Cryptococcal genotype influences immunologic response and human clinical outcome after meningitis

Abstract

In sub-Saharan Africa, cryptococcal meningitis (CM) continues to be a predominant cause of AIDS-related mortality. Understanding virulence and improving clinical treatments remain important. To characterize the role of the fungal strain genotype in clinical disease, we analyzed 140 Cryptococcus isolates from 111 Ugandans with AIDS and CM. Isolates consisted of 107 nonredundant Cryptococcus neoformans var. grubii strains and 8 C. neoformans var. grubii/neoformans hybrid strains. Multilocus sequence typing (MLST) was used to characterize genotypes, yielding 15 sequence types and 4 clonal clusters. The largest clonal cluster consisted of 74 isolates. The results of Burst and phylogenetic analysis suggested that the C. neoformans var. grubii strains could be separated into three nonredundant evolutionary groups (Burst group 1 to group 3). Patient mortality was differentially associated with the different evolutionary groups (P = 0.04), with the highest mortality observed among Burst group 1, Burst group 2, and hybrid strains. Compared to Burst group 3 strains, Burst group 1 strains were associated with higher mortality (P = 0.02), exhibited increased capsule shedding (P = 0.02), and elicited a more pronounced Th(2) response during ex vivo cytokine release assays with strain-specific capsule stimulation (P = 0.02). The results of these analyses suggest that cryptococcal strain variation can be an important determinant of human immune responses and mortality.

Importance: Cryptococcus neoformans is a common life-threatening human fungal pathogen that is responsible for an estimated 1 million cases of meningitis in HIV-infected patients annually. Virulence factors that are important in human disease have been identified, yet the impacts of the fungal strain genotype on virulence and outcomes of human infection remain poorly understood. Using an analysis of strain variation based on in vitro assays and clinical data from Ugandans living with AIDS and cryptococcal infection, we report that strain genotype predicts the type of immune response and mortality risk. These studies suggest that knowledge of the strain genotype during human infections could be used to predict disease outcomes and lead to improved treatment approaches aimed at targeting the specific combination of pathogen virulence and host response.

FIG 1

Multilocus sequence typing of Ugandan clinical isolates. (A) Pseudocolor representation of the allelic structure of the UgCl strain population. Alleles at the seven loci are indicated by different colors. Each row indicates a single strain. Vertical lines on the left represent Burst groups. (B) Consensus tree from 1,000 bootstrapped replicates of a maximum parsimony analysis of the concatenated sequences of all informative loci. Bootstrap values over 50% are shown. (C) Analytical separation of UgCl strains into nonredundant evolutionary groups based on Burst analysis of the sequence types. The colors in the circles indicate the 3 Burst groups. Outlier strains are not shaded.

FIG 2

One-year survival of culture-positive patients presenting at Mulago Hospital in Kampala, Uganda, based on strain genotype. Patients infected with Burst group 3 strains lived longer than patients infected with other genotypes (P = 0.007 by log rank test). No patients infected with hybrid strains survived the infection.

FIG 3

Cryptococcal virulence factors and genotype. (A and B) Association between Burst groups and temporal melanization as indicated by K-means cluster rank on niger seed (A) or l-DOPA (B) medium. (C) Association between Burst groups and shed capsule as measured by cryptococcal antigen titer. Each symbol represents the value for the individual strain. The short vertical bars represent the means for the different groups. Mean values that are statistically significantly different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG 4

Immune response of healthy volunteers to ex vivo stimulation with capsule antigens from genetically distinct strains. (A to D) Production of IFN-γ (IFNg) (A), IL-4 (B), IL-10 (C), and IL-8 (D) cytokines in response to shed capsule antigen from a representative clonal cluster strain within each Burst group. Antigens were standardized to a physiologically relevant final assay CRAG titer of 1:1,024 as determined by cryptococcal antigen agglutination assay. Data are representative from assays of 6 independent strains. Mean values that are statistically different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.005. Mean values that are not statistically different are indicated by bars labeled n.s. (for nonsignificant) with P = 0.08 (A) and P = 0.99 (D).