Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients

Marcela Gaviria, Vanessa Rivera, Cesar Muñoz-Cadavid, Luz Elena Cano, Tonny Williams Naranjo

    Research output: Contribution to journalArticlepeer-review

    11 Scopus citations

    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 1. fg of P. brasiliensis DNA.

    Original languageEnglish
    Pages (from-to)376-383
    Number of pages8
    JournalBrazilian Journal of Infectious Diseases
    Volume19
    Issue number4
    DOIs
    StatePublished - 1 Jul 2015

    Bibliographical note

    Funding Information:
    This study was supported by: COLCIENCIAS, Bogotá, Colombia (Project No. 2213-519-28916), the Corporación para Investigaciones Biológicas (CIB), the Universidad de Antioquia (UdeA), the Universidad Pontificia Bolivariana (UPB), and the Research Committee (CODI) of the Universidad de Antioquia through the Sustainability Strategy for Groups A1 and A Program 2013–2014. Medellín, Colombia. Convocatoria Nacional Jóvenes Investigadores e Innovadores of Colciencias , Number 501, year 2010 (supporting Marcela Gaviria). Convocatoria Nacional Jóvenes Investigadores e Innovadores of Colciencias, Number 645, year 2014 (supporting Vanessa Rivera). Mycotic Research Branch, Centers for Disease Control and Prevention (CDC), for providing purified DNAs (see Table 2 ).

    Publisher Copyright:
    © 2015 Elsevier Editora Ltda.

    Keywords

    • Molecular diagnosis
    • Nested PCR
    • Paracoccidioides brasiliensis
    • Paracoccidioidomycosis

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