Investigation of acute encephalitis syndrome with implementation of metagenomic next generation sequencing in a low-middle-income setting

Abstract BackgroundAcute Encephalitis Syndrome is characterised by acute onset of fever and altered sensorium followed by a rapidly worsening clinical condition and even death. The causative agents vary with season and geographic location, while the etiologies remain unknown in 68-75% of the cases. In Nepal, the cases of acute encephalitis syndrome are tested only for Japanese encephalitis, which constitutes about 15% of the cases globally. However, there could be several causative organisms, including vaccine-preventable etiologies that cause acute encephalitis, which upon identification could direct public health efforts for prevention, including expanded use of vaccines or address gaps in vaccine coverage. ObjectiveThis study employs metagenomic next generation sequencing in the exploration of infectious etiologies contributing to acute encephalitis syndrome in a low-and-middle-income setting. MethodsIn this study, we have investigated 90, Japanese encephalitis-negative, banked retrospective cerebrospinal fluid samples that were collected as part of a national surveillance network in 2016 and 2017. The randomisation was done to include three age groups of <5 years, 5-14 years, and >15 years. Only some metadata related to the subjects (age and gender) were known. The analysis was performed in two batches which included total nucleic acid extraction, followed by individual library preparation for DNA and RNA and sequencing on Illumina iSeq100. The generated genomic data were interpreted using CZID and confirmed with polymerase chain reaction. ResultsHuman alphaherpesvirus 2 and Enterovirus B were seen in two of the ninety samples. These hits were confirmed by qPCR and seminested PCR respectively. Most of the other samples were marred by low sample quality, lack of clinical metadata, storage issues, and possible ambiguous collection procedures. ConclusionFrom this study, two documented, causative agents were revealed through metagenomic next generation sequencing. Insufficiency of clinical metadata, process controls, and absence of standard operating procedure to collect and store samples in nucleic acid protectants impeded the study and incorporated ambiguity while correlating the identified hits to infections. Therefore, there is dire need of implementing stringent collection procedures for sample collection, including proper process controls. Despite the challenging conditions, this study highlights the usefulness of mNGS to investigate diseases with unknown etiologies, such as AES, and guide the development of adequate clinical management algorithms and outbreak investigations in low-middle income settings.