Center for Infectious Disease Research and Surveillance, Dhulikhel Hospital Kathmandu University Hospital, Dhulikhel, Nepal.
Department of Pharmacology, Kathmandu University School of Medical Sciences, Dhulikhel, Nepal.
BMC Infect Dis. 2024 Jul 25;24(1):734. doi: 10.1186/s12879-024-09628-y.
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.
This study employs metagenomic next-generation-sequencing in the investigation of underlying causative etiologies contributing to acute encephalitis syndrome in Nepal.
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.
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.
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.
在 68-75%的急性脑炎综合征病例中，其病因仍不清楚。在尼泊尔，只对日本脑炎进行检测，而日本脑炎仅占病例的 15%左右。然而，可能有几种病原体，包括可通过疫苗预防的病因，导致急性脑炎，如果能够确定这些病因，可以指导开展预防工作，包括解决疫苗覆盖方面的差距。
本研究采用宏基因组下一代测序技术调查导致尼泊尔急性脑炎综合征的潜在病因。
本研究调查了 90 份日本脑炎阴性的存档脑脊液样本，这些样本是在 2016 年和 2017 年作为国家监测网络的一部分收集的。通过随机化纳入三个年龄组（<5 岁；5-14 岁；>15 岁）。仅提供了一些元数据（年龄和性别）。该研究分两批进行，包括总核酸提取，然后分别进行 DNA 和 RNA 文库制备和 Illumina iSeq100 测序。使用 Chan Zuckerberg-ID 对基因组数据进行解释，并通过聚合酶链反应进行确认。
在两个样本中检测到人类疱疹病毒 2 型和肠病毒 B。这两个结果分别通过 qPCR 和半巢式 PCR 得到确认。大多数其他样本受到病原体丰度低、可能的冻融循环、缺乏过程控制以及相关临床元数据的影响。
通过这项研究，通过宏基因组下一代测序揭示了两种已确定的病因。临床元数据、过程控制、病原体丰度低以及缺乏采集和储存样本的标准程序来保护核酸，可能会阻碍研究并在将鉴定出的靶点与感染相关联时产生歧义。因此，需要制定标准化的样本采集程序，包括过程控制和临床元数据。尽管条件具有挑战性，但本研究强调了 mNGS 在调查病因不明的疾病和指导制定适当的临床管理方案以及在尼泊尔开展暴发调查方面的有用性。
