Cryptococcus gattii in AIDS Patients, Southern California

The encapsulated basidiomycete Cryptococcus neoformans was recently divided into 2 species, C. neoformans (Cn) and C. gattii (Cg) (1). Cn consists of 2 varieties, grubii (CnVG) and neoformans (CnVN), which are opportunistic pathogens and predominantly infect immunocompromised persons (2,3). CnVG is the major causative agent of cryptococcosis worldwide, except in central Europe, where CnVN infection is most prominent. In contrast, Cg is a primary pathogen, which predominantly infects immunocompetent persons (4). Cg was previously thought to be restricted to tropical and subtropical climates with a special ecologic niche on Eucalyptus trees (5,6). However, the recent outbreak of Cg infection in healthy humans and animals in the temperate climate of Vancouver Island, British Columbia, Canada, and its isolation from several species of trees other than Eucalyptus have raised the strong possibility that this fungus might have broader geographic distribution (7–9). The mechanisms underlying pathogenic and environmental differences between Cn and Cg are not known. Within Cn species, CnVN infections are more likely to display skin involvement and to afflict older patients, whereas CnVG infections are reported to cause a higher mortality rate (3,10). In contrast, infections caused by Cg result in a lower mortality rate but are frequently complicated by neurologic sequelae and require surgery and prolonged therapy (4,11). Our recent studies with the Cu,Zn SOD (SOD1) and MnSOD (SOD2) knockout mutants of Cg indicated that these antioxidants are crucial for Cg pathogenesis (12,13). In contrast, the antioxidant function of SOD1 in CnVG is less crucial for pathogenesis (14). These observations are the first molecular evidence of a likely divergence in the pathogenic mechanisms used by Cn and Cg. Both Cn and Cg have a single locus, 2-allele mating system comprising MATα and MATa strains. The MATα strains of Cn and Cg predominate in nature and in clinical settings, and this predominance over MATa strains is linked to high virulence and reproduction by haploid fruiting (3,15). Generally, Cryptococcus strains are haploid, but hybrid strains have also been characterized from both clinical and environmental sources (16–20). Thus, characterizing clinical Cryptococcus isolates to the individual species or varieties and according to mating and hybrid types could be useful for managing cryptococcosis cases and for further understanding the epidemiology of this disease. Several laboratory typing methods have been used in epidemiologic studies of cryptococcosis, including serotyping, electrophoretic karyotyping, use of mitochondrial DNA probes, use of genomic DNA probes, determination of allelic variations at the URA5 locus, multilocus enzyme typing, measurement of creatinine utilization, polymerase chain reaction (PCR), fingerprinting and amplified fragment-length polymorphism (reviewed in [2]). Previously, we described a PCR–restriction fragment length polymorphism (PCR-RFLP) typing scheme for Cn and Cg pheromone genes, which could be used for characterizing mating types, hybrids, and variety (21). In the present study, we developed a capillary electrophoresis–fragment length analysis (CE-FLA) test, and a CE–single stranded conformation polymorphism (CE-SSCP) test by using the pheromone genes MFα1 and MFa1. These tests were used in parallel with more traditional specialized culture medium and a commercial serotyping kit to characterize Cryptococcus isolates from AIDS patients in southern California.