Neurological complications of pandemic A(H1N1)2009pdm, postpandemic A(H1N1)v, and seasonal influenza A

Abstract Objectives Not much is known about influenza‐associated neurological complications. We aimed to describe the case series of hospitalized patients who were confirmed with influenza A and presented with neurological symptoms in order to capture the broad spectrum of influenza clinical manifestation and suggest including influenza diagnostic in some neurological conditions. Materials and methods The inclusion criteria were age ≥ 18 and laboratory‐confirmed influenza presenting with neurological symptoms. Influenza‐associated neurological complication was described as a development of neurological symptom with no other origin. The outcomes were classified into 5 categories: 1. recovery with no significant disability; 2. minor disability (able to manage on their own); 3. moderate disability (requiring some help but able to walk without assistance); 4. severe disability (unable to walk without assistance and perform daily activities); 5. death. Results In total, 12 patients (five women and seven men) were enrolled, with age range 18–71 years old. Neurological complications of pandemic A(H1N1)2009pdm influenza developed in seven out of 69 (10.1%) hospitalized patients. The most common neurological complication was encephalopathy. Neurological complications developed in two out of 24 (8.3%) hospitalized patients during postpandemic (H1N1)V period. One patient presented with encephalopathy, another with meningoencephalitis. During the 2018 influenza season, there was one patient who has developed influenza A neurological complications. Overall, two out of 104 (1.9%) influenza A patients developed influenza‐associated neurological complications in 2019. Conclusions Every patient with unexplained neurological symptoms and signs similar to aseptic and septic meningitis/encephalitis has to be tested for influenza virus during epidemics and pandemics.


| INTRODUC TI ON
Influenza is a highly contagious disease caused by RNA viruses of the family Orthomyxoviridae. Although influenza affects the respiratory system and most often causes pulmonary complications, it can also cause a variety of neurological complications which are severe enough to require hospitalization. All types of influenza, including seasonal A(H 1 N 1 ) and pandemic A(H 1 N 1 )2009pdm influenza, can affect central (CNS) and peripheral (PNS) nervous systems (Blut, 2009;Paksu et al., 2018).
Unlike seasonal influenza, pandemic influenza causes more severe illness, including more frequent neurological complications, in older children and young adults, with only several cases among the elderly. In etiological studies of encephalitis, influenza, both A and B, has been identified in about 10% of cases in children and 8.5% of cases in adults (Studahl, 2003). In comparison, herpes simplex virus (HSV) accounts for approximately 13.8%, Epstein-Barr virus (EBV) for 1.36%, and Mycobacterium tuberculosis for 5% of encephalitis cases (Gnann and Whitley, 2017;Granerod et al., 2010;Kumar et al., 2018). Neurological complications of pandemic influenza A (H 1 N 1 )2009pdm have been reported in 6%-10% of pediatric cases (Takkar et al., 2015). Since neurological complications are rare, there is a lack of published studies covering this topic. However, smaller studies and case reports describe such complications as Guillain-Barre syndrome (GBS), transverse myelitis, meningoencephalitis, and, the most troubling complication, encephalopathy or encephalitis, which might rapidly evolve to an acute necrotic encephalitis, causing lesions in thalami and cerebral cortex (Ambrozaitis et al., 2016;Davis, 2010;Rothberg & Haessler, 2010). Severe encephalitis can cause lethargy, coma, or even death of the patient (Ferrari et al., 2009). The pathogenesis of neurological complications of influenza is not fully understood, and diagnostic methods are not standardized.
The aim of this study is to describe the cases of pandemic A(H 1 N 1 )2009pdm, postpandemic (H 1 N 1 ) V, and seasonal influenza A presented with neurological manifestation in order to help capture the wide spectrum of influenza clinical presentation and propose to include influenza diagnostic in some neurological conditions.

