Molecular and microbiological diagnosis

Abstract

Introduction and Objectives: Paracoccidioidomycosis (PCM), caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycoses in Latin America. The diagnostic of PCM is sometimes difficult in outside endemic areas. The LAMP has the advantages of rapid reaction, simple operation, and easy detection that required few hours from fungal DNA extraction to identification. In the present study, we have evaluated a LAMP method to unambiguously PCM diagnosis detecting gp43 gene of P. brasiliensis in clinical sputa specimens obtained from a group of Brazilian PCM patients. Methods and Results: A total of 23 sputa samples obtained from 21 patients with chronic PCM (male, aged 28 to 72 years) with a diagnostic established by x-ray, clinical data, and isolation by culture and/or serological tests were used in this study. By using one set of primers F3 for LAMP, B3, FIP, and BIP designed from the partial sequence of gp43 (GenBank accession number U26160), the LAMP assay rapidly amplified the target gene in less than 4 h. We obtained positive results in 11 out of 23 sputa samples of PCM patients (47.8%). Conclusion: Detection of gp43 gene specie specific in sputum can be very useful because it is a nonculture method, can be performed rapidly and do not require invasive sampling procedures. The present study was the first one to apply LAMP method to investigate P. brasiliensis gp43 gene in sputum specimens. Although further study is necessary for its widespread use, we believe that LAMP method might be applicable to diagnosis of PCM.