CSF Samples Research Articles

Abstract B015: CSF liquid biopsies reveal disease status and resistance mechanisms in children undergoing targeted therapy for malignant brain tumors

Abstract BACKGROUND: Patients with relapsed or refractory brain tumors face poor outcomes due to the aggressive nature of their disease. Recent studies suggest that administration of cell cycle inhibitors may help slow tumor progression. In the Phase I trial SJDAWN (NCT03434262) conducted at St. Jude Children’s Research Hospital, CDK4/6 inhibitor therapy (ribociclib) was assessed for safety and efficacy in such patients. To understand how tumors evolve in response to this treatment, longitudinal CSF liquid biopsies from SJDAWN were retrospectively analyzed by cell-free DNA (cfDNA) sequencing. METHODS: CSF samples (n=143) were collected by lumbar puncture from 34 of 68 (50%) patients before and during treatment. cfDNA was extracted and analyzed using an innovative DNA methylation-based platform that concurrently detects tumor-associated copy number alterations, classifies tumor subtypes, and distinguishes malignant from non-malignant methylation signatures. cfDNA inputs ranged from 50 to 500 pg. CSF profiles were compared to patient-matched tumor tissue DNA methylation arrays and subsequently correlated with MRI findings at corresponding timepoints. RESULTS: Circulating tumor DNA (ctDNA) was detected in 29 of 34 (85%) patients, yielding accurate tumor classification even in the setting of diploid genomes. CNV profiles from CSF liquid biopsies strongly correlated with patient-matched tumor tissue methylation arrays and often revealed genetic events not previously captured in the tumor tissue. Resistance to CDK4/6 inhibition was characterized by focal amplification of positive cell cycle regulators, including CDK6, CCND2, MYCN, and RRM2 that were detectable in CSF ∼6 months prior to radiographic progression. Remarkably, three high-risk Group 3/4-MB patients were progression-free for >1.5 years. The trend of their longitudinal cfDNA profiles varied: one had persistent ctDNA detected throughout therapy with resistance emerging at ∼1.5 years; another had undetectable ctDNA for ∼2 years before reemergence of tumor; and the third remained MRD-negative, potentially owing to reduced ctDNA release resulting from CDK4/6 inhibition. Methylation changes in response to treatment were also observed, providing unprecedented insights into epigenetic landscapes associated with tumor evolution concurrent with targeted therapy. CONCLUSIONS: Methylation-based analysis of CSF liquid biopsies provides an ultra-sensitive and comprehensive method for serial disease monitoring. Even with sub-nanogram cfDNA inputs, tumor reemergence and resistance mechanisms were detectable months before MRI changes, enabling timely clinical intervention. Resistance to CDK4/6 inhibition is mediated by focal amplification of cell cycle regulators, suggesting alternative approaches will be required to overcome this path of resistance. CDK4/6 inhibitor therapy offers clinical benefit to a specific subset of high-risk Group 3/4-MB patients, motivating expanded evaluation of this population in future trials. Citation Format: Katie Han, Kyle S Smith, Anna Kostecka, Sandeep Dhanda, Hong Lin, Margit Mikkelsen, Giles W Robinson, Paul A Northcott. CSF liquid biopsies reveal disease status and resistance mechanisms in children undergoing targeted therapy for malignant brain tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B015.

Prevalence of comorbid autoimmune diseases and antibodies in newly diagnosed multiple sclerosis patients.

Diagnosing multiple sclerosis (MS) is challenging due to diverse symptoms and the absence of specific biomarkers. Concurrent autoimmune diseases (AID) or non-specific antibodies further complicate diagnosis, progression monitoring, and management. Data on AID prevalence in MS patients are sparse. This study aims to identify concurrent AIDs alongside MS. In this retrospective single-center study, we analyzed patient records at our university hospital from 2010 to 2017, focusing on cases suspected of inflammatory demyelinating disease. The 2017 McDonald criteria were applied. Additionally, we measured neurofilament light (NfL) levels from available CSF samples in our biobank. We identified a total of 315 patients, of whom 66% were women. In total, 13.7% of all patients had concurrent AID, while 20.3% had isolated antibody findings without AID. The most common AID was autoimmune thyroiditis (8.9%), followed by chronic inflammatory skin diseases (1.6%), arthritis (1%), type 1 diabetes (1%), Sjögren's syndrome (0.6%), and inflammatory bowel diseases (0.6%). Cardiolipin antibodies were the most frequent isolated antibodyfinding (8.6%). Our data showed that, from the perspective of the initial demyelinating event, neither comorbid AID nor isolated antibodies significantly influenced relapses or MS progression over a median follow-up of 9months. Standard CSF parameters and NfL levels were similar between the groups at the time of MS diagnosis. Our study shows that AIDs, particularly autoimmune thyroiditis, frequently occur at the onset of MS. The proportion of AIDs commonly treated with immunomodulatory therapy in our cohort was similar to that observed in the general population. Comorbid AID did not affect NfL levels, indicating similar disease activity. Future research should explore new AID emergence during the course of MS, especially considering the increased incidence of rheumatic diseases later in life.

