Comparison between PCR-RFLP and sequencing techniques in the analysis of Paracoccidioides spp. biodiversity: limitations and insights into species and variant differentiation.

Abstract

The study of Paracoccidioides spp. faces significant challenges due to limitations inherent in the molecular biology techniques employed. Recently, new species were described whose geographical and genetic distributions were investigated. The phylogenetic studies have revealed that genotypes originally thought to be exclusive in specific regions from South American countries are now being found in other areas of the continent. This finding indicates a broader geographic distribution of these genotypes than previously recognized. To evaluate two molecular biology techniques employed to identify genotypes of Paracoccidioides spp. strains from a Brazilian culture collection previously identified only by mycological methods. DNA samples from 35 Paracoccidioides spp. strains maintained in a Brazilian culture collection were subjected to amplification and enzymatic digestion with PCR-RFLP of tub1 gene, followed by sequencing of gp43 Exon 2 loci. Strains with species identification discrepancies had their tub1 sequences determined to verify possible nucleotide mutations. The genotypic characterization of Paracoccidioides spp. using PCR-RFLP of the tub1 gene identified 22 isolates as P. brasiliensis sensu stricto, two as P. americana, four as P. restrepiensis, and eight as P. lutzii. Sequencing of the gp43 Exon 2 loci revealed discrepancies in the identification of four P. venezuelensis isolates, previously characterized as P. brasiliensis sensu stricto by PCR-RFLP of tub1. The sequencing of tub1 from P. brasiliensis sensu stricto and P. venezuelensis isolates revealed nucleotide differences in the pyrimidine class (C and T) in their sequences, specifically at the position 176bp. These molecular tools were able to establish the genetic diversity within the Paracoccidioides genus, crucial for taxonomy and epidemiology studies. The finding of presence of P. venezuelensis in Brazil, previously thought to be exclusive to Venezuela, highlights genetic connections and evolutionary divergences within the genus. While the PCR-RFLP of tub1technique showed limitations in identifying P. venezuelensis, sequencing of the gp43 Exon 2 loci was able to accurately identify this genotype. Thus, our findings contribute to the understanding of the molecular epidemiology of PCM and emphasize the need for precise species characterization in mycological research.