SRA

Background: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). A portion of the EV-A71 viral protein 1 gene was highly similar to a neuroinvasive German EV-A71 isolate. Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF).Methods: 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 on an Illumina HiSeq 4000. Microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Statistics were performed using McNemar's statistical test.Results:Of the EV-positive samples by conventional qPCR (n=25), metagenomic next-generation sequencing (mNGS) was 100% concordant (n=25). In virus-negative samples by polymerase chain reaction (n=35), virus was found in 31.4% (n=11) by mNGS, including 5 CSF samples, 4 of which had EV-A71. Overall, EV-A71 was identified by mNGS in 80% of patients (n=16), Echovirus 30 in 25% (n=5), Coxsackievirus B in 15% (n=3) and human herpesvirus 7 (HHV-7) in 1 patient. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, we increased the proportion of enterovirus-positive samples from 42% (25/60) to 58% (35/60) (McNemar's test; p-value = 0.0044). For CSF in particular, mNGS doubled the number of pathogen-positive samples from 5 (25%) to 10 (50%) (McNemar's test; p-value = 0.074). Using phylogenetic analysis, the full length EV-A71 viral protein 1 gene clustered with a neuroinvasive German EV-A71 isolate. Non-synonymous EV-A71 single nucleotide variants that segregated with brainstem encephalitis patients were not identified.Conclusion: mNGS is a powerful tool for pathogen detection in outbreak scenarios. mNGS showed a 100% concordance with clinical qPCR for pathogen detection by conventional means during an outbreak of EV-related brainstem encephalitis, and we significantly increased the detection of enterovirus across all samples. In the CSF, we doubled the number of samples with detectable pathogen including identification of EV-A71 in the CSF of 1 brainstem encephalitis case, 1 encephalitis case and 2 children with meningitis. Our findings make it more likely the neurologic complications observed in the outbreak were virus-induced rather than a result of para-infectious mechanisms.