Despite the increased availability of antiretroviral therapy, cryptococcal meningitis remains a
neglected disease that is responsible for up to 30% of AIDS related deaths. In the light of
next generation sequencing technologies, this thesis investigates the molecular epidemiology
of Cryptococcus neoformans var. grubii (Cng) in southern Africa. Sampling in the field and
laboratory isolation yielded 37 environmental Cng and 38 C. gattii (Cg) from various ecotypes
in Zambia and South Africa. In Zambia, the two sister-species were associated with different
ecoregions (p <0.001). Cng was predominantly found inhabiting the Zambezi Mopane Wood-
lands, a previously described ecological niche for the pathogen, whereas Cg was more frequently
isolated from wet Miombo Woodlands. The fungal community structure associated with these
two saprophytes was explored using a high-throughput metabarcoding approach. Changes in
the microbial assemblages were observed between the different ecoregions (RANOSIM >0.350, p
<0.001) and these differences were more pronounced when climatic (temperature and precipi-
tation) and spatial factors were considered. This work confirmed the local adaptation of these
fungal species to their specific ecoregions, both at the level of the fungal community and their
associated environmental variables. The characterisation of Cryptococcus biotic environments
will help to understand where the pathogen might emerge and to predict a potential spillover of
the pathogen into susceptible host populations as well as uninfected ecoregions. Whole-genome
sequencing of 50 environmental Cng genomes revealed the presence of 26 isolates that grouped
into lineage VNI, and 24 isolates that grouped into lineage VNB. Genome-scans were performed
to uncover genetic ‘outliers’that were putatively under selection within each molecular type.
Genomes of environmental VNB isolates were found to group into two statistically-supported
clades and 32% of VNB isolates possessed the MAT a mating-type locus. Recombination was
shown to occur frequently within the VNB molecular type, whereas VNI isolates displayed a
more clonal profile with the MAT a mating-type being much more rare (4%). VNI infections
were shown to be more often associated with urban centres while VNB Cng are mainly present
across the Colophospermum mopane region, therefore VNB infection most likely occurs in rural
settings. Subsequently, a comparison between environmental and clinical VNB isolates was
performed to understand whether the genetic makeup of Cryptococcus neoformans isolated in
patients is linked to specific environmental genotypes. Genetic variants associated with clinical
isolates were uncovered. Nineteen genes with a known-virulence function were found to be un-
der selection in the environment and were also found to be also associated with clinical isolates.
In vitro phenotypic assays were performed to identify variations between clinical and environ-
mental isolates. Clinical isolates were found to be thermotolerant, produced larger capsule and
were more easily phagocytosed than environmental isolates. Clinical VNI isolates replicated
faster at 37◦ C than clinical VNB isolates (p <0.002). This finding might be a reflection of the
evolutionary relationship between the VNI molecular type and its association with domestic
and feral pigeons. Finally, the epidemiology of the VNI molecular type with isolates from the
African and Asian continents (n=155) was investigated in the light of whole-genome sequence
data. Phylogenetic analyses revealed eight genetic clusters. A ten-fold increase in population
size was observed in each VNI cluster in the past 200 to 8,000 years, suggesting the role of
the domestication of the pigeon in the global spread of the VNI molecular type. These find-
ings indicate that each cluster is likely to contribute differently to the neglected epidemic of
Cryptococcus neoformans var. grubii worldwide.