Conventional Cerebrospinal Fluid Research Articles

CSF liquid biopsy by low-pass whole genome sequencing for clinical disease monitoring in pediatric embryonal tumors

Abstract Background Liquid biopsy assays that detect cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) are a promising tool for disease monitoring in pediatric patients with primary central nervous system (CNS) tumors. As a compliment to tissue-derived molecular analyses, CSF liquid biopsy has the potential to transform risk stratification, prognostication, and precision medicine approaches. Methods In this pilot study, we evaluated a clinical pipeline to determine feasibility and sensitivity of low-pass whole genome sequencing (LP-WGS) of CSF-derived cfDNA from patients with CNS embryonal tumors. Thirty-two longitudinal CSF samples collected from 17 patients with molecularly characterized medulloblastoma (12), embryonal tumor with multilayered rosettes (2), CNS embryonal tumor, NEC (2), and atypical teratoid/rhabdoid tumor (1) were analyzed. Results Adequate CSF-derived cfDNA for LP-WGS analysis was obtained in 94% of samples (30/32). Copy number variants compatible with neoplasia were detected in 90% (27/30) and included key alterations, such as isodicentric ch17, monosomy 6, and MYCN amplification, among others. Compared to tissue specimens, LP-WGS detected additional aberrations in CSF not previously identified in corresponding primary tumor specimens, suggesting a more comprehensive profile of tumor heterogeneity and evolution of cfDNA profiles over time. Among the 12 CSF samples obtained at initial staging, only 2 (17%) were cytologically positive, compared to 11 (92%) that were copy number positive by LP-WGS. Conclusions LP-WGS of CSF-derived cfDNA is feasible using a clinical platform, with greater sensitivity for tumor detection compared to conventional CSF cytologic analysis. Large prospective studies are needed to further evaluate LP-WGS as a predictive biomarker.

Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases.

Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Improved Diagnostics in Bacterial Neonatal Meningitis Using a Next-Generation Sequencing Platform.

Bacterial meningitis in infants is an infrequent but life-threatening condition. Empiric therapy should begin as soon as meningitis is thought likely. Consequently, the causative microorganisms may not always be detected using culturing techniques, as cerebrospinal fluid (CSF) cultures are influenced by antibiotics. Nucleic acid amplification tests, such as polymerase chain reaction (PCR) (multiplex panels), may overcome this limitation but require a priori knowledge of the likely pathogen present within the sample. With this in mind, we investigated to what extent a culture-free, broad-range 16S rRNA gene next-generation sequencing (NGS) platform (MYcrobiota) could add to the microbiological diagnosis of meningitis. Retrospective cohort study at level III neonatal intensive care unit. Included were all infants with suspected meningitis admitted between 10 November 2017 and 31 December 2020. A comparison was made of the bacterial pathogen detection rate between MYcrobiota and conventional bacterial culture. In a 3-year period, 37 CSF samples (diagnostic and follow-up) from 35 infants with proven or possible meningitis were available for MYcrobiota testing. MYcrobiota detected the presence of bacterial pathogens in 11 samples (30%), in contrast with the conventional CSF culture, which detected bacteria in 2 of 36 samples (5.6%). Addition of 16S rRNA sequencing to conventional culturing greatly improved the identification of the aetiology of bacterial meningitis compared to culturing of CSF samples alone.

Peripheral Blood Brain Microvascular Endothelial Cells As a Novel Marker in the Detection of Blood-Brain Barrier Injury in EV71 Severe Encephalopathy

