Sixty clinical isolates of Cryptococcus neoformans var. neoformans were analyzed by random amplification of polymorphic DNA (RAPD) using 12- to 22-mer primers in pairs. Five major profiles, which clearly distinguished between serotypes A (profiles I-III), AD (profile IV), and D (profile V), were identified. Forty-two of 58 serotype A isolates were assigned to profile I, 13 to profile II, and 3 to profile III. Profile I compromised 5 subtypes (profiles Ia-Ie), with 37 to 42 isolates in profile Ia. Twenty-seven of 28 isolates from patients with AIDS belonged to profile Ia (P<.001), as did 7 of 10 isolates from otherwise immunocompromised patients. Isolates from immunocompetent hosts were broadly distributed (profile I, 8 isolates; profile II, 10 isolates; profile III, 2 isolates). RAPD profiles were independent of body site and geographic origin of isolates. Isolates pairs from 3 patients produced identical profiles. A predominant genetic profile among serotype A strains from AIDS patients has not been reported previously.
Sixty-one clinical and forty-nine environmental isolates ofCryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12-to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5 GAGGGTGGCGGTTCT 3). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.
Random amplified polymorphic DNA (RAPD) was optimized and used to distinguish between the varieties and serotypes of Cryptococcus neoformans. The RAPD technique distinguished between serotypes A, D or AD within C. neoformans var. neoformans, and revealed further differentiation within each serotype. Four RAPD profiles were clearly recognizable within C. neoformans var. gattii, although its two serotypes, B and C, were only differentiated with one primer combination out of seven.
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