Next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a Chinese tertiary pediatric neurology center
NGS can be used as a new method for the detection of pathogens, especially for unexplained encephalitis/meningitis. 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
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.
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.
Cerebrospinal fluid NGS was carried out by the Binhai Genomics Institute.
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) 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) 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.
Fig. 1. NGS (next-generation Sequencing), SAT (simultaneous amplification and testing), BC(bacterial culture).
(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.
Fig. 2. Next-generation sequencing of listeria monocytogenes and adenovirus.
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 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.
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.
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.