Cerebral Spinal Fluid Samples Research Articles

Molecular Detection of Canine Distemper Virus Among Dogs Showing Neurologic and Non-neurologic Forms of Disease

Background: Canine distemper (CD) is the dogs’ most contagious and lethal viral disease. Despite the widespread use of vaccines to control CD, the prevalence of the CD virus (CDV) has increased at an alarming rate in recent years. Objectives: To identify the genotypes responsible for the neurological and non-neurological clinical forms of CD and to investigate the presence of the virus in the neurological and non-neurological forms of the disease. Methods: In this descriptive-analytical study, the samples were collected from 70 CD suspected unvaccinated dogs with clinical signs of distemper. All cases were tested with rapid tests and separated into 3 groups based on clinical signs and symptoms. Cerebral spinal fluid (CSF), respiratory secretion, and fecal samples of allall 70 cases were examined for reverse transcription polymerase chain reaction (RT-PCR). After sequencing the hemagglutinin gene (H gene), phylogenetic analysis of the gene isolated from CDVs was carried out using MEGA software, version 7. Results: The RT-PCR results showed that the respiratory secretion sample in the non-neurological CDV group (85%) and the neurological CDV group (80%) had the highest level of virus contamination. However, in the non-neurological CDV group, the CSF sample (40%) had a high level of infection. In neurotic groups, cases older than 12 months showed the highest percentage of distemper contamination, and in the non-neurologic CDV group, those between 3 and 6 months were more involved. Sequencing and phylogenetic analysis of the H gene revealed the CDV as a member of the endemic Arctic-like genetic lineage. Conclusion: The genotypic examination of the hemagglutinin gene of the distemper virus reveals that the recent isolates of neurologic and non-neurologic clinical forms of CDV in Iran are similar. In positive rapid test cases, the PCR test of respiratory secretions for virus detection ranks with the highest sensitivity. In neurologic cases with negative rapid test results, PCR of CSF had the highest sensitivity, so it may serve as a diagnostic criterion.

Determination of Ideal Factors for Early Adoption and Standardization of Metagenomic Next-Generation Sequencing for Respiratory System Infections.

Metagenomic next-generation sequencing (mNGS) demonstrates great promise as a diagnostic tool for determining the cause of pathogenic infections. The standard diagnostic procedures (SDP) include smears and cultures and are typically viewed as less sensitive and more time-consuming when compared to mNGS. There are concerns about the logistics and ease of transition from SDP to mNGS. mNGS lacks standardization of collection processes, databases, and sequencing. Additionally, there is the burden of training clinicians on interpreting mNGS results. Until now, few studies have explored factors that could be used as early adoption candidates to ease the transition between SDP and mNGS. This study evaluated 123 patients who had received both SDP and mNGS and compared several variables across a diagnostic test evaluation. The diagnostic test evaluation observed metrics such as sensitivity, specificity, positive and negative likelihood ratios (PLR, NLR), positive and negative predictive values (PPV, NPV), and accuracy. Factors included various sample sources such as bronchoalveolar lavage fluid (BALF), lung tissue, and cerebral spinal fluid (CSF). An additional factor observed was the patient's immune status. Pathogen detection was found to be significantly greater for mNGS for total patients, BALF sample source, CSF sample source, and non-immunocompromised patients (p<0.05). Pathogen detection was found to be insignificant for lung tissue sample sources and immunocompromised patients. Sensitivity, PLR, NLR, PPV, NPV, and accuracy appeared to be higher with mNGS for the total patients, BALF sample source, and non-immunocompromised patients when compared with SDP (p<0.05). With higher metrics in sensitivity, specificity, PLR, NLR, PPV, NPV, and accuracy for overall patients, mNGS may prove a better diagnostic tool than SDP. When addressing sample sources, mNGS for BALF-collected samples appeared to have higher scores than SDP for the same metrics. When patients were in a non-immunocompromised state, mNGS also demonstrated greater diagnostic benefits to BALF and overall patients compared to SDP. This study demonstrates that using BALF as a sample source and selecting non-immunocompromised patients may prove beneficial as early adoption factors for mNGS standard protocol. Such a study may pave the road for mNGS as a routine clinical method for determining the exact pathogenic etiology of lung infections.

