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.

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.

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.

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.

Evolving etiologies, comorbidities, survival, and costs of care in adult encephalitis.

Encephalitis is a central nervous system disorder, often caused by infectious agents or aberrant immune responses. We investigated causes, comorbidities, costs, and outcomes of encephalitis in a population-based cohort. ICD-10 codes corresponding to encephalitis were used to identify health services records for all adults from 2004 to 2019. Data were cross-validated for identified diagnoses based on laboratory confirmation using univariate and multivariate statistical analyses. We identified persons with a diagnosis of encephalitis and abnormal cerebrospinal fluid (CSF) results (n = 581) in whom viral genome was detected (n = 315) in a population of 3.2 million adults from 2004 to 2019. Viral genome-positive CSF samples included HSV-1 (n = 133), VZV (n = 116), HSV-2 (n = 34), enterovirus (n = 4), EBV (n = 5), and CMV (n = 3) with the remaining viruses included JCV (n = 12) and HHV-6 (n = 1). The mean Charlson Comorbidity Index (2.0) and mortality rate (37.6%) were significantly higher in the CSF viral genome-negative encephalitis group although the mean costs of care were significantly higher for the CSF viral genome-positive group. Cumulative incidence rates showed increased CSF VZV detection in persons with encephalitis, which predominated in persons over 65 years with a higher mean Charlson index. We detected HSV-2 and VZV more frequently in CSF from encephalitis cases with greater material-social deprivation. The mean costs of care were significantly greater for HSV-1 encephalitis group. Encephalitis remains an important cause of neurological disability and death with a viral etiology in 54.2% of affected adults accompanied by substantial costs of care and mortality. Virus-associated encephalitis is evolving with increased VZV detection, especially in older persons.

Metagenomic next-generation sequencing and proteomics analysis in pediatric viral encephalitis and meningitis.

Introduction: Early and accurate identification of pathogens is essential for improved outcomes in patients with viral encephalitis (VE) and/or viral meningitis (VM). Methods: In our research, Metagenomic next-generation sequencing (mNGS) which can identify viral pathogens unbiasedly was performed on RNA and DNA to identify potential pathogens in cerebrospinal fluid (CSF) samples from 50 pediatric patients with suspected VEs and/or VMs. Then we performed proteomics analysis on the 14 HEV-positive CSF samples and another 12 CSF samples from health controls (HCs). A supervised partial least squaresdiscriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) model was performed using proteomics data. Results: Ten viruses in 48% patients were identified and the most common pathogen was human enterovirus (HEV) Echo18. 11 proteins overlapping between the top 20 DEPs in terms of P value and FC and the top 20 proteins in PLS-DA VIP lists were acquired. Discussion: Our result showed mNGS has certain advantages on pathogens identification in VE and VM and our research established a foundation to identify diagnosis biomarker candidates of HEV-positive meningitis based on MS-based proteomics analysis, which could also contribute toward investigating the HEV-specific host response patterns.

A case report of long-delayed diagnosis of pseudorabies virus encephalitis with endophthalmitis: lessons from metagenomic next generation sequencing.

BACKGROUND: Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human. CASE PRESENTATION: We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss. CONCLUSIONS: This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.

Diagnosing viral encephalitis and emerging concepts.

PURPOSE OF REVIEW: This review offers a contemporary clinical approach to the diagnosis of viral encephalitis and discusses recent advances in the field. The neurologic effects of coronaviruses, including COVID-19, as well as management of encephalitis are not covered in this review. RECENT FINDINGS: The diagnostic tools for evaluating patients with viral encephalitis are evolving quickly. Multiplex PCR panels are now in widespread use and allow for rapid pathogen detection and potentially reduce empiric antimicrobial exposure in certain patients, while metagenomic next-generation sequencing holds great promise in diagnosing challenging and rarer causes of viral encephalitis. We also review topical and emerging infections pertinent to neuroinfectious disease practice, including emerging arboviruses, monkeypox virus (mpox), and measles. SUMMARY: Although etiological diagnosis remains challenging in viral encephalitis, recent advances may soon provide the clinician with additional tools. Environmental changes, host factors (such as ubiquitous use of immunosuppression), and societal trends (re-emergence of vaccine preventable diseases) are likely to change the landscape of neurologic infections that are considered and treated in clinical practice.

Development and Validation of a Risk Score to Differentiate Viral and Autoimmune Encephalitis in Adults.

