Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing.

Samuel R Dominguez,Alexander L Greninger,Samia N Naccache,Craig A Press,Michael Vollmer,Kevin Messacar,Jerome Bouquet,Shigeo Yagi,Thelma Dunnebacke,Yosuke Nomura,Charles Y Chiu,Bette K Kleinschmidt-Demasters,Carol Glaser,David Mirsky,Scot Federman

doi:10.1186/s13073-015-0235-2

Abstract

BackgroundPrimary 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.MethodsIn 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.Results and DiscussionComparative 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0235-2) contains supplementary material, which is available to authorized users.

Highlights

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
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 next-generation sequencing (NGS)
We demonstrate the ability of metagenomic NGS to rapidly detect Balamuthia mandrillaris from the cerebrospinal fluid (CSF) of a critically ill 15-year-old girl, and highlight the importance of genomic reference sequences by retrospective analysis of a hospital day (HD) 1 sample

Summary

Introduction

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. Balamuthia mandrillaris is a free-living amoeba that is a rare, almost uniformly lethal, cause of primary amoebic meningoencephalitis (PAM) in humans [1]. Our lab has demonstrated the capacity of metagenomic next-generation sequencing (NGS) to provide clinically actionable information in a number of acute infectious diseases, most notably encephalitis [15, 16]. This approach enables the rapid and simultaneous detection of viruses, bacteria, and eukaryotic parasites in clinical samples [17]. The utility of diagnostic NGS is highly dependent on the breadth and quality of databases that contain wholegenome sequence information of reference strains needed for alignment [19]

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