Single-end reads of serial isolates from cryptococcal meningitis cases

BACKGROUND: Cryptococcal meningitis (CM) causes an estimated 200,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLU) monotherapy. Whilst relapse after FLU monotherapy with resistant strains is frequently observed, the clinical impact and mechanisms of emergence of FLU resistance in human CM are poorly understood. Heteroresistance – a resistant subpopulaton within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS: A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLU monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient CSF. Saved isolates underwent whole genome sequencing and phenotypic characterisation. RESULTS: Heteroresistance was detectable in the CSF of all patients at baseline. During FLU monotherapy, the proportion of resistant colonies in the CSF increased during the first two weeks of treatment. In contrast, those receiving a combination of FLU and 5FC had no resistant subpopulation detectable in CSF by day 14. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with Chromosome 1 disomy predominating. This is significant due to the presence on Chr1 of ERG11 (the FLU drug target) and AFR1, which encodes a drug efflux pump. Efflux levels in vitro positively correlated with the level of heteroresistance. CONCLUSION: Our findings demonstrate for the first time the presence and emergence of aneuploidy-driven FLU heteroresistance in human CM, and the successful suppression of heteroresistance with 5FC/FLU combination therapy.