title: Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients.
abstract: Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA.
authors.affiliation: Corporación para Investigaciones Biológicas, Medellín, Colombia.
authors.forename: Marcela
authors.initials: M
authors.lastname: Gaviria
authors.affiliation: Corporación para Investigaciones Biológicas, Medellín, Colombia.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Rivera
authors.affiliation: Corporación para Investigaciones Biológicas, Medellín, Colombia.
authors.forename: Cesar
authors.initials: C
authors.lastname: Muñoz-Cadavid
authors.affiliation: Corporación para Investigaciones Biológicas, Medellín, Colombia; Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia.
authors.forename: Luz Elena
authors.initials: LE
authors.lastname: Cano
authors.affiliation: Corporación para Investigaciones Biológicas, Medellín, Colombia; Escuela de Ciencias de la Salud, Universidad Pontifica Bolivariana, Medellín, Colombia. Electronic address: tnaranjo@cib.org.co.
authors.forename: Tonny Williams
authors.initials: TW
authors.lastname: Naranjo
mesh_terms: D003105:Colombia; D004271:DNA, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D010228:Paracoccidioides / Q000235:genetics* / Q000302:isolation & purification; D010229:Paracoccidioidomycosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins
keywords: Molecular diagnosis; Nested PCR; Paracoccidioides brasiliensis; Paracoccidioidomycosis