二代测序技术结合常规方法检测脑炎/脑膜炎病原体：
中国三级甲等儿童医院神经中心的临床经验
Next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a Chinese tertiary pediatric neurology center
二代测序技术作为检测病原体的新方法，在不明原因的脑炎/脑膜炎中的应用颇受关注[1]。脑炎是一种严重的中枢神经系统炎症性疾病，累及脑实质。脑膜炎是一种累及脑膜的疾病，可引起头痛、颅内压增高和严重的脑损伤。尽管在脑炎/脑膜炎的诊断和治疗方面取得了显著进展，但许多病例预后仍不令人满意。在过去的几十年里，加州脑炎项目研究表明，超过一半的感染儿童没有获得病原诊断。在1570名脑炎患儿中，近63%的脑炎患者病原体不明[2]。
NGS can be used as a new method for the detection of pathogens, especially for unexplained encephalitis/meningitis[1]. Encephalitis is a serious central nervous system (CNS) inflammatory disease involving the brain parenchyma. Meningitis is a disease involving the meninges, inducing headache, increased intracranial pressure, and severe brain involvement. Despite significant advances in the diagnosis and treatment of encephalitis/meningitis, many cases still have unsatisfactory outcomes. In the past few decades, more than half of infected children in the California Encephalitis Project did not receive a pathogenic diagnosis. Among 1570 children with encephalitis enrolled, nearly 63% of encephalitis patients had unidentified pathogens.2
许多病原体可导致脑炎/脑膜炎，然而，在世界范围内，高达50%的病例的病因不明[3-4]。近年来，高通量DNA测序（HTS），又称二代测序技术（NGS），由于其在病原体检测方面的广泛覆盖，逐渐引起人们的关注[5-8]。目前关于二代测序技术的报道主要是病例报告，还没有关于病原体阳性率的研究发表。本研究结合常规方法检测脑炎/脑膜炎患儿脑脊液病原体，并分析其病原分布。
Many pathogens can cause encephalitis/meningitis, however, the pathogeny of up to 50% of cases worldwide is unknown.[3-4] In recent years, high-throughput DNA sequencing (HTS), also known as next-generation sequencing (NGS), has gradually gained attention because of its wide coverage in pathogen detection.5, 6, 7, 8 Current reports incorporating NGS are mainly case reports, and no studies on the positive rate of pathogens were published. This study will combine NGS with routine methods to detect the pathogens of cerebrospinal fluid in children with encephalitis/meningitis, and will analyze the distribution of these pathogens.
患儿为于2017年2月至2018年8月来自中国不同地区的、在首都儿科研究所附属儿童医院神经内科住院患儿。脑炎/脑膜炎诊断标准是指2013年脑炎共识和北京脑炎协作组的脑炎诊断标准[9]。
We recruited patients from February 2017 to August 2018 from different regions of China; all were inpatients of the Department of Neurology, Children's Hospital affiliated with the Capital Institute of Pediatrics. The diagnostic criteria for encephalitis/meningitis refer to the consensus on the definition of encephalitis in 2013 and the diagnostic criteria for encephalitis in the Beijing encephalitis group.
由滨海华大基因业研究院进行脑脊液NGS检测。
Cerebrospinal fluid NGS was carried out by the Binhai Genomics Institute.
本研究共有153名符合入选标准的脑炎/脑膜炎患儿，其中男性88名，女性65名，平均年龄5.45±3.8岁（0.17-15岁）。在90例患者中，明确的病原体被发现，并且大多数的病原体是肠道病毒。
In our study, there were 153 children with encephalitis/ meningitis who met the enrollment criteria, 88 males and 65 females, with an average age of 5.45±3.8 years (0.17-15 years). In 90 cases clear pathogens were detected, and the most pathogen is enterovirus.
（1）常规方法：153例脑脊液细菌培养，阳性9例，占5.88%。采用肺炎支原体RNA扩增技术检测138例，阳性病例23例，占16.67%。在153例患者中，实时PCR检测肠道病毒42例，占27.45%。
(1) Routine methods: of 153 cases with cerebrospinal fluid bacterial culture, the number of positive cases was 9, accounting for 5.88%. Of 138 cases analyzed by Mycoplasma pneumoniae RNA-constant amplification technology detection, the number of positive cases was 23, accounting for 16.67%. Of 153 cases, the number of cases of enterovirus detected by real-time PCR was 42, accounting for 27.45%.
（2）脑脊液NGS：109例NGS患者中，27例阳性，占24.77%。采用常规方法结合NGS对95例儿童进行检测，共检出阳性病例60例。总阳性率为63.16%，其中NGS阳性率为24.77%，常规方法阳性率为49.02%，如图1所示。
(2) Cerebrospinal fluid NGS: Of 109 cases with NGS, 27 cases were positive, accounting for 24.77%. Routine methods combined with NGS were used in 95 children, resulting in a total of 60 positive cases. The total positive rate was 63.16%, of which the NGS positive rate was 24.77% and the routine method positive rate was 49.02%, as shown in Figure 1.
图1 NGS（二代测序）、SAT（同时扩增和检测）、BC（细菌培养）。
Fig. 1. NGS (next-generation Sequencing), SAT (simultaneous amplification and testing), BC(bacterial culture).