PATH-52. DIFFERENCES IN GENE EXPRESSION, GENOME-WIDE METHYLATION AND METABOLITES IN ADULT DIFFUSE GLIOMAS

Abstract Metabolic reprogramming is a hallmark of cancer, including diffuse gliomas. IDH1 and IDH2 are key enzymes linking cellular metabolism to epigenetic regulation and redox states. IDH1/IDH2 enzymes catalyze the conversion of isocitrate to α-ketoglutarate, while the mutant enzymes increase the production of D-2-hydroxyglutarate, an oncometabolite that induces epigenetic changes, leading to gene expression alterations. Understanding how epigenetic alterations affect metabolic gene expression and metabolite levels in diffuse gliomas could help identify diagnostic biomarkers and therapeutic targets. We studied the expression of metabolism-related genes, metabolites, and genome-wide methylation status, which will facilitate an integral understanding of the metabolic changes in IDH-mutant and IDH-wildtype gliomas. Study design included 47 fresh frozen tissue samples. RNA was extracted and used for gene expression analysis using the NanoString platform (nCounter® Metabolic Pathways Panel), while extracted DNA was used for targeted sequencing with a panel interrogating 600 genes and for genome-wide methylation analysis using the Illumina Epic v2. In addition, unbiased metabolomic analysis by mass spectrometry was performed in 25 tissue and CSF samples out of 47 samples. Methylation status, gene expression and metabolite levels were compared between IDH-mutant and IDH-wildtype gliomas. Results showed significant differences in DNA methylation, gene expression and metabolites between IDH-mutant and IDH-wildtype gliomas. UPP1, an enzyme involved in uracil metabolism, was studied in depth to understand the interplay between DNA methylation, gene expression, and metabolic reprogramming in diffuse glioma. Epigenetic analysis revealed that the CpG sites within the UPP1 gene displayed reduced methylation in IDH wild-type compared to IDH-mutant tumors. IDH wild-type tumors showed higher UPP1 expression compared to their IDH-mutant counterparts. Consequently, we observed higher levels of uracil in tissue and CSF samples from patients with IDH wild-type gliomas. These data provide a framework to study the relationship between mutations, methylation, gene expression and metabolism in diffuse gliomas.

CTIM-02. PIONEERING QUAD-TARGETING CAR T CELL THERAPY IN PEDIATRIC CNS TUMORS – ANALYSIS FROM THE INITIAL PATIENTS TREATED ON THE FIRST-IN-HUMAN PHASE 1 TRIAL BRAINCHILD-04

