261. Balamuthia mandrillaris encephalitis detected by next generation sequencing of cerebrospinal fluid
Session: Poster Abstract Session: Diagnostics: Typing and Sequencing
Thursday, October 8, 2015
Room: Poster Hall


Amoebic encephalitis is a rare, often fatal, cause of encephalitis which requires a high index of suspicion for diagnosis.  We report detection of Balamuthia mandrillaris by next generation sequencing (NGS) of cerebrospinal fluid (CSF) in a child with rapidly progressive encephalitis. 

Case Report

A 15 year old girl with type 1 diabetes mellitus and celiac disease presented with seven days of headache, vomiting, arm weakness, clumsiness, and confusion.  Brain MRI showed hemorrhagic lesions with surrounding edema in the left frontal and occipital lobes (Figure 1 A-C).  CSF showed 377 leukocytes/μL with negative bacterial and viral studies.  IV methylprednisolone (1g/day) was given hospital day (HD) 2-5 for suspected acute hemorrhagic leukoencephalitis.  The patient clinically deteriorated on HD 5.  Repeat MRI on HD 5 (Figure 1 D-F) showed multiple rim-enhancing lesions and brain biopsy pathology from HD 6 showed a hemorrhagic necrotizing process with numerous amoeba (Figure 2).  Despite combination amoebicidal therapy, she developed intracranial hypertension, cardiac arrest, and died.    Informed consent was obtained.  Ventricular CSF from HD 6 was analyzed by NGS.


Total nucleic acid was extracted from CSF and half was treated with Turbo DNAse. RNA was reverse-transcribed with random hexamers.  Double-stranded cDNA or extracted DNA from CSF underwent 14-18 cycles of amplification using half-reactions of Nextera XT.  Libraries were cleaned using Ampure XP beads, quantitated on the BioAnalyzer, and run on Illumina MiSeq (1x160nt run).   Reads were taxonomically analyzed via SURPI (June 2014 NCBI NT/NR database). 


A total of 2,813,691 160 bp reads were recovered from the RNA library prepared from CSF.  381,663 reads were removed by preprocessing and 879,711 reads were removed by human subtraction.  A total of 173,980 reads aligned to NCBI NT database by SNAP, comprising 2,022 reads to viruses (primarily phages), and 46,380 reads to bacteria (primarily skin contaminants). Of the reads aligning to non-chordate eukaryotes, 6,540 reads aligned to 16S mitochondrial rRNA and 18S rRNA sequence from B. mandrillaris.


            Encephalitis has a broad differential.  NGS has potential clinical utility through the unbiased detection of rare, potentially treatable, infectious etiologies.


Yosuke Nomura, MD1, Alex Greninger, PhD2,3, Kevin Messacar, MD1, Thelma Dunnebacke, AB, MS, PhD4, Samia N Naccache, Ph.D.2,3, Jerome Bouquet, PhD2,3, David Mirsky, MD5, Craig Press, MD, PhD6, B.K. Kleinschmidt-Demasters, MD7, Sarah Parker, MD1, Samuel Dominguez, MD, PhD1 and Charles Y. Chiu, M.D./Ph.D.3,8, (1)Pediatric Infectious Diseases, University of Colorado School of Medicine and Children's Hospital Colorado, Aurora, CO, (2)Laboratory Medicine, University of California, San Francisco, San Francisco, CA, (3)UCSF Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, (4)California Department of Public Health, Richmond, CA, (5)Pediatric Radiology, University of Colorado School of Medicine, Children's Hospital Colorado, Aurora, CO, (6)Pediatric Neurology, University of Colorado School of Medicine, Children's Hospital Colorado, Aurora, CO, (7)Pathology, Neurology, Neurosurgery, University of Colorado School of Medicine, Aurora, CO, (8)Laboratory Medicine and Medicine / Infectious Diseases, University of California, San Francisco, San Francisco, CA


Y. Nomura, None

A. Greninger, None

K. Messacar, None

T. Dunnebacke, None

S. N. Naccache, None

J. Bouquet, None

D. Mirsky, None

C. Press, None

B. K. Kleinschmidt-Demasters, None

S. Parker, None

S. Dominguez, None

C. Y. Chiu, Abbott: Investigator , Research grant

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