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Updated: Jan 30, 2026
Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body
Published on: January 11, 2018
Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
Neil Rh Stone1, Johanna Rhodes2, Matthew C Fisher2
1Centre for Global Health, Institute for Infection and Immunity, St. George's, University of London, United Kingdom.
Background:
Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation 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 FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization.
Results:
Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance.
Conclusion:
Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy.
Funding:
This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.
Insights
Fluconazole resistance in cryptococcal meningitis emerges due to aneuploidy, but combination therapy with flucytosine suppresses this resistance. This finding is crucial for improving treatment outcomes in affected patients.
Area of Science:
- Mycology
- Infectious Diseases
- Genetics
Background:
- Cryptococcal meningitis (CM) is a major cause of death, particularly in sub-Saharan Africa.
- Current treatment often involves fluconazole (FLC) monotherapy, with observed relapses linked to drug resistance.
- Mechanisms of FLC resistance emergence in human CM, including heteroresistance (HetR), are poorly understood.
Purpose of the Study:
- To investigate the presence and emergence of FLC heteroresistance in HIV-associated CM.
- To understand the genetic mechanisms driving FLC resistance in Cryptococcus spp.
- To evaluate the impact of combination therapy (FLC + 5FC) on FLC heteroresistance.
Main Methods:
- Prospective observation of 20 HIV-associated CM patients in Tanzania.
- Quantification of total and resistant Cryptococcus spp. subpopulations in cerebrospinal fluid (CSF).
- Whole genome sequencing and phenotypic characterization of isolates.
Main Results:
- Heteroresistance was detected in all patients at baseline.
- FLC monotherapy led to an increase in resistant subpopulations within 2 weeks.
- Combination therapy with FLC and 5FC suppressed detectable resistant subpopulations by day 14.
- Aneuploidy, particularly Chr1 disomy involving ERG11 and AFR1, was associated with heteroresistance and relapse isolates.
- In vitro efflux levels correlated with heteroresistance levels.
Conclusions:
- Demonstrated aneuploidy-driven FLC heteroresistance in human CM for the first time.
- Established an association between efflux levels and heteroresistance.
- Showed successful suppression of heteroresistance using 5FC/FLC combination therapy.
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