Amsterdam UMC, Laboratory of Experimental Virology, Department of Medical Microbiology and Infection Prevention, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
Amsterdam Institute for Infection and Immunity, Postbus 22660, 1100 DD, Amsterdam, The Netherlands.
Fluids Barriers CNS. 2022 Dec 22;19(1):102. doi: 10.1186/s12987-022-00400-5.
In patients with central nervous system (CNS) infections identification of the causative pathogen is important for treatment. Metagenomic next-generation sequencing techniques are increasingly being applied to identify causes of CNS infections, as they can detect any pathogen nucleic acid sequences present. Viromic techniques that enrich samples for virus particles prior to sequencing may simultaneously enrich ribosomes from bacterial pathogens, which are similar in size to small viruses.
We studied the performance of a viromic library preparation technique (VIDISCA) combined with low-depth IonTorrent sequencing (median ~ 25,000 reads per sample) for detection of ribosomal RNA from common pathogens, analyzing 89 cerebrospinal fluid samples from patients with culture proven bacterial meningitis.
Sensitivity and specificity to Streptococcus pneumoniae (n = 24) before and after optimizing threshold parameters were 79% and 52%, then 88% and 90%. Corresponding values for Neisseria meningitidis (n = 22) were 73% and 93%, then 67% and 100%, Listeria monocytogenes (n = 24) 21% and 100%, then 27% and 100%, and Haemophilus influenzae (n = 18) 56% and 100%, then 71% and 100%. A higher total sequencing depth, no antibiotic treatment prior to lumbar puncture, increased disease severity, and higher c-reactive protein levels were associated with pathogen detection.
We provide proof of principle that a viromic approach can be used to correctly identify bacterial ribosomal RNA in patients with bacterial meningitis. Further work should focus on increasing assay sensitivity, especially for problematic species (e.g. L. monocytogenes), as well as profiling additional pathogens. The technique is most suited to research settings and examination of idiopathic cases, rather than an acute clinical setting.
在中枢神经系统（CNS）感染患者中，确定病原体对于治疗非常重要。元基因组下一代测序技术越来越多地用于识别 CNS 感染的原因，因为它们可以检测到存在的任何病原体核酸序列。在测序之前富集用于病毒颗粒的病毒组技术可能同时富集与小病毒大小相似的细菌病原体的核糖体。
我们研究了一种病毒组文库制备技术（VIDISCA）与低深度 IonTorrent 测序（中位数~25,000 个读段/每个样本）相结合，用于检测经培养证实的细菌性脑膜炎患者的 89 例脑脊液样本中常见病原体的核糖体 RNA。
在优化阈值参数之前和之后，针对肺炎链球菌（n=24）的灵敏度和特异性分别为 79%和 52%，然后为 88%和 90%。对于脑膜炎奈瑟菌（n=22）的相应值分别为 73%和 93%，然后为 67%和 100%，李斯特菌（n=24）分别为 21%和 100%，然后为 27%和 100%，流感嗜血杆菌（n=18）分别为 56%和 100%，然后为 71%和 100%。更高的总测序深度、腰椎穿刺前无抗生素治疗、疾病严重程度增加和更高的 C 反应蛋白水平与病原体检测相关。
我们提供了一个原理证明，即病毒组方法可用于正确识别细菌性脑膜炎患者的细菌核糖体 RNA。进一步的工作应集中在提高检测灵敏度上，特别是对于有问题的物种（例如李斯特菌），以及对其他病原体进行分析。该技术最适合研究环境和检查特发性病例，而不是急性临床环境。