（3）常规方法检测的病原体有：肠道病毒、肺炎支原体、1型单纯疱疹病毒、念珠菌、肺炎链球菌、粪肠球菌、金黄色葡萄球菌、巨细胞病毒、EB病毒共9种。NGS检测阳性病原菌27例（阳性率24.77%，27/109）。NGS检测的病原体有：水痘带状疱疹病毒、人疱疹病毒6a、人疱疹病毒6b、人疱疹病毒7型、单纯疱疹病毒1型、腺病毒、EB病毒、巨细胞病毒、细小病毒B19、单核细胞增生李斯特菌、结核分枝杆菌、肺炎链球菌、流感嗜血杆菌、粪肠球菌、葡萄球菌、金黄色葡萄球菌和念珠菌共有16种病原体。二代测序检测到病原体，但常规方法未检测到：单核细胞李斯特菌、流感嗜血杆菌、结核分枝杆菌、水痘带状疱疹病毒、单纯疱疹病毒6型（A型和B型）、单纯疱疹病毒7型、腺病毒和细小病毒B19型。下一代测序如图2所示。
(3) The pathogens detected by the routine methods were: enterovirus, Mycoplasma pneumoniae, herpes simplex virus type 1, Candida, Streptococcus pneumoniae, Enterococcus faecium, Staphylococcus aureus, cytomegalovirus, and Epstein-Barr virus, a total of 9 types of pathogens. The number of positive pathogens detected by NGS were 27 (positive rate 24.77%, 27/109). The pathogens detected by NGS were: Varicella zoster virus, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus type 7, herpes simplex virus type 1, adenovirus, EB virus, cytomegalovirus, parvovirus B19, Listeria monocytogenes, Mycobacterium tuberculosis, Streptococcus pneumoniae, Haemophilus influenzae, Enterococcus faecium, Staphylococcus aureus, and Candida, a total of 16 types of pathogens. The Next-generation sequencing detected pathogens but not detected by routine methods included: Listeria monocytogenes, Haemophilus influenzae, Mycobacterium tuberculosis, Varicella zoster virus, herpes simplex virus type 6 (Type A and Type B), herpes simplex virus type 7, adenovirus, and parvovirus B19 type. Next-generation sequencing of the two pathogens is shown in Figure 2.
图2 单核细胞李斯特菌和腺病毒的二代测序
Fig. 2. Next-generation sequencing of listeria monocytogenes and adenovirus.
这是我国首次在相对较大的样本中，采用常规方法结合NGS检测脑炎/脑膜炎儿童病原体的前瞻性研究。以常规方法为基础，对儿童脑炎/脑膜炎进行NGS病原学检测。研究表明，常规方法结合NGS可提高阳性检出率，扩大已知病原谱。
This is the first prospective study in China to use NGS combined with routine methods to detect pathogens in children with encephalitis/meningitis in a relative large sample. Based on the routine methods, NGS was used to detect pathogens in children with encephalitis/meningitis. Our research showed that routine methods combined with NGS increased the positive detection rate, and the known-pathogen spectrum was expanded.
NGS具有广泛的病原体覆盖范围，可以同时检测数千种病原体。本研究中，单核细胞李斯特菌、结核分枝杆菌、腺病毒、单纯疱疹病毒6型和人细小病毒B19均为NGS检测，不是由常规方法检出的。其中单核细胞李斯特菌和结核分枝杆菌为胞内细菌，临床细菌培养阳性率不高。NGS可以通过破坏细胞壁分离细胞内的病原体，从而检测核酸片段并提高阳性率。本研究中的病原菌总阳性率高于以往报道的大多数病原菌，并且检测到的病原菌种类多于常规方法。（鉴于某些病原体的发病率较低，本研究中的病毒常规检测不包括一些RNA病毒，如日本脑炎病毒、西尼罗河病毒、风疹病毒和登革热病毒)。与常规方法相比，NGS检测到7种以上的病原菌，为临床精确治疗提供了依据。
NGS has a wide coverage of pathogens and can detect thousands of pathogens at the same time. In this study, Listeria monocytogenes, Mycobacterium tuberculosis, adenovirus, herpes simplex virus type 6, and human parvovirus B19 were all detected by NGS, but not by routine methods. Among these, Listeria monocytogenes and Mycobacterium tuberculosis are intracellular bacteria, and the positive rate of clinical bacterial culture is not high. NGS can separate pathogens in the cell by breaking the cell wall, thereby allowing detection of nucleic acid fragments and improving the positive rate. The overall pathogen positive rate in this study was higher than rates of most pathogens previously reported, and more types of pathogens were detected than by routine methods alone. (In view of the low incidence of some pathogens, the routine detection of viruses in this study does not include some RNA viruses, such as Japanese encephalitis virus, West Nile virus, rubella virus, and dengue virus. If these were detected, the pathogen positive rate may have been higher.) Compared with routine methods, NGS has detected more than seven additional pathogens, providing evidence for clinical precision treatment.
与常规方法相比，NGS检测的病原菌阳性率不高。一个可能的原因是常规方法主要针对已知的病原体，这些病原体占了脑炎/脑膜炎儿童感染的大多数。已知最常见的脑炎病原体是肠道病毒，肠道病毒是RNA病毒。本研究中的NGS方法主要用于DNA病毒的检测。
Compared with routine methods, the positive rate of pathogens detected by NGS was not higher. One possible reason is that the routine methods are mainly for known pathogens, which account for the majority of infections in children with encephalitis/meningitis. The most common known pathogen of encephalitis is enteric viruses, and the enterovirus is an RNA virus. The NGS method in this study was mainly for detection of DNA viruses.
研究发现，NGS阳性率低于常规方法，但检出的病原菌数量多于常规方法，因此NGS更适合于不明原因脑炎/脑膜炎的病原诊断。为实现中枢神经系统感染的全病原检测，需要常规方法与一NGS相结合。
The study found that the positive rate of NGS is lower than the rutine methods, but the number of pathogens detected is more than the routine methods, so the NGS is more suitable for the pathogenic diagnosis of unexplained encephalitis/meningitis. To achieve full pathogen detection of CNS infection, routine methods are needed in combination with NGS.
