Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing.

Objective: Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods: We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results: The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion: Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

A Specific Pattern of Routine Cerebrospinal Fluid Parameters Might Help to Identify Cases of West Nile Virus Neuroinvasive Disease.

BACKGROUND: Viral meningitis/encephalitis (ME) is a rare but potentially harmful disease. The prompt identification of the respective virus is important to guide not only treatment but also potential public health countermeasures. However, in about 40% of cases, no virus is identified despite an extensive diagnostic workup. The aim of the present study was to analyze demographic, seasonal, and routine cerebrospinal fluid (CSF) parameters in cases of viral ME and assess their utility for the prediction of the causative virus. METHODS: Demographic data, season, and routine CSF parameters (total leucocytes, CSF cell differentiation, age-adjusted CSF/serum albumin ratio, and total immunoglobulin ratios) were retrospectively assessed in cases of viral ME. RESULTS: In total, 156 cases of acute viral ME (74 female, median age 40.0 years) were treated at a tertiary-care hospital in Germany. Specific viral infections were detected in 93 (59.6%) cases. Of these, 14 (9.0%) cases were caused by herpes simplex virus (HSV), 36 (23.1%) by varicella-zoster virus (VZV), 27 (17.3%) by enteroviruses, 9 (5.8%) by West Nile virus (WNV), and 7 (4.5%) by other specific viruses. Additionally, 64 (41.0%) cases of ME of unknown viral etiology were diagnosed. Cases of WNV ME were older, predominantly male, showed a severe disruption of the blood-CSF-barrier, a high proportion of neutrophils in CSF, and an intrathecal total immunoglobulin M synthesis in the first CSF sample. In a multinominal logistic regression analysis, the accuracy of these CSF parameters together with age and seasonality was best for the prediction of WNV (87.5%), followed by unknown viral etiology (66.7%), VZV (61.8%), and enteroviruses (51.9%). CONCLUSIONS: Cases with WNV ME showed a specific pattern of routine CSF parameters and demographic data that allowed for their identification with good accuracy. These findings might help to guide the diagnostic workup in cases with viral ME, in particular allowing the timely identification of cases with ME due to WNV.

Blood and CSF findings of cellular immunity in anti-NMDAR encephalitis.

OBJECTIVES: To investigate the immunopathogenic mechanisms of anti-N-methyl-D-aspartate receptor encephalitis (NMDAR-E) by characterizing the changes of immune cells in both peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with NMDAR-E. METHODS: Cytology and flow cytometry were used to explore and compare different immunological parameters in PB and CSF of patients with NMDAR-E, viral encephalitis (VE) and healthy volunteers. Moreover, different models were established to assess the possibility of identifying NMDAR-E patients based on PB and CSF parameters. RESULTS: The neutrophil counts and monocyte-to-lymphocyte ratios (MLR) in PB are higher in NMDAR-E patients than in both VEs and controls (P < 0.001, respectively), while the percentages of CD3 + T, CD4 + T lymphocytes, and the leukocytes count in CSF were lower in NMDAR-Es than in VEs (P < 0.01, respectively). The higher percentages of CD8 + T cells in blood and CSF were both correlated with more severe NMDAR-E (P < 0.05, respectively). The poor neurological status group had significantly higher PB leukocytes but lower CSF leukocyte count (P < 0.05). Longitudinal observations in patients with NMDAR-E showed a decreasing trend of leukocyte count, neutrophils count, neutrophil-to-monocyte ratios (NMR), and neutrophil-to-lymphocyte ratios (NLR) with the gradual recovery of neurological function. CONCLUSIONS: The expression patterns of T lymphocyte subsets were different in patients with NMDAR-E and viral encephalitis. The changing trends of leukocyte and lymphocyte populations in peripheral blood and cerebrospinal fluid may provide clues for the diagnosis of different types of encephalitides, including NMDARE, and can be used as immunological markers to assess and predict the prognosis.

The incidence and risk factors of secondary epilepsy after viral encephalitis in children: A 10-year single-center retrospective analysis.

Secondary epilepsy is a common concomitant disease of viral encephalitis (VE) in children. However, the risk factors for secondary epilepsy after VE remain debated. The aim of this study was to perform a 10-year single-center retrospective analysis to investigate the incidence and risk factors of secondary epilepsy after VE in children. A total of 8691 patients suffered from VE in our hospital between December 2011 and February 2022 were included. The patients were divided into control group (Group C) and epilepsy group (Group E) according to whether they followed secondary epilepsy. Information about treatment process was collected from medical records to determine the incidence. Univariate analysis and multivariate logistic regression analysis were performed to identify the independent risk factors. In the current study, the occurrence of secondary epilepsy after VE in pediatric patients was 10.99% (385 of 3503). The results of univariate and multivariate analysis showed that unconsciousness, convulsions, times of epilepsy >2, epileptiform discharge of Electroencephalogram (EEG), and cortical and subcortical damage of magnetic resonance imaging/computer tomography were the significant risk factors for secondary epilepsy after VE. Nearly one tenth of pediatric patients suffered from secondary epilepsy after VE. Interventions for identified risk factors should be used to prevent the occurrence of secondary epilepsy.

