Characteristics and Mortality Risk Factors of Influenza-Associated Encephalopathy/Encephalitis in Children in a Tertiary Pediatric Hospital in China, 2016-2019

Objective: Seasonal influenza associated neurological complications had high mortality and morbidity rates in children. In this study, we aimed to investigate the clinical characteristics and mortality risk factors in children with influenza-associated encephalopathy. Methods: Retrospectively analyze the clinical data, laboratory tests, and imaging examinations of 68 children diagnosed with influenza-associated encephalopathy from January 2016 to December 2019 at Guangzhou Women and Children’s Medical Center, and the cases were divided into survival and non-survival groups by disease outcome and analyzed between two groups Results: Among the 68 children with influenza-associated encephalopathy, 40 were male, and 28 were female, aged from 3 months to 13 years, of which 66.18% (45/68) were under 5 years old. Pathogenetic tests showed that influenza virus type A accounted for 63.24% (43/68), and influenza virus type B accounted for 36.76% (25/68). Typical brain MRI changes in childhood influenza-associated encephalopathy were bilateral symmetrical lesions of the thalamus, basal ganglia, brainstem, and cerebellum. 68 patients had a mortality rate of 20.59% (14/68), with a significantly higher proportion of fever peak >39°C, Acute Disturbance Of Consciousness (ADOC), and cardiac arrest in the non-survival group than in the survival group (P<0.05). Laboratory tests showed significantly higher Alanine Aminotransferase (ALT), Aspartate Transaminase (AST), Creatinine Kinase (CK), and Lactate Dehydrogenase (LDH), Lactate, C-Reactive Protein (CRP), and CSF protein levels in the non-survival group compared with the survivor (P<0.05), and among them, elevated ALT, AST, LDH, and CSF protein were independent high-risk factors for death from influenza- associated encephalopathy. Conclusions: Children under 5 years of age with influenza are prone to combine neurological complications and have a higher mortality rate. Significant elevations in ALT, AST, LDH, and CSF proteins predict death from influenza- associated encephalopathy in children.


Introduction
Influenza, belonging to the family Orthomyxoviridae, primarily affects the respiratory system and represents one of the most frequent causes of acute upper respiratory tract infections during the winter season [1,2]. Although the infection is usually self-limiting, children have a higher risk of complications [3]. Neurological complication, often defined as Influenza-Associated Encephalitis/Encephalopathy (IAE), is rare but is an important complication of influenza infection with approximatively three-quarters of cases regarding children and leading up to 30% of mortality in children [1,4,5].
For including a variety of acute encephalopathy syndromes and lacking available biomarkers, it is difficult to diagnose or predict the outcome of IAE. Neurological signs/symptoms caused by influenza include mild altered mental status, vertigo, epileptic status, meningitis, and demyelinating disease [6][7][8][9]. Age less than 5 years,

Research Article
Characteristics and Mortality Risk Factors of Influenza-Associated Encephalopathy/Encephalitis in Children in a Tertiary Pediatric Hospital in China, 2016-2019

Definition of neurological complications
The definition of IAE includes the following criteria [10][11][12]: sudden onset of symptoms of influenza, and respiratory specimen positive for an influenza virus; the emergence or development of a neurological symptom in influenza infection, such as convulsion, alteration of consciousness, rapidly progressive coma; the abnormal neuroradiological finding in Neuroimaging, including edema, hemorrhage, and bilateral thalamic lesions, as well as electroencephalography showing a diffuse slowing of the background activity; exclusion of infection from other pathogens or non-infectious diseases such as metabolic disorders, trauma, cerebrovascular disorders, and/or brain tumors.

