A MINORITY OF CELLS CAUSING A MAJOR PROBLEM? HETERORESISTANCE TO FLUCONAZOLE IN CRYPTOCOCCUS NEOFORMANS

BACKGROUND: Cryptococcal meningitis (CM), caused by the fungal pathogen Cryptococcus neoformans (Cn), is responsible for an estimated 500,000 deaths per year. Most CM patients are treated with fluconazole (FLU) despite poor clinical outcomes. The phenomenon of heteroresistance, in which a minor subpopulation of cells are drug-resistant in an otherwise susceptible strain, has been described in vitro in association with aneuploidy events. The role of heteroresistance in the clinical setting has not been reported previously for CM.

METHODS: A cohort of patients in Tanzania was studied prospectively. Patients were treated with FLU, as monotherapy or in combination with 5FC. Patient’s cerebro-spinal fluid (CSF) was plated immediately onto plain and FLU-containing agar. Stored Cn isolates underwent population assay profiling (PAP) to determine the parameters of heteroresistance, as well as whole genome DNA sequencing. Drug efflux pump activity in vitro was evaluated using rhodamine-6G.

RESULTS: 20 strains of Cn were obtained from the clinical study. A FLU resistant subpopulation was found at baseline in all isolates. By day 7, the proportion of resistant cells in the population increased (“amplification”) in 83% of patients on FLU monotherapy, whilst in patients receiving the 5FC/FLU combination, the resistant subpopulation disappeared. Importantly, aneuploidy (most commonly disomy of Chr1) was frequently detected in heteroresistant colonies and in isolates from patients whose CM relapsed, and was associated with elevated fluconazole MIC levels. Efflux activity in vitro correlated with the level of heteroresistance.

CONCLUSIONS: Heteroresistant colonies have elevated MIC compared to the parental strain and are associated with disomy and increased drug efflux activity. Our results suggest that heteroresistance is a common feature of Cn isolates in Tanzania, and that the addition of 5FC can prevent amplification of these resistant subpopulation in clinical CM.