Paracoccidiodomycosis (PCM) is the most important systemic mycosis in Brazil, and is usually associated with rural work. PCM is caused by inhalation of infective propagules of thermodimorphic fungi from the genus Paracoccidioides. In the past, it was believed that Paracoccidioides brasiliensis was the single species responsible for PCM cases. However, recent advances in molecular methods allowed the description of several new species, using phylogenetic concordance as the gold standard. Aside from P. brasiliensis sensu stricto, Paracoccidioides americana is also endemic in Rio de Janeiro state, Brazil. This study aimed to evaluate intraspecific genetic variability of Paracoccidioides isolates from patients diagnosed with PCM at a reference center for endemic mycoses in Rio de Janeiro state, from 2015 to 2021. Among the sixteen retrieved isolates, three (18.75%) were identified as P. americana and thirteen (81.25%) as P. brasiliensis sensu stricto. No intraspecific genetic variation was observed by the M-13 primer in P. americana isolates from this geographic region. However, P. brasiliensis sensu stricto isolates were clustered into two distinct molecular profiles, despite being grouped in a single clade in the phylogenetic tree after partial sequencing of arf and gp43 genes. The results suggest a single P. americana lineage and two P. brasiliensis populations causing PCM in Rio de Janeiro, Brazil.
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