BIOM-52. PROTEOMIC PROFILE OF CEREBROSPINAL FLUID IN MEDULLOBLASTOMA AND THEIR RELATED EXTRACELLULAR VESICLE PROTEIN

Abstract The development of a sensitive diagnostic method for medulloblastoma (MBL), a common malignant brain tumor in children, is still a challenge. Cerebral spinal fluid (CSF) contains various substances such as soluble proteins and extracellular vesicles (EVs) and may reflect pathological changes in the central nervous system. Especially, EVs within CSF have great potential in the dynamic monitoring and intervention strategies of MBL. This study attempted to determine whether the total CSF protein selected based on proteome information distinguished from the CSF in MBL is also considered in EVs cargo proteins. CSF samples from patients with MBL (N = 44) and hydrocephalus (HC, control, N = 18) were analyzed with LC-MS. A machine-learning approach was used to identify protein biomarkers discriminating MBL CSF. The biomarker potential of the selected proteins was verified through ELISA and ROC analysis. TEM, Nanosight, and Exoview systems were used to analyze the physiological and biological characteristics of proteins in EVs from MBL CSF. Proteomic analysis revealed four elevated soluble proteins (Transketolase (TKT), SPTBN1, HSP90AA1 and NME1-NME2) in MBL CSF. Verification of the selected proteins in whole CSF confirms that TKT was significantly elevated in MBL than in HC. Importantly, the AUC value of TKT was 0.94, which was recognized as an excellent diagnostic marker. In additional TKT analysis, when MBL was divided into subtypes, Group 4 was the most significant compared to HC, and when divided into metastasis group, metastasis was more significant than non-metastatic compared to HC. Consistently, TKT was more abundant in MBL CSF EVs compared to HC CSF EVs. Our results provide insights into the relevance of the proteins in CSF to the cargo proteins reflected in EVs, while also proposing TKT in CSF EVs as an MBL diagnostic marker.

Evaluation of Laboratories Supporting Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance in the World Health Organization African Region, through the Performance of Coordinated External Quality Assessment.

(1) Background: Laboratories supporting the invasive bacteria preventable disease (IB-VPD) network are expected to demonstrate the capacity to identify the main etiological agents of pediatric bacterial meningitis (PBM) (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) on Gram stains and in phenotypic identification. Individual reports of sentinel site (SSL), national (NL) and regional reference (RRL) laboratories participating in the World Health Organization (WHO)-coordinated external quality assessment, distributed by the United Kingdom National External Quality Assessment (EQA) Services (UK NEQAS) for Microbiology between 2014 and 2019 were analyzed. (2) Methods: The panels consisted of (1) unstained bacterial smears for Gram staining, (2) viable isolates for identification and serotyping/serogrouping (ST/SG) and (3) simulated cerebral spinal fluid (CSF) samples for species detection and ST/SG using polymerase chain reaction (PCR). SSLs and NLs tested for Gram staining and species identification (partial panel). RRLs, plus any SSLs and NLs (optionally) also analyzed the simulated CSF samples (full panel). The passing score was ≥75% for NLs and SSLs, and ≥90% for RRLs and NLs/SSLs testing the full panel. (3) Results: Overall, 63% (5/8) of the SSLs and NLs were able to correctly identify the targeted pathogens, in 2019; but there were challenges to identify Haemophilus influenzae either on Gram stains (35% of the labs failed 2014), or in culture. Individual performance showed inconsistent capacity, with only 39% (13/33) of the SSLs/NLs passing the EQA exercise throughout all surveys in which they participated. RRLs performed well over the study period, but one of the two failed to reach the minimal passing score in 2016 and 2018; while the SSLs/NLs that optionally tested the full panel scored between 75% and 90% (intermediate pass category). (4) Conclusions: We identified a need for implementing a robust quality management system for timely identification of the gaps and then implementing corrective and preventive actions, in addition to continuous refresher training in the SSLs and NLs supporting the IB-VPD surveillance in the World Health Organization, Regional Office for Africa (WHO AFRO).