Data collection
We take the retrospective method in this study. Data on demographics, clinical presentation, laboratory findings, microbiologic and neuroimaging findings, treatment, response to therapy and outcome were collected. Demographic data included age, gender, onset month. Clinical data included fever (peak temperature and time of fever lasting), respiratory symptoms, headache, vomiting, and neurological symptoms (convulsion, ADOC, rapidly progressive coma). Results of routine blood test, the comprehensive metabolic panel (CMP), routine and biochemical test in CSF, and pathogenetic test in blood/ nasal swabs/CSF were collected on admission day as laboratory and microbiologic data. And neuroimaging data included CT and MRI, diagnosed by a senior pediatric radiologist (more than ten years experiences), and were reviewed again for IAE.

Data analyses
All statistical analyses were performed using the SPSS 22.0 software (IBM Corp., Armonk, NY, USA). Categorical data were presented as frequency with the corresponding percentage, and continuous data were presented as median with the Interquartile Range (IQR). The Chi-squared or Fisher exact test was used to compare categorical variables. To determine the independent contribution of each factor to the case outcomes, multiple logistic regression analysis was performed. Two-tailed P-values of <0.05 were considered statistically significant.

Demographics and clinical characteristics
There was a total of 68 patients with IAE in this study, 40 males and 28 females, aged 3 months to 13 years, of whom 66.18% (45/68) were younger than 5 years old, were not vaccinated against influenza and had no underlying disease. The patients' main clinical manifestations at the time of admission were fever, convulsions, Acute Disturbance of Consciousness (ADOC), and vomiting. IAE occurred mainly in January (7 cases), February (8 cases), March (11 cases), June (9 cases), and December (13 cases). The patients were divided into survival and non-survival groups according to clinical outcome. We observed 20.5% patients (14 cases)

Laboratory findings
In this study, the median blood while cell (WBC) count in laboratory tests was 8.55 (2.4-29.6)×10 9 Table 1. The median CSF protein in the non-survival group and survival group was respectively 1.90g/L (0.12-34.5) and 0.34g/L (0. 12-12.4). The difference between the two groups was statistically significant (P<0.05).

Neuroimaging findings
In this study, except for 4 patients who failed to complete neuroimaging examination due to their unstable condition, the other 64 cases underwent brain CT or MRI. The typical brain MRI in children with IAE could be found in some patients, presenting symmetric high signal in the bilateral thalamus, basal ganglia, medulla oblongata, pontocerebellar and cerebellum as shown in Figure 1. But the other showed various and non-specific manifestations, which may show scattered abnormal signal shadows in the cerebral hemispheres, cerebral edema, and some, did not show apparent abnormalities ( Figure 2).

Treatment
After admission, the patients were routinely monitored and given temperature reduction and anticonvulsive treatment. All patients were treated with oseltamivir, methylprednisolone (10mg/kg.d), and gammaglobulin (1g/kg.d*2d) immediately after diagnosis. Twentyfour patients were ventilated with tracheal intubation due to central respiratory failure, and were given mannitol, concentrated sodium, and nutritional support therapy. After the above treatment, 39.7% (27/

