Evaluation of metagenomic and pathogen-targeted next-generation sequencing for diagnosis of meningitis and encephalitis in adults: A multicenter prospective observational cohort study in China.

BACKGROUND: Next-generation sequencing (NGS) might aid in the identification of causal pathogens. However, the optimal approaches applied to cerebrospinal fluid (CSF) for detection are unclear, and studies evaluating the application of different NGS workflows for the diagnosis of intracranial infections are limited. METHODS: In this multicenter, prospective observational cohort study, we described the diagnostic efficacy of pathogen-targeted NGS (ptNGS) and metagenomic NGS (mNGS) compared to that of composite microbiologic assays, for infectious meningitis/encephalitis (M/E). RESULTS: In total, 152 patients diagnosed with clinically suspected M/E at four tertiary hospitals were enrolled; ptNGS and mNGS were used in parallel for pathogen detection in CSF. Among the 89 patients who were diagnosed with definite infectious M/E, 57 and 39 patients had causal microbial detection via ptNGS and mNGS, respectively. The overall accuracy of ptNGS was 65.1%, with a positive percent agreement (PPA) of 64% and a negative percent agreement (NPA) of 66.7%; and the overall accuracy of mNGS was 47.4%, with a PPA of 43.8% and an NPA of 52.4% after discrepancy analysis. There was a significant difference in the detection efficiency between these two methods both for PPA (sensitivity) and overall accuracy for pathogen detection (P < 0.05). CONCLUSIONS: NGS tests have provided new information in addition to conventional microbiologic tests. ptNGS seems to have superior performance over mNGS for common causative pathogen detection in CSF for infectious M/E.

Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

A retrospective observational study of 1000 consecutive patients tested with the FilmArray® Meningitis/Encephalitis panel: clinical diagnosis at discharge and microbiological findings.

FilmArray® Meningitis/Encephalitis panel (FAME-p) is used to diagnose central nervous system (CNS) infections. In this study, we investigated performance of FAME-p compared to comparator assays (CA), and for the first time, clinical diagnosis at discharge (CDD). 1000 consecutive patients with a cerebrospinal fluid (CSF) sample analyzed with FAME-p were identified. As CA, culture, polymerase chain reaction and cryptococcal antigen test were used. Medical records of patients were obtained. A CDD of CNS infection was made in 139 of 1000 CSF samples. FAME-p was positive in 66 samples with 44 viral and 22 bacterial agents. Thirteen FAME-p findings were not confirmed by CA, with four discrepant results remaining after comparison with the CDD. Positive percentage agreement (PPA) calculated against CA was 100%. Negative percentage agreement (NPA) calculated against CA was 94.4-99.8% for Haemophilus influenzae, Listeria monocytogenes, Streptococcus agalactiae, S. pneumoniae and varicella-zoster virus (VZV). NPA calculated against CDD was higher (compared to CA) for L. monocytogenes, S. agalactiae and VZV (100%), and lower for Escherichia coli, enterovirus and herpes simplex virus 2 (50-83.3%). NPA of FAME-p for human herpes virus 6 was difficult to interpret. Eighty-four cases received diagnosis of CNS-infection despite negative FAME-p. The four most common non-infectious etiologies were primary headache disorders, cranial nerve palsies, neuroinflammatory disorders and seizure. Although FAME-p shows good performance in diagnosis of CNS infections, result of FAME-p should be interpreted carefully. Considering infectious diseases not covered by FAME-p as well as non-infectious differential diagnoses is important in this context.

A multicenter evaluation of the QIAstat-Dx meningitis-encephalitis syndromic test kit as compared to the conventional diagnostic microbiology workflow.

PURPOSE: Rapid diagnosis and treatment of infectious meningitis and encephalitis (ME) is critical to minimize morbidity and mortality. Recently, Qiagen introduced the CE-IVD QIAstat-Dx ME panel (QS-ME) for syndromic diagnostic testing of meningitis and encephalitis. Some data on the performance of the QS-ME in comparison to the BioFire FilmArray ME panel are available. In this study, the performance of the QS-ME is compared to the current diagnostic workflow in two academic medical centers in the Netherlands. METHODS: A total of 110 cerebrospinal fluid samples were retrospectively tested with the QS-ME. The results obtained were compared to the results of laboratory-developed real-time PCR assays (LDTs), IS-pro, bacterial culture, and cryptococcal antigen (CrAg) testing. In addition, the accuracy of the QS-ME was also investigated using an external quality assessment (EQA) panel consisting of ten samples. RESULTS: Four of the 110 samples tested failed to produce a valid QS-ME result. In the remaining 106 samples, the QS-ME detected 53/53 viral targets, 38/40 bacterial targets, and 7/13 Cryptococcus neoformans targets. The discrepant bacterial results consisted of two samples that were previously tested positive for Listeria monocytogenes (CT 35.8) and Streptococcus pneumoniae (CT 40), respectively. The QS-ME detected one additional result, consisting of a varicella-zoster virus signal (CT 35.9), in a sample in which both techniques detected Streptococcus pyogenes. Finally, 100% concordance was achieved in testing a blinded bacterial ME EQA panel. CONCLUSION: The QS-ME is a relevant addition to the syndromic testing landscape to assist in diagnosing infectious ME.