Current Advances in Japanese Encephalitis Virus Drug Development.

Japanese encephalitis virus (JEV) belongs to the Flaviviridae family and is a representative mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. Despite the availability of vaccines, JEV remains a major public health threat with the potential to spread globally. According to the World Health Organization (WHO), there are an estimated 69,000 cases of JE each year, and this figure is probably an underestimate. The majority of JE victims are children in endemic areas, and almost half of the surviving patients have motor or cognitive sequelae. Thus, the absence of a clinically approved drug for the treatment of JE defines an urgent medical need. Recently, several promising and potential drug candidates were reported through drug repurposing studies, high-throughput drug library screening, and de novo design. This review focuses on the historical aspects of JEV, the biology of JEV replication, targets for therapeutic strategies, a target product profile, and drug development initiatives.

Multiplex Serology for Sensitive and Specific Flavivirus IgG Detection: Addition of Envelope Protein Domain III to NS1 Increases Sensitivity for Tick-Borne Encephalitis Virus IgG Detection.

Tick-borne encephalitis is a vaccine-preventable disease of concern for public health in large parts of Europe, with EU notification rates increasing since 2018. It is caused by the orthoflavivirus tick-borne encephalitis virus (TBEV) and a diagnosis of infection is mainly based on serology due to its short viremic phase, often before symptom onset. The interpretation of TBEV serology is hampered by a history of orthoflavivirus vaccination and by previous infections with related orthoflaviviruses. Here, we sought to improve TBEV sero-diagnostics using an antigen combination of in-house expressed NS1 and EDIII in a multiplex, low-specimen-volume set-up for the detection of immune responses to TBEV and other clinically important orthoflaviviruses (i.e., West Nile virus, dengue virus, Japanese encephalitis virus, Usutu virus and Zika virus). We show that the combined use of NS1 and EDIII results in both a specific and sensitive test for the detection of TBEV IgG for patient diagnostics, vaccination responses and in seroprevalence studies. This novel approach potentially allows for a low volume-based, simultaneous analysis of IgG responses to a range of orthoflaviviruses with overlapping geographic circulations and clinical manifestations.

Immune-mediated encephalitis following SARS-CoV-2 vaccinations.

OBJECTIVES: one of the neurological side effects of SARS-CoV-2 vaccinations is immune encephalitis. This review aims at summarising previous and current findings on the frequency, clinical presentation, diagnosis, treatment, and outcome of SARS-CoV-2 vaccination-associated encephalitis (SC2VIE). METHODS: narrative review of eligible articles meeting defined search criteria and published between January 2021 and January 2024. RESULTS: A total of 21 patients with SC2VIE reported in 18 articles were included. The AstraZeneca vaccine (ChAdOx1) was the trigger in 10 cases, the Biontech Pfizer vaccine (BNT162b2) in 8 cases, and the Moderna (mRNA1273), CoronaVac, and Sinopharm vaccine (BBIBP-CorV) in one case each. The ages ranged from 21 to 82 years. Twelve patients were female. SC2VIE developed after the first dose in eight patients, after the second in six patients, and in two after the third dose. The latency between vaccination and onset of clinical manifestations ranged from 1 to 56d. Eighteen patients received steroids, one patient intravenous immunoglobulins, one patient plasmapheresis, and two patients rituximab. Complete recovery was achieved in nine patients and incomplete recovery in ten. CONCLUSIONS: SC2VIE is not an uncommon complication of SARS-CoV-2 vaccinations. The clinical presentation and treatment of SC2VIE do not differ from those of autoimmune encephalitis of other causes. Since SC2VIE can manifest only as a psychiatric disease, patients with post-SARS-CoV-2 vaccination psychosis should be evaluated for SC2VIA. The outcome of SC2VIE depends largely on the severity of the immune response and comorbidities.

Elevated levels of ß2-microglobulin in cerebrospinal fluid in adult patients with viral encephalitis/meningitis.

