Comparative genomics of Cryptococcus neoformans var. grubii associated with meningitis in HIV infected and uninfected patients in Vietnam

Abstract

The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNIγ). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNIγ cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher's exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii.

Fig 1. Population structure of Vietnamese clinical isolates (MLST).

Minimum-spanning tree of the 14 detected STs and their relative distribution between HIV infected and HIV uninfected patients of 136 Vietnamese clinical isolates of C. neoformans var. grubii. Circle sizes are proportional to the number of isolates; red = isolate from HIV infected patient, grey—isolate from HIV uninfected patient. ST—multi locus sequence type.

Fig 2. Effect of infection sequence type on survival of Vietnamese HIV infected patients with cryptococcal meningitis.

Kaplan-Meier survival curves by infecting sequence type (ST5 (solid line) versus non-ST5 (dashed line)) for 290 HIV infected patients enrolled in a randomized controlled trial of combination antifungal therapy for cryptococcal meningitis over 6 months following randomization. No significant difference in the risk of death was detected by either 10 weeks or 6 months following randomization. Figures below the time axis are the number of patients at risk.

Fig 3. BRIG plot showing the relatedness of an ST5 isolate (BMD700) and an ST4 isolate (BMD1415) to the H99 reference genome.

On the inner two rings, the coloured regions represent high pairwise similarity with H99 (>70%) according to BLASTn; lighter regions show areas of difference with H99. The outer two rings plot the number of SNPs per 1000 base pairs. The bar scale is limited to a maximum frequency of 10 SNPs per 1000 bp; any window with greater than this frequency is coloured blue. The figure illustrates that SNP density varies widely across the genome between areas of high and low frequencies; some of these are common to both STs compared with H99, others are ST4 or ST5 specific.

Fig 4. The phylogenetic relationship of eight ST5 and non-ST5 Vietnamese C. neoformans in comparison with the H99 reference genome.

Genome-wide SNP derived maximum likelihood tree of eight Vietnamese strains of C. neoformans var. grubii and the H99 reference strain. Scale bar = genetic distance. All bootstrap values were greater than 0.9.