Investigation of acute encephalitis syndrome with implementation of metagenomic next generation sequencing in Nepal.

BACKGROUND: The causative agents of Acute Encephalitis Syndrome remain unknown in 68-75% of the cases. In Nepal, the cases are tested only for Japanese encephalitis, which constitutes only about 15% of the cases. However, there could be several organisms, including vaccine-preventable etiologies that cause acute encephalitis, when identified could direct public health efforts for prevention, including addressing gaps in vaccine coverage. OBJECTIVES: This study employs metagenomic next-generation-sequencing in the investigation of underlying causative etiologies contributing to acute encephalitis syndrome in Nepal. METHODS: In this study, we investigated 90, Japanese-encephalitis-negative, banked cerebrospinal fluid samples that were collected as part of a national surveillance network in 2016 and 2017. Randomization was done to include three age groups (< 5-years; 5-14-years; >15-years). Only some metadata (age and gender) were available. The investigation was performed in two batches which included total nucleic-acid extraction, followed by individual library preparation (DNA and RNA) and sequencing on Illumina iSeq100. The genomic data were interpreted using Chan Zuckerberg-ID and confirmed with polymerase-chain-reaction. RESULTS: Human-alphaherpes-virus 2 and Enterovirus-B were seen in two samples. These hits were confirmed by qPCR and semi-nested 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 procedures to collect and store samples in nucleic-acid protectants could have impeded the study and incorporated ambiguity while correlating the identified hits to infection. Therefore, there is need of standardized procedures for sample collection, inclusion of process controls and clinical metadata. Despite challenging conditions, this study highlights the usefulness of mNGS to investigate diseases with unknown etiologies and guide development of adequate clinical-management-algorithms and outbreak investigations in Nepal.

Stakeholder Engagement in Planning the Design of a National Needs Assessment for Cardiovascular Disease Prevention and Management in Nepal.

BACKGROUND: There is growing support for stakeholder engagement in health research, but the actual impact of such engagement has not been well established. OBJECTIVES: This paper describes the stakeholder engagement process and evaluation during the planning of the national needs assessment for cardiovascular disease in Nepal. METHODS: We used personal and professional networks to identify relevant stakeholders within the 7Ps framework (Patients and the Public, Providers, Purchasers, Payers, Public Policy Makers and Policy Advocates, Product Makers and the Principal Investigators) to develop a plan for assessing cardiovascular health needs in Nepal. We consulted 40 stakeholders through 2 meetings in small groups and a workshop in a large group to develop the study methods, conceptual framework, and stakeholder engagement process. We interviewed 33 stakeholders to receive feedback on the stakeholder engagement process. RESULTS: We engaged 80% of the targeted stakeholders through small group discussions and a workshop. Three of 5 recommendations from the small group discussion were aimed at improving the stakeholder engagement process and 2 were aimed to improve the research methods. Eleven of 27 recommendations from the workshop aimed to improve the research methods, 4 aimed to improve stakeholder engagement, and 2 helped to expand the scope of dissemination. Ten were irrelevant or could not be incorporated due to resource limitation. Most stakeholders noted that the workshop provided an open platform for a multisectoral group to colearn from one another and share ideas. Others highlighted that the discussion generated insights to enhance research by incorporating expertise and ideas from different perspectives. The major challenges discussed were about committing the time for engagement. CONCLUSIONS: The stakeholder engagement process positively affected the design of our research. This study provides important insights for future researchers that aim to engage stakeholders in national-level assessment programs in the health care system in the context of Nepal.