Infectious Diseases Center of Vilnius University Hospital Santaros
Klinikos, which is the reference center for adult infectious diseases in Vilnius district. It serves the population of 809,000, which is 27% of the nation's population (Ambrozaitis et al., 2016). In total, 12 patients who presented with neurological influenza complications were included for analysis. The inclusion criteria were age ≥ 18 and laboratory-confirmed influenza manifesting with neurological complications.

| Data collection
The demographic and clinical characteristic of the patients were recorded. The variables for the characterization of the cases were age, sex, traveling history, exposure, underlying conditions, vaccination status, clinical presentations, dates (onset of symptoms, manifestation of first neurological symptom, treatment started), laboratory tests, and outcomes.

| Data availability
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. An influenza-related neurological complication was defined as the emergence of neurological symptom with no explanation of other etiology. Encephalitis was defined as the presence of parenchymatous brain involvement signs such as focal neurological signs, seizures, decreased consciousness, and delirium concomitant with CSF leukocytes >5 × 10 6 /L. Encephalopathy was defined as the presence of signs listed above with normal leukocytes count in CSF. Meningitis was defined as CSF leukocytes >5 × 10 6 /L.

| Classification of outcomes
The outcomes were classified into 5 categories: 1. recovery with no significant disability; 2. minor disability (able to manage on their own); 3. moderate disability (requiring some help, but able to walk without assistance); 4. severe disability (unable to walk without assistance and perform daily activities); 5. death.

| Statistical analysis
Descriptive statistics were used for data analysis. Vilnius Regional Bioethics Committee approved the pandemic A(H 1 N 1 )2009pdm and postpandemic (H 1 N 1 ) V ) study in 2009-2011 protocol; this was deemed as a minimal risk, and a written consent was waived. Since 2018, a study "Etiology and sequelae of infectious encephalitis" has been started in Infectious Diseases Center of Vilnius University Hospital Santaros Klinikos. Vilnius Regional Bioethics Committee approved this study protocol.

| RE SULTS
A total number of 12 patients with neurological complications of influenza A virus infection were included in this study. The majority of the study participants were young (median age: 27, range: 18-71 years old). The baseline study characteristics are summarized in Table 1.
None of the patients were vaccinated with either seasonal or pandemic influenza vaccine, also none of them were vaccinated with pneumococcal vaccine, and they did not receive anti-influenza antiviral therapy  The most common complication was influenza encephalopathy (Cases 1-5, Table 2); other patients were diagnosed with bacterial meningitis (Case 6) and tetraparesis (Case 7). Patients diagnosed with encephalopathy (Cases 1-5, Table 2) were young, from 18 to 27 years old, and most of them had no comorbidities, except one patient with previous head trauma and epilepsy. Four patients out of seven were students. The patients presented with altered consciousness (Glasgow coma scale (GCS) 9-13), which lasted 1-2 days (Cases 1-5, Table 2). One patient developed bacterial meningitis (case 6), the other one delirium and polyneuropathy (case 7).

| Case 6
A 24-year-old patient was hospitalized on day 5 of the disease onset,

| Case 7
A 52-year-old man with a history of diabetes mellitus was admitted to the intensive care unit on the fifth day from the onset of the

| Case 10
A 58-year-old patient presented with fever up to 39°C, headache, analysis showed lymphocytic pleocytosis (320 × 10 6 ), moderate elevation of protein, and normal concentration of glucose; also, blood test showed signs of bacterial infection (Table 2). Nose and throat swabs confirmed influenza A. rRT-PCR assay on CSF for herpes simplex virus (HSV), human herpes virus 6 (HHV-6), HHV-7, HHV-8 DNA was performed. There was no bacterial growth in both the blood and The patient was treated with oseltamivir 150 mg twice a day for 3 days and 75 mg twice a day for 5 days with a short course of intravenous acyclovir, as well as antibiotics such as ceftriaxone and ampicillin. He was transferred to neurosurgery unit for cranioplasty with minor disability.

Neurological manifestations of influenza were first reported in
1918 (Goenka et al., 2014). The incidence of influenza neurological complications has increased after 2009 H 1 N 1 pandemic (Meijer et al., 2016). It has been found that neurological complications appear to 0.21/1,000,000 symptomatic patients per year (Hjalmarsson et al., 2009). A study of influenza-related neurological complications in the USA discovered a higher incidence rate in Asian/Pacific Islands population (12.79/1,000,000) compared to non-Hispanic/white patients (3.09/1,000,000), implying a plausible genetic predisposition (Sellers et al., 2017). Moreover, in Japan, the most commonly identified pathogen causing an acute encephalopathy is influenza virus (Goenka et al., 2014). According to Lee et al., neurological complications ap-pear to be more common in patients with pandemic influenza (6.6%) than those with seasonal influenza (4.5%) (Lee et al., 2011). In total, 59,000 influenza cases were reported during the peak of influenza  (Bengualid & Berger, 2017). The neurological complications of influenza B are considered to be rather mild than severe and less frequent compared to influenza A; for example, influenza B accounts for approximately 10% of influenza-associated encephalopathy (IAE) (Popescu et al., 2017;Surtees & DeSousa, 2006).