Enterovirus 71 (EV71), the main causative agent of hand, foot, and mouth disease (HFMD), is also considered a neuroinvasive virus. EV71 causes sporadic epidemics and outbreaks with more than 13 million people worldwide, predominantly occurring in the Asia-Pacific, and elicited more than 3000 deaths, including 2457 deaths in China from 2008 to 2014. However, there are no available noninvasive biomarkers in peripheral blood for neuro-EV71 infection and optimizing therapeutic options. Brain microvascular endothelial cells (BMECs) are a special type of cells that constitute the blood-brain barrier (BBB), which dynamically shed associated with BBB injury. A mouse model of BBB injury induced by microbial and non-microbial agents was performed, using magnetic bead extraction (MBE) to identify circulating endothelial cells (CECs,CD146+/DAPI+), circulating BMECs (cBMECs, CD146+/S100B+/DAPI+) and endothelial progenitor cells (EPCs, CD146+/CD133+/DAPI+), which are identified based on their S100B (brain marker)+/ CD146 (EC marker) +/CD133+ (PC marker) /DAPI (nuclei)+phenotype with significantly higher levels in the experimental group than in the control group. These results indicate that it is feasible to evaluate BBB injury by quantifying BMECs and EPCs with MBE. The whole cell proteins were prepared by us from EV71 infected BMECs, and among the samples infected with EV71, MALDI TOF / TOF mass spectrometry successfully identified 28 proteins, 4 proteins including vimentin (VIM) were significantly up-regulated, 6 proteins were down regulated, and the other 18 proteins were expressed to various degrees at different incubation times. Furthermore, we performed a glioblastoma model with low vimentin expression and found that EV71 may activate the NLRP3 inflammasome via the VIM-ERK-NFκB pathway during EV71 CNS infection, which promotes caspase-1 activation and the inflammatory factor IL-1β release, causing an inflammatory response that ultimately leads to BBB/CNS disorders and neuronal death. The same results for those experiments with neonatal mice of EV71 infection were also observed. The results showed that cBMECs may be used as potential cell-based biomarkers for indexing of BBB injury. Then peripheral blood samples were obtained from the children with severe HFMD infection from a medical center for women and children, and examination showed that the counts of cBMECs were significantly higher in severe HFMD children than in the control children, indicating that the detection of cBMECs may be used for the early diagnosis of severe HFMD. In addition, Mfsd2 antibody specifically on BMECs were attached to magnetic beads and used directly to isolate BMECs from blood, followed by immunofluorescence staining with CD146 antibody, and the two corresponding fluorophores, CD146 and DPAI, appeared microscopically to preliminarily determine BMECs. The use of mfsd2 antibody to isolate BMECs both simplifies experimental procedures and improves detection precision and efficiency. In conclusion, in this study, cBMECs are demonstrated to serve as cellular biomarkers for BBB injury caused by microbial and non-microbial factors. Proteomics combined with mechanistic studies indicate that cBMECs are important indicators of BBB/CNS disorders in severe HFMD. Ultimately, this study successfully established a method to detect EV71 severe encephalopathy in human peripheral blood, which is safer and noninvasive than conventional cerebrospinal fluid puncture and pathological collection methods, causes minimal physical trauma to patients, and is more intuitive and accurate than conventional imaging examination. Therefore, this assay has good clinical application and may provide a new means for noninvasive and visual detection of EV71 severe encephalopathy. However, further cases were also collected to verify the accuracy of peripheral blood BMEC count for diagnosing severe HFMD, while establishing suitable cut-off values for application in early diagnosis. (Acknowledgements: Corresponding author: Hong Cao, gzhcao@smu.edu.cn; The Basic Research Project of Key Laboratory of Guangzhou, China (No.202102100001) and Grant from School of Public Health of Southern Medical University, China, No.GW202222 to H.C.)

Detection of Circulating Tumor Cells in Cerebrospinal Fluid of Patients with Suspected Breast Cancer Leptomeningeal Metastases: A Prospective Study.

The diagnosis of breast cancer (BC)-related leptomeningeal metastases (LM) relies on the detection of tumor cells in cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, the sensitivity of this technique is low. Our goal was to evaluate whether circulating tumor cell (CTC) detection in CSF using the CellSearch® system could be used for LM diagnosis. This prospective, monocentric study included adult patients with suspected BC-related LM. The clinical sensitivity and specificity of CTC detection in CSF for LM diagnosis were calculated relative to conventional CSF cytology. Forty-nine eligible patients were included and 40 were evaluable (CTC detection technical failure: n = 8, eligibility criteria failure: n = 1). Cytology was positive in 18/40 patients. CTCs were detected in these 18 patients (median: 5824 CTC, range: 93 to 45052) and in 5/22 patients with negative cytology (median: 2 CTC, range: 1 to 44). The detection of ≥1 CSF CTC was associated with a clinical sensitivity of 100% (95% CI, 82.4-100) and a specificity of 77.3% (95% CI, 64.3-90.3) for LM diagnosis. HER2+ CTCs were detected in the CSF of 40.6% of patients with HER2- BC (median: 500 CTC, range: 13 to 28 320). The clinical sensitivity of CTC detection in CSF with the CellSearch® system for LM diagnosis is higher than that of CSF cytology. CTC detection in patients with negative cytology, however, must be further investigated. The finding of HER2+ CTCs in patients with HER2- BC suggests that the HER2 status of LM should be evaluated to increase the treatment opportunities for these patients.

Non-Invasive Nasal Discharge Fluid and Other Body Fluid Biomarkers in Alzheimer's Disease.

