Identificación de Histoplasma capsulatum en muestras clínicas mediante la técnica de PCR en dos rondas

• Main Authors: Colella María Teresa, Mata Sofía, Hartung Claudia, Pérez Celina, Roselló Arantza, Olaizola Carolina, Fernández Carmen, Magaldi Silvia, Toro Félix
• Format: Article in Journal/Newspaper
• Language: English; Spanish
• Published: Universidad del Zulia,Facultad de Medicina,Departamento de Enfermedades Infecciosas y Tropicales 2007
• Subjects: Histoplasmosis; PCR; ADN; RCP; DNA; Arctic medicine. Tropical medicine; RC955-962; Public aspects of medicine; RA1-1270; Arctic; Hongo; Triton
• Online Access: https://doaj.org/article/a1d80ee4a9584124958d057553be3e33

Abstract: Histoplasmosis is a systemic mycosis whose diagnosis is based on clinical, mycological and immunological criteria. Identification of its etiological agent in biological samples is difficult to perform. We evaluated a nested-PCR technique to identify Histoplasma capsulatum in clinical specimens (blood and bone marrow) and reference strains. Three different methods for DNA isolation were tested based on the reagents Guanidine thiocyanate, Benzoyl chloride and Triton X-100. Among these procedures, Guanidine thiocyanate-based method yielded by utmost good quality DNA and was easy to perform when used with clinical specimens. However, a better recovery of DNA was accomplished with the Triton-X-100 method. Reference strains of Paracoccidioides brasiliensis, Candida glabrata, Cryptococcus neoformans and Coccidioides immitis (C posadasii)were tested to assess PCR assay specificity. The samples were amplified by nested-PCR and analyzed by 3% agarose gel electrophoresis. Each of the reference strains showed the same signal upon amplification with a DNA band of 210 bases pairs, as well as 5 blood samples and 2 bone marrow specimens, with high sensitivity (10 picograms of Histoplasma capsulatum DNA) and specificity (no cross reactivity with P. brasiliensis, C. glabrata, C. neoformans and C. immitis). This technique has proved to be useful as a diagnostic technique. Resumen: La histoplasmosis es una micosis profunda de distribución mundial cuyo diagnóstico se fundamenta en estudios clínicos, micológicos e inmunológicos. La identificación del agente etiológico en muestras biológicas es difícil de realizar. Se evaluó la técnica de PCR en dos rondas para identificar ADN de Histoplasma capsulatum en muestras clínicas (sangre y médula ósea) y cultivos del hongo. Se ensayaron 3 métodos de aislamiento de ADN basados en los reactivos Tiocianato de Guanidina, Cloruro de Bencilo y Triton-X-100. La técnica de aislamiento con Tiocianato de guanidina resultó de fácil aplicación en muestras clínicas. Sin embargo, se obtuvo un ...