Serological and metagenomic interrogation of cerebrospinal fluid implicates enteroviruses in pediatric acute flaccid myelitis

Abstract

BackgroundSince 2014, the United States has experienced a biennial spike in pediatric acute flaccid myelitis (AFM). Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF) and only inconsistently identified from the respiratory tract, serum, or stool.MethodsWe interrogated CSF from children with AFM (n=42) and pediatric controls with other neurologic diseases (OND) (n=58). Samples were incubated with T7 bacteriophage expressing 481,966 sixty-two amino acid peptides with a fourteen amino acid overlap tiled across all known vertebrate virus and arbovirus genomes, an adaption of the VirScan method. Antibody-bound phage were deep sequenced to quantify enriched peptides with normalized counts expressed as reads per hundred thousand (rpK). EV antibody findings were confirmed with ELISA using whole viral protein 1 (VP1) from contemporary enterovirus (EV) A71 and D68 strains. Separately, metagenomic next-generation sequencing (mNGS) of CSF RNA, both unbiased and with targeted enrichment for EVs, was performed.ResultsThe most significantly enriched viral family by VirScan of CSF in AFM versus OND controls was Picornaviridae (mean rpK 11,266 versus mean rpK 950, p-adjusted < 0.001, Wilcoxon signed-rank test with Bonferroni adjustment). Enriched Picornaviridae peptides belonged almost entirely to the genus Enterovirus. The mean EV VP1 ELISA signal in AFM (mean OD 0.51) was significantly higher than OND controls (mean OD 0.08, p-value < 0.001, Mann-Whitney test). mNGS did not detect additional enterovirus RNA in CSF.ConclusionDespite the rare detection of EV RNA in the CNS of patients with AFM, a pan-viral serologic assay identified high levels of CSF EV antibodies in AFM CSF compared to CSF from OND controls. These results provide further evidence for a causal role of non-polio enteroviruses in AFM.

Diagnostic accuracy of VIDISCA-NGS in patients with suspected central nervous system infections

“…flaviviruses), one could determine the infection by analysing specific antibodies, which can be detected for a much longer time after infection. Recently, micro arrays containing epitopes to all known human viruses or even the entire human epitome have been developed [26], which allow for the detection of all viruses at once. Nonetheless, detection of (the genome of) a pathogen is often preferred, as serological assays often lack diagnostic accuracy because of high cross reactivity and ambiguous cut off criteria.…”

Acute Flaccid Myelitis Surveillance: A Signal Through the Noise

“…Although detection of a pathogen in the CSF would provide definitive proof of causation, in this study and others, no pathogen has been consistently identified in CSF from AFM cases, even with the use of highly sensitive unbiased research technologies for pathogen detection and discovery. 1,19 Development of intrathecal enterovirus antibody tests for CSF may further facilitate diagnosis of enterovirus-associated AFM, 20 even when the virus is no longer present in CSF, similar to diagnosis of neuroinvasive arboviral infections. Additionally, improving AFM awareness to ensure early diagnosis with prompt and complete specimen collection (including respiratory sampling in addition to CSF and stool) will improve the potential for pathogen detection, as evidenced by increased detection rates from specimens collected within 5 days of prodromal symptom onset.…”