Leon, Kristoffer ESchubert, Ryan DCasas-Alba, DidacHawes, Isobel ARamachandran, Prashanth SRamesh, AkshayaPak, John EWu, WesleyCheung, Carly KCrawford, Emily DKhan, Lillian MLaunes, CristianSample, Hannah AZorn, Kelsey CCabrerizo, MariaValero-Rello, AnaLangelier, CharlesMuñoz-Almagro, CarmenDeRisi, Joseph LWilson, Michael R2020-03-182020-03-182020-05Neurol Neuroimmunol Neuroinflamm. 2020 Mar 5;7(3). pii: e703.2332-7812http://hdl.handle.net/20.500.12105/9279OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/Attribution-NonCommercial-NoDerivatives 4.0 Internacional3213944073e70310.1212/NXI.00000000000007032332-7812Neurology(R) neuroimmunology & neuroinflammationopen access