BACKGROUND: Encephalitis represents a challenging condition to diagnose and treat. To assist physicians in considering autoimmune encephalitis (AE) sooner, we developed and validated a risk score. METHODS: The study was conducted as a retrospective cohort of patients with a diagnosis of definite viral encephalitis (VE) and AE from​​ February 2005 to December 2019. Clinically relevant and statistically significant features between cases of AE and VE were explored in a bivariate logistic regression model and results were used to identify variables for inclusion in the risk score. A multivariable logistic model was used to generate risk score values and predict risk for AE. Results were externally validated. RESULTS: A total of 1310 patients were screened. Of the 279 enrolled, 36 patients met criteria for definite AE and 88 criteria for definite VE. Patients with AE compared with VE were more likely to have a subacute to chronic presentation (odds ratio [OR] = 22.36; 95% confidence interval [CI], 2.05-243.7), Charlson comorbidity index <2 (OR = 6.62; 95% CI, 1.05-41.4), psychiatric and/or memory complaints (OR = 203.0; 95% CI, 7.57-5445), and absence of robust inflammation in the cerebrospinal fluid defined as <50 white blood cells/µL and protein <50 mg/dL (OR = 0.06; 95% CI, .005-0.50). Using these 4 variables, patients were classified into 3 risk categories for AE: low (0-1), intermediate (2-3), and high (4). Results were externally validated and the performance of the score achieved an area under the curve of 0.918 (95% CI, .871-.966). DISCUSSION: This risk score allows clinicians to estimate the probability of AE in patients presenting with encephalitis and may assist with earlier diagnosis and treatment.

Inherited IRAK-4 Deficiency in Acute Human Herpesvirus-6 Encephalitis.

Human herpesvirus-6 (HHV-6) infection can rarely cause life-threatening conditions, such as encephalitis, in otherwise healthy children, with unclear pathogenesis. We studied a child who presented with acute HHV-6 encephalitis at the age of 10 months and who was homozygous for a novel missense mutation in IRAK4, encoding interleukin-1 receptor-associated kinase 4, identified by whole-exome sequencing. We tested the damaging impact of this mutation in silico by molecular dynamics simulations and in vitro by biochemical and functional experiments utilizing cell lines and patient's cells. We found that the mutation is severely hypomorphic, impairing both the expression and function of IRAK-4. Patient's leukocytes had barely detectable levels of IRAK-4 and diminished anti-viral immune responses to various stimuli inducing different Toll-like receptors and cytosolic nucleic acid sensors. Overall, these findings suggest that acute HHV-6 encephalitis can result from inborn errors of immunity to virus. This study represents the first report of isolated acute HHV-6 infection causing encephalitis in an inherited primary immunodeficiency, notably autosomal recessive (AR) partial IRAK-4 deficiency, and the first report of AR IRAK-4 deficiency presenting with a severe viral disease, notably HHV-6 encephalitis upon an acute infection, thereby expanding the clinical spectrum of IRAK-4 deficiency.

Comparisons in the changes of clinical characteristics and cerebrospinal fluid cytokine profiles between varicella-zoster virus meningitis/encephalitis and other central nervous system infections. / 水痘-å¸¦çŠ¶ç–±ç–¹ç— æ¯’è„‘è†œç‚Ž/è„‘ç‚Žä¸Žå ¶ä»–ä¸­æž¢ç¥žç»ç³»ç»Ÿæ„ŸæŸ“çš„ä¸´åºŠç‰¹å¾å’Œè„‘è„Šæ¶²ç»†èƒžå› å­æ¯”è¾ƒï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Varicella-zoster virus (VZV) is one of the most common etiologies of viral meningitis/encephalitis. The early clinical manifestations and cerebrospinal fluid (CSF) changes of VZV meningitis/encephalitis lack specificity, and it is easy to be misdiagnosed as other viral encephalitides or tuberculous meningitis. This study aims to investigate whether the clinical characteristics, CSF analysis findings, and CSF cytokine levels could distinguish VZV meningitis/encephalitis from central nervous system (CNS) herpes simplex virus (HSV) and Mycobacterium tuberculosis (MTB) infections. METHODS: The medical records from 157 CNS infections, including 49 HSV (45 HSV-1, 4 HSV-2), 55 VZV, and 53 MTB infections between January 2018 and June 2021 in the Cytology Laboratory, Department of Neurology, Third Xiangya Hospital of Central South University were retrospectively reviewed. The data of 3 groups included demographic characteristics, laboratory results, radiographic findings, and outcomes. The levels of 12 cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, IFN-γ, IFN-α, and TNF-α) in the CSF of 68 patients (13 HSV, 22 VZV, and 33 MTB infection cases) were quantified. Clinical and laboratory data were compared among the 3 groups. RESULTS: The most common clinical manifestations in the 3 groups were fever, headache, vomiting, and neck stiffness. The clinical manifestations of HSV and VZV CNS disease were similar, although fever and altered consciousness were less common in the VZV group than those in the HSV and MTB groups (63.6% vs 87.8% vs 96.2%, P<0.001, and 14.5% vs 26.5% vs 47.2%, P=0.004, respectively). Seven patients (7/55, 12.7%) presented cutaneous zoster in the VZV group. CSF leukocyte count was significantly higher in the VZV group (230×106 cells/mL) and MTB groups (276×106 cells/mL) than that in the HSV group (87×106 cells/mL, P=0.002). CSF protein level was significantly higher in the VZV than that in the HSV group (1 034 mg/L vs 694 mg/L, P=0.011) but lower than that in the MTB group (1 744 mg/L, P<0.001). IL-6 (VZV vs HSV vs MTB: 2 855.93 pg/mL vs 2 128.26 pg/mL vs 354.77 pg/mL, P=0.029) and IL-8 (VZV vs HSV vs MTB: 4 001.46 pg/mL vs 1 578.11 pg/mL vs 1 023.25 pg/mL, P=0.046) levels were significantly different among the 3 groups and were elevated in the VZV group.Post hoc analysis revealed that IL-6 and IL-8 were significantly higher in the VZV group than those in the MTB group (P=0.002 and P=0.035, respectively), but not in the HSV group (P>0.05). CONCLUSIONS: VZV meningitis/encephalitis presents with CSF hypercellularity and proteinemia, challenging the classical view of CSF profiles in viral encephalitis. CSF IL-6 and IL-8 levels are elevated in patients with VZV meningitis/encephalitis, indicating a more intense inflammatory response in these patients.

