishmaniasis visceral. Por costo-efectividad, se concluyó que las muestras entomológicas para estudios de incriminación vectorial utilizando la PCR, se pueden preservar a temperatura ambiente en etanol al 70%.Palabras clave: Lutzomyia, Leishmania, preservación, PCR, Chelex 100. Definition of temperature and storage adequate conditions in Leishmania DNA detection by PCR in phlebotomine samplesFor epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (=L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 °C, -20 °C and room temperature. To determine infection rat...

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Diagnosis of Leishmania Using the Polymerase Chain Reaction: a Simplified Procedure for Field Work

Cited by 180 publications

References 0 publications

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

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