Geographic grouping of Cryptococcus neoformans var. gattii by random amplified polymorphic DNA fingerprint patterns and ITS sequence divergence.

Eleven reference strains of Cryptococcus neoformans var. gattii were analyzed by random amplified polymorphic DNA (RAPD) patterns using three oligo primers. Three major RAPD pattern profiles (profiles I, II and III) were identified with A-1 oligo primer. Each profile was found to relate to a geographic region. Since the strains which belong to profiles I and II were mainly the isolates from America, and profile III from Asia with A-1 primer, these three profiles were assigned to the geographic grouping of America-1, America-2 and Asia, respectively. Analysis of rDNA sequences coding an internal transcribed spacer (ITS) region of C. neformans var. gattii revealed that the fungus with each RAPD profile has characteristic base sequences at the four positions (10 and 15 positions in ITS1 and 8 and 56 positions at ITS2) of the ITS regions. On the basis of the combinations of the four bases specific for the ITS regions, four ITS types, AAGG (America-1), AAAC (America-2), GGGC (Asia-1) and AGGC (Asia-2) were identified: the geographic group of Asia was further classified into two subgroups of Asia-1 and Asia-2 based on the ITS typing. Clinical isolates from Thailand (6 strains) and Brazil (7 strains) were found to belong to the geographic group of America-1 nd America-2, respectively. Five reference strains of C. neoformans var. gattii from the CBS culture collection were classified into two America-2, one Asia-1 and two Asia-2 groups. This ITS region analysis allowed us to distinguish all isolates of C. neoformans var. gattii into four geographic groups based on the ITS base sequence, and further molecular epidemiological and ecological research on this fungus is recommended.