Abstract
Classified as a critical public health threat by the World Health Organization, Cryptococcus neoformans infections with significant morbidity and mortality. Reports of cryptococcosis persistence, relapse, and reinfection date back to the 1950s, yet the factors driving chronic infections remain poorly understood. A major challenge is the scarcity of serial patient specimens and detailed medical records to study the simultaneous evolution of the pathogen and host health status. This study provides the first genomic and phenotypic analysis of in-host evolution of C. neoformans during chronic infections lasting over a year in six immunocompromised patients. We find fungal genome evolution during persistent infection is characterized by large-scale genome restructuring and increasing genomic heterogeneity. Phenotypic changes show diversification in virulence traits and antifungal susceptibility. Genotypically and phenotypically distinct sub-lineages arise and co-persist within the same tissues, consistent with a model of diversifying selection and niche partitioning in the complex environment of human hosts.