Geographically structured populations of Cryptococcus neoformans Variety grubii in Asia correlate with HIV status and show a clonal population structure

Abstract

Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.

Figure 1. Minimum spanning trees using the goeBURST algorithm showing MLST relationships among Asian C. neoformans var. grubii isolates.

(A) Tree represents 476 C. neoformans var. grubii isolates from different countries. Each circle represents a unique genotype/sequence type (STs). The size of the circle corresponds to the number of isolates within that genotype. Different colors correspond to different countries; (B) Same as A, but now showing the genotypes from clinical and environmental sources; (C) Same as A and B, but with the addition of the genotypes of 179 C. neoformans var. grubii isolates from different continents (data from http://mlst.mycologylab.org and previous reports by Cogliati et al., 2013 and Mihara et al., 2012 [16]).

Figure 2. Unrooted Neighbor-joining phylogenetic analysis of the concatenated MLST sequences found among Asian isolates.

Numbers at each branch indicate bootstrap values >50%, based on 1,000 replicates. Each color rectangle represents the origin of isolates according to geographic origin, and clinical and environmental origin. Number inside each color rectangle indicates number of isolates.

Figure 3. Unrooted Neighbor-joining phylogenetic analysis of the concatenated sequences of each sequence type (ST) found among the global isolates.

Numbers at each branch indicate bootstrap values >50%, based on 1,000 replicates. Each color rectangle represents the geographic origin of isolates.

Figure 4. Diagram showing DNA polymorphisms of C. neoformans var. grubii from different Asian regions.

(A) DNA polymorphism, genetic differentiation and gene flow; (B) same as A, but comparing African, American, Asian and European populations.

Figure 5. Population structure analysis among Asian C. neoformans var. grubii isolates obtained with the program STRUCTURE.

(A) Comparing isolates from different countries and (B) global C. neoformans var. grubii isolates (data from http://mlst.mycologylab.org). The population groups are indicated by different colors. Each bar represents the individual isolates. Mixed color bar represents to share haplotypes in the individual isolate.