Quantification of nine antiretroviral drugs in cerebrospinal fluid: An approach to overcome sample collection tube adsorption

A highly sensitive LC-MS/MS methods were developed and validated to quantify nine antiretrovirals (atazanavir [ATV], tenofovir [TFV], emtricitabine [FTC], darunavir [DRV], dolutegravir [DTG], efavirenz [EFV], lamivudine [3TC], raltegravir [RAL], and ritonavir [RTV]) in human cerebral spinal fluid (CSF). The approach remedies adsorption issues caused by polypropylene based sample collection tubes. 1% ammonium hydroxide in methanol was added in an amount equal to the volume of each quality control (QC) or patient sample. Protein precipitation was utilized with a CSF sample volume of 100 μL and a 100 μL of methanol:ACN and vortexed. Chromatographic separation was achieved with a 3 × 100 ACE® C18 column for ATV, DRV, DTG, EFV, RTV and RAL, and a 2 × 100 Polar RP column for TFV/FTC/3TC. Mobile phase was methanol:water:formic acid (70:30:0.1, v/v/v) for ATV, DRV, DTG, EFV and RTV (10 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (35:65:0.1, v/v/v) for RAL (50 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (2:98:0.1, v/v/v) for TFV, FTC and 3TC (50 uL injection, flow rate: 0.35 mL/min). Column temperature was 40° C across all assays. The mass spectrometer was operated in positive, multiple-reaction-monitoring (MRM) mode with electrospray ionization (ESI) for all analytes with the exception of EFV, which was operated in negative, MRM mode with ESI. The assay was linear over the calibration range of 1 to 250 ng/mL for all analytes. The addition of 1% ammonium hydroxide in sample tubes overcame up to 44% negative bias in QC samples and allowed the methods to meet full validation criteria.

An association of CSF apolipoprotein E glycosylation and amyloid-beta 42 in individuals who carry the APOE4 allele

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer’s disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aβ42 levels (r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aβ42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aβ42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aβ metabolism and can be a potential target of treatment.

Association between Cerebrospinal Fluid and Serum Biomarker Levels and Diagnosis, Injury Severity, and Short-Term Outcomes in Patients with Acute Traumatic Spinal Cord Injury.

Acute traumatic spinal cord injury (SCI) is recognized as a global problem that can lead to a range of acute and secondary complications impacting morbidity and mortality. There is still a lack of reliable diagnostic and prognostic biomarkers in patients with SCI that could help guide clinical care and identify novel therapeutic targets for future drug discovery. The aim of this prospective controlled study was to determine the cerebral spinal fluid (CSF) and serum profiles of 10 biomarkers as indicators of SCI diagnosis, severity, and prognosis to aid in assessing appropriate treatment modalities. CSF and serum samples of 15 SCI and ten healthy participants were included in the study. The neurological assessments were scored on admission and at discharge from the hospital using the American Spinal Injury Association Impairment Score (AIS) grades. The CSF and serum concentrations of SBDP150, S100B, GFAP, NF-L, UCHL-1, Tau, and IL-6 were significantly higher in SCI patients when compared with the control group. The CSF GBDP 38/44K, UCHL-L1, S100B, GFAP, and Tau levels were significantly higher in the AIS A patients. This study demonstrated a strong correlation between biomarker levels in the diagnosis and injury severity of SCI but no association with short-term outcomes. Future prospective controlled studies need to be done to support the results of this study.

Biotin accounts for less than half of all biotin and biotin metabolites in the cerebrospinal fluid of children

Background:Biotin is likely transported into cerebral spinal fluid (CSF) via one or more specific transporters. Concentrations of biotin in CSF measured by using modern analytic techniques that are specific for biotin and biotin metabolites have not previously been reported. Objectives:We aimed to accurately measure the concentration of biotin and major biotin metabolites, biotin sulfoxide (BSO) and bisnorbiotin (BNB), in the CSF of children. Design:Concentrations of biotin were determined initially as total avidin-binding substances (TABS) in CSF obtained by lumbar puncture from 55 children. Biotin, BSO, and BNB were quantitated by HPLC and an avidin-binding assay in CSF samples from a subset of 11 children. Results:Concentrations of TABS in CSF averaged 1.6 nmol/L with substantial variability (SD = 1.3 nmol/L). CSF concentrations of biotin and biotin analogs varied widely, but substantial amounts of BSO were detected in every sample. Biotin accounted for 42 ± 16%, BSO for 41 ± 12%, and BNB for 8 ± 14% of the total. It was surprising that the molar sum of biotin, BSO, and BNB on average was >200-fold the TABS concentrations from the same CSF sample. Using several analytic approaches, we found no masking of detection, nor did we find degradation of biotin or BSO. Gel electrophoresis and streptavidin Western blot detected several biotinylated proteins in CSF. Conclusions:Biotin appears to be bound to protein covalently, reversibly, or both, and this binding likely accounts for the increase in detectable biotin after HPLC. Protein-bound biotin may play an important role in biotin nutriture of the brain.

