The molecular ecology of cryptococcus neoformans and cryptococcus gattii in Zambia - implications for clinical epidemiology, virulence and drug resistance

Understanding the distribution and population genomics of sapronoses in the environment can lend important insights into their clinical threat. Environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Zambia collected in 2013/14 revealed a north-south ecological divide of the two species complexes, high diversity of molecular types, and clinical phenotype enrichment in VNB-II. However, the number of environmental genomes available for analysis was limited and analytical methods can be improved. To address this, additional environmental isolates were collected in 2019 to better understand the molecular epidemiology, species distribution and niche preference of C. neoformans and C. gattii in Zambia. Combined analysis of 2013/14 and 2019 isolates revealed high environmental diversity, but a paucity of the main clinical threat, VNI, and abundance of lineages of less clinical significance (VNB and C. gattii). Chromosome copy number variation was identified as an important adaptation mechanism observed solely in clinical C. neoformans isolates. Phenotyping assays suggested clinical VNI to be more heavily melanised, although melanisation was associated with a loss of fitness. Tolerance and heteroresistance to fluconazole were observed in environmental isolates, suggesting these to be inherent internal mechanisms. SDM largely supported an ecological split between the two species complexes, but for the first time this was attributed in large part to soil mineral composition as opposed to climate factors. Hyrax middens were confirmed as preferred niches for C. gattii, but C. neoformans was also identified here for the first time. Analysis of ancient hyrax midden samples suggested possible presence of cryptococcal genomes up to 60,000 calibrated years before the present. First attempts, to our knowledge, of air sampling in Zambia could not confirm presence of aerosolised cryptococcal spores. Findings have implications for geographic risk stratification to inform diagnostic practices, knowledge of occupied niches, and mechanisms of virulence as well as antifungal resistance and tolerance.