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

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

Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously “African” VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen

In Vitro Activities of Eight Antifungal Drugs against 55 Clinical Isolates of Fonsecaea spp.▿

The in vitro activities of eight antifungal drugs against clinical isolates of Fonsecaea pedrosoi (n = 21), Fonsecaea monophora (n = 25), and Fonsecaea nubica (n = 9) were tested. The resulting MIC90s for all strains (n = 55) were as follows, in increasing order: posaconazole, 0.063 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.25 μg/ml; voriconazole, 0.5 μg/ml; amphotericin B, 2 μg/ml; caspofungin, 2 μg/ml; anidulafungin, 2 μg/ml; and fluconazole, 32 μg/ml

In Vitro Activity of the New Azole Isavuconazole (BAL4815) Compared with Six Other Antifungal Agents against 162 Cryptococcus neoformans Isolates from Cuba▿

Cuban Cryptococcus isolates (n = 165) were tested in vitro against amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, giving MIC90 values of 0.25, 8, 4, 0.25, 0.125, 0.016, and 0.016 μg/ml, respectively. Isavuconazole and posaconazole seem to be potentially active drugs for treating cryptococcal infections

In Vitro Antifungal Susceptibilities and Amplified Fragment Length Polymorphism Genotyping of a Worldwide Collection of 350 Clinical, Veterinary, and Environmental Cryptococcus gattii Isolates▿

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC90 (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log2 dilution step increase (MIC90, 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC90 of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC90s of 8 μg/ml

Comparison of <i>C. gattii</i> isolates and interspecies hybrids with <i>C. neoformans</i> isolates and hybrids between both <i>C. neoformans</i> varieties.

<p>The forty heat-killed <i>Cryptococcus</i> isolates are grouped according to (sub)species. Cytokine production by human PBMCs after 24 h (IL-1β, TNF-α, IL-6 and IL-1Ra) and 7 d (IL-17 and IL-22) incubation is shown. Mean values (n = 5 to 7) ± SE of three independent experiments are presented. *, p 0.01 to 0.05; **, p 0.001 to 0.01; ***, p<0.001. The horizontal line represents the lower detection limit.</p

Signature profiles of the 4 most prevalent microsatellite complexes and the reference isolate H99 upon whose genome the selection of markers was made.

<p>Signature profiles of the 4 most prevalent microsatellite complexes and the reference isolate H99 upon whose genome the selection of markers was made.</p

The role of TLR2, TLR4 and TLR9 in IL-1β and IL-17 induction by <i>C. gattii</i>.

<p>Cytokine production by human PBMCs preincubated for one hour with culture medium (white bar) or PRR blocking reagents (dark gray bars) or their control (light gray bar) prior to stimulation with heat-killed <i>C. gattii</i> (strain B5742) [10⁷/ml]. IL-1β is determined after 24 h incubation, IL-17 is determined after 7 d incubation. Mean values ± SE of eight individuals in 4 independent experiments (IL-17) or six individuals in 5 independent experiments (IL-1β) (with exclusion of additional four individuals with undetectable cytokine induction by <i>C. gattii</i>) are presented. *, p 0.01 to 0.05. The horizontal line represents the lower detection limit.</p

All forty <i>Cryptococcus</i> strains induce low amounts of IL-17, but high amounts of IL-22.

<p>IL-17 and IL-22 production after 7 d by PBMCs stimulated with RPMI+, either one of 40 different heat-killed <i>Cryptococcus</i> strains [10⁷ microorganisms/mL] or heat-killed <i>Candida albicans</i> [10⁵ microorganisms/mL] is shown respectively. Mean values ± SE (n = 5) of three independent experiments are presented.</p