PT  - JOURNAL ARTICLE
AU  - Kristoffer E. Leon
AU  - Didac Casas-Alba
AU  - Akshaya Ramesh
AU  - Lillian M. Khan
AU  - Cristian Launes
AU  - Hannah A. Sample
AU  - Kelsey C. Zorn
AU  - Ana Valero-Rello
AU  - Charles Langelier
AU  - Carmen Muñoz-Almagro
AU  - Joseph L. DeRisi
AU  - Michael R. Wilson
TI  - Pediatric Brainstem Encephalitis Outbreak Investigation with Metagenomic Next-Generation Sequencing
AID  - 10.1101/414979
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 414979
4099  - http://biorxiv.org/content/early/2018/09/12/414979.short
4100  - http://biorxiv.org/content/early/2018/09/12/414979.full
AB  - In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF). RNA was extracted from CSF (n=20), plasma (n=9), stool (n=15) and nasopharyngeal samples (n=16) from 10 children with brainstem encephalitis or encephalomyelitis and 10 contemporaneous pediatric controls with presumed viral meningitis or encephalitis. Unbiased complementary DNA libraries were sequenced, and microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Metagenomic next-generation sequencing (mNGS) was concordant with qRT-PCR for all samples positive by PCR (n=25). In virus-negative samples (n=35), mNGS detected virus in 28.6% (n=10), including 5 CSF samples. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, mNGS increased the proportion of EV-positive samples from 42% (25/60) to 57% (34/60) (McNemar’s test; p-value = 0.0077). For CSF, mNGS doubled the number of pathogen-positive samples (McNemar’s test; p-value = 0.074). Using phylogenetic analysis, the outbreak EV-A71 clustered with a neuroinvasive German EV-A71 isolate. Brainstem encephalitis specific, non-synonymous EV-A71 single nucleotide variants were not identified. mNGS demonstrated 100% concordance with clinical qRT-PCR of EV-related brainstem encephalitis and significantly increased the detection of enteroviruses. Our findings increase the probability that neurologic complications observed were virus-induced rather than para-infectious. A comprehensive genomic analysis confirmed that the EV-A71 outbreak strain was closely related to a neuroinvasive German EV-A71 isolate. There were no clear-cut viral genomic differences that discriminated between patients with differing neurologic phenotypes.