BACKGROUND: Increased cerebrospinal fluid (CSF) ß2-microglobulin (ß2-MG) values are attributed to immune activation, lymphoid cell turnover and release of tissue destruction in the central nervous system (CNS). We investigated plasma and CSF ß2-MG levels in adult patients with viral encephalitis/meningitis and their correlations with clinical parameters. METHOD: CSF samples from 26 patients with viral encephalitis/meningitis were collected. Moreover, 24 CSF samples from patients with non-inflammatory neurological disorders (NIND) as controls were collected. Plasma samples from 22 enrolled patients and 20 healthy individuals were collected. The ß2-MG levels were measured by immunoturbidimetry on an automatic biochemical analyzer. Clinical data were extracted from an electronic patient documentation system. RESULT: CSF levels of ß2-MG, adenosine deaminase (ADA), white blood cell (WBC), lactate dehydrogenase (LDH), protein and lactate were significantly increased in patients with viral encephalitis/meningitis respectively (p < 0.001, p < 0.001, p < 0.001, p = 0.001, p < 0.001, p = 0.013). In contrast, no statistically significant difference was found in plasma levels of ß2-MG. Furthermore, CSF levels of ß2-MG were weakly correlated with WBC (r = 0.426, p = 0.030), lymphocyte percentage (r = 0.599, p = 0.018), ADA (r = 0.545, p = 0.004) and LDH (r = 0.414, p = 0.036), but not with lactate (r = 0.381, p = 0.055), protein (r = 0.179, p = 0.381) and plasma levels of ß2-MG (r = -0.156, p = 0.537) in viral encephalitis/meningitis patients. CONCLUSION: CSF ß2-MG may be a potential inflammatory marker for viral encephalitis/meningitis in adult patients diagnosed with viral encephalitis/meningitis.

Metagenomic Next-Generation Sequencing (mNGS) of cerebrospinal fluid for diagnosis of human herpesvirus 6B encephalitis following transplantation for severe aplastic anemia.

INTRODUCTION: Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation sequencing (mNGS) may facilitate early diagnosis of HHV-6B encephalitis. Herein, we described a case of HHV-6B encephalitis following transplantation for severe aplastic anemia (SAA) diagnosed by mNGS. CASE SUMMARY: A 31-year-old male underwent myeloablative haploid hematopoietic stem cell transplantation for the treatment of SAA. On day + 21 after transplantation, the patient developed symptoms such as sudden epilepsy, drowsiness, memory dislocation, and memory loss. HHV-6B encephalitis was confirmed based on cranial MRI and mNGS of cerebrospinal fluid. Following antiviral therapy with sodium foscarnet, the symptoms improved and HHV-6B was negative by mNGS. There were no serious sequelae. Currently, the patient is in good health and is still under follow-up. CONCLUSIONS: A case of HHV-6B encephalitis after SAA transplantation was diagnosed by mNGS of cerebrospinal fluid in time and was effectively treated with sodium foscarnet.

Acyclovir-Induced Nephrotoxicity in Adults with Viral Encephalitis.

OBJECTIVE: To determine the frequency of parenteral Acyclovir-induced Acute Kidney Injury (AKI) in patients with viral encephalitis. STUDY DESIGN: Descriptive study. Place and Duration of the Study: Department of Neurology, Liaquat National Hospital, Karachi, from January to December 2021. METHODOLOGY: A total of 89 suspected and proven cases of encephalitis receiving IV Acyclovir were collated. All had extensive medical histories and underwent CSF studies with +/- brain imaging. CSF routine and viral PCR were done. Acyclovir-induced AKI was defined as a rise in serum creatinine of >0.3 mg/dl in 48 h or by ≥1.5 times the baseline value, and its severity was staged into 1 (risk), 2 (injury), and 3 (failure) according to the KDIGO guidelines (Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group, 2012). Patients' variables, including age, gender, presenting features, comorbid conditions, and CSF findings, were divided into two groups, i.e. with and without AKI. RESULTS: This research included 89 patients with a mean age of 48 years. AKI occurred in 34 patients (38.2%). The frequency of AKI with Stage 1 was 24%, Stage 2 was 44%, and Stage 3 was 32%; approximately two-thirds of cases were in Stage 2 and 3 (p >0.05). Five patients (5.6%) from Stage 3, required dialysis. CONCLUSION: AKI is an important adverse effect of parenteral acyclovir, which necessitates its early identification and timely management. Renal function monitoring is essential for patients on Acyclovir treatment as they are at risk for AKI. KEY WORDS: Acyclovir, Acute kidney injury, Viral encephalitis, Creatinine, Kidney Disease Improving Global Outcomes.

Clinical epidemiology, determinants, and outcomes of viral encephalitis in Ghana; a cross-sectional study.