The key to current Alzheimer’s disease (AD) therapy is the early diagnosis for prompt intervention, since available treatments only slow the disease progression. Therefore, this lack of promising therapies has called for diagnostic screening tests to identify those likely to develop full-blown AD. Recent AD diagnosis guidelines incorporated core biomarker analyses into criteria, including amyloid-β (Aβ), total-tau (T-tau), and phosphorylated tau (P-tau). Though effective, the accessibility of screening tests involving conventional cerebrospinal fluid (CSF)- and blood-based analyses is often hindered by the invasiveness and high cost. In an attempt to overcome these shortcomings, biomarker profiling research using non-invasive body fluid has shown the potential to capture the pathological changes in the patients’ bodies. These novel non-invasive body fluid biomarkers for AD have emerged as diagnostic and pathological targets. Here, we review the potential peripheral biomarkers, including non-invasive peripheral body fluids of nasal discharge, tear, saliva, and urine for AD.

CXCL13, CXCL10 and CXCL8 as Indicators of Ocular and Neurological Involvement in Patients With Ocular Syphilis: An Observational Descriptive Study.

To investigate the role of the chemokines CXCL13, CXCL10 and CXCL8 in the diagnosis of ocular- and neurosyphilis by examining the serum, aqueous humour (AH) and cerebrospinal fluid (CSF) of patients with ocular syphilis. An observational descriptive study was performed prospectively at Tygerberg Academic Hospital in Cape Town, South Africa from 1 February 2018 till 31 January 2021 which enrolled 23 participants. 14 Patients were male and 9 female, 15 patients were HIV positive, and all patients were newly diagnosed with ocular syphilis. Upon diagnosis of ocular syphilis, the HIV status of each patient was determined, and 3 samples (AH, serum and CSF) were collected to measure the levels of CXCL13, CXCL10 and CXCL8 in each. All patients were treated with 14 days of intravenous Penicillin G and topical corticosteroid drops for uveitis. The mean concentrations of all 3 biomarkers were higher in the AH and CSF than in the serum. The mean concentrations of the 3 measured biomarkers were markedly different when comparing both AH and CSF levels to serum levels. The level of CXCL13 measured in the AH correlated well with the concentrations found in the CSF of patients with neurosyphilis. In patients with neurosyphilis, mean AH levels of CXCL13 and CXCL10 were markedly higher than in serum while mean CSF levels of CXCL10 were also markedly higher than in serum. Also, the AH/serum ratio of CXCL13 and CXCL10, as well as the CSF/serum ratio of CXCL10, was much higher in patients with neurosyphilis than without. In patients with HIV infection, mean AH CXCL13 levels were much higher than in patients without HIV infection. The levels of CXCL13, CXCL10 and CXCL8 in the AH of patients with neurosyphilis are similar to previously reported levels in the CSF of patients with neurosyphilis and can potentially be an adjunct in the diagnosis of ocular syphilis. Patients with ocular syphilis who tested negative for neurosyphilis with conventional CSF testing showed features of neurosyphilis when analysing the CSF chemokines.

IMG-13. Role of diffusion weighted imaging of spine in pediatric brain tumors showing diffusion restriction and CSF metastases

Abstract INTRODUCTION: Early detection of leptomeningeal metastases (LM) with paediatric brain tumors cases can change the prognosis as well as course of treatment. Conventional MR imaging and CSF (cerebrospinal fluid) analysis plays a vital role in detection of metastases, however, due to certain limitations these can be missed. In our study, diffusion weighed imaging (DWI) of spine was able to bridge this gap and increased the detection rate in MRI equivocal cases in which CSF study was negative. AIMS: To evaluate the incremental value of DWI over the conventional sequences in detection of LM. MATERIALS & METHODS: Paediatric patients with primary brain tumors known to show restriction and propensity for CSF metastasis underwent MRI on 1.5T Philip’s machine. CSF analysis and radiological follow up were used to confirm the diagnosis. RESULTS: Of the 26 patients proven to have LM, linear leptomeningeal metastases (LLP) were seen in 7 patients and nodular leptomeningeal metastases (NLM) were seen in 6 patients and both were seen in 13 patients. Of the 20 patients with LLM, 18 were detected on contrast; however, these were not detected on T2W and DWI except one patient with thick LLP. In 19 patients with NLM, a total of 84 lesions were detected. All were appreciated on contrast with additional 6 lesions which were false positive (FP). Total 96 lesions were detected on T2W, of which 80 were true positive and 16 were FP. About 80 lesions were detected on DWI. Thus, on contrast sequence FP were 6 while on T2W 16 were FP. DISCUSSION: DWI showed a lower FP rate than conventional sequences. CONCLUSION: Contrast sequence remains most reliable sequence for the detection of LM. However, DWI is useful in equivocal cases. The role of DWI in Group 3 and 4 Medulloblastoma with non-enhancing metastases needs to be further explored.

Nanopore 16S sequencing enhances the detection of bacterial meningitis after neurosurgery.