Safety and Tolerability of Concentrated Intraventricular Nicardipine for Poor-Grade Aneurysmal Subarachnoid Hemorrhage-Related Vasospasm.

Objective: To report the preliminary safety, tolerability, and cerebral spinal fluid (CSF) sampling utility of serial injections of concentrated intraventricular nicardipine (IVN) in the treatment of aneurysmal subarachnoid hemorrhage (aSAH). Methods: We report the clinical, radiographic, and laboratory safety and tolerability data of a retrospective case series from a single academic medical center. All patients with aSAH developed vasospasm despite enteral nimodipine and received serial injections of concentrated IVN (2.5 mg/mL). CSF injection safety, tolerability, and utility are defined and reported. Results: A total of 59 doses of concentrated IVN were administered to three patients with poor-grade SAH. In Case 1, a 33-year-old man with modified Fisher scale (mFS) grade 4 and Hunt-Hess scale (HH) score 4 received 26 doses; in Case 2, a 36-year-old woman with mFS grade 4 and HH score 5 received 13 doses; and in Case 3, a 70-year-old woman with mFS grade 3 and HH score 4 received 20 doses. No major safety or tolerability events occurred. Two patients were discharged to a rehabilitation facility, and one died after discharge from the hospital. Conclusions: A concentrated 4 mg IVN dose (2.5 mg/mL) in a 1.6 mL injection appears relatively safe and tolerable and potentially offers a second-line strategy for treating refractory vasospasm in poor-grade SAH without compromising intracranial pressure or cerebral perfusion pressure.

Meningitis Outbreak Caused by &amp;lt;i&amp;gt;Neisseria meningitidis&amp;lt;/i&amp;gt; Serogroup C ST 10217 in 2019 in Diapaga, Burkina Faso

Introduction: Meningitis caused by Neisseria meningitidis constitutes a burden for the countries in the meningitis belt of sub-Saharan in general and particularly for Burkina Faso. In 2019 the Diapaga health district experienced a meningitis epidemic due to N. meningitidis serogroup C. Methods: This is a cross-sectional study with a descriptive aim in the health district of Diapaga where all cases of meningitis were included in this work. Rapid diagnostic tests (RDTs), culture as well as real-time PCR were used for the biological analysis of cerebral spinal fluid (CSF) samples. Results: Of 155 CSF samples analysed, 42% (65/155) were tested positve. Of them, N. meningitidis C accounted for 83% of all positive cases. Likewise, all thirteen (13) NmC strains were susceptible to oxacillin, ceftriaxone, penicillin and chloramphenicol. All strains of NmC belonged to the sequence type (ST) 10 217 and to the clonal complex (CC) 10 217. These CCs belonged to the same variant PorA type: P1.21-15.16; FetA type: F1-7; PorB type: 3-463. Conclusion: Burkina Faso had known an epidemic of meningitis caused by NmC in 2019 in the health district of Diapaga. This outbreak was contained in time due to the performance of the epidemiological surveillance system which made it possible to investigate on time and introduce the vaccine against the pathogen NmC.

DNER-IgG screening of 3290 cerebral spinal fluid samples: Two autoimmune cerebellar ataxia cases without tumors in long-term follow-up

BackgroundThis study aimed to retrospectively report the findings of delta/notch-like epidermal growth factor-related receptor (DNER)-immunoglobin G (IgG) in cerebral spinal fluid (CSF) in two cases with cerebellar ataxia. Methods: CSF samples were collected from 3290 patients with possible autoimmune encephalitis. The immunofluorescence pattern was reviewed using a tissue-based assay. Results: An immunofluorescence pattern with a specific IgG-binding pattern in the cytoplasm of cerebellar Purkinje cells, as well as a fine-dotted pattern in the molecular layer, was found in two samples (0.06%, 2/3290), each from one patient. DNER-IgG was confirmed by cell-based assays. Both of the patients were male and did not have tumors. This suggests that cerebellar ataxia was the main manifestation. Brain magnetic resonance imaging showed non-specific lesions in both patients. One patient responded well to steroid treatment, but the other patient demonstrated a weak response to treatment. During the two-year follow-up period, both patients were stable, and no tumors developed in either patient. ConclusionThe DNER-IgG antibody is rarely found in patients with possible autoimmune encephalitis, and therefore it is not necessarily associated with tumors.

