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Next-generation sequencing in the diagnosis of viral encephalitis: sensitivity and clinical limitations
Scientific Reports ( IF 3.8 ) Pub Date : 2020-09-30 , DOI: 10.1038/s41598-020-73156-3
Karol Perlejewski 1 , Iwona Bukowska-Ośko 1 , Małgorzata Rydzanicz 2 , Agnieszka Pawełczyk 1 , Kamila Caraballo Cortѐs 1 , Sylwia Osuch 1 , Marcin Paciorek 3 , Tomasz Dzieciątkowski 4 , Marek Radkowski 1 , Tomasz Laskus 3
Affiliation  

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 105 to 102 and from 105 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.



中文翻译:

病毒性脑炎诊断中的新一代测序:敏感性和临床局限性

鉴定引起病毒性脑炎的病原体仍然具有挑战性,并且在超过 50% 的病例中,病因因素仍未确定。基于新一代测序 (NGS) 的宏基因组学已成功用于检测新的和罕见的感染,但其对脑炎常规诊断的价值仍不清楚。本研究的目的是确定鸟枪法宏基因组测序方案的敏感性,包括预扩增,并针对脑炎患者的脑脊液 (CSF) 样本进行测试。对于敏感性测试,HIV 和 HBV 阳性血清在来自未感染患者的 CSF 中连续稀释。NGS 反复检测到 HIV 和 HBV 序列,浓度范围为 10 5至 10 2和 10 5分别为 10 个病毒拷贝/反应。然而,当对来自 6 名肠道病毒性脑炎患者(中位病毒载量 47 拷贝/毫升)和 15 名 HSV、CMV 或 VZV 脑炎患者(中位病毒载量 148 拷贝/毫升)的 RT-PCR/PCR 阳性 CSF 样本应用相同的方案时),只有 7 个 (28.6%) 被确定为阳性。总之,虽然 NGS 的优势在于能够识别范围广泛的潜在病原体,但与诊断脑炎的标准扩增检测相比,它似乎不太敏感,脑炎的低病毒载量很常见。

更新日期:2020-09-30
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