Clinical features and influencing prognostic factors in patients with anti-NMDAR encephalitis: a cohort follow-up study in Chinese patients

Objectives The clinical manifestations of patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis in East China and factors associated with prognosis were analyzed. Methods A retrospective study of 106 patients (58 females; 48 males) with anti-NMDAR encephalitis in East China was carried out from June 2015 to February 2019. Clinical features and factors influencing outcomes were reviewed. Results Behavioral changes were observed in 74.5% (79/106) of patients, and comprised the initial symptoms in 61.3% (65/106). Seizures were observed in 67% (71/106) of patients, and served as initial symptoms in 31.1% (33/106). A total of 54.9% (39/71) of seizures were focal seizures. More clinical symptoms were observed in female patients than in male patients ( P =0.000). Similarly, background activity (BA) with cerebrospinal fluid (CSF) antibody titers at the peak stage was more severe in female patients than in male patients ( P = 0.000). The Binary logistic regression and ROC curve analyses revealed the factors associated with poor outcomes included consciousness disturbance (OR: 4.907, 95% CI: 1.653-14.562, P =0.004; area: 65.4%, sensitivity: 44.2%, specificity: 86.5%, P =0.014 ), EEG BA (OR: 3.743, 95% CI: 1.766-7.932, P =0.001; area: 76.6%, sensitivity: 73%, specificity: 75%, P =0.000), number of symptoms (OR: 2.911, 95% CI: 1.811-4.679, P =0.000; area: 77.1%, sensitivity: 59.5%, specificity: 78.6%, P =0.000) and CSF antibody titer (OR: 31.778, 95% CI: 8.891-113.57, P =0.000; area: 83.9%, sensitivity: 89.2%, specificity: 78.6%, P =0.000). independent-samples t test used to compare between and male patients, and between patients with low and high CSF antibody titers, as well as to compare CSF white cell and protein levels between patients with abnormal T2/ fluid-attenuated inversion recovery (FLAIR) image and patients with normal T2/FLAIR image. The binary logistic regression analysis and receiver operating characteristic (ROC) curve were used to analyze the association between age, sex, consciousness disturbance, CSF antibody titers, EEG BA during peak stage, number of symptoms, imaging results, relapse, intensive care unit (ICU) admissions, follow-up period and prognosis in patients with anti-NMDAR encephalitis. P<0.05 indicated statistical significance.

titers served as predictors of poor outcomes.

Introduction
Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disease associated with serum antibodies against functional NMDARs [1,2]. This syndrome usually develops with a sequential presentation of symptoms, including headache and fever, followed by behavioral changes and psychosis. Seizures can occur at any stage but most commonly manifest early [3]. Magnetic resonance imaging (MRI) remains one of the most important examinations for the diagnosis of central nervous system diseases. As reported, normal brain MRI results have been observed in most patients [3][4][5]. Electroencephalography (EEG) may be useful for diagnosing anti-NMDAR encephalitis [6][7][8]. The number of clinical symptoms and electroencephalography (EEG) results were associated with cerebrospinal fluid (CSF) antibody titers [9]. To date, no study has established whether EEG background activity (BA) during the peak stage of anti-NMDAR encephalitis is associated with prognosis. Currently available data are also unclear regarding the associations between CSF antibody titers and outcomes.
The purpose of our study is to provide a summary of the clinical features, and analyze the association between clinical features and prognosis in patients with anti-NMDAR encephalitis.
Patients and methods Patients A total of 106 patients from the Department of Neurology of Nanjing Brain Hospital were included. The inclusion criteria were as follows: (1) patients who fulfilled the anti-NMDAR encephalitis diagnostic criteria with rapid development (disease course < 3 months) of one or more symptoms, including mental/behavioral disorders, cognitive impairment, language impairment, consciousness disturbance, epilepsy, involuntary movement, autonomic nervous system dysfunction, or central hypoventilation; and (2) patients with positive anti-NMDAR IgG in CSF with or without positive anti-NMDAR IgG in serum. Patients were excluded if they are diagnosed with other diseases, such as viral encephalitis, brain tumor, metabolic diseases, and drug poisoning [10].

Patient data
The following patient data were collected: age; sex; presence or absence of malignancy (teratomas and others); neurological symptoms such as seizures and psychiatric symptoms; high (1:10 or 1:32) or low (1:1 or 1:3.2) CSF anti-NMDAR titers; EEG BA including mild diffuse polymorphic slowing (mild DPS), moderate diffuse polymorphic slowing (moderate DPS), severe diffuse polymorphic slowing (SDPS) and extreme delta brush (EDB); MRI; arterial spin labeling (ASL); lumbar puncture; and modified Rankin scale (mRS) scores after 12 to 50 months of immunosuppressive treatment [8]. The initial stage was within 14 days of symptom onset, the peak stage was 14 to 60 days after symptom emergence, the improvement stage was 60 to 180 days after disease onset, and the recovery stage was 180 days after disease onset [11,12].