ObjectiveNosocomial bacterial meningitis is one of the major complications after neurosurgery. We performed nanopore 16S amplicon sequencing from cerebrospinal fluid (CSF) to evaluate bacterial meningitis in patients who underwent neurosurgery.MethodsAmong the patients who visited the neurosurgery department of Seoul National University Hospital between July 2017 and June 2020, those with clinically suspected bacterial meningitis were included. 16S rDNA PCR was performed from the CSF, and nanopore sequencing was performed for up to 3 h. The reads were aligned to the BLAST database. In each case, the culture and the 16S rRNA gene amplicon analysis were simultaneously performed and compared with each other, and we retrospectively reviewed the medical records. Genuine infection was determined by the identical results between conventional culture study and the sequencing, or clinically determined in cases with inconsistent results between the two methods.ResultsOf the 285 samples obtained from 178 patients who had 16S rDNA PCR, 41 samples (14.4%) were diagnosed with genuine infection. A total of 56.1% (23/41) of the samples with genuine infection showed a false‐negative culture test. In particular, 16S amplicon sequencing was useful in evaluating patients at the initial tests who had infection with intraventricular hemorrhage (Culture false‐negative rate = 100%), subarachnoid hemorrhage (Culture false‐negative rate = 77.8%), and systemic cancer (Culture false‐negative rate = 100%), which are risk factors for central fever. Moreover, 16S amplicon sequencing could suggest the possibility of persistent bacterial meningitis in empirical antibiotic use.ConclusionCSF nanopore 16S sequencing was more effective than conventional CSF culture studies in postoperative bacterial meningitis and may contribute to evidence‐based decisions for antibiotic maintenance and discontinuation.

Diagnosis of Leptomeningeal Metastasis in Women With Breast Cancer Through Identification of Tumor Cells in Cerebrospinal Fluid Using the CNSide™ Assay

Diagnosis of LM is limited by low sensitivity of cerebrospinal fluid (CSF) cytopathology. Detecting tumor cells in CSF (CSF-TCs) might be more sensitive. We evaluated if CNSide (CNSide), a novel assay for tumor cell detection in CSF, can detect CSF-TCs better than conventional CSF cytology. We enrolled adults with metastatic breast cancer and clinical suspicion for LM to undergo lumbar puncture (LP) for CSF cytopathology and CNSide. CNSide captured CSF-TCs using a primary 10-antibody mixture, streptavidin-coated microfluidic channel, and biotinylated secondary antibodies. CSF-TCs were assessed for estrogen receptor (ER) expression by fluorescent antibody and HER2 amplification by fluorescent in situ hybridization (FISH). CSF cell-free DNA (cfDNA) was extracted for next-generation sequencing (NGS). Leptomeningeal disease was defined as positive CSF cytology and/or unequivocal MRI findings. We calculated sensitivity and specificity of CSF cytology and CNSide for the diagnosis of LM. Ten patients, median age 51 years (range, 37-64), underwent diagnostic LP with CSF evaluation by cytology and CNSide. CNSide had sensitivity of 100% (95% Confidence Interval [CI], 40%-100%) and specificity of 83% (95% CI, 36%-100%) for LM. Among these patients, concordance of ER and HER2 status between CSF-TCs and metastatic biopsy were 60% and 75%, respectively. NGS of CSF cfDNAidentified somatic mutations in three patients, including one with PIK3CA p.H1047L in blood and CSF. CNSide may be a viable platform to detect CSF-TCs, with potential use as a diagnostic tool for LM in patients with metastatic breast cancer. Additional, larger studies are warranted.

Serum neurofilament light chain as a surrogate of cognitive decline in sporadic and familial frontotemporal dementia.

Neurofilament light chain (NfL) has recently been proposed as a promising biomarker in frontotemporal dementia (FTD). We investigated the correlation of both cerebrospinal fluid (CSF) and serum NfL with detailed neuropsychological data and cognitive decline in a cohort of sporadic and familial FTD. CSF and serum NfL, as well as conventional CSF Alzheimer's disease (AD) biomarkers (Aβ42, t-Tau, p-Tau181), were determined in 63 FTD patients (30 sporadic-FTD, 20 with progranulin (GRN) mutations [FTD-GRN], 13 with chromosome 9 open reading frame 72 [C9orf72] expansions [C9orf72-FTD]), 37 AD patients, and 31 neurologic controls. Serum NfL was also quantified in 37 healthy individuals. Correlations between baseline CSF and serum NfL levels, standardized neuropsychological tests, and the rate of cognitive decline in FTD patients were assessed. CSF and serum NfL presented with significantly higher levels in FTD than in AD patients and both control groups. Within FTD subtypes, genetic cases, and particularly FTD-GRN, had higher CSF and serum NfL levels. Significant correlations between NfL levels and overall cognitive function, abstract reasoning (CSF and serum), executive functions, memory, and language (serum) were found. A relationship between increased baseline CSF and serum NfL and a decay in cognitive performance over time was also observed. Our findings highlight the potential of serum NfL as a useful surrogate end point of disease severity in upcoming targeted treatments.