Central Nervous System Infections Associated with the SARS-CoV-2 Virus

Neurological manifestations in addition to the typical clinical symptoms of COVID-19, have been reported around the world. A case series of clinical suspect of SARS-CoV-2 infection in the central nervous system (n=8) were analyzed. Cerebral Spinal Fluid (CSF) and nasopharyngeal swab were analyzed by RT-PCR. Our results showed SARS-CoV-2 in five CSF samples and three patients testing positive for COVID-19 also on nasopharyngeal swab. The patients showed different clinical manifestations, including &lt;95% saturation, fever, cough, dyspnea and abdominal pain. The findings of this study alert to the existence of nonspecific symptoms associated with central nervous system infection caused by SARS-CoV-2.

Ultrasensitive dopamine detection with graphene aptasensor multitransistor arrays

Detecting physiological levels of neurotransmitters in biological samples can advance our understanding of brain disorders and lead to improved diagnostics and therapeutics. However, neurotransmitter sensors for real-world applications must reliably detect low concentrations of target analytes from small volume working samples. Herein, a platform for robust and ultrasensitive detection of dopamine, an essential neurotransmitter that underlies several brain disorders, based on graphene multitransistor arrays (gMTAs) functionalized with a selective DNA aptamer is presented. High-yield scalable methodologies optimized at the wafer level were employed to integrate multiple graphene transistors on small-size chips (4.5 × 4.5 mm). The multiple sensor array configuration permits independent and simultaneous replicate measurements of the same sample that produce robust average data, reducing sources of measurement variability. This procedure allowed sensitive and reproducible dopamine detection in ultra-low concentrations from small volume samples across physiological buffers and high ionic strength complex biological samples. The obtained limit-of-detection was 1 aM (10–18) with dynamic detection ranges spanning 10 orders of magnitude up to 100 µM (10–8), and a 22 mV/decade peak sensitivity in artificial cerebral spinal fluid. Dopamine detection in dopamine-depleted brain homogenates spiked with dopamine was also possible with a LOD of 1 aM, overcoming sensitivity losses typically observed in ion-sensitive sensors in complex biological samples. Furthermore, we show that our gMTAs platform can detect minimal changes in dopamine concentrations in small working volume samples (2 µL) of cerebral spinal fluid samples obtained from a mouse model of Parkinson’s Disease. The platform presented in this work can lead the way to graphene-based neurotransmitter sensors suitable for real-world academic and pre-clinical pharmaceutical research as well as clinical diagnosis.

Detection Analysis of Perioperative Plasma and CSF Reveals Risk Biomarkers of Postoperative Delirium of Parkinson's Disease Patients Undergoing Deep Brain Stimulation of the Subthalamic Nuclei.

This study aimed to explore possible biomarkers of postoperative delirium (POD) of Parkinson's disease (PD) patients received deep brain stimulation (DBS) of the subthalamic nuclei. This nested case control study analyzed perioperative plasma and cerebral spinal fluid (CSF) of patients (n = 40) who developed POD undergone DBS surgery (n = 10) and those who did not (n = 30). Blood sample was collected before surgery and on the first day postoperative, CSF sample was collected at the beginning of the operation. POD was assessed by the Confusion Assessment Method (CAM) twice a day between 7:00 am and 7:00 pm after the surgery until discharge. Plasma and CSF sample from the two groups were analyzed to investigate possible biomarkers for POD in PD patients. There was no difference between POD and Non-POD groups on the concentration of Interleukin 6 and Tumor Necrosis Factor-α in CSF, preoperative plasma and postoperative plasma. There was no difference between POD and Non-POD groups on the concentration of S100 calcium-binding protein β protein (S100β) and Neurofilament light chain (NFL) in preoperative plasma and postoperative plasma. The concentration of C-reactive protein (CRP), NFL and S100β were significant higher in POD group than non-POD group in CSF. The concentration of CRP was significantly higher in POD group than non-POD group in preoperative plasma and postoperative plasma. CSF concentration of S100β might be a potential biomarker for POD via the receiver operating characteristic curve analysis and the area under the curve value of 0.973. For PD patients received DBS surgery, CSF S100β might be a marker for aiding detection of high-risk patients with delirium. This requires further confirmation in clinical trials.

