Resumo

C. gattii is an encapsulated yeast that can cause severe human infection, even in immunocompetent patients. Before the recent outbreak in the US and Canada, C. gattii was thought to be restricted to tropical areas. It is endemic to many countries of South America, especially northeastern Brazil. In collaboration with the Centers for Disease Control and Prevention (Atlanta, GA, USA), multi-locus sequence typing (MLST) was performed on 56 C. gattii isolates (1 per patient) from the collection (1994-2012) of Instituto Adolfo Lutz (Sao Paulo, SP, Brazil). The isolates were purified and plated on yeast extract-peptone dextrose agar (YPD) plus 0.5% NaCl and DNA was extracted using the UltraClean kit (MO BIO Laboratories). We performed PCR for both mating type determination (a and α) and MLST using the 7 loci (CAP59, URA5, IGS1, PLB1, SOD1, GPD1, LAC1) according to the International Consensus Multi-Locus Sequence Typing Scheme for C. neoformans and C. gattii). DNA sequencing was performed using Big Dye terminator and the same primers used for the PCR reaction. The MLST results were compared to the online C. gattii MLST database (http://mlst.mycologylab.org). For molecular type VGII isolates, 12 alleles had no matches in the database and were determined to be new alleles. We found four new alleles for IGS1, three new alleles for CAP59, two new alleles for GDP1 and SOD1, and one new allele for PLB1. There were no new alleles for LAC1 or URA5. For our molecular type VGI isolates we found only a single new allele for SOD1. Of the 56 isolates in our study, 20 (35,7%) were new sequence types (STs) generated by the new alleles or by a new combination of previously known alleles. Twelve (21,4%) of the isolates were mating-type a and a single isolate had the diploid mating-type a/α . In contrast to what is seen in North America, high molecular diversity among C. gattii isolates from Brazil was observed. Comparison to ST of US isolates showed that the clonal isolates VGIIa, VGIIb, and VGIIc seen in the US were not observed in Brazil, but phylogenetic analysis revealed possible ancestral strains to VGIIa and VGIIc. This study showed that genotypic diversity among Brazilian C. gattii isolates is much higher than in North America, possibly due to increased recombination between a and α mating type isolates. Our results increase our knowledge of the genotypic diversity of C. gattii and possibly reveal the ancestry of the North American VGIIa and VGIIc genotypes.