The significance of cerebrospinal fluid analysis in the differential diagnosis of 564 psychiatric patients: Multiple sclerosis is more common than autoimmune-encephalitis.

The analysis of cerebrospinal fluid (CSF) is an essential tool for the differential diagnosis of psychiatric disorders caused by autoimmune inflammation or infections. Clear guidelines for CSF analysis are limited and mainly available for schizophrenia and dementia. Thus, insights into CSF changes in psychiatric patients largely derive from research. We analyzed the clinical and CSF data of 564 psychiatric patients without pre-existing neurological diagnoses from March 1998 to April 2020. Primary aim was to detect previously undiagnosed neurological conditions as underlying cause for the psychiatric disorder. Following CSF analysis, 8 % of patients (47/564) were diagnosed with a neurological disorder. This was the case in 12.0 % (23/193) of patients with affective disorders, 7.2 % (19/262) of patients with schizophrenia, and 4.0 % (23/193) of patients with anxiety disorders. The predominant new diagnoses were multiple sclerosis (19/47) and autoimmune encephalitis (10/47). Abnormal CSF findings without any implications for further treatment were detected in 17.0 % (94/564) of patients. Our data indicates that CSF analysis in patients suffering from psychiatric disorders may uncover underlying organic causes, most commonly multiple sclerosis and autoimmune encephalitis. Our findings imply that the incorporation of CSF analysis in routine psychiatric assessments is potentially beneficial.

case of autoimmune GFAP astrocytopathy with eosinophils in the cerebrospinal fluid.

Cerebrospinal fluid (CSF) eosinophilia is associated with a narrow differential, primarily including parasitic and fungal infections, neoplasm, and chemical meningitis. It has rarely been reported in neuroinflammatory conditions including as a finding of CSF cytology in two autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy cases. Here we describe a case of autoimmune GFAP astrocytopathy with classic clinical and radiographic features as well as presence of eosinophils in the CSF. This case highlights a potential association of eosinophils in the CSF with autoimmune GFAP astrocytopathy, which may suggest its inclusion in the differential diagnosis of eosinophilic meningitis, encephalitis, or myelitis.

Case report: A toxoplasmic encephalitis in an immunocompromised child detected through metagenomic next-generation sequencing.

There exist numerous pathogens that are capable of causing infections within the central nervous system (CNS); however, conventional detection and analysis methods prove to be challenging. Clinical diagnosis of CNS infections often depends on clinical characteristics, cerebrospinal fluid (CSF) analysis, imaging, and molecular detection assays. Unfortunately, these methods can be both insensitive and time consuming, which can lead to missed diagnoses and catastrophic outcomes, especially in the case of infrequent diseases. Despite the application of appropriate prophylactic regimens and evidence-based antimicrobial agents, CNS infections continue to result in significant morbidity and mortality in hospital settings. Metagenomic next-generation sequencing (mNGS) is a novel tool that enables the identification of thousands of pathogens in a target-independent manner in a single run. The role of this innovative detection method in clinical pathogen diagnostics has matured over time. In this particular research, clinicians employed mNGS to investigate a suspected CNS infection in a child with leukemia, and unexpectedly detected Toxoplasma gondii. Case: A 3-year-old child diagnosed with T-cell lymphoblastic lymphoma was admitted to our hospital due to a 2-day history of fever and headache, along with 1 day of altered consciousness. Upon admission, the patient's Glasgow Coma Scale score was 14. Brain magnetic resonance imaging revealed multiple abnormal signals. Due to the patient's atypical clinical symptoms and laboratory test results, determining the etiology and treatment plan was difficulty.Subsequently, the patient underwent next-generation sequencing examination of cerebrospinal fluid. The following day, the results indicated the presence of Toxoplasma gondii. The patient received treatment with a combination of sulfamethoxazole (SMZ) and azithromycin. After approximately 7 days, the patient's symptoms significantly improved, and they were discharged from the hospital with oral medication to continue at home. A follow-up polymerase chain reaction (PCR) testing after about 6 weeks revealed the absence of Toxoplasma. Conclusion: This case highlights the potential of mNGS as an effective method for detecting toxoplasmic encephalitis (TE). Since mNGS can identify thousands of pathogens in a single run, it may be a promising detection method for investigating the causative pathogens of central nervous system infections with atypical features.