Abstract BACKGROUND BrainChild-04 is a first-in-human clinical trial of quad-chimeric antigen receptor (CAR) T cell therapy for children and young adults with central nervous system (CNS) tumors. This trial administers repeated locoregional CAR T cells targeting B7-H3, EGFR, HER2, and IL-13Ralpha2 (“quad-CAR T cells”), leveraging multi-antigen targeting to address the tumor heterogeneity of high-grade CNS tumors. METHODS The primary endpoints are feasibility and safety, and secondary endpoints are disease response and correlative studies of CAR T cell activity. The trial utilized a Bayesian Optimal Interval (BOIN) statistical design with three planned dose regimens(DR). There are 2 arms with weekly delivery for 3 weeks of a 4-week cycle:(A) –diffuse intrinsic pontine glioma (DIPG), (B) –non-pontine diffuse midline glioma (DMG) or other relapsed CNS tumors. RESULTS Enrolled patients include DMG N=6, DIPG N=5, other CNS tumors N=4. Of the 15 enrolled patients, 7 have been infused with 8 awaiting infusions. A total of 33 intracranial CAR T doses have been delivered, including Arm A DR 1 (N=1), Arm B DR 1 (N=3), DR2 (N=3). The most common adverse events have been grade 1-2 fever (7/7 patients), grade 1-3 headache (6/7), and grade 1-2 nausea/vomiting (6/7). There have been no DLTs and no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity (ICANS). CTCAE and tumor inflammation-associated neurotoxicity are both being captured. All treated patients are alive with median follow up time post-initial infusion of 93(14-198) days. CAR T cells were detected by flow cytometric analysis of CSF post-infusion in 14/24 CSF samples. Radiographic response, CSF circulating tumor DNA, targeted mass spectrometry, and cytokine analysis will be presented. CONCLUSIONS Our preliminary experience suggests tolerability of locoregional delivery of quad-CAR T cells at doses currently evaluated. The trial remains ongoing and these data support continued evaluation of this product to better assess anti-tumor activity in patients with CNS tumors, including DIPG/DMG.

BIOM-28. WHOLE EXOME TUMOR MOLECULAR PROFILING FROM CEREBROSPINAL FLUID (CSF): PRELIMINARY RESULTS OF A PILOT STUDY

Abstract BACKGROUND Tissue sampling for molecular profiling has become an integral part of the diagnosis, prognosis, and management of CNS malignancies. However, many tumors are surgically inaccessible, and even biopsy of accessible lesions conveys some risk, and the possibility of sampling error. We evaluated the accuracy and potential utility of CSF liquid biopsy for identifying mutations in patients with intracranial tumors. METHODS CSF samples were collected from ventricular reservoirs and stored at -80 degrees Celsius until analyzed. Cell-free nucleic acids were extracted, concentrated, and quantified. Samples over 110ng quantification were normalized to 110ng and input into library prep. Library prep was done using v3.5 of Caris Assure and whole exome sequencing in an S2 FC on a Novaseq 6000. Bioinformatic analysis was performed. RESULTS CSF samples were obtained from 20 patients (10 gliomas, 5 CNS lymphomas, 5 metastases), 19 of whom also had tumor specimens available. Two hundred eighty-one mutations were identified: 17 (6.6%) in tumor only, 184 (72.4%) in CSF only, and 80 (31.5%) in both. Key alterations found in CSF include: IDH, TERT, ATRX in gliomas, and MYD88, PIM1, BCL2, TERT in lymphomas. Glioma CSF with IDH1 mutations did not have PTEN mutations (with one exception, in which the CSF was collected 3 years after the tissue). Targetable mutations in metastases included PIK3CA, NF1, and BRCA. CONCLUSION These preliminary results suggest that CSF liquid biopsy may be useful for identifying mutations in primary and metastatic brain tumors. Serial sampling is feasible, and may disclose changes in diagnostic and/or driver mutations over time, with important therapeutic and diagnostic implications. Many mutations were found in CSF alone. CSF may capture the entire molecular landscape of the tumor, overcoming the sampling error inherent in tissue biopsies. Prospective studies are underway to further elucidate the clinical applications of this technique.