P 42. ALS patients show increased T-cell activation in peripheral blood and cerebrospinal fluid

Objective. We investigated intrathecal and peripheral immune profiles in amyotrophic lateral sclerosis (ALS) patients compared to controls free of neurological disorders (CTRL) and patients with dementia (DEM) or primary progressive multiple sclerosis (PPMS) to elucidate whether compartment-specific inflammation contributes to ALS pathophysiology. Methods. Conventional cerebrospinal fluid (CSF) parameters were examined in 308 patients including 132 ALS patients. In a subgroup of 41 ALS patients, extensive flow-cytometric immune cell profiling in peripheral blood (PB) and CSF was performed and compared with data from 26 CTRL, 57 DEM and 27 PPMS patients. Results. ALS patients presented with significantly altered proportions of monocyte subsets in PB and increased frequencies of CD4+ and CD8+ T cells expressing the activation marker HLA-DR in PB and CSF compared to CTRL. The increased intrathecal levels of T-cell activation were comparable to DEM and PPMS and resulted from in situ differentiation rather than trans-migration from the PB. Moreover, peripheral CD4+ and CD8+ T cell HLA-DR expression significantly exceeded DEM and PPMS. While T-cell activation did not correlate with clinical measures, patients with high disease disability and rapid progression showed reduced intrathecal levels of immune-regulatory CD56bright natural killer (NK) cells. The integration of these parameters into a composite score facilitated the differentiation of patients with high ALS severity and rapid disease progression from patients from all other cohorts. Conclusion. Increased peripheral and intrathecal activation of CD4+ and CD8+ T cells concomitant with diminished immune regulation by CD56bright NK cells suggest an involvement of these immune cells in ALS pathophysiology.

Evaluation of the Utilization of FilmArray Meningitis/Encephalitis in Children With Suspected Central Nervous System Infection: A Retrospective Case Series.

The etiology of central nervous system infections is often difficult to establish. FilmArray meningitis/encephalitis (ME) panel is a multiplex polymerase chain reaction for rapid identification of 14 pathogens. The aim of this study was to evaluate potential real-life contributions of the use of this method in the pediatric population. We herein report the results obtained with FilmArray ME in a retrospective case series of 367 children with suspected central nervous system infection. We identified viral and bacterial agents by FilmArray, and we evaluated the potential diagnostic contributions of the use of the panel taking into account the cytological, biochemical, and microbiological results of the cerebrospinal fluid (CSF) analysis. The FilmArray ME panel detected a viral infection in 186 cases (50.7%) and a bacterial infection in 12 cases (3.3%). Fifty-three cases (28.4%) of viral infection had at least 1 CSF finding that could be mistaken for bacterial meningitis. Enterovirus was identified in 2 cases with normal CSF findings. Among 12 bacterial infection cases, only 6 (50%) had a positive result with conventional microbiology analysis (Gram stain and culture). The CSF findings suggestive of bacterial meningitis were found in all 6 cases in which FilmArray was the only method to identify bacterial etiological agent. FilmArray ME panel identified an etiological agent in cases in which conventional CSF analysis failed, providing potential clinical contributions to the management of such cases.

Evaluating Infectious, Neoplastic, Immunological, and Degenerative Diseases of the Central Nervous System with Cerebrospinal Fluid-Based Next-Generation Sequencing.

Cerebrospinal fluid (CSF) is a clear and paucicellular fluid that circulates within the ventricular system and the subarachnoid space of the central nervous system (CNS), and diverse CNS disorders can impact its composition, volume, and flow. As conventional CSF testing suffers from suboptimal sensitivity, this review aimed to evaluate the role of next-generation sequencing (NGS) in the work-up of infectious, neoplastic, neuroimmunological, and neurodegenerative CNS diseases. Metagenomic NGS showed improved sensitivity—compared to traditional methods—to detect bacterial, viral, parasitic, and fungal infections, while the overall performance was maximized in some studies when all diagnostic modalities were used. In patients with primary CNS cancer, NGS findings in the CSF were largely concordant with the molecular signatures derived from tissue-based molecular analysis; of interest, additional mutations were identified in the CSF in some glioma studies, reflecting intratumoral heterogeneity. In patients with metastasis to the CNS, NGS facilitated diagnosis, prognosis, therapeutic management, and monitoring, exhibiting higher sensitivity than neuroimaging, cytology, and plasma-based molecular analysis. Although evidence is still rudimentary, NGS could enhance the diagnosis and pathogenetic understanding of multiple sclerosis in addition to Alzheimer and Parkinson disease. To conclude, NGS has shown potential to aid the research, facilitate the diagnostic approach, and improve the management outcomes of all the aforementioned CNS diseases. However, to establish its role in clinical practice, the clinical validity and utility of each NGS protocol should be determined. Lastly, as most evidence has been derived from small and retrospective studies, results from randomized control trials could be of significant value.

