Microsatellite Typing of Clinical and Environmental Cryptococcus neoformans var. grubii Isolates from Cuba Shows Multiple Genetic Lineages

Abstract

BackgroundHuman 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 FindingsWe 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/SignificanceA 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.

Most Cases of Cryptococcal Meningitis in HIV-Uninfected Patients in Vietnam Are Due to a Distinct Amplified Fragment Length Polymorphism-Defined Cluster of Cryptococcus neoformans var. grubii VN1

“…However, a subgroup of C. neoformans var. grubii that is associated with HIV-uninfected individuals has been described only once before, despite extensive study using numerous typing methods (4,6,21,32,37,52). This may be explained by the relatively small number of isolates from HIV-uninfected patients, particularly from those without underlying disease.…”

Extensive Genetic Diversity within the Dutch Clinical Cryptococcus neoformans Population

“…The genetic relatedness of C. neoformans variety grubii (serotype A) isolates was further investigated using nine short tandem repeat markers (STRs) as described recently (25). The C. neoformans var.…”

“…grubii genome-sequenced reference strain H99 (CBS8710) was used as a control. Fragment analysis on a MegaBACE 500 capillary electrophoresis platform (GE Healthcare Life Sciences, Diegem, Belgium) was carried out as described by Illnait-Zaragozi et al (25). Bionumerics version 6.1 (Applied Maths, Sint-Martens-Latem, Belgium) was used to calculate a minimum spanning tree.…”

“…Bionumerics version 6.1 (Applied Maths, Sint-Martens-Latem, Belgium) was used to calculate a minimum spanning tree. A multistate categorical similarity coefficient was used, and microsatellite complexes (MCs) were defined by a minimum group of two genotypes that contain at least five isolates and with a maximum neighbor distance of a two-locus difference among the nine loci studied (25).…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Thus, IllnaitZaragozi et al [84] applied PCR-CE to study this relationship. The genotypic analysis from pigeon guano samples suggested that additional sources contribute to human cryptococcal infections, and PCR-CE analysis based on the selected microsatellite markers distinguished 104 genotypes with the discriminatory power was 0.993, indicating that this PCR-CE was an effective method for the epidemiologic study of Cryptococcus neoformans var.…”