Statistics
Data input and statistical analyses were performed using the Statistical Package for Social Sciences (IBM Corporation, Armonk, NY, USA). The Mann-Whitney U test was adopted to compare EEG categorical variables at the peak stage between female and male patients and between patients with low and high CSF antibody titers. The independent-samples t test was used to compare symptoms between female and male patients, and between patients with low and high CSF antibody titers, as well as to compare CSF white cell and protein levels between patients with abnormal T2/ fluid-attenuated inversion recovery (FLAIR) image and patients with normal T2/FLAIR image. The binary logistic regression analysis and receiver operating characteristic (ROC) curve were used to analyze the association between age, sex, consciousness disturbance, CSF antibody titers, EEG BA during peak stage, number of symptoms, imaging results, relapse, intensive care unit (ICU) admissions, follow-up period and prognosis in patients with anti-NMDAR encephalitis. P<0.05 indicated statistical significance.

Clinical characteristics
High CSF antibody titers were observed in 60 patients with anti-NMDAR encephalitis and low CSF antibody titers in 37 patients. Ovarian teratomas were observed in 4  Fig.2). During the peak stage of the disease, focal high blood flow with normal MRIs were observed from 56.52% (13/23) ASL of anti-NMDAR encephalitis patients in Table 1. Focal high blood flow with brain lesions were observed in 39.13% (9/23) ASL of patients (  Fig.4 B, D, F and H). The BA during the peak stage in female patients with high CSF antibody titers was more severe than that in female patients with low CSF antibody titers ( Fig.4B VS Fig.4C; Mann-Whitney U test, P = 0.000). BA in those with high CSF antibody titers at the peak stage was more severe in female patients than in male patients ( Fig.4B VS Fig.4F; Mann-Whitney U test, P = 0.000) ( Table 1, Fig.4).

Factor-related prognosis
A total of 103 (97.2%) patients were treated with first-line immunotherapy, 3 (2.8%) with second-line immunotherapy, and 1 with tumor removal and immunotherapy.

