MOLECULAR CHARACTERIZATION AND PHENOTYPIC OF Cryptococcus neoformans AND Cryptococcus gattii ISOLATES IN ARAÇATUBA, SÃO PAULO STATE, BRAZIL.

Cryptococcosis is a disease caused by an opportunistic fungus of the Cryptococcus genus present in nature, mainly in soil and trees. Cryptococcosis is distributed worldwide but has only become recognized as a major health threat with the emergence of the AIDS pandemic in 1980s. This genus has two pathogenic species, Cryptococcus neoformans (serotype A, VNI/AFLP1 and VNII/AFLP1A, AFLP1B,VNB, formerly C. neoformans var. grubii), C. deneoformans (serotype D, VNIV/AFLP2, formerly C. neoformans var. neoformans), and a C. neoformans × C. deneoformans hybrid (formerly VNIII/AFLP3 or AD hybrids). C. gattii has five separate species, namely, C. gattii (VGI/AFLP4), C. deuterogattii (VGII/AFLP6), C. bacillisporus (VGIII/AFLP5), C. tetragattii (VGIV/AFLP7), and C. decagattii (VGIV and VGIIIc/AFLP10). Transmission is caused by inhalation of desiccated yeast cells in the environment, associated with various organic substrates, particularly decomposing wood. The study was conducted in the city of Araçatuba, located in the northwest region of the state of São Paulo, 534km from the State capital. For this study, 100 samples were randomly collected from trunks and hollows of trees (Cassia sp., Ficus sp., Caesalpinea peltophorides) of 10 unprotected locations, such as public parks and areas near a significant flow of people and animals, to investigate the occurrence of pathogenic yeast. To avoid contamination, the material was stored in sterile plastic containers. Briefly, one gram of each sample was suspended in 50ml of NaCL 0.9% containing 0.04% of chloramphenicol and vortexed for 5 minutes. After a 30 minutes rest period, 1ml of the supernatant was placed over 5 petri dishes of Niger agar and 5 plates containing Sabouraud dextrose with Clorafenicol (0.1 ml each). The plates were then incubated at 30ºC and verified daily for collection of pure isolates. Each beige and brown colony was individually inoculated in Niger medium for phenotypic and molecular characterization. These samples were collected for phenotypic and molecular identification of the species through the use of polymerase chain reaction (PCR). Out of 100 samples collected, 18% were positive for Cryptococcus, where ten samples were C. neoformans var. grubii (VNI/AFLP1), one was C. deneoformans (serotype D, VNIV/AFLP2), one was C.gattii (VGI/AFLP4), one sample of C. deuterogattii (VGII/AFLP6) and one sample C. bacillisporus (VGIII/AFLP5). The presence of Cryptococccus spp. was confirmed in biodegradable material of hollow and trunks of trees and, the molecular types identified (VNI and VGII), correspond to the types responsible for most cases of human infection in Brazil.