title: Microevolution of Serial Clinical Isolates ofCryptococcus neoformansvar.grubiiandC. gattii.
abstract: The pathogenic species ofCryptococcusare a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification ofERG11at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution ofCryptococcusspecies under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.IMPORTANCEOpportunistic infections caused by species of the pathogenic yeastCryptococcuslead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates ofCryptococcusfrom cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity ofCryptococcusor resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.|Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Yuan
authors.identifier: 0000-0001-7652-153X
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Rhys A
authors.initials: RA
authors.lastname: Farrer
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Giamberardino
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Sharadha
authors.initials: S
authors.lastname: Sakthikumar
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Jones
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Yang
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Tenor
authors.affiliation: European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.
authors.forename: Omar
authors.initials: O
authors.lastname: Wagih
authors.affiliation: National Institute for Communicable Diseases (Centre for Opportunistic, Tropical, and Hospital Infections), A Division of the National Health Laboratory Service, Johannesburg, South Africa.
authors.forename: Marelize
authors.initials: M
authors.lastname: Van Wyk
authors.affiliation: National Institute for Communicable Diseases (Centre for Opportunistic, Tropical, and Hospital Infections), A Division of the National Health Laboratory Service, Johannesburg, South Africa.
authors.forename: Nelesh P
authors.initials: NP
authors.lastname: Govender
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Mitchell
authors.affiliation: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Anastasia P
authors.initials: AP
authors.lastname: Litvintseva
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA cuomo@broadinstitute.org john.perfect@dm.duke.edu.
authors.forename: Christina A
authors.identifier: 0000-0002-5778-960X
authors.initials: CA
authors.lastname: Cuomo
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA cuomo@broadinstitute.org john.perfect@dm.duke.edu.
authors.forename: John R
authors.initials: JR
authors.lastname: Perfect
mesh_terms: D000220:Adaptation, Biological; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D056285:Cryptococcus gattii / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D003455:Cryptococcus neoformans / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D025141:Drug Resistance, Fungal; D019143:Evolution, Molecular; D005838:Genotype; D006801:Humans; D008137:Longitudinal Studies; D016919:Meningitis, Cryptococcal / Q000382:microbiology*; D010641:Phenotype; D012008:Recurrence; D013019:South Africa; D013696:Temperature; D014774:Virulence