Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin

Abstract

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.

Figure 1. eBURST illustration comparing the isolates from Thailand with the global population of Cng used in this study.

No. isolates = 176, no. STs = 53, no. re-samplings for bootstrapping = 1000, no. loci per isolate = 7, no. identical loci for group def = 1, no. groups = 1. STs identified by eBURST as present in Thailand and elsewhere in the global dataset are highlighted pink text, those only found in Thailand highlighted green and those only in the global population and not in Thailand are black. Founding genotypes are in blue, and the size of the dots are representative of the number of isolates of that ST.

Figure 2. Principle Components Analysis of the allelic profiles of the Thai Cng genotypes typed in this study.

Individual genotypes (dots) are linked by coloured lines to form clusters which are summarised by coloured ellipses proportional in size to the number of isolates represented. The three groups depicted are numbered and defined according to Thai region: 1 = North (red; n = 91), 2 = Northeast (blue; n = 79) and 3 = South (purple; n = 9). P - value is shown and eigenvalues represented in the bar plot.

Figure 3. Neighbour-joining tree inferring the evolutionary relationships of the Thai isolates typed in this study (n = 183).

Each circle represents a Sequence Type (ST) of the Thai isolates and is proportional in size to the number of isolates of this ST. The isolates are grouped according to three regions of Thailand, Northern province in dark blue (n = 91), Northeastern province in light blue (n = 79) and Southern province in red (n = 9). The four Thai isolates of unknown origin are in black (n = 4). The percentage replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) more than 70% of the time (n≥70%) are indicated. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.

Figure 4. Principle Components Analysis of the allelic profiles of the global Cng genotypes analysed in this study.

Individual genotypes (dots) are linked by coloured lines to form clusters which are summarised by coloured ellipses proportional in size to the number of isolates represented. The four groups are numbered and defined according continent: 1 = Asia (pink; n = 191), 2 = South America (grey; n = 5), 3 = North America (light blue; n = 19), 4 = Africa (dark blue; n = 44). P - value is shown and eigenvalues represented in the bar plot.

Figure 5. Neighbour-joining tree inferring the evolutionary relationships of the global Cng isolates included in this study (n = 261).

The geographical origins of the isolates are represented by coloured rectangles: green  =  Africa (n = 44), red  =  Thailand (isolates typed in this study; n = 186), purple  =  remaining Asian isolates (n = 5), dark blue  =  North America (n = 19), light blue  =  South America (n = 5) and yellow  =  Europe (n = 2). Black rectangles represent reference strains of known VN molecular types that are detailed on the figure for VNI (WM148, H99; n = 232), VNII (WM626; n = 11) and VNB (n = 21). Reference strains of the C. gattii complex (molecular groups VGI – IV) are labelled and serve as an outgroup: WM179, WM178, WM175 and WM779. The percentage replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are indicated if supported by significant bootstrap values (n≥80%). The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.

Figure 6. Haplotype networks of the 53 STs of the global Cng population at each of the seven loci.

Sampled haplotypes are indicated by circles or rectangles colored according to the geographical region from which the sample was collected. STs unique to the African population are shown in green and consist only of clinical isolates. Haplotypes found both in Africa and elsewhere are in brown, while those not found in Africa are represented in blue. Rectangles depict the haplotype with the highest ancestral probability. Each branch indicates a single mutational difference and black dots on the lines are representative of the number of mutational steps required to generate allelic polymorphisms. Circle size is proportional to observed haplotype frequency.