title: Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain.
abstract: Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.|Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Gago
authors.affiliation: Sección Micología, Hospital San Juan de Dios del Aljarafe, Sevilla, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Serrano
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.|Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Ana
authors.identifier: http://orcid.org/0000-0001-8651-4405
authors.initials: A
authors.lastname: Alastruey-Izquierdo
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.|Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Cuesta
authors.affiliation: Unidad de Gestión de Enfermedades Infecciosas y Microbiología, Hospital de Valme, Sevilla, Spain.
authors.forename: Estrella
authors.initials: E
authors.lastname: Martín-Mazuelos
authors.affiliation: Unidad de Gestión de Enfermedades Infecciosas y Microbiología, Hospital de Valme, Sevilla, Spain.
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Aller
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.|Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Gómez-López
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.|Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Emilia
authors.initials: E
authors.lastname: Mellado
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000150:complications* / Q000382:microbiology; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D003453:Cryptococcosis / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology*; D003454:Cryptococcus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D016172:DNA Fingerprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics; D026141:Drug Resistance, Multiple, Fungal / Q000235:genetics; D015725:Fluconazole / Q000494:pharmacology; D006801:Humans; D007814:Larva / Q000382:microbiology; D016919:Meningitis, Cryptococcal / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D058889:Molecular Typing; D009036:Moths / Q000382:microbiology; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012703:Serotyping; D013030:Spain / Q000453:epidemiology; D014774:Virulence
chemical_list: D000935:Antifungal Agents; D004271:DNA, Fungal; D021901:DNA, Intergenic; D015725:Fluconazole
keywords: Cryptococcus species; azole resistance evolution; mixed infection