RNA-sequencing-based detection of human viral pathogens in cerebrospinal fluid and serum samples from children with meningitis and encephalitis.

Encephalitis and meningitis are notable global public health concerns, especially among infants or children. Metagenomic next-generation sequencing (mNGS) has greatly advanced our understanding of the viruses responsible for these diseases. However, the detection rate of the aetiology remains low. We conducted RNA sequencing and virome analysis on cerebrospinal fluid (CSF) and serum samples commonly used in the clinical diagnosis to detect viral pathogens. In total, 226 paired CSF and serum samples from 113 children with encephalitis and meningitis were enrolled. The results showed that the diversity of viruses was higher in CSF, with a total of 12 viral taxa detected, including one case each of herpesvirus, coronavirus and enterovirus, and six cases of adenovirus related to human diseases. In contrast, the Anelloviridae was the most abundant viral family detected in serum, and only a few samples contained human viral pathogens, including one case of enterovirus and two cases of adenovirus. The detection rate for human viral pathogens increases to 10.6 %(12/113) when both types of samples are used simultaneously, compared to CSF along 7.9 % (9/113) or serum alone 2.6 % (3/113). However, we did not detect these viruses simultaneously in paired samples from the same case. These results suggest that CSF samples still have irreplaceable advantages for using mNGS to detect viruses in patients with meningitis and encephalitis, and serum can supplement to improve the detection rate of viral encephalitis and meningitis. The findings of this study could help improve the etiological diagnosis, clinical management and prognosis of patients with meningitis and encephalitis in children.

Mechanical filtration of the cerebrospinal fluid: procedures, systems, and applications.

INTRODUCTION: Mechanical methods aimed at the filtration of the cerebrospinal fluid (CSF) are a group of therapies that have been proposed to treat neurological conditions where pathogens are present in the CSF. Even though the industry of medical devices has not been very active in this field, there is a lack of systematization of the different systems and procedures that can be applied. AREAS COVERED: First, we systematize the classification and definitions of procedures and systems for mechanical filtration of the CSF. Then, we made a literature review in search of clinical or preclinical studies where any system of mechanical CSF clearance was proposed or applied. EXPERT OPINION: We found mechanical filtration of the CSF has been explored in subarachnoid hemorrhage, CNS infections (bacterial, viral, and fungal), meningeal carcinomatosis, multiple sclerosis, autoimmune encephalitis, and polyradiculomyelitis. Brain aging and neurodegenerative diseases are additional potential conditions of interest. While there is some preliminary positive evidence for many of these conditions, more advanced systems, detailed descriptions of procedures, and rigorous validations are needed to make these therapies a reality in the next decades.

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

How do we reduce acyclovir overuse? Impact of FilmArray meningitis/encephalitis panel tests for pediatric patients.

BACKGROUND: Few Japanese hospitals can perform in-house cerebrospinal fluid (CSF) polymerase chain reaction (PCR) to screen for herpes simplex virus, leading to patients being administered acyclovir (ACV) for several days. The FilmArray Meningitis/Encephalitis Panel (ME Panel) is a multiplex PCR test that can identify 14 major pathogens within 1 h. We aimed to investigate the efficacy of the ME Panel in children admitted with central nervous system infections in Japan. METHODS: We conducted a single-center, quasi-experimental study. The ME panel was introduced in April 2020. We outsourced the CSF samples to a laboratory during the pre-intervention period (April 2016 to March 2020) and performed the ME panel at our hospital during the post-intervention period (April 2020 to December 2021). Duration and dose of ACV and antibiotic use, length of stay (LOS) in the pediatric intensive care unit (PICU), and total LOS after testing were compared using the Mann-Whitney U test. RESULTS: The number of cases in the pre- and post-intervention periods was 67 and 22 cases, respectively. The median duration of ACV decreased significantly from 6 days to 0 day (p < 0.001), and the median dose of ACV use decreased significantly from 14 vials to 0 vial (p < 0.001). No significant differences were noted in the total duration and dose of antibiotic use, LOS in PICU, and the total LOS after testing. CONCLUSION: The introduction of ME panel may contribute to appropriate ACV use; however, there was no significant change in the duration and dose of antibiotic use or LOS.