Viral encephalitis is a rare, yet severe neurological disorder. It poses a significant public health threat due to its high morbidity and mortality. Despite the disproportionate burden of the disease in impoverished African countries, the true extent of the problem remains elusive due to the scarcity of accurate diagnostic methods. The absence of timely and effective diagnostic tools, particularly Real-time Polymerase Chain Reaction, has led to misguided treatment, and an underestimation of the disease burden in Ghana. We conducted a prospective cross-sectional study to determine the viral aetiologies of encephalitis among patients presenting to a major referral hospital in Ghana from May 2019 and August 2022. The study aimed at providing a comprehensive information on the clinical epidemiology, and outcomes of viral encephalitis in Ghana. Clinical samples were collected from patients presenting with signs and symptoms of encephalitis and tested for viral agents using real-time polymerase chain reaction. We assessed the clinical epidemiology, risk factors and outcome of individuals using descriptive and logistic regression analysis. Seventy-seven (77) patients were enrolled unto the study. The participants frequently presented with fever (85.7%), seizures (80.5%), lethargy (64.9%) and headache (50.6%). Viruses were detected in 40.3% of the study participants in either cerebrospinal fluid, rectal or oral swab samples. The most frequently detected viruses were cytomegalovirus (48.4%), enteroviruses (38.7%) and HSV (29.0%). Twenty-one (27.3%) of the patients died while on hospital admission. Gender (OR = 5.70 (1.536-1.172), p = 0.01), and negative polymerase chain reaction test results were identified as significant factors associated with death. Antiviral treatment increased the chance of survival of viral encephalitis patients by 21.8%. Our results validate the crucial role of molecular tools as essential for the rapid diagnosis of viral encephalitis, enabling effective treatment and improved patient outcomes. This study contributes valuable epidemiological and clinical insight into viral encephalitis in Ghana.

Effects of adjunctive Chinese patent medicine on outcome of viral encephalitis in children: A multicenter retrospective study in China.

BACKGROUND: Some patients with viral encephalitis in China seek treatment with Chinese patent medicine (CPM) to improve their symptoms, but few studies have focused on the impact of CPM on the prognosis of viral encephalitis (VE). The aim of this multicenter retrospective study was to assess the benefit of adjunctive CPM therapy on the outcome of children with VE in China. METHODS: This study retrospectively included 834 children with viral encephalitis who were hospitalized at five medical institutions from 2018 to 2021. Univariate and multivariate logistic regression was used to assess the effect of CPM on sequelae in patients with VE. 1:1 propensity score matching was used to exclude the effect of confounding factors. Forest plots were used to observe the effect of CPM on the prognosis of VE in different subgroups. RESULTS: There were fewer patients with sequelae in the group of patients using CPM regardless of whether they were matched or not. The results of multivariate logistic regression analysis showed that the use of CPM was an independent protective factor for the development of sequelae in VE patients (OR = 0.063, 95 % CI: 0.011-0.350, p = 0.002). Subgroup analyses showed that CPM was a protective factor for the development of sequelae regardless of the presence or absence of coma and comorbidities. In addition, we evaluated other outcome indicators and found shorter duration of illness, fever and headache in children with EV in the CPM group. CONCLUSION: Adjunctive CPM therapy may significantly reduce sequelae in children with VE, as well as effectively alleviate patients' clinical symptoms. However, more prospective studies and clinical trials are needed to further evaluate its efficacy and safety.

Tick-Borne Encephalitis, Lombardy, Italy.

Tick-borne encephalitis was limited to northeast portions of Italy. We report in Lombardy, a populous region in the northwest, a chamois displaying clinical signs of tickborne encephalitis virus that had multiple virus-positive ticks attached, as well as a symptomatic man. Further, we show serologic evidence of viral circulation in the area.

A nationwide survey of adenovirus-associated encephalitis/encephalopathy in Japan.

BACKGROUND: Adenovirus is a major pathogen causing febrile illness among children. It may also cause acute encephalitis/encephalopathy. This study aimed to elucidate the clinical features of adenovirus-associated encephalitis/encephalopathy (AdVE) among children in Japan. METHODS: A nationwide survey of children with AdVE was conducted. An initial survey was distributed among pediatricians to obtain information about children with AdVE treated between January 2014 and March 2019. A second survey was used to obtain the clinical information of children with AdVE from hospitals that responded to the initial survey and those identified from a literature search of the reported cases. We collected demographic data and information about symptoms of infection, neurological symptoms, laboratory parameters, treatment, and outcomes. Outcomes were determined using the Pediatric Cerebral Performance Category Score. RESULTS: Clinical information was available for 23 children with a median age of 39 months. Two had preexisting neurological disorders and six had a history of febrile seizures. The outcome was good in 15 patients and poor in eight patients. Serum lactate dehydrogenase, glucose, and ammonia levels were higher among children with a poor outcome compared to those with a good outcome. Clinically mild encephalitis/encephalopathy with a reversible splenial lesion was the most common type (n = 8), followed by acute encephalopathy with biphasic seizures and late reduced diffusion (n = 7). CONCLUSION: A prior history of febrile seizures was frequent in children with AdVE. Several different subtypes of acute encephalopathy were seen in children with AdVE, and the outcome was poor in those with acute encephalopathy with biphasic seizures and late reduced diffusion and hemorrhagic shock and encephalopathy syndrome. Elevated lactate dehydrogenase, glucose, and ammonia levels on admission were found to correlate with a poor outcome.