This might be due to a slower mutation rate compared to influenza
A. We had no cases of influenza B neurological complications in our study, although in 2018, 176 patients with influenza B were treated.
However, there is a lack of extensive studies on the neurological complications of influenza B (Moon et al., 2013).
In our study, neurological influenza complications developed more often during the pandemic. Young patients (age 18-27, except one 52-year-old patient) were affected. The majority of them (five out of seven) had no comorbidities. The two patients with comorbidities (diabetes mellitus and post-traumatic epilepsy) developed more serious neurological complications. The most common complication was encephalopathy. IAE is initially characterized by altered level of consciousness appearing within a few days of influenza infection (Sellers et al., 2017). A variety of clinical syndromes are associated with IAE, including acute necrotizing encephalopathy, acute encephalopathy with biphasic seizures and late restricted diffusion, and mild encephalopathy with reversible splenial lesion (Ferrari et al., 2009;Hjalmarsson et al., 2009). In our study, patients with encephalopathy presented with short-lasting stupor or coma. All of them have completely recovered.
There is no single mechanism which would explain the origin of neurological complications of influenza. Theories include immune response susceptibility (cytokine storm theory), influenza virus invasion to CNS, postinfectious immune-mediated process, genetic factors, RANBP2 gene mutations (Goenka et al., 2014;Meijer et al., 2016;Sellers et al., 2017;Takkar et al., 2015). According to cytokine storm theory, respiratory viral infection provokes pro-inflammatory cytokines that travel to the brain through blood (Takkar et al., 2015). This theory is supported by significantly elevated levels of serum cytokines IL-6, TNF-alpha, and IL-10 in patients with IAE compared to those with no neurological disorders or not infected with influenza virus. The correlation between the severity of neurological complications and serum levels of pro-inflammatory cytokines also supports the hypothesis of a cytokine driven mechanism (Algahtani & Shirah, 2016;Hasegawa et al., 2011;Kawada et al., 2003;Sellers et al., 2017). Pro-inflammatory cytokines may increase the permeability of hematoencephalic barrier, trigger an injury of vascular endothelium as well as apoptosis of neurons and glia cells, and provoke acute brain edema and necrosis. Prolonged activation of microglia may damage neurons and impair synaptic transmission and structure (Hosseini et al., 2018;Ito et al., 1999).
Initially, influenza viruses do not show a direct tropism to the nervous system. However, virus detection in retina, the olfactory bulb, and trigeminal nerve has been described in animal models and may be favored by the free nerve endings near the influenza-infected epithelial cells in the upper respiratory tract (Cárdenas et al., 2014;Studahl, 2003). Additionally, there are data on influenza virus being found on brain tissue in neuropil, ependymal, Purkinje cells, other neurons, and cerebrospinal fluid (Cárdenas et al., 2014). Tomonaga et al. described that neurotropic strains of influenza A virus may enter the CNS through the hematoencephalic barrier and vascular endothelial cells (Tomonaga, 2004). Viral RNA in brain tissue and CSF implies a direct viral invasion of the CNS (Sellers et al., 2017;Steininger et al., 2003). In our study, the influenza encephalopathy was most likely associated with an increase of pro-inflammatory cytokines in blood, as direct influenza virus-induced neuronal damage would lead to a more severe presentation. The patients presented as early as 2-3 days since the onset of illness and all of them were treated with oseltamivir immediately after hospitalization.
Another theory suggesting that neurological manifestations are secondary to a postinfectious immune-mediated process in the CNS is based on the absence of the virus RNA in the CSF according to studies (Meijer et al., 2016;Sellers et al., 2017). GBS, acute disseminated encephalomyelitis, and transverse myelitis are immune-mediated complications of influenza. Molecular mimicry of influenza virus epitopes and normal human brain antigens is yet to be discovered (Lei et al., 2012;Sellers et al., 2017). A Canadian study demonstrated a lower risk of GBS in individuals vaccinated with trivalent inactivated influenza vaccine compared to unvaccinated individuals (Kwong et al., 2013). We hypothesize that in our study, polyneuropathy was associated with postinfectious immune mechanisms. The neurological complications developed late on the 19th day of illness.
Bacterial co-infection is observed in up to 65% of adult influenza cases (Goenka et al., 2014;Klein et al., 2016 Neither of them were vaccinated against influenza or the most common bacterial meningitis agents, despite the fact that both were at risk for both influenza complications and bacterial meningitis. There is no specific time for the onset of neurological complications as they may arise early within the first week of illness or late after the infection (Ferrari et al., 2009;Goenka et al., 2014;Paksu et al., 2018). In our study, neurological complications developed mostly within the first week. Neurological complications are associated with prolonged hospitalization, and there is a possibility for permanent neurological sequelae such as life-long motor weakness, cognitive defects, or even death (Paksu et al., 2018). The median duration of hospitalization in our study was 10 days (ranging from 3 to 37 days). The influenza meningoencephalitic patients were treated longer at hospital compared to encephalopathic patients (15 versus 5 days). The patient with polyneuropathy was treated for the longest period of 37 days and was discharged with severe residual events, leading to disability and a significant impairment on the quality of his life.
The diagnosis of influenza-related encephalitis remains complicated due to a lack of clear evaluating criteria. At initial presentation, blood, CSF analysis, influenza RNR in nasopharynx samples and CSF, EEG, computed tomography (CT), MRI should be performed to confirm influenza encephalopathy or encephalitis (Ambrozaitis et al., 2016;Meijer et al., 2016). The combination of rapid ELISA-based tests with rRT-PCR-based diagnostic tests is especially recommended during the influenza season for diagnosing (Peteranderl et al., 2016). Up to 50%-55% of patients with IAE present normal CT scans. CT or MRI usually show lesions in the corpus callosum, white matter basal ganglia, and occasionally in the cortical and subcortical regions (Algahtani & Shirah, 2016;Ferrari et al., 2009;Goenka et al., 2014). The limitation of our study is that we did not test CSF for influenza RNR, as this method has not yet been standardized in Lithuania. It will be introduced in the future because of the real possibility of the influenza virus to cause neurological complications.
Influenza is mostly treated with antiviral drugs such as oseltamivir, zanamivir, and rimantadine. Neither of them cross the bloodbrain barrier well (Ambrozaitis et al., 2016). Although the particular mechanism of antiviral therapy effect in the treatment of neurological complications is unclear, it is assumed that antiviral drugs suppress the viral expression leading to a reduction of the inflammatory response (Klein et al., 2016;Kwong et al., 2013;Paksu et al., 2018).