Α case of seronegative autoimmune encephalitis associated with human herpesvirus-7 (HHV-7).

In the last 10 years, an increased number of patients presenting with acute encephalitis is being observed, a finding that is attributed to autoimmune mechanisms. Despite the fact that autoantibodies usually target the neuronal cell surface or synaptic proteins in the central nervous system (CNS), in many cases these remain undetectable, constituting a future diagnostic and therapeutic challenge. Human herpesvirus-7 (HHV-7) is proven to be a neurotropic virus, causing various neurological complications mostly in the adult population. We present the case of a 10-year-old girl, with confirmed active HHV-7 infection of the CNS, who developed acute seronegative autoimmune encephalitis. To our best knowledge, there is no literature concerning pediatric cases of autoimmune encephalitis following HHV-7 infection.

Increased cytokines/chemokines and hyponatremia as a possible cause of clinically mild encephalitis/encephalopathy with a reversible splenial lesion associated with acute focal bacterial nephritis.

OBJECTIVE: Clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), the second most common encephalopathy syndrome in Japan, is most often associated with viral infection. Bacterial MERS has been rarely reported but is mostly associated with acute focal bacterial nephritis (AFBN) for an unknown reason. We examined cytokines and chemokines in four MERS patients with AFBN to determine if they play an important role in the pathogenesis. METHODS: We examined the clinical charts and MRI results in four MERS patients with AFBN, and measured 10 cytokines and chemokines in serum and cerebrospinal fluid in the acute phase. These were analyzed using the Mann-Whitney U test, compared with the control group (cases with a non-inflammatory neurological disease). Longitudinal changes in the serum cytokine and chemokine levels were evaluated in two patients. RESULTS: Hyponatremia was observed in all four patients with MERS associated with AFBN (128-134 mEq/L). CSF analysis revealed increased cytokines/chemokines associated with Th1 (CXCL10, TNF-α, IFN-γ), T reg (IL-10), Th17 (IL-6), and neutrophil (IL-8 and CXCL1). In serum, upregulation was observed in those associated with Th1 (CXCL10, TNF-α, IFN-γ), Th17 (IL-6), and inflammasome (IL-1ß). The increased serum cytokines/chemokines in the acute stage normalized within 2 weeks in patients 1 and 2, so examined, in accordance with their clinical improvement. CONCLUSION: Increased cytokines/chemokines and hyponatremia may be factors that explain why AFBN is likely to cause MERS.

Clinical Significance of Positive Results of the BioFire Cerebrospinal Fluid FilmArray Meningitis/Encephalitis Panel at a Tertiary Medical Center in the United States.

CONTEXT.­: The FilmArray Meningitis/Encephalitis (ME) panel is the first US Food and Drug Administration-cleared multiplex polymerase chain reaction panel for the detection of central nervous system infections. While the assay's performance characteristics have been described, the real-world significance of positive results has not been fully characterized. OBJECTIVE.­: To evaluate the clinical significance of positive ME panel results in a tertiary care medical center in New York, New York. DESIGN.­: Four physicians independently performed retrospective clinical assessments of all positive ME panel results at Columbia University Irving Medical Center, including the Children's Hospital of New York, during an 18-month period. Each reviewer determined the likelihood of central nervous system infection for all cases and whether cases fit Brighton diagnostic criteria for meningitis, encephalitis, or meningoencephalitis. RESULTS.­: Among 119 cases, there was 75% positive agreement (95% CI, 54%-89%) between ME panel results and clinical consensus, which varied among panel targets. CONCLUSIONS.­: The ME panel showed good agreement with expert clinical consensus for patients presenting with acute meningitis/encephalitis. Factors contributing to clinically insignificant ME positive results included low pretest probability, traumatic lumbar puncture, specimen contamination, and detection of incidental viral targets such as human herpesvirus 6. Notably, the ME panel detected more than twice the number of cases of bacterial meningitis detected by culture alone, particularly among patients receiving empiric antimicrobial therapy before lumbar puncture. Appropriate test use and contextual interpretation of results are critical to leveraging the advantages of the platform while avoiding potential pitfalls.

Assessment of the FilmArray ME panel in 4199 consecutively tested cerebrospinal fluid samples.

