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

Abstract

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue 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 fingerprinting 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 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MAT a/ MATα hybrids, 17 (44.7%) were MATα , and five (13.2%) possessed MAT a mating type. We used conventional 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.

Molecular Diagnostics of Cryptococcus spp. and Immunomics of Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome

“…One Denmark study reported the combination of AFLP with multilocus sequence typing (MLST of nuclear loci including LAC1, CAP59, IGS1, PLB1, URA5, SOD1, and GPD1). Selected Cryptococcus isolates subsequently underwent antifungal susceptibility testing and a comparison with isolates across the world [30]. Similarly, the AFLP and MLST methods were utilized for subsequent multilocus microsatellite typing (MLMT) and phylogenetic analysis.…”