Considering that some influenza A viruses have neurotropic prop-
erties, new antiviral drugs that cross the blood-brain barrier are needed (Davis, 2010;McCullers, 2006).

| CON CLUS IONS
Our study, based on case reports, demonstrates that influenza A neurological complications are more common during pandemics. The most common neurological complication, caused by A(H 1 N 1 )2009pdm virus, was encephalopathy, affecting young patients. We speculate that it was immune mediated-related to cytokine storm. The most severe complication during pandemic was polyneuropathy with tetraparesis. The most common symptoms and signs of postpandemic (H 1 N 1 ) v and seasonal influenza A encephalitis were headache, dizziness, and coordination disorders. We suggest that every patient with unexplained neurological symptoms and signs similar to aseptic and septic meningitis/encephalitis should be tested for influenza virus during epidemics and pandemics. We are calling for more extensive international research on the pathogenesis, laboratory diagnosis, and treatment of influenza neurological complications.

ACK N OWLED G M ENT
We thank Marija Grakalskienė for English Language editing.

CO N FLI C T S O F I NTE R E S T
The authors state no conflict of interest.

AUTH O R CO NTR I B UTI O N
Daiva Radzišauskienė is the main researcher who organized this study, participated in data collection and analysis, and wrote the manuscript. Monika Vitkauskaitė and Karolina Žvinytė wrote the manuscript. Rūta Mameniškienė revised the manuscript and supported with helpful discussions.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1916.