Aseptic meningitis is the most common CNS infection with cerebrospinal fluid (CSF) negative for bacteria, and EVs, human parechovirus (HPeV), Varicella zoster virus (VZV) and Herpes simplex virus (HSV) 1 and 2 are the most common viral etiologies. [21, 22]. Specifically, EVs are widely recognized as the main causal agent of aseptic meningitis and association is proved by virus genotype from CSF specimen, which occur in both children and adults. [23]. Various studies over several decades have shown children are the primary victims of this disease, and numerous enterovirus meningitis outbreaks associated with EVs have been described [23, 24, 25, 26]. Though, the surveillance system of population-based EVs in mainland China is limited, several related studies of enterovirus meningitis outbreaks have been reported frequently in the provinces of Jiangsu [27], Gansu [28], Anhui [29], Zhejiang [30, 31, 32], Guangdong [8, 33, 34], Yunnan [10], Hebei [35] and Shandong [14, 15, 16, 17, 18, 36, 37, 38] in recent years. These outbreaks were characterized by a large number of hospitalized children, which has become a a pressing issue to public health in current China. Hence, rapid EV identification and genotyping from the CSF samples are critical to investigate the EV circulation and understand the social burden of EV infection.
In this study, we described the molecular epidemiology and enterovirus genotypes from CSF samples of children hospitalized for aseptic meningitis in Liaocheng, China. Our results showed that 107 out of 504 (21.2%) cases of aseptic meningitis were found positive for enteroviruses and 8 different enteroviruses genotypes were responsible for enterovirus meningitis. Several previous studies showed the identification rate of EV types from the CSF ranged from 19.5–54.1% using previously developed assays [39, 40, 41, 42]. The findings in our study are consistent with these studies and the success rate of direct EV typing was low (21.2%). This could be attributed to the low viral load of CSF samples, the high variability of the VP1 region or other virus infection.
The majority of EV cases (53.3%, 57/107) in this study were children aged between three and six, which occurs most frequently in preschool children. Previous studies showed that a higher prevalence of enteroviral meningitis in males vs. females [18, 32]. In our study, the findings are similar with these reports and the male-to-female ratio was found to be 1.97:1. In the present study, 105 EV cases (98.1%, 105/107) were detected in the summer and autumn seasons, which appears with a peak in July. Only 2 cases were detected in spring. Our results revealed that EV infection has typical seasonal feature in temperate climates. This is consistent with other studies showing that most aseptic meningitis occur in summer and autumn, and less frequent in the spring and winter [11, 32].
The clinical signs and symptoms of enteroviral meningitis in children were mostly nonspecific, with fever and headache being the most common symptoms in this study followed by vomiting. Among the 107 patients with enteroviral meningitis, 101 cases (94.4%, 101/107) suffered from fever at the time of hospital admission, which is in congruence with previous studies [43]. Although the EV-positive patients and the negative patients exhibited different clinical symptoms, these symptoms were not enough to distinguish between different etiologies. CSF WBC and protein levels were elevated in most EV-positive cases in this study, and in comparison to the EV-negative groups, significant differences were observed. Whereas, 8 EV cases did not have pleocytosis, which is in congruence with other previous studies [44, 45, 46].
In this study, a total of the 8 enterovirus genotypes were detected by sequencing the VP1 region of the EV genome from 2018 to 2019, which were found to belong to EV species B (E18, E6, E11, CVB5, CVA9, E30, E5 and E20). During the 2-year period, the predominant enterovirus types were E18 (45.8%), E6 (23.8%) and E11 (20.5%), and these are associated most commonly with aseptic meningitis. The enterovirus positive rate was 18.2% in 2018 and 24.9% in 2019, and the proportion of predominant genotype varied significantly between years. We found that E6, E18, and E11 was the primary causative agent in 2018, while only E18 as the dominant pathogen in 2019.
