Environmental isolation and characterisation of Cryptococcus species from living trees in Havana city, Cuba.

Cryptococcus isolates from Cuban patients were identified as C. neoformans var. grubii. Although this species has since long been associated with bird droppings, a recent genotyping study provided strong evidence for additional origins of exposure. We sampled different species of trees in Havana, Cuba to identify other potential sources of exposure to this fungus. A total of 662 samples were collected from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty-five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified.