title: Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing.
abstract: BACKGROUND
  Primary amoebic meningoencephalitis (PAM) is a rare, often lethal, cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes.
  
  
  METHODS
  In this study, we sequenced a full draft assembly of the Balamuthia mandrillaris genome (44.2 Mb in size) from a rare survivor of PAM, and recovered the mitochondrial genome from six additional Balamuthia strains. We also used unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year-old girl.
  
  
  CONCLUSIONS
  Comparative analysis of the mitochondrial genome and high-copy number genes from six additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, and the closest Balamuthia homologs corresponded to other amoebae, hydroids, algae, slime molds, and peat moss. Real-time NGS testing of hospital day 6 CSF and brain biopsy samples detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. The presumptive diagnosis of PAM by visualization of amoebae on brain biopsy histopathology and NGS analysis was subsequently confirmed at the US Centers for Disease Control and Prevention (CDC) using a Balamuthia-specific PCR assay. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better clinical outcome, would have required availability of the complete genome sequence.
  
  
  CONCLUSIONS
  These results underscore the diverse evolutionary origins of Balamuthia mandrillaris, provide new targets for diagnostic assay development, and will facilitate further investigations of the biology and pathogenesis of this eukaryotic pathogen. The failure to identify PAM from a day 1 sample without a fully sequenced Balamuthia genome in the database highlights the critical importance of whole-genome reference sequences for microbial detection by metagenomic NGS.
authors.affiliation: Department of Laboratory Medicine, University of California, 185 Berry Street, Box 0134, San Francisco, CA, 94107, USA.|UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Greninger
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Messacar
authors.affiliation: California Department of Public Health, Richmond, CA, USA.
authors.forename: Thelma
authors.initials: T
authors.lastname: Dunnebacke
authors.affiliation: Department of Laboratory Medicine, University of California, 185 Berry Street, Box 0134, San Francisco, CA, 94107, USA.|UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
authors.forename: Samia N
authors.initials: SN
authors.lastname: Naccache
authors.affiliation: Department of Laboratory Medicine, University of California, 185 Berry Street, Box 0134, San Francisco, CA, 94107, USA.|UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
authors.forename: Scot
authors.initials: S
authors.lastname: Federman
authors.affiliation: Department of Laboratory Medicine, University of California, 185 Berry Street, Box 0134, San Francisco, CA, 94107, USA.|UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
authors.forename: Jerome
authors.initials: J
authors.lastname: Bouquet
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mirsky
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Nomura
authors.affiliation: California Department of Public Health, Richmond, CA, USA.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yagi
authors.affiliation: Kaiser Permanente Hospital, Oakland, CA, USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Glaser
authors.affiliation: John Muir Hospital, Walnut Creek, CA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Vollmer
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Press
authors.forename: Bette K
authors.initials: BK
authors.lastname: Kleinschmidt-DeMasters
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: Bette K
authors.initials: BK
authors.lastname: Klenschmidt-DeMasters
authors.affiliation: Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: Samuel R
authors.initials: SR
authors.lastname: Dominguez
authors.affiliation: Department of Laboratory Medicine, University of California, 185 Berry Street, Box 0134, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.|UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA. charles.chiu@ucsf.edu.|Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.
authors.forename: Charles Y
authors.initials: CY
authors.lastname: Chiu
mesh_terms: D000293:Adolescent; D000562:Amebiasis / Q000134:cerebrospinal fluid* / Q000175:diagnosis; D059611:Balamuthia mandrillaris / Q000235:genetics*; D001921:Brain / Q000378:metabolism; D004660:Encephalitis / Q000134:cerebrospinal fluid* / Q000175:diagnosis; D005260:Female; D064349:Genome, Microbial; D054629:Genome, Mitochondrial; D006801:Humans; D056186:Metagenomics; D017422:Sequence Analysis, DNA