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Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis

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 emerge...

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Autores principales: Stone, Neil R.H., Rhodes, Johanna, Fisher, Matthew C., Mfinanga, Sayoki, Kivuyo, Sokoine, Rugemalila, Joan, Segal, Ella Shtifman, Needleman, Leor, Molloy, Síle F., Kwon-Chung, June, Harrison, Thomas S., Hope, William, Berman, Judith, Bicanic, Tihana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Clinical Investigation 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391087/
https://www.ncbi.nlm.nih.gov/pubmed/30688656
http://dx.doi.org/10.1172/JCI124516
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author Stone, Neil R.H.
Rhodes, Johanna
Fisher, Matthew C.
Mfinanga, Sayoki
Kivuyo, Sokoine
Rugemalila, Joan
Segal, Ella Shtifman
Needleman, Leor
Molloy, Síle F.
Kwon-Chung, June
Harrison, Thomas S.
Hope, William
Berman, Judith
Bicanic, Tihana
author_facet Stone, Neil R.H.
Rhodes, Johanna
Fisher, Matthew C.
Mfinanga, Sayoki
Kivuyo, Sokoine
Rugemalila, Joan
Segal, Ella Shtifman
Needleman, Leor
Molloy, Síle F.
Kwon-Chung, June
Harrison, Thomas S.
Hope, William
Berman, Judith
Bicanic, Tihana
author_sort Stone, Neil R.H.
collection PubMed
description 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.
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spelling pubmed-63910872019-03-09 Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis Stone, Neil R.H. Rhodes, Johanna Fisher, Matthew C. Mfinanga, Sayoki Kivuyo, Sokoine Rugemalila, Joan Segal, Ella Shtifman Needleman, Leor Molloy, Síle F. Kwon-Chung, June Harrison, Thomas S. Hope, William Berman, Judith Bicanic, Tihana J Clin Invest Clinical Medicine 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. American Society for Clinical Investigation 2019-01-28 2019-03-01 /pmc/articles/PMC6391087/ /pubmed/30688656 http://dx.doi.org/10.1172/JCI124516 Text en Copyright © 2019 Stone et al. http://creativecommons.org/licenses/by/4.0/ This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Clinical Medicine
Stone, Neil R.H.
Rhodes, Johanna
Fisher, Matthew C.
Mfinanga, Sayoki
Kivuyo, Sokoine
Rugemalila, Joan
Segal, Ella Shtifman
Needleman, Leor
Molloy, Síle F.
Kwon-Chung, June
Harrison, Thomas S.
Hope, William
Berman, Judith
Bicanic, Tihana
Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title_full Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title_fullStr Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title_full_unstemmed Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title_short Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
title_sort dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis
topic Clinical Medicine
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391087/
https://www.ncbi.nlm.nih.gov/pubmed/30688656
http://dx.doi.org/10.1172/JCI124516
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