Contamination of CSF culture: Serious risk to patient safety

Background: Acute bacterial meningitis is a medical emergency, and early initiation of appropriate therapy is important to improve outcomes. Culture-based methods are still the standards for detection of microorganisms in cerebrospinal fluid (CSF). It is proven that automated blood culture systems yield better results in culturing of sterile body fluids including CSF. However high sensitivity of this technology is not without the danger of contamination compromising final outcome. Thus we tried to study the culture yield in suspected meningitis and determine the rate of contamination. Methods and materials: A retrospective cohort study conducted over one year period from January 2017 to December 2017 for CSF samples collected for culture from the patients presented with sign and symptoms indicative of meningitis. The bacterial aetiologies’ and rates of contamination were extracted from laboratory records. Descriptive statistics from Microsoft Excel (Microsoft Corporation, Redmond, WA) were used to analyze patient data. Limited statistical analysis (Fisher's exact test) was performed using SPSS for Windows version 18 (SPSS, Chicago, IL, USA). Results: A total of 1053 CSF samples were received for aerobic culture, out of which maximum was conventional culture (685/1053, 65%) whereas BacT/ALERT culture was requested for 35% (368/1053) cases. Out of 685 conventional CSF culture, pathogens were isolated in (28/685, 4.1%), most common being Acinetobacter baumannii complex (10/28, 35.7%) and contaminants in 0.58% samples (4/685). In 368 BacT/ALERT cultures, pathogens were detected in (15/368, 4.1%), most common being Streptococcus spp (5/15, 33.3%), whereas contamination rate was 51.1% cases (188/368). The overall rate of contamination was 18.2% (192/1053). Maximum CSF cultures were received from patients in neurosurgery (350/1053, 33.2%) followed by medicine wards (270/1053, 25.6%). Aerobic spore bacilli (109/1053, 10.35%) was by far the most common contaminant. Conclusion: We believe efforts to decrease the contamination rate are among the most cost-effective, while targeted clinical re-evaluation for all patients with positive CSF cultures remains vital. Due to high sensitivity of automated culture system, it is a requisite to maintain strict aseptic conditions while collecting CSF samples, failure of which may lead to overgrowth of contaminants masking the growth of the true pathogens in culture.

Cerebrospinal Fluid (CSF) Analysis of Tumor-Specific Cell-Free DNA (cfDNA) As a Diagnostic and Prognostic Tool for Central Nervous System (CNS) Invasion in Lymphoma

