title: Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles.
abstract: OBJECTIVE
  MDR Candida strains are emerging. Next-generation sequencing (NGS), which enables extensive and deep genome analysis, was used to investigate echinocandin and azole resistance in clinical Candida isolates.
  
  
  METHODS
  Six genes commonly involved in antifungal resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1 and FKS2) were analysed using NGS in 40 Candida isolates (18 Candida albicans, 15 Candida glabrata and 7 Candida parapsilosis). The strategy was validated using strains with known sequences. Then, 8 clinical strains displaying antifungal resistance and 23 sequential isolates collected from 10 patients receiving antifungal therapy were analysed.
  
  
  RESULTS
  A total of 391 SNPs were detected, among which 6 coding SNPs were reported for the first time. Novel genetic alterations were detected in both azole and echinocandin resistance genes. A C. glabrata strain, which was resistant to echinocandins but highly susceptible to azoles, harboured an FKS2 S663P mutation plus a novel presumed loss-of-function CgPDR1 mutation. This isolate was from a patient with deep-seated and urinary candidiasis. Another C. glabrata isolate, with an MDR phenotype, carried a new FKS2 S663A mutation and a new putative gain-of-function CgPDR1 mutation (T370I); this isolate showed mutated (80%) and WT (20%) populations and was collected after 75 days of exposure to caspofungin from a patient who underwent complicated abdominal surgery.
  
  
  CONCLUSIONS
  This study shows that NGS can be used for extensive assessment of genetic mutations involved in antifungal resistance. This type of wide genome approach will become very valuable for detecting mechanisms of resistance in clinical strains subjected to multidrug pressure.
authors.affiliation: Laboratoire de Parasitologie-Mycologie, Institut de Biologie et de Pathologie, CHU de Grenoble, Grenoble, France Laboratoire TIMC-TheREx, UMR 5525 CNRS-UJF, Université Grenoble Alpes, Grenoble, France cgarnaud@chu-grenoble.fr.
authors.forename: Cécile
authors.initials: C
authors.lastname: Garnaud
authors.affiliation: Unité de Parasitologie-Mycologie, Département de Virologie, Bactériologie-Hygiène, Mycologie-Parasitologie, CHU Henri Mondor, APHP, DHU VIC, Créteil, France EA Dynamyc UPEC-ENVA, Créteil, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Botterel
authors.affiliation: Unité Biologie et Pathogénicité Fongiques-INRA USC2019, Institut Pasteur, Paris, France.
authors.forename: Natacha
authors.initials: N
authors.lastname: Sertour
authors.affiliation: Unité Biologie et Pathogénicité Fongiques-INRA USC2019, Institut Pasteur, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Necker Enfants Malades, Service de Microbiologie, Unité de Parasitologie-Mycologie, F-75015 Paris, France.
authors.forename: Marie-Elisabeth
authors.initials: ME
authors.lastname: Bougnoux
authors.affiliation: EA Dynamyc UPEC-ENVA, Créteil, France Université Paris-Descartes, Faculté de Médecine, Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Dannaoui
authors.affiliation: Laboratoire de Virologie, Institut de Biologie et de Pathologie, CHU de Grenoble, Grenoble, France UVHCI UMI 3265 CNRS-UJF-EMBL, Université Grenoble Alpes, Grenoble, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Larrat
authors.affiliation: Assistance Publique-Hôpitaux de Paris, Hôpital St Antoine, Service de Parasitologie-Mycologie, F-75012 Paris, France Centre d'Immunologie et de Maladies Infectieuses (CIMI-Paris), Inserm U1135, CNRS ERL 8255, Sorbonne Universités, UPMC Univ Paris 06 CR7, Paris, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Hennequin
authors.affiliation: Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañon, Madrid, Spain.
authors.forename: Jesus
authors.initials: J
authors.lastname: Guinea
authors.affiliation: Laboratoire de Parasitologie-Mycologie, Institut de Biologie et de Pathologie, CHU de Grenoble, Grenoble, France Laboratoire TIMC-TheREx, UMR 5525 CNRS-UJF, Université Grenoble Alpes, Grenoble, France.
authors.forename: Muriel
authors.initials: M
authors.lastname: Cornet
authors.affiliation: Laboratoire de Parasitologie-Mycologie, Institut de Biologie et de Pathologie, CHU de Grenoble, Grenoble, France Laboratoire TIMC-TheREx, UMR 5525 CNRS-UJF, Université Grenoble Alpes, Grenoble, France.
authors.forename: Danièle
authors.initials: D
authors.lastname: Maubon
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D002177:Candidiasis / Q000188:drug therapy / Q000382:microbiology; D025141:Drug Resistance, Fungal; D054714:Echinocandins / Q000494:pharmacology*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D054714:Echinocandins
keywords: NGS; antifungals; mutations