Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey

Abstract

A total of 356 clinical isolates of the encapsulated basidiomycetous fungus Cryptococcus neoformans var. neoformans, obtained from Australia, Argentina, Brazil, India, Italy, New Zealand, Papua New Guinea, South Africa, Thailand and the USA, were analyzed to lay the basis for a comprehensive evaluation of the global genetic structure of C. neoformans. Two polymerase chain reaction (PCR)-based typing techniques were standardized: PCR fingerprinting using a single primer specific to minisatellite or microsatellite DNA, and randomly amplified polymorphic DNA (RAPD) analysis using two combinations of three 20- to 22-mer random primers. Previous studies showed that the resultant profiles are reproducible and stable over time. Identical results were obtained in two different laboratories and by different scientists in the same laboratory. Both typing techniques separated the isolates into four major groups (VNI and VNII, serotype A; VNIII, serotype A/D; and VNIV, serotype D). The majority (78%) of isolates belonged to VNI, compared with 18% VNII, 1% VNIII and 3% VNIV. All US isolates could be differentiated by a unique, strain-specific PCR fingerprint or RAPD pattern in contrast to most of the non-US isolates, which showed a substantially higher degree of genetic homogeneity, with some clonality, in different parts of the world. Isolates obtained from the same patient at different times and from different body sites, had identical banding patterns. Both typing techniques should provide powerful tools for epidemiological studies of medically important fungi.