Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana

Abstract

Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis, and its prevalence is highest in sub-Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees and soil. To investigate the prevalence and population structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MATa allele were observed in both clinical and environmental isolates; however, the mating-type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MATa and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans.

Keywords: Cryptococcus neoformans; genotyping; population structure; recombination.

Figure 1

(A) An outline map of Botswana showing the three geographic locations where environmental samples of Cryptococcus neoformans var. grubii were obtained and analysed in this study. (B) The ratios of MATα and MAT a isolates in different populations. Similar mating‐type ratios were found in all clinical and environmental isolates, but different ratios were found in environmental isolates collected from different areas.

Figure 2

splitstree obtained using concatenated sequences of the eight loci for 139 Botswana Cryptococcus neoformans var. grubii isolates (without VNII isolates). The observation that isolates are linked to each other by multiple pathways, thereby forming an interconnected network rather than a single bifurcating tree, is suggestive of recombination. VNI isolates are labelled blue; VNB‐I isolates are labelled orange; VNB‐II isolates are labelled green; and VNI/VNB admixture genotypes are labelled red.

Figure 3

The genetic relationships among 134 Botswana Cryptococcus neoformans var. grubii (Cng) isolates (two VNII isolates and five VNI isolates with global genotypes, which are distantly related to the Botswana isolates were removed) are visualized by principle components analysis. (A) Circles represent clinical isolates, and triangles represent environmental isolates. Different colours represent different Cng molecular types: blue, VNI; orange, VNB‐I; green, VNB‐II; red, VNB/VNI admixture genotypes. (B) Different colours represent the sources of the isolates. Black dots represent clinical isolates, and the other coloured symbols represent environmental isolates from different areas: blue, Francistown; green, Maun; red, Gaborone.

Figure 4

Results of Botswana Cryptococcus neoformans var. grubii isolates ancestry analysis (K = 3) for all clinical and environmental isolates (A) and ancestry analysis for environmental isolates with different geographical origins: Francistown, Gaborone (Gabs) and Maun (B). Each individual is represented by a thin vertical line, which is partitioned into three coloured segments that represent the individual's estimated membership fractions in three clusters. Black dashed lines separate individuals of different populations. Molecular type of each isolate was labelled at the bottom.