Determination of the main mechanisms associated with fluconazole resistance in Colombian clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii

Over the past 40 years, throughout the HIV/AIDS pandemic, the number of cases of cryptococcosis caused by Cryptococcus neoformans has increased considerably worldwide, due to its status as an opportunistic pathogen that mainly affects immunosuppressed patients. Specifically in Colombia, 1.1 cases per thousand HIV patients are affected by cryptococcosis meningitis, whose mortality rate reaches almost 50%. Additionally, cryptococcosis caused by Cryptococcus gattii, which mainly affects patients without an apparent risk factor, is associated with serious sequelae and prolonged treatment. Remarkably, both C. neoformans and C. gattii frequently recover from the environment, including soil samples, bird excreta, various species of trees and decaying wood.

Of the few antifungal agents available for the treatment of mycosis, only the amphotericin B deoxycholate and fluconazole are available in developing countries to treat cryptococcal infections. While amphotericin is used together with fluconazole in induction therapy, fluconazole is used as the sole medication in consolidation and maintenance therapies, and is also recommended as primary prophylaxis for cryptococcosis. In developed countries, however, 5-fluorocytocin is used instead of fluconazole in induction therapy because of its higher efficacy.

Although antifungal resistance in C. neoformans and C. gattii is generally rare, in Colombia not only clinical but also environmental isolates resistant to fluconazole have been reported, which has been associated with therapeutic failures and relapses. In these two Cryptococcus species, resistance to azoles such as fluconazole is associated with the molecular mechanism of point mutations in the ERG11 gene, which encodes an enzyme that converts lanosterol into ergosterol, the target of action of azole compounds. In addition, the phenotypic phenomenon of intrinsic heteroresistance to fluconazole has been described, which is characterized by the ability of the fungus to adapt to high concentrations of the antifungal gradually.

Because the decreased susceptibility or resistance of C. neoformans and C. gattii to fluconazole influences the management of cryptococcosis and worsens the prognosis, it is important to know the mechanisms of the origin of such resistance, which in turn could lead to define methodologies that improve the diagnosis of resistant isolates and direct therapeutic strategies, making them more appropriate and timely.

Products of new scientific or technological knowledge expected:
1. Identification and inventory of ERG11 gene polymorphisms associated with fluconazole resistance in Colombian clinical and environmental isolates of C. neoformans and C. gattii.
2. Creation of a database with the microbiological and molecular information of the C. neoformans and C. gattii isolates included in the study, as well as a collection of their DNA.
3. Standardization of methodologies for the amplification and sequencing of the ERG11 gene and for the evaluation of fluconazole heteroresistance of C. neoformans and C. gattii.
4. Contribution with the collection of nucleotide sequences of public access for the study of pathogens of importance in Public Health, such as C. neoformans and C. gattii, through the deposit of the sequences obtained in the GenBank databases.
5. Preparation of a manuscript for publication in an international indexed journal.
6. Participation and presentation of study results at a national or international scientific event

Expected training products:
1.Articulation of a young researcher in the project