OBJECTIVES: In central nervous system infections, early and correct management is of utmost importance. Rapid syndromic panel testing can potentially provide valuable guidance. The FilmArray meningitis/encephalitis (ME) panel detects 14 pathogens through multiplex PCR. Our study objectives were to assess its performance compared with established diagnostic procedures, especially real-time quantitative PCR for detection of viruses, and to determine the diagnostic and clinical significance of discrepant results. METHODS: All cerebrospinal fluid samples sent for viral diagnostics to our microbiological laboratory over 34 months were analysed with the ME panel and in-house real-time PCR for herpes simplex virus type 1 (HSV-1), HSV-2, varicella zoster virus and enteroviruses. Other pathogens detected by the panel were confirmed by routine diagnostic procedures. Discrepant results were analysed through interpretation of biological and clinical data, and performance data were calculated for individual pathogens. RESULTS: Altogether, 315 pathogens were detected by the ME panel in 4199 cerebrospinal fluid samples (7.5%) and an additional 21 viral targets were identified using real-time PCR. Thirty-four ME panel detections were not confirmed, totalling 55 discrepant results. After discrepancy analysis, 20 false-positive and 21 false-negative ME panel results remained. Performance varied between pathogens. Sensitivity for HSV-1 was calculated at 82.4% (59.0%-93.8%) with three false-negative results. Also noteworthy were 13 false-negative enterovirus and eight false-positive Streptococcus pneumoniae results. CONCLUSIONS: Our analysis shows good performance for the ME panel in diagnosing central nervous system infection. The risk of false-negative HSV-1 results, however, warrants additional testing when encephalitis is suspected. Uncertainties in interpretation of enterovirus and S. pneumoniae results represent other limitations.

Evaluation of the selection of cerebrospinal fluid testing in suspected meningitis and encephalitis.

Diagnostic stewardship interventions can decrease unnecessary antimicrobial therapy and microbiology laboratory resources and costs. This retrospective cross-sectional study evaluated factors associated with inappropriate initial cerebrospinal fluid (CSF) testing in patients with suspected community-acquired meningitis or encephalitis. In 250 patients, 202 (80.8%) and 48 (19.2%) were suspected meningitis and encephalitis, respectively. 207 (82.8%) patients had inappropriate and 43 (17.2%) appropriate testing. Any inappropriate CSF test was greatest in the immunocompromised (IC) group (n = 54, 91.5%), followed by non-IC (n = 109, 80.1%) and HIV (n = 44, 80%). Ordering performed on the general ward was associated with inappropriate CSF test orders (adjOR 2.81, 95% CI [1.08-7.34]). Laboratory fee costs associated with excessive testing was close to $300,000 per year. A stepwise algorithm defining empiric and add on tests according to CSF parameters and patient characteristics could improve CSF test ordering in patients with suspected meningitis or encephalitis.

The changing spectrum of antibody-mediated encephalitis in China.

In the past 5 years, the positivity rate of autoimmune encephalitis antibody panels has significantly decreased in patients with clinically suspected encephalitis in an encephalitis center in China. Furthermore, the spectrum of patients with autoantibodies related to autoimmune encephalitis has changed significantly, exhibiting a decreased percentage of patients with anti-N-methyl-d-aspartate receptor antibodies and an increased percentage of patients with infrequently observed autoantibodies. Meanwhile, a small but non-negligible proportion of patients with autoantibodies against cell surface and synaptic proteins exhibited positivity for more than one autoantibody.

CSF Findings in Acute NMDAR and LGI1 Antibody-Associated Autoimmune Encephalitis.