Background: CNS invasion in aggressive lymphoma (including diffuse large B-cell [DLBCL], double-hit [DHL], Burkitt [BL] and plasmablastic lymphoma [PBL]) remains a major diagnostic and therapeutic challenge. Parenchymal CNS invasion requires a brain biopsy to diagnose, and conventional CSF evaluations lack sensitivity. Furthermore, determining which patients (pts) without CNS disease at diagnosis might be at high risk of future CNS relapse, and thus would most benefit from aggressive CNS prophylaxis, is difficult. Predictors that use clinical variables (e.g. the CNS-IPI) can select a high-risk group with ~10% risk of CNS relapse, but ~50% of events occur in low/intermediate-risk groups. We evaluated a novel CSF assay that detects clonotype-specific cell-free DNA (cfDNA) by next-generation sequencing (NGS) combined with multiplex PCR (Chang T et al, BMC Cancer 2020). This assay can identify minimal residual disease (MRD) in the plasma of pts with DLCBL and can predict systemic relapse (Roschewski et al, Lancet Oncol 2015). Methods: To examine the diagnostic sensitivity of the NGS-MRD assay in the CSF, we prospectively collected CSF from 6 pts with lymphomas who had known parenchymal or leptomeningeal CNS invasion. We also tested stored DNA from historical CSF samples of 8 pts with CNS lymphoma who tested negative by conventional IGH-PCR. We then enrolled 19 pts with newly diagnosed high-risk lymphomas without CNS invasion to examine the prognostic value of the NGS-MRD assay in the CSF collected at diagnosis. In the two-step assay, tumor-specific clonotype sequences were first identified from genomic DNA in primary tumor specimens using NGS of rearranged IGK, IGH (VJ or DJ), and IGL loci. Tumor-specific clonotypes were then selected for tracking in the CSF (split into acellular CSF fluid and cell pellet) and in paired plasma samples. The assay quantitated copy numbers of each tracked sequence per mL of acellular CSF or per 106 cells in the pellet, as well as tumor clonotype frequency (%) per all B-cells. Results: The NGS-MRD detected tumor-specific clonotype in 100% of CSF samples from pts with known CNS lymphoma (n=6; Fig A), including pts with isolated parenchymal-only CNS disease that was not detected by CSF cytology, flow cytometry, or IGH-PCR. For pts with CNS parenchymal disease, median cfDNA copy count in the CSF fluid was 2 /mL (range, 0.4-929) and median clonotype frequency was 9.0% (range, 0.03-68.9%). For pts with leptomeningeal disease, these values were 2233 /mL (1.2-5620) and 37% (1.7-98.4%), respectively (Fig. B). Furthermore, the NGS-MRD assay detected tumor clonotypes in DNA isolates from 6 historical CSF samples (collected in 2014-2018) of pts with known CNS disease, in which lymphoma was not detectable by conventional IGH-PCR (2 additional historical samples failed DNA quality check). Among 19 newly diagnosed pts with no known CNS invasion (median age 57, 42% women, median LDH 352 IU/L), 12 (63%) had DLBCL with high risk of CNS relapse by virtue of high CNS-IPI or epidural tumor, and 7 had other high-risk histologies (2 DHL, 2 BL, HIV+ PBL, and 2 lymphoblastic leukemias). The CSF NGS-MRD assay was positive for tumor-specific clonotype in 8 pts (42%). Median cfDNA copy count in the CSF fluid was 3 /mL (range, 0.9-15.6) and median clonotype frequency was 18.6% (range, 1.4-28.5%). We observed no significant correlation between the amount of cfDNA and number of red cells /mL of CSF (a measure of potential CSF contamination by blood during collection procedure; Fig. C) or between the amount of cfDNA in plasma and in CSF (Fig. D). With median follow-up of 11 months, no pts with negative baseline NGS-MRD assay in CSF relapsed, whereas 2 of 10 with a positive assay had a CNS relapse (12-month incidence, 29%; Fig. E), despite both pts having negative CNS imaging, CSF cytology, flow cytometry, and IGH-PCR at diagnosis. Conclusions: In this proof-of-concept study, the NGS-MRD CSF assay showed 100% sensitivity for diagnosing intraparenchymal CNS invasion in aggressive lymphomas that were not detected in the CSF using conventional methods. In a prospective cohort of newly diagnosed pts without baseline CNS invasion but at high clinical risk for CNS relapse, the assay identified lymphoma-derived cfDNA in 42% of cases. The 100% sensitivity and 100% negative predictive value of the assay for subsequent CNS relapse warrants further prospective evaluation as a potential tool for personalizing CNS-directed prophylaxis. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech, Inc.: Research Funding. Mullins:Adaptive Biotechnologies: Current Employment, Other: shareholder.

Contamination of CSF Culture: Serious Risk to Patient Safety

Background: Acute bacterial meningitis is a medical emergency, and early initiation of appropriate therapy is important to improving outcomes. Culture-based methods are still the standards for the detection of microorganisms in cerebrospinal fluid (CSF). Automated blood culture systems yield better results in cultures sterile body fluids, including CSF. However, the high sensitivity of this technology does not negate the danger of contamination compromising the final outcome. Thus, we tried to study the culture yield in suspected meningitis to determine the rate of contamination. Methods: We conducted a retrospective cohort study of CSF samples collected for culture over 1 year from January 2018 to December 2018 from patients who presented with signs and symptoms indicative of meningitis. The bacterial etiologies and rates of contamination were extracted from laboratory records. Descriptive statistics from Microsoft Excel software were used to analyze patient data. Limited statistical analysis (ie, the Fisher exact test) was performed using SPSS for Windows version 18 software. Results: In total, 1,053 CSF samples were received for aerobic culture, of which the most frequent were conventional cultures (685 of 1053, 65%), whereas BacT/ALERT cultures were requested for 368 of 1,053 cases (35%). Of 685 conventional CSF cultures, pathogens were isolated in 28 of 685 (4.1%), most commonly Acinetobacter baumannii complex (10 of 28, 35.7%). Contaminants were identified in 0.58% samples (4 of 685). In the 368 BacT/ALERT cultures, pathogens were detected in 15 (4.1%), most commonly Streptococcus spp (5 of 15, 33.3%). The contamination rate was 51.1% (188 of 368 cases). The overall rate of contamination was 18.2% (192 of 1,053 cases). The most CSF cultures were received from patients in neurosurgery (350 of 1,053, 33.2%) followed by medicine wards (270 of 1,053, 25.6%). Aerobic spore bacilli was by far the most common contaminant (109 of 1,053, 10.35%).Conclusions: We believe efforts to decrease contamination are among the most cost-effective, but targeted clinical re-evaluation for all patients with positive CSF cultures remains vital. Due to the high sensitivity of the automated culture system, it is a imperative to maintain strict aseptic conditions while collecting CSF samples. Failure to do so may lead to overgrowth of contaminants masking the growth of the true pathogens in culture.Disclosures: NoneFunding: None

Metabolic Profiles Help Discriminate Mild Cognitive Impairment from Dementia Stage in Alzheimer's Disease.

