Microsatellite typing of clinical and environmental Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages

Abstract

Background: Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods.

Methodology/principal findings: We selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68; from pigeon guano) C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5-51). A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70%) fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical). Clinical isolates were segregated over multiple microsatellite complexes.

Conclusions/significance: A large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s) of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii.

Figure 1. Origin of samples and relationships between genotypes.

A: Map of Cuba. Colored dots indicate provinces from which clinical and/or environmental samples were available. B: Minimum spanning tree (MST) based on a multistate categorical analysis representing the genotypes of 190 C. neoformans var. grubii isolates from Cuba. Each circle represents a unique genotype. The size of the circle corresponds to the number of isolates with that genotype. Genotypes are linked to their closest relative. Numbers and connecting lines correspond to the number of different markers between genotypes. Genotypes with identical colors and connected by a shaded background are part of a microsatellite complex (MC). In yellow are unique genotypes that are not part of a MC. C: Same as B, but now showing cross-links between all genotypes that differ in no more than 2 markers. D: Same MST as in B, but now showing genotypes obtained from clinical and environmental samples.