BACKGROUND AND OBJECTIVES: CSF in antibody-defined autoimmune encephalitis (AE) subtypes shows subtype-dependent degrees of inflammation ranging from rare and often mild to frequent and often robust. AEs with NMDA receptor antibodies (NMDAR-E) and leucine-rich glioma-inactivated protein 1 antibodies (LGI1-E) represent opposite ends of this spectrum: NMDAR-E with typically frequent/robust and LGI1-E with rare/mild CSF inflammation. For a more in-depth analysis, we characterized CSF findings in acute, therapy-naive NMDAR-E and LGI1-E in a multicentric, retrospective, cross-sectional setting. METHODS: Eighty-two patients with NMDAR-E and 36 patients with LGI1-E from the GErman NEtwork for Research of AuToimmune Encephalitis (GENERATE) with lumbar puncture within 90 days of onset and before immunotherapy were included. CSF parameters comprised leukocytes, oligoclonal bands (OCBs), and CSF/serum ratios for albumin, immunoglobulin G (IgG), A (IgA), and M (IgM), the latter 3 converted to Z scores according to Reiber formulas. The MRZ reaction was tested in 14 patients with NMDAR-E and 6 patients with LGI1-E, respectively. RESULTS: CSF was abnormal in 94% of NMDAR-E but only in 36% of LGI1-E patients. Robust quantitative intrathecal immunoglobulin synthesis (IIS, IgG > IgM >> IgA) was characteristic for NMDAR-E, but absent in LGI-E. In NMDAR-E, CSF leukocytes were higher when IIS was present or more pronounced. In addition, in NMDAR-E, CSF leukocytes were lower and IIS occurred less often and if so to a lesser degree at older age. Patients with NMDAR-E with severe functional impairment more often had positive OCBs. In CSF obtained later than 3 weeks of onset, leukocytes were lower. In parallel, the correlation of leukocytes with IIS disappeared as IIS was partially independent of disease duration. The MRZ reaction was positive in 5 (36%) patients with NMDAR-E. All these associations were completely absent in LGI1-E. Here, younger patients showed more blood-CSF barrier dysfunction. In LGI1-E, but not in NMDAR-E, the blood-CSF barrier was more dysfunctional when CSF leukocytes were higher. DISCUSSION: NMDAR-E and LGI-E differ in their typical extent of CSF inflammation. In addition, the patterns formed by the different inflammatory CSF parameters and their relationship with disease severity, age, and disease duration are subtype-characteristic. Moreover, signs for multiple sclerosis-like chronic inflammation are present in a subgroup of patients with NMDAR-E. These CSF patterns might be markers for the different immunopathogeneses of LGI1-E and NMDAR-E.

Enhanced Virus Detection and Metagenomic Sequencing in Patients with Meningitis and Encephalitis.

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific tests, sometimes over days to weeks, whereas metagenomic next-generation sequencing (mNGS) profiles all nucleic acid in a sample. In this single-center, prospective study, 68 hospitalized patients with known (n = 44) or suspected (n = 24) CNS infections underwent mNGS from RNA and DNA to identify potential pathogens and also targeted sequencing of viruses using hybrid capture. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects, 3 of whom had no clinical diagnosis by routine workup. Among subjects diagnosed with infection by serology and/or peripheral samples, we demonstrated the utility of mNGS to detect pathogen nucleic acid in CSF, importantly for the Ixodes scapularis tick-borne pathogens Powassan virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. We also evaluated two methods to enhance the detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed the detection of varicella-zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with efficacy similar to that of pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. IMPORTANCE Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40 to 60% of people with meningitis or encephalitis have no clear cause of CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 prospectively enrolled patients with known (n = 44) or suspected (n = 24) CNS viral infection from a single center in New England and evaluated enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become integrated into the diagnostic toolkit for CNS infections.

Limitations of a Commercial Assay as Diagnostic Test of Autoimmune Encephalitis.

Detection of neuronal surface antibodies (NSAb) is important for the diagnosis of autoimmune encephalitis (AE). Although most clinical laboratories use a commercial diagnostic kit (Euroimmun, Lübeck, Germany) based on indirect immunofluorescence on transfected cells (IIFA), clinical experience suggests diagnostic limitations. Here, we assessed the performance of the commercial IIFA in serum and CSF samples of patients with suspected AE previously examined by rat brain immunohistochemistry (Cohort A). Of 6213 samples, 404 (6.5%) showed brain immunostaining suggestive of NSAb: 163 (40%) were positive by commercial IIFA and 241 (60%) were negative. When these 241 samples were re-assessed with in-house IIFA, 42 (18%) were positive: 21 (9%) had NSAb against antigens not included in the commercial IIFA and the other 21 (9%) had NSAb against antigens included in the commercial kit (false negative results). False negative results occurred more frequently with CSF (29% vs 10% in serum) and predominantly affected GABABR (39%), LGI1 (17%) and AMPAR (11%) antibodies. Results were reproduced in a separate cohort (B) of 54 AE patients with LGI1, GABABR or AMPAR antibodies in CSF which were missed in 30% by commercial IIFA. Patients with discordant GABABR antibody results (positive in-house but negative commercial IIFA) were less likely to develop full-blown clinical syndrome; no significant clinical differences were noted for the other antibodies. Overall, NSAb testing by commercial IIFA led to false negative results in a substantial number of patients, mainly those affected by anti-LG1, GABABR or AMPAR encephalitis. If these disorders are suspected and commercial IIFA is negative, more comprehensive antibody studies are recommended.