Accurate differentiation between neurodegenerative diseases is developing quickly and has reached an effective level in disease recognition. However, there has been less focus on effectively distinguishing the prodromal state from later dementia stages due to a lack of suitable biomarkers. We utilized the Disease State Index (DSI) machine learning classifier to see how well quantified metabolomics data compares to clinically used cerebrospinal fluid (CSF) biomarkers of Alzheimer’s disease (AD). The metabolic profiles were quantified for 498 serum and CSF samples using proton nuclear magnetic resonance spectroscopy. The patient cohorts in this study were dementia (with a clinical AD diagnosis) (N = 359), mild cognitive impairment (MCI) (N = 96), and control patients with subjective memory complaints (N = 43). DSI classification was conducted for MCI (N = 51) and dementia (N = 214) patients with low CSF amyloid-β levels indicating AD pathology and controls without such amyloid pathology (N = 36). We saw that the conventional CSF markers of AD were better at classifying controls from both dementia and MCI patients. However, quantified metabolic subclasses were more effective in classifying MCI from dementia. Our results show the consistent effectiveness of traditional CSF biomarkers in AD diagnostics. However, these markers are relatively ineffective in differentiating between MCI and the dementia stage, where the quantified metabolomics data provided significant benefit.

Enhanced tES and tDCS computational models by meninges emulation

Objective. Understanding how current reaches the brain during transcranial electrical stimulation (tES) underpins efforts to rationalize outcomes and optimize interventions. To this end, computational models of current flow relate applied dose to brain electric field. Conventional tES modeling considers distinct tissues like scalp, skull, cerebrospinal fluid (CSF), gray matter and white matter. The properties of highly conductive CSF are especially important. However, modeling the space between skull and brain as entirely CSF is not an accurate representation of anatomy. The space conventionally modeled as CSF is approximately half meninges (dura, arachnoid, and pia) with lower conductivity. However, the resolution required to describe individual meningeal layers is computationally restrictive in an MRI-derived head model. Emulating the effect of meninges through CSF conductivity modification could improve accuracy with minimal cost. Approach. Models with meningeal layers were developed in a concentric sphere head model. Then, in a model with only CSF between skull and brain, CSF conductivity was optimized to emulate the effect of meningeal layers on cortical electric field for multiple electrode positions. This emulated conductivity was applied to MRI-derived models. Main results. Compared to a model with conventional CSF conductivity (1.65 S m−1), emulated CSF conductivity (0.85 S m−1) produced voltage fields better correlated with intracranial recordings from epilepsy patients. Significance. Conventional tES models have been validated using intracranial recording. Residual errors may nonetheless impact model utility. Because CSF is so conductive to current flow, misrepresentation of the skull-brain interface as entirely CSF is not realistic for tES modeling. Updating the conventional model with a CSF conductivity emulating the effect of the meninges enhances modeling accuracy without increasing model complexity. This allows existing modeling pipelines to be leveraged with a simple conductivity change. Using 0.85 S m−1 emulated CSF conductivity is recommended as the new standard in non-invasive brain stimulation modeling.

Evaluation of the BioFire FilmArray meningitis/encephalitis panel for the detection of bacteria and yeast in Chinese children.

Meningitis and encephalitis are life-threatening syndromes with high morbidity and mortality in children. Due to limitations of traditional laboratory approaches in etiological diagnosis, the rate of misdiagnoses is unacceptably high. We retrospectively compared the potential clinical impact of the FilmArray meningitis/encephalitis (ME) panel vs. conventional cerebrospinal fluid (CSF) culture in children with central nervous system (CNS) infections. Sixty-eight pediatric patients (<18 years of age) with an initial diagnosis of meningitis or encephalitis were enrolled at 2 children's hospital from January to October 2017. Fifteen specimens were found to be positive after CSF culture, with a positive rate of 22.1% (15/68). For the FilmArray ME panel, 26 bacteria and fungi from 25 samples were detected, and the positive rate was 36.8% (25/68). The FilmArray ME panel identified 14 pathogens in previously pathogen-negative patients. This study demonstrated the capability of the FilmArray ME panel in the diagnosis of bacterial and fungal meningitis and therefore its potential use in facilitating enhanced patient care.