Resumen del taller sobre el uso de la reacción en cadena de la polimerasa (PCR) para distinguir entre Trypanosoma cruzi y tripanosoma rangeli

This workshop on the use of the polymerase chain reaction (PCR) to detect and differentiate T. cruziand T. rangeli had as its main purpose the transfer of this technology to laboratories in Colombia. To demonstrate this technique, we used clinical samples or isolates obtained from endemic areas of Colombia. In order to evaluate the possible effect of different culture media components on the sensitivity of PCR, analyses were performed on trypanosomes that were grown in different culture media. The DNA was extracted from these cells by boiling, Geneclean, and hypotonic Iysis. The DNA was amplified using a conserved 22 synthetic oligonucleotide sequence within the tandemly repeated mini-exon gene from T. cruzi and T. rangel;. The two organisms were distinguished by the electrophoretic mobilities of their respective amplification products. The confirmation of the identity of the PCR products was obtained using species-specific probes from intergenic regions. Hybridisation was visualised by the NBT colour reaction. From a total of 28 samples analysed, 17 identifications were in agreement with their prior identification. From 5 unknown samples, three were identified as T. rangeli and two as mixed infections. We obtained only two ambiguous identificationscaused by contamination in samples or PCR reaction. Only two samples could not be identified by PCR because of DNA extraction problems. The results from this workshop support the potential of the PCR technique as a usefuI additional tool for the detection and diagnosis of Chagas' disease in Colombia.Este taller se realizó con el propósito de transferir la tecnología de la reacción en cadena de la polimerasa (PCR) para la detección de T. cruzi y T. rangeli a laboratorios en Colombia involucrados en el diagnóstico y estudios epidemiológicos de la enfermedad de Chagas. Para demostración de la técnica se utilizaron muestras clínicas y epidemiológicas de áreas endémicas colombianas. En los ensayos se emplearon muestras de tripanosomas provenientes de diferentes medios de cultivo para evaluar el posible efecto de los componentes de estos medios sobre la sensibilidad del PCR. Se hizo extracción de ADN utilizando los métodos de ebullición, lisis hipotónica y geneclean. El ADN se amplificó utilizando oligonucleótidos sintéticos, correspondientes a una secuencia conservada de 22 nucleótidos dentro de un gen mini-exón. Los dos organismos fueron distinguidos por las movilidades electroforéticas de sus respectivos productos de amplificación, confirmando su identidad con sondas intergénicas específicas de especie, marcadas con digoxigenina dUTP. La hibridación se visualizó con la reacción de color del NBT. De un total de 28 muestras analizadas, se lograron 17 identificaciones que coincidieron con la clasificación original. De cinco muestras desconocidas, tres fueron identificadas como T. rangeli y dos como infecciones mixtas. Se presentaron resultados ambiguos en dos muestras, ocasionados por contaminación en el PCR. Solamente dos muestras no se pudieron identificar mediante PCR por problemas en la extracción del ADN de la muestra. Teniendo en cuenta estos resultados preliminares se abre la posibilidad de utilizar esta técnica como una herramienta útil o método adicional a las técnicas de rutina para detectar y diagnosticar la enfermedad de Chagas en Colombia

PCR-Based Diagnosis of Acute and Chronic Cutaneous Leishmaniasis Caused by Leishmania (Viannia)

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis

Programa para la caracterización de la enfermedad de Chagas en el Magdalena medio antioqueño

IP 1115-05-017-8