A Unique Case of COVID-19 Presenting as Focal Seizures With Impaired Awareness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has very rapidly become a global pandemic with millions of confirmed cases worldwide. In early 2021, viral encephalitis was the first neurological complication associated with COVID-19 and since then rise in cases has been reported with this association. A review highlighting 3 potential mechanisms linking a correlation between seizures and COVID-19 was previously reported. Herein described is a unique case of SARS-CoV2 infection that presented with focal seizure with impaired awareness.

A case of successful hormone therapy for refractory hypotension following viral encephalitis: Case report.

RATIONALE: Refractory hypotension is a life-threatening condition that can result from various causes. We report a rare case of refractory hypotension following herpes simplex virus type 1 encephalitis that was successfully treated with hormone therapy. PATIENT CONCERNS: The patient was a 66-year-old male who was admitted to the hospital because of fever, chills, convulsions, and impaired consciousness. He developed respiratory failure and was intubated. Cerebrospinal fluid metagenomic sequencing confirmed herpes simplex virus type 1 infection. He received piperacillin-tazobactam for anti-infection, acyclovir for antiviral therapy, and dexamethasone for anti-inflammatory therapy. He had repeated episodes of hypotension despite fluid resuscitation and vasopressor therapy. DIAGNOSIS: The diagnosis of herpes simplex virus type 1 encephalitis complicated by refractory hypotension was based on the patient's epidemiological history, clinical manifestations, laboratory tests, and imaging studies. Cerebrospinal fluid examination was the most important diagnostic method, which could detect viral nucleic acids. Head magnetic resonance imaging showed a large recent lesion in the right temporal-parietal and insular lobes. INTERVENTIONS: The treatment of refractory hypotension mainly included anti-infection, antiviral, anti-inflammatory, and hormone therapy. Hormone therapy used methylprednisolone shock treatment until tapering withdrawal. Other treatments included fluid resuscitation, vasopressors, anticonvulsants, etc. OUTCOMES: The patient's blood pressure stabilized after receiving methylprednisolone shock treatment, and his mean arterial pressure increased from 73 mm Hg to 92 mm Hg within 24 hours. Three months later, the patient's blood pressure was normal without medication, and he had a good social and physical recovery. LESSONS: This case illustrates the possible role of hormone therapy in restoring blood pressure in patients with refractory hypotension following viral encephalitis. It suggests that adrenal insufficiency or autonomic dysfunction may be involved in the pathophysiology of this condition. Further studies are needed to confirm the efficacy and safety of hormone therapy in this setting.

Autoimmune GFAP astrocytopathy after viral encephalitis: a case report of bimodal overlapping encephalitis.

Autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy is a treatable autoimmune disorder affecting the central nervous system. Despite extensive research, the exact etiology and pathogenesis of this condition remain unclear. In recent years, autoimmune encephalitis (AE) after viral encephalitis (VE) has gathered significant attention. Here, we present a case report of autoimmune GFAP astrocytopathy after VE in a 43-year-old Asian male with a history of oral and labial herpes. The patient presented with high-grade fever, headache, urinary retention, unresponsiveness, and apathy. Elevated levels of protein and GFAP-IgG were observed in the cerebrospinal fluid (CSF), and enhanced brain magnetic resonance imaging (MRI) revealed linear enhancement oriented radially to the ventricles. Treatment with intravenous immunoglobulin (IVIG) resulted in symptom relief, reduced lesion enhancement, and decreased protein levels. This case report highlights bimodal encephalitis with no discernible interval between VE and autoimmune GFAP astrocytopathy, which poses diagnostic challenges. Notably, autoimmune GFAP astrocytopathy is a novel form of autoimmune encephalitis, and its treatment lacks sufficient clinical experience. Intriguingly, our patient demonstrated sensitivity to IVIG, a treatment that differed from past reports. Therefore, further exploration of treatment strategies for this condition is warranted.