Discussion
Our study focused on clinical features and factors related to prognosis in patients with anti-NMDAR encephalitis. The male patients were older than the female patients (P = 0.000) ( Table 1). There was no sex difference in this sample, which is similar to a previous report on adult-onset anti-NMDAR encephalitis in Korea [13].
The prevalence of teratomas in females over 18 years old was 56%, but only 31% in females under 18 years old, and only 9% in females under 14 years old [3]. In short, the younger the female patients, the lower the incidence of teratomas [3]. There were only four female patients with tumors in this study.
Behavioral changes and seizures were the major symptoms. Behavioral changes comprised the initial symptoms in 61.3% (65/106) of the patients with anti-NMDAR encephalitis in this study. Seizures were observed as the onset symptoms in 31.3% (33/106) of the patients. Moreover, 54.9% (39/71) of the seizures were focal (Table   1). Most patients initially presented with behavioral changes and seizures, which aligned with previous findings [14,15]. In an observational cohort study, 87% of patients exhibited 4 or more categories of symptoms by the end of the first month [16].Our study investigated whether clinical symptoms were associated with sex and CSF antibody titers in patients with anti-NMDAR encephalitis. A greater number of clinical symptoms were noted in patients with high CSF antibody titers than in those with low CSF antibody titers (Table 1, Fig.1), which aligned with our previous findings [9]. The peak-stage BA was worse in the female patients with high CSF antibody titers than in those with low CSF antibody titers (Mann-Whitney U test, P = 0.000) (Table 1, Fig.4). The clinical symptoms were more severe in the female patients with high CSF antibody titers than in male patients with high CSF antibody titers (mean: 3.97±0.17 vs 2.33±0.20, respectively, P = 0.000) (Table 1, Fig.1).
Meanwhile, the female patients with high CSF antibody titers showed worse peak stage BA than the male patients with high CSF antibody titers (Mann-Whitney U test, P = 0.000) (Table 1, Fig.4). The constant chewing observed in the female patients with high CSF antibody titers during the peak stage was unlikely to be caused by temporal or frontal seizures because no epileptiform discharges (EDs) were observed in the EEG recordings, and antiepileptic drugs (AEDs) were ineffective [9].
Constant chewing may be a useful marker of the peak period of the disease.
During the peak stage of the disease, brain lesions were observed in 27.45% of the patients (28/102) in this study. Moreover, 13 patients with normal MRIs showed focal high blood flow in Table 1. In several previous studies, 37.2%-50% of patients showed abnormal brain MR imaging results [3][4][5]. On MRI, hyperintensities involving the hippocampus, temporal cortex, insula, parietal cortex, frontal cortex, and white matter (frontal region) were noted in 11.8% (12/102), 12.7% (13/102), 6.9% (7/102), 7.8% (8/102), 3.9% (4/102), and 3.9% (4/102) of the patients in this study, respectively ( Table 1, Fig. 2). In 2017 resting-state functional connectivity in patients with anti-NMDAR encephalitis revealed widespread alterations of functional connectivity correlated with clinical measures [17].These alterations included impaired hippocampal functional connectivity, decoupling of the medial temporal and the default-mode networks, and an overall impairment in frontotemporal connections. Furthermore, functional connectivity was impaired within distributed large-scale networks, including the sensorimotor, frontoparietal, lateral-temporal, and visual networks. Memory impairment correlated with hippocampal and medialtemporal-lobe network connectivity, whereas schizophrenia-like symptoms were associated with functional connectivity changes in frontoparietal networks. Machinelearning analyses corroborated these findings and identified frontoparietal and frontotemporal connections as reliably discriminating features between patients and controls, yielding an overall accuracy of 81% [17]. In a previous study, patients with normal MRIs were younger than patients with abnormal MRIs [18]. There were no statistically significant dfferences in MRI findings (normal or abnormal) between patients with or without tumors, patients experiencing seizures, patients experiencing hypoventilation, or patients experiencing loss of consciousness.
Abnormal MRIs did not affect prognosis evaluated by mRS [18]. In this study, CSF white cells in the patients with abnormal T2/FLAIR were higher than those in the patients with normal T2/FLAIR (mean: 89.63±29.53 vs 18.44±7.42, respectively, P = 0.000). Similarly, there were higher CSF protein levels in patients with abnormal T2/FLAIR than in patients with normal T2/FLAIR (mean: 0.63±0.06 vs 0.4±0.03, respectively, P = 0.002).
In a large unselected cohort of adults, a normal posterior rhythm in the first EEG recording at a median of 19 days from disease onset predicted a favorable clinical outcome, while a severely abnormal EEG was associated with a poor outcome [19].
Ordinal logistic regression showed that the presence of a normal posterior rhythm was associated with lower mRS at final follow-up [19]. Binary logistic regression analysis and ROC curve analysis showed that EEG BA during the peak clinical state (median: 19.5 days) was associated with poor outcomes in this study. The number of symptoms and CSF antibody were also predictors of poor outcomes (  Fig.5). A greater number of clinical symptoms were noted in patients with high CSF antibody titers than in those with low CSF antibody titers (Table 1, Fig. 1). The main epitope targeted by the antibodies is in the extracellular N-terminal domain of the NR1 subunit. Patients' antibodies decreased the numbers of cell-surface NMDA receptors and NMDA-receptor clusters in postsynaptic dendrites, an effect that could be reversed by antibody removal. The severity of anti-NMDAR encephalitis was associated with antibody titers [5]. Furthermore, consciousness disturbance was a factor associated with nonfavorable outcomes in this study ( Table 2 and Fig.   5), which was similar to a previous study [20]. In previous studies, the predictors of good outcomes were early treatment and lack of ICU admission [4]. In our study, 11 patients were admitted to the ICU. ICU stay was not a predictor of poor outcomes.
Many patients are not admitted to the ICU due to the shortage of ICU beds. All patients admitted to the ICU had poor outcomes (mRS≥2). Relapses were defined as the new onset of symptoms, or worsening of symptoms after at least 2 months of improvement or stabilization. Although relapses were not a predictor of poor outcomes in our study, all patients with relapse had poor outcomes (mRS≥2). The follow-up period may have been short in our study. Titulaer et al. followed their anti-NMDAR encephalitis patients for a median duration of 24 months and 7.8% of patients in that study experienced one or more clinical relapses [4].

Conclusion
EEG BA and the number of symptoms were associated with CSF antibody titers. CSF white cell and protein levels in patients with abnormal T2/FLAIR were higher than in patients with normal T2/FLAIR. Consciousness disturbance, EEG BA, number of symptoms and CSF antibody titers served as predictors of poor outcomes.

Availability of Data and Materials
Not applicable

Acknowledgements
We would like to acknowledge the physicians and nurses at Department of Neurology, Nanjing Brain Hospital.

Ethics approval and consent to participate
Our study was reviewed and approved by the ethical boards of the Affiliated Brain   One hundred and fourteen EEG or VEEG recordings were obtained from 92 patients. EEG BA w Figure 5 In the ROC curve analysis, predictors for poor outcomes included consciousness disturbance