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

Background Acute 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. Objectives This study employs metagenomic next generation sequencing in the exploration of infectious etiologies contributing to acute encephalitis syndrome in a low-and-middle-income setting. Methods In 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. Results Human 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 abundance of pathogen, possible freeze thaw cycles, lack of process controls and associated clinical metadata. Conclusion From this study, two documented, causative agents were revealed through metagenomic next generation sequencing. Insufficiency of clinical metadata, process controls, low pathogen abundance and absence of standard operating procedure to collect and store samples in nucleic acid protectants could have impeded the study and incorporated ambiguity while correlating the identified hits to infections. Therefore, there is need of implementing standardized collection procedures for sample collection, including proper process controls and clinical metadata. 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by Bill and Melinda Gates Foundation under Grand Challenges. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was ethically cleared from Nepal Health Research Council (NHRC) under id: 903 - 2019. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the author