Prevalence, molecular and phenotypic profiles of arboreal associated Cryptococcus neoformans in Botswana.

Mopane tree (Colophospermum mopane) is one of the main ecological niches of Cryptococcus neoformans, an opportunistic fungal pathogen that causes cryptococcosis primarily on immunocompromised hosts after inhalation of basidiospores from the environment. Hence, we investigated the prevalence, and phenotypically (antifungal resistance and biofilm formation capacity) and genotypically (mating type and genetic structure) characterized C. neoformans isolated from C. mopane, Acacia tortilis, Adansonia digitata and Ziziphus mucronata in Botswana. We report 7.1% and 2.9% prevalence of C. neoformans in C. mopane and other trees, respectively. All tested C. neoformans isolates were determined to be non-WT to fluconazole. Most isolates (65%) of C. neoformans isolates were biofilm producers. Mating type determination revealed a higher proportion of the globally rare MATa allele (53%) and a single MATα/MATa hybrid. The observed genotypeswere VNI (71%), VNB (23%) and VNB/VNB hybrids (6%). Native trees other than C. mopane are alternative ecological niches of antifungal resistant C. neoformans, and this represents a serious public health concern,and this represents a serious public health concern, especially for high-risk populations. Prevalence of C. neoformans on native trees and the observed emergence of hybrids (evidence of sexual recombination) highlight the need for increased surveillance and risk assessment within a One Health paradigm.

Determining Potential Link between Environmental and Clinical Isolates of Cryptococcus neoformans/Cryptococcus gattii Species Complexes Using Phenotypic and Genotypic Characterisation

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes con tinue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR finger printing and sequencing (Oxford Nanopore Technology) of the intergenic spacer region.Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans x C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n= 3), and 5.3% of C. tetragattii (n = 2) in 38 representa tive isolates that have been characterized. Mating type-specific PCR performed on 38 repre sentative environmental and clinical isolates revealed that 16 (42.1%) were MATa/MATα hybrids, 17 (44.7%) were MATα, and five (13.2%) possessed MATa mating type. We used con ventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.