Efficacy of urine reagent strips for analysis of cerebrospinal fluid cellularity and biochemical parameters

Background: Meningitis is a severe infection affecting the central nervous system requiring early management to prevent debilitating consequences and death. Detection of meningitis can be done by the analysis of cerebrospinal fluid cellularity and biochemistry (glucose and protein concentration). Urine reagent strips can be used to assess these variables and reach to the diagnosis of meningitis early, thereby, reducing the delay in the initiation of its treatment. Aims and Objectives: The current study aimed to determine the efficacy of urine reagent strips for the analysis of CSF. Materials and Methods: The current prospective study was done in a tertiary care center in Rewa, M.P., on 100 cerebral spinal fluid (CSF) samples obtained from the children suspected of meningitis, which were, then, analyzed by both the conventional methods of CSF analysis (including routine biochemical and microscopic analysis) the index test, utilizing the Combur-10 urine reagent strips for the estimation of CSF cell, protein, and glucose. These results were, then, correlated to calculate the diagnostic utility of urine reagent strips for the rapid diagnosis of meningitis. Results: The sensitivity, specificity, and diagnostic accuracy of urine reagent strips for the detection of cell count &gt;10/cumm were 81.81%, 98.21%, and 91%, respectively, and 78.57%, 81.25%, and 79% for detection of protein &gt;30 mg/dL. Reagent strips showed a sensitivity and specificity of 96.85% and 79.41% for the detection of glucose levels &lt;50 mg/dL. The two methods showed an almost perfect degree of agreement for the detection of leucocytes with Cohen’s kappa value of 0.86. Conclusion: Reagent strips can be a helpful bedside screening test for the detection of meningitis and also in resource-limited settings.

PATH-16. Noninvasive diagnosis of gliomas through CSF cfDNA sequencing in pediatric and adolescent and young adult (AYA) patients

Abstract PURPOSE: A subset of pediatric, adolescent and young adult (AYA) gliomas are located in the brainstem, eloquent locations, or present with diffuse/leptomeningeal disease, and are associated with high risk and low yield of biopsy. At the same time, accurate molecular diagnosis is necessary to direct optimal therapy. In such cases, analysis of cell free DNA (cfDNA) form cerebral spinal fluid (CSF) may represent a diagnostic alternative to biopsy. METHODS: We investigated the utility of CSF cfDNA sequencing through a stepwise approach, using clinically validated, targeted molecular assays. Testing was performed using a broad hybrid capture next generation sequencing assay (MSK-IMPACT) and subsequent targeted digital droplet PCR in a subset of cases. RESULTS: We analyzed 17 CSF samples from 17 pediatric (n=6) and AYA (n=11) glioma patients with primary or recurrent disease. Thirteen had tumors located within the brainstem, and four had leptomeningeal involvement. Somatic alterations were detected in 12/17 samples (71%). In 3/4 patients with leptomeningeal involvement, cfDNA testing revealed a BRAF fusion consistent with the diagnosis of diffuse leptomeningeal glioneuronal tumor (DLGNT). Among the 13 patients with brainstem involvement, four had somatic H3 K27M mutations, three had IDH mutations, and one had TP53 and ATRX mutations; five patients had no detectable mutations. CONCLUSION: In our analysis, we found that established glioma hotspot mutations were able to be detected within the CSF. We propose that in patients for whom tissue biopsy is high risk, not feasible, or tissue was nondiagnostic, CSF cfDNA sequencing has a substantial diagnostic yield and should be considered as a valuable novel diagnostic tool. Ongoing research is aiming to further increase the sensitivity of cfDNA testing, especially in patients with very low levels of CSF cfDNA.

IgG kappa multiple myeloma with isolated central nervous system relapse.