Detection and Diagnostic Value of Cytokines in Serum of Children with Acute Viral Encephalitis.

Acute viral encephalitis is one of the serious infectious diseases. In order to analyze the diagnostic efficacy of serum procalcitonin (PCT), C-reactive protein (CRP), and S100B protein in acute viral encephalitis, a total of 100 children with acute viral encephalitis from July 2019 to December 2021 are selected and included in the viral encephalitis group. The results show that S100B protein model has high specificity and sensitivity and is simple to operate. It provides new ideas and directions for differential diagnosis, improvement, and optimization of relevant clinical diagnosis and treatment schemes and has high clinical value.

Case report: Novel treatment regimen for enterovirus encephalitis in SCID.

Most non-polio enterovirus infections in immunocompetent individuals are acute and self-limiting in nature; however, infection can be severe, chronic and have devastating outcomes in immunocompromised hosts. Therapeutic strategies have predominantly involved supportive care, with the lack of approved antiviral treatments proving challenging for management. We report a case of an 8-month-old child who presented with severe enterovirus encephalitis following gene therapy for X-linked severe combined immunodeficiency (X-SCID) and who demonstrated clinical and microbiological improvement after a novel regimen of favipiravir, fluoxetine, and high-dose intravenous immunoglobulin (IVIg). The patient presented 6 weeks post-gene therapy with rapid neurological deterioration in the context of incomplete immune reconstitution, with microbiological and radiological evidence confirming enterovirus encephalitis. His neurologic examination stabilised 8 weeks after treatment, and he subsequently demonstrated excellent immune recovery. This is the first case report of combined therapy with favipiravir, fluoxetine, and high-dose IVIg in the context of severe enterovirus encephalitis in an immunocompromised host. This case highlights the importance of considering enterovirus encephalitis in immunocompromised patients presenting with both acute and chronic neurological signs, as well as developmental regression. The demonstrated treatment success and the associated low risk of toxicity warrant further investigation of this therapeutic regimen.

Epstein-Barr virus encephalitis with excessive daytime sleepiness as the main manifestation: Two case reports.

RATIONALE: Excessive daytime sleepiness (EDS) is a clinical manifestation of various disorders. Here, we report 2 cases of EDS related to Epstein-Barr virus (EBV) encephalitis. PATIENT CONCERNS: Both the patients were elderly men. Case 1 presented with EDS with headache and fever. Case 2 was presented with EDS only. The 2 patients slept normally at night without taking sleeping pill. They were able to get up and go to the toilet and eat by themselves during the day, but they almost slept at other times. DIAGNOSIS: After admission, a lumbar puncture was performed to collect the cerebrospinal fluid, and next-generation sequencing showed that EBV infection was detected. Combined with the patient's head magnetic resonance imaging and clinical features, a diagnosis of EBV encephalitis was made. INTERVENTIONS: Both patients received antiviral therapy. OUTCOMES: Case 1 had a rapid improvement in headache and fever and was discharged from the hospital after the symptoms of EDS gradually improved. In case 2, EDS symptoms gradually improved. Two patients were followed up for 3 months after discharge, and the outcome was good. LESSONS: EDS can also be the main clinical manifestation of viral encephalitis, and we should diagnose and identify it early and treat it promptly.

SARS-CoV-2-associated haemorrhagic encephalitis mimicking Herpes encephalitis.

Although acute encephalopathy is quite commonly seen in patients of SARS-CoV-2 infection, encephalitis characterised by brain inflammation is relatively rare. Encephalitis caused by Herpes simplex type 1 is the most common cause of identified sporadic encephalitis, and early diagnosis and prompt treatment can prevent the devastating outcome. In this brief communication, we report a case of SARS-CoV-2 associated haemorrhagic encephalitis mimicking herpes encephalitis. In today's pandemic era, it is especially important to distinguish herpes encephalitis from SARS-CoV-2-associated encephalitis as treatment and prognosis of both the conditions differ greatly. This case highlights the importance of suspecting SARS-CoV-2 infection in a patient presenting with clinical symptoms and brain imaging suggestive of Herpes encephalitis.