Molecular typing of global isolates ofCryptococcus 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.

Molecular characterization of environmental Cryptococcus neoformans isolated in Vitoria, ES, Brazil

“…Oligonucleotides of the minisatellite-specific core sequence of the wild-type phage M13 (5'-GAGGGTGGCGGTTCT-3') were used as single primers for PCR amplification, according to MEYER et al 23 . PCR products were separated by electrophoresis in 1.4% agarose gel in 1XTBE buffer stained with ethidium bromide at 0.5 ng mL -1 and visualized under UV light.…”

“…neoformans (serotype AD), whereas the species C. gattii comprises the serotypes B and C 17 . The Cryptococcus neoformans complex has been divided into eight major molecular types by M13 fingerprinting, URA5-RFLP and AFLP analysis 23,24 . These include VNI and VNII (C. neoformans var.…”

“…In Brazil, the epidemiology of serotype A in the southern and southeastern regions reproduces the picture observed worldwide. On the contrary, serotype B is the most frequent agent of cryptococcosis in the northeastern region 23,27 . While C. gattii is found to be associated with trees, C. neoformans is mostly recovered from bird excreta 4 .…”

Determinación in vitro de la pareja sexual en aislamientos del complejo Cryptococcus neoformans

“…grubii is the major fungal pathogen in patients with AIDS (1,2). The second species: C. gattii (serotypes B and C, genotypes VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5 and VGIV/ AFLP7) is a primary pathogen that mainly affects patients with normal immunity (3)(4)(5). Recently, a natural occurring hybrid was described between C. neoformans and C. gattii serologically characterized as BD (6).…”

“…Recently, a natural occurring hybrid was described between C. neoformans and C. gattii serologically characterized as BD (6). These serotypes/molecular types differ in their epidemiological, ecological, virulence and molecular characteristics (1,4,(7)(8)(9)(10)(11). Infections are assumed to be acquired via inhalation of infectious propagules, assumed to be desiccated yeast cells (blastoconidia) or basidiospores, from environmental niches (1,7,8).…”