A 76-year-old male presented with acute renal failure and cast nephropathy was diagnosed with IgG kappa multiple myeloma (MM). His paraprotein was 40 g/L, kappa-free light chain level was 17,058 mg/L, kappa: lambda light chain ratio was 1445. An automated full blood count showed hemoglobin 90 g/L, white blood cells 2.96 × 109/L, neutrophil 1.7 × 109/L, and platelet 179 × 109/L. Bone marrow trephine sample was hypercellular with nearly 100% plasma cells, which were positive for CD138, CD56, and cyclin D1, and showed kappa light chain restriction. Fluorescence in situ hybridization analysis with dual color/dual probes for immunoglobulin heavy chain (IGH) and MYC genes (Cytocell) and whole genome screening using 8 × 60K oligonucleotide arrays (Agilent) performed on the CD138 enriched cells (CD138 microbeads Miltenyi Biotech) from the liquid bone marrow sample showed no evidence of IGH rearrangement, MYC rearrangement, 1q gain, 1p loss, 17p loss, or any clinically significant imbalance. He was treated with eight cycles of bortezomib, cyclophosphamide, and dexamethasone (VCD) to very good partial response. He relapsed 30 months after diagnosis with paraprotein of 36 g/L, kappa light chains of 10,000 mg/L, and creatinine of 498 μmol/L. He was treated with eight cycles of bortezomib, thalidomide, and dexamethasone (VTD) to paraprotein undetectable and kappa light chain 29 mg/L. Seven months post completion of VTD (4 years after myeloma diagnosis), he presented with abnormal gait, falls, and a left-sided facial droop. His paraprotein was 2 g/L and serum-free kappa light chain was 81 mg/L. Magnetic resonance imaging (MRI) head and whole spine showed multifocal diseases in spinal canal and intracranially, involving the cranial nerves most prominent the fifth, seventh, eighth, and hypoglossal nerve on the left, anterior surface of the inferior pons medulla (Figure 1A). There were multiple lesions in the cervical spine as well as in the thoracic spine (Figure 1B). Computerized tomography (CT) chest abdomen pelvis did not show the evidence of other new diseases. Plasma cells were detected in cerebral spinal fluid (CSF) (Figure 1C, May–Grünwald–Giemsa stain x100 objective). He was treated with pomalidomide and dexamethasone. Weekly cytarabine intrathecal (IT) treatment was given until the clearance of plasma cells from CSF samples for three times. Four months later, he achieved the complete remission on MRI head and whole spine with undetectable paraprotein and normal light chain ratio. Serial MRI scans confirmed ongoing remission. A year later, he relapsed again with worsening mobility and reappearance of myeloma cells in CSF. By flow cytometry, these cells were positive for CD138, CD38, CD56, CD117, and negative for CD19. MRI head showed leptomeningeal disease with a new 2.2 cm frontal lobe lesion. His paraprotein remained undetectable and kappa light chain remained at below 100 mg/L. He continued systemic pomalidomide and dexamethasone chemotherapy and received palliative whole brain radiotherapy 20 Gy in 5 fractions for symptom control. His disease continued progressing and he passed away 3 months later (5.5 years after myeloma diagnosis). Here, we reported a myeloma patient with isolated CNS relapse, who achieved 1 year of remission with combination of intrathecal chemotherapy and systemic pomalidomide and dexamethasone treatment, but sadly passed away 4 months after second CNS relapse. CNS MM is a rare form of extramedullary disease, but carries a very poor prognosis. The optimal approach to treatment of CNS MM is not currently known. The current approach includes systemic therapy with agents known to cross the blood brain barrier, intrathecal therapy, and CNS irradiation. Innovative approach to treatment is urgently needed. The authors received no specific funding for this work. Ke Xu and Lucy Kamuriwo wrote the manuscript. Ke Xu, Lucy Kamuriwo, Claire Anderson and Parag Jasani critically revised the final version of the manuscript. The authors declare they have no conflicts of interest. No written or verbal consent obtained from patient. Patient has passed away.

Fully automated immunoassays for measuring beta‐amyloid peptide (1‐42) and total tau in cerebral spinal fluid