The phylogenetic analysis of VP1 gene in the present study has been widely used for typing EVs and molecular epidemiological investigations. Some EV serotypes (e.g., E6, E11, E18, E30 and CVB5) are still causing global epidemic outbreaks, while others (e.g., CVA9, E5 and E20) are primarily endemic infections [18, 47, 48, 49, 50, 51]. E18 has caused many aseptic meningitis outbreaks in Germany, the United States, Japan, Korea, and other countries or regions [50, 52, 53, 54, 55, 56]. However, in mainland China, E18 meningitis was less frequently reported. In China, E18 encephalitis/meningitis outbreak was first reported in Hebei Province in 2015 [35]. Subsequently, E18-associated aseptic meningitis occurred in Zhejiang province in 2014 and 2017 [32]. Previous studies showed that all E18 strains segregated into three genotypes: A, B, and C. Genotype C could be further divided into subgenotypes C1 and C2 [35]. Genotypes A and subgenotypes C2 had been circulated in mainland China. The subgenotype C2 had a wide geographical distribution and was the absolute dominant subgenotype in mainland China in recent five years, whereas genotype A disappeared after 2005. In this study, all Liaocheng E18 isolates belonged to subgenotype C2. Chen et al. reported that the meningitis outbreak in Hebei Province was caused by a new C2 E18 strain, our study supported this observation. Liaocheng E18 had evolutionally close relationship with the reference E18-314 strain circulating in the neighboring Hebei Province, which suggested that E18-314 formed an exclusive transmission chain in China. As frequent travel or the returning of migrant workers to Hebei Province might increase importation of E18, continuous surveillance is needed.
In China, E6 was first reported to be associated with aseptic meningitis outbreak in 2005 [29]. Then Zhejiang province has also detected E6 meningitis outbreak in 2014 and 2017 [32]. In Shandong, sporadic E6 cases were occasionally observed, and environmental surveillances on sewage had proved that E6 was the predominant serotype in certain years [15, 16, 57, 58]. Furthermore, E6 was one of the predominant types that are responsible for enterovirus meningitis in 2014 [18]. Previous studies showed that all known E6 strains divided into three genotypes-A, B and C. Genotype C could be subdivided into four subgenotypes C1 to C4 [29]. Phylogenetic analyses showed that the subgenotype C2 became the most frequently detected subgenotype in mainland China after 2014, while subgenotype C4 appeared extinct after 2005. Liaocheng E6 isolates belonged to C2 subgenotype, which was most closely related to the meningitis strains isolated in 2014 in Shandong province, suggesting far less variety and continuous circulation in Shandong province.
Echovirus 11 (E11) is one of the most common cause of meningitis in Russia, America, India, Japan and Israel, but the meningitis outbreak of E11 has not been found in mainland China [59, 60, 61, 62]. Though in China, no E11 meningitis outbreak has been reported, EV surveillance has been performed, and E11 cases with meningitis, AFP (acute flaccid paralysis) and HFMD were observed on sewage or CSF conducted in provinces of Shandong, Fujian, Yunnan, and so on [48, 63, 64, 65]. In this study, phylogenetic analysis revealed that Liaocheng E11 grouped into subgenotype D5. In previous epidemiology study on worldwide E11, genotype A was the predominant genotype in mainland China, and D5 was the predominant subgenotype circulated in American, European and Russia [66]. However, E11 meningitis caused by subgenotype D5 was reported in Fujian Province in China in 2011, and E11 isolated from the HFMD surveillance system in China in 2016–2017 were also particularly classified into subgenotype D5 [48, 65]. In this study, no Liaocheng E11 belonged to genogroup A, and a great number of subgenotype D5 were identified, suggesting meningitis-related E11 circulating in China is mainly composed of isolates of this subgenotype in recent years.
There are some limitations to this study. Firstly, the study was conducted in a tertiary hospital and the number of cases was low. Secondly, the findings are limited by the use of nest RT-PCR performed for the detection of EVs in CSF samples, the viral load of the samples was low and other virus infection has not been involved in our study. In the future, to elucidate further the epidemiology of the pathogens for aseptic meningitis, a prospective multicentric larger studies will be developed and other pathogens such as HPeV, VZV, HSV-1 and HSV-2 should be taken and evaluated.