Discussion
Influenza is a common infectious disease worldwide [13]. While mainly invading the respiratory tract, influenza viruses can cause neurological damage in some cases, leading to a series of neurological syndromes. The morbidity and mortality rates of neurological damages due to influenza are high, especially in children [4,14], and The results indicate that children under 5 years of age with influenza are prone to combine neurological complications and have a higher mortality rate, significant elevations in ALT, AST, LDH, and CSF proteins predict death from influenza-associated encephalopathy in children.
It has been previously reported that influenza in children at a younger age or with underlying diseases is more likely to combine neurological complications; especially in children younger than 5 years of age [16][17][18]. In this study, 66.18% (45/68) of the patients were ≤5 years old, which is consistent with the reports. All patients in this study were previously healthy and without underlying diseases. Therefore, it should be given close attention when the onset of neurological symptoms in influenza in healthy children. In this study, we find that the onset of IAE occurred mainly from December to March, which is consistent with the high influenza season. It is currently unknown whether influenza vaccines can prevent IAE or reduce the disease's extent. There is a report showing that patients with IAE were unvaccinated against influenza [19]. In this study, none of the 68 patients were vaccinated against influenza, suggesting the potential importance of influenza vaccine for children, and therefore vaccination of healthy preschool children in the pre-high influenza season is necessary.
Influenza-associated encephalopathy lacks specific clinical manifestations, and convulsions and impaired consciousness have been previously reported as the most common neurological symptoms of influenza-associated encephalopathy [6]. The main clinical manifestations of the cases in this study are fever, convulsions, ADOC, and respiratory symptoms could be mild or even absent. Moreover, the number of convulsions in the non-survival group did not differ from that in the survival group, indicating that the number of convulsions cannot be used to indicate disease severity.
Previous studies have reported that IAE is mainly caused by influenza virus type A, especially H1N1 [2,20,21]. In this study, we detected influenza virus type A at 63.2% (43/68) in the patients and influenza virus type B at 36.8% (25/68), showing that influenza virus type A is the primary infection source of IAE. However, there was no significant difference in the pathogenic distribution between the non-survival and survival groups, indicating that influenza virus type B may be causing relatively minor neurological damage, but leading to death in severe cases.
Some laboratory tests were found to be markedly abnormal in this study, such as reduced platelets, elevated ALT, AST, CK, LDH, and CSF proteins level. The above indicators were more abnormal in the non-survival group than the survival group, which was statistically significant. Furthermore, Logistic regression analysis showed that elevated ALT, AST, LDH, and CSF protein were independent risk     [5,[22][23][24]. Therefore, patients found to have abnormalities of the above indicators in clinical practice should be given intensive care and immediate treatment to minimize death occurrence.
Brain MRI of IAE can show characteristic changes and has an important role in diagnosing and clinical staging of neurological complications of influenza. The typical brain MRI manifestations of children with IAE were previously reported symmetrical abnormalities of bilateral thalamus, basal ganglia area, medulla oblongata, and pons [20,25,26], which is consistent with the present study. The brain MRI of pediatric IAE is specific and contributes to the disease's early diagnosis.
The pathogenesis of IAE is still unclear, and it is generally believed that it may result from immune damage or direct viral invasion. Similar to previous studies reported [19], few patients in this study had elevated CSF leukocytes. At the same time, CSF protein was elevated in this study, and it was significantly higher in the non-survival group compared with the survival group, while logistic analysis showed elevated CSF protein as a high-risk factor for death in IAE. This suggests that neurological injury may be immunemediated, or direct viral invasion, or vascular inflammation, rather than a simple inflammatory response. However, whether influenza viruses can invade the nervous system is controversial, and some studies have reported positive results for viral RNA in CSF and brain tissue in only a few patients [27]. As reported, all patients in this study had negative results for influenza virus RNA on CSF.
There is no standard treatment protocol for IAE, and currently used protocols include antiviral therapy, immunotherapy (pulse therapy with corticosteroids, immunoglobulins), plasma replacement and hypothermia, but it is unclear which protocol is more effective [28][29][30][31]. For oseltamivir inhibiting the viral expression and reducing the inflammation, some studies report that antiviral therapy may be effective, while guidelines of Infectious Diseases Society of America recommend oseltamivir in the presence of IAE [32]. Although pulse therapy with corticosteroids and immunoglobulins remains controversial, it is currently used widely in clinical practice, for considering IAE is related to immune-mediated damage to the nervous system. All cases in this study were given oseltamivir, and pulse therapy with corticosteroids and immunoglobulins after diagnosis of influenza infection, so it was impossible to compare the difference in efficacy between the survival and non-survival groups.
Our study presents several limitations. The present study is a single-center retrospective analysis with a relatively small number of cases and a lack of long-term assessment in children, which has limitations, and more studies are needed to address these issues.

Conclusion
In conclusion, IAE should be considered when neurological symptoms are present for influenza in children under 5 years of age. Elevated ALT, AST, LDH, and CSF proteins are high risk factors for death from IAE. Future larger studies are needed to understand the pathogenesis of IAE, explore biomarkers for assessing neurological involvement and disease severity, and follow the long-term prognosis of the disease.