AbstractBackgroundBeta‐amyloid1‐42 (Aβ42) and tau protein are cerebral spinal fluid (CSF) biomarkers for progression to AD. The sensitivity requirements for Aβ42 and total tau assays are in the pg/mL range, a challenge for any methodology. The use of these biomarkers for clinical purposes requires assays that can reliably deliver accurate and reproducible results.MethodNew prototype immunoassays for Aβ42 and total tau on a fully automated clinical analyzer [Siemens Healthineers ADVIA Centaur® XPT Immunoassay System] are under development to enable routine testing of AD biomarkers in clinical laboratories. Each assay uses an antigen/antibody sandwich format and chemiluminescence detection. Standard curves from 0 to 4000 pg/mL and 0 to 6500 pg/mL were generated for the Aβ42 and Tau assays, respectively. Synthetic Aβ42 and recombinant tau‐441 were spiked into an immunodepleted (ID) CSF pool and tested. Precision was evaluated over 6 days by testing a CSF pool and buffer controls on two analyzers. Functional sensitivity was estimated using the precision data. Clinical utility was evaluated by testing 30 clinical CSF samples from subjects with and without AD.ResultRecoveries of spiked Aβ42 from CSF ranged from ‐13% to +0.7% over 113 to 3179 pg/mL. Recoveries of spiked tau‐441 from CSF ranged from ‐11% to +13% over 35 to 3826 pg/mL. The repeatability and within‐laboratory precision of the Aβ42 assay ranged from 1.3% to 8.6% and 1.9% to 9.9%, respectively, over 107 to 2005 pg/mL. The repeatability and within‐laboratory precision of the total tau assay ranged from 1.2% to 10.8% and 1.6% to 12.5%, respectively, over 108 to 3300 pg/mL. The functional sensitivities of the Aβ42 and total tau assays were estimated to be &lt; 200 pg/mL and &lt; 80 pg/mL, respectively. Using ROC analysis, a ratio of Aβ42 and total tau results was able to differentiate AD from non‐AD subjects with 90% sensitivity and 95% specificity (AUC 0.95).ConclusionWhen finalized, the fully automated Aβ42 and total tau assays will have performance characteristics suitable for routine use in a clinical laboratory. The performance of the assays in combination with automation will make testing for these important biomarkers more reliable and routine.

Acute Cortisol Profile Associations With Cognitive Impairment After Severe Traumatic Brain Injury

Background Cognitive impairments commonly occur after traumatic brain injury (TBI) and affect daily functioning. Cortisol levels, which are elevated during acute hospitalization for most individuals after severe TBI, can influence cognition, but this association has not been studied previously in TBI. Objective We hypothesized that serum and cerebral spinal fluid (CSF) cortisol trajectories over days 0–5 post-injury are associated with cognition 6-month post-injury. Methods We examined 94 participants with severe TBI, collected acute serum and/or CSF samples over days 0–5 post-injury, and compared cortisol levels to those in 17 healthy controls. N = 88 participants had serum, and n = 84 had CSF samples available for cortisol measurement and had neuropsychological testing 6 months post-injury. Group based trajectory analysis (TRAJ) was used to generate temporal serum and CSF cortisol profiles which were examined for associations with neuropsychological performance. We used linear regression to examine relationships between cortisol TRAJ groups and both overall and domain-specific cognition. Results TRAJ analysis identified a high group and a decliner group for serum and a high group and low group for CSF cortisol. Multivariable analysis showed serum cortisol TRAJ group was associated with overall cognitive composites scores (P = .024) and with executive function (P = .039) and verbal fluency (P = .029) domain scores. CSF cortisol TRAJ group was associated with overall cognitive composite scores (P = .021) and domain scores for executive function (P = .041), verbal fluency (P = .031), and attention (P = .034). Conclusions High acute cortisol trajectories are associated with poorer cognition 6 months post-TBI.

Long-term intrathecal administration of morphine vs. baclofen: Differences in CSF glycoconjugate profiles using multiglycomics

Opioid use for treatment of persistent pain has increased dramatically over the past two decades, but it has not resulted in improved pain management outcomes. To understand the molecular mechanisms of opioids, molecular signatures that arise from opioid exposure are often sought after, using various analytical methods. In this study, we performed proteomics, and multiglycomics via sequential analysis of polysialic acids, glycosaminoglycans, N-glycans and O-glycans, using the same cerebral spinal fluid (CSF) sample from patients that had long-term (>2years), intrathecal morphine or baclofen administered via an indwelling pump. Proteomics and N-glycomics signatures between the two treatment groups were highly conserved, while significant differences were observed in polysialic acid, heparan sulfate glycosaminoglycan and O-glycan profiles between the two treatment groups. This represents the first study to investigate the potential relationships between diverse CSF conjugated glycans and long-term intrathecal drug exposure. The unique changes, observed by a sequential analytical workflow, reflect previously undescribed molecular effects of opioid administration and pain management.