CTNI-15. A PERIOPERATIVE STUDY OF SAFUSIDENIB IN PATIENTS WITH IDH1 MUTATED GLIOMA

Abstract BACKGROUND IDH mutations cause aberrant accumulation of (R)-2-hydroxyglutarate (2-HG), an oncometabolite that promotes tumorigenesis through abnormal hypermethylation of histones and DNA as well as suppression of normal cellular differentiation. IDH inhibition significantly improves progression free survival in patients with IDH mutant WHO grade 2 glioma, however most patients progress, and there is a lack of understanding on mechanistic reasons for failure. METHODS We tested Safusidenib, an oral, blood brain barrier penetrant IDH1 R132X enzyme inhibitor in a single arm, open label perioperative study. Patients with IDH1 mutated glioma, treatment naïve to radiation or chemotherapy, received Safusidenib (250 mg BID) for four weeks following surgical biopsy and prior to definitive resection (Part A). After resection patients continued Safusidenib until toxicity or progression (Part B). Translational endpoints include paired whole genome transcriptome sequencing (WGTS), single nuclei RNA sequencing (snRNAseq), spatial transcriptomics and spatial metabolomics as well as longitudinal sampling of blood and CSF for circulating tumour DNA (ctDNA). Here we are reporting Part A of the study. RESULTS As of May/27/2024 10 patients (mean age 37, 40% female) have been enrolled: seven with astrocytoma, three with oligodendroglioma; six had prior surgical resection. Pharmacokinetic sampling showed a mean tumor concentration 2457 ng/mL (range 957.0 – 4810) with tumor:plasma ratio 0.33. Consistent with mechanism, tumor 2-HG reduced 86% from biopsy (mean 75.0μM, range 7.8-203) to surgery (10.3μM, range 2.3 – 30.8), as well as identification of novel biomarkers of response. Paired tumour snRNAseq reveals transcriptional shift with changing cell-to-cell communication, and increased immune infiltration post-treatment, validated by spatial transcriptomics. Additionally, we find reduced heterogeneity consistent with a more differentiated tumour state. Treatment was well tolerated with ongoing follow-up. CONCLUSIONS This innovative trial facilitates intra-patient comparisons and confirmation of the on-target effects of Safusidenib, enhancing understanding of the mechanisms of response and resistance. The approach serves as a proof of concept for advancing drug development in glioma through perioperative trials.

CTNI-62. EVALUATION OF SAFER-SEQS CTDNA ASSAY OPTIMIZED FOR DETECTING LOW CTDNA COUNTS FROM CSF AND PLASMA: ASSESSING SENSITIVITY AND FEASIBILITY FOR CLINICAL IMPLEMENTATION

Abstract BACKGROUND Gliomas with BRAF alterations are often difficult to treat in the recurrent setting due to emergent resistance to FDA-approved targeted therapies. Additionally, it is difficult to assess response to therapy given the limitations of radiographic techniques and the infeasibility of serial tissue sampling. This protocol will serve as a prototype for determining the feasibility of using CSF and plasma circulating tumor DNA (ctDNA) as biomarkers for response to a novel-BRAF inhibitor, plixorafenib. Plixorafenib is a small-molecule selective inhibitor of Class 1 and 2 BRAF mutations that does not induce paradoxical reactivation of MAPK signaling. METHODS This clinical trial is designed as a single institution, signal-finding study to demonstrate the feasibility and sensitivity of detecting ctDNA at baseline and after one month of treatment with plixorafenib (primary endpoint). In addition, we will evaluate the correlation of ctDNA with disease status over time and response rate to plixorafenib. Patients aged 18 years or older with measurable recurrent BRAF-V600 mutant glioma (by RANO 2.0), who have received prior BRAF and/or MEK inhibitor therapy will be screened and consented for the study prior to surgery. Leptomeningeal disease allowed. A total of 12 evaluable patients will be enrolled. Patients will undergo clinically-indicated resection or biopsy for confirmation of disease progression and characterization of putative resistance alterations. All patients will have a ventricular reservoir placed at time of surgery with CSF and plasma sampling. Patients will initiate the study drug (oral plixorafenib 900mg daily with cobicistat 150mg daily) when clinically recovered from surgery. Patients will take the drug continuously for 28-day cycles. MRI, CSF, and plasma assessments will occur approximately every two months to evaluate disease status. Enrollment is planned to begin November, 2024.

NCMP-09. SPONTANEOUS RESOLUTION OF PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML) DEVELOPED AFTER RITUXIMAB TREATMENT FOR LOW-GRADE B-CELL LYMPHOMA

Abstract Progressive multifocal leukoencephalopathy (PML) is a rare viral infection caused by the reactivation of the JC virus, which causes significant morbidity and mortality. Rituximab, a CD20 inhibitor used for B-cell neoplasms as well as autoimmune diseases, is associated with the development of PML. Here, we report a case of a 74-year-old woman with a history of low-grade B-cell lymphoma with biopsy-proven PML with spontaneous immune reconstitution with clinical and radiographic improvement. For her low-grade B-cell lymphoma, she received six cycles of rituximab and bendamustine, followed by one cycle of maintenance rituximab, which was discontinued due to intolerance. Ten months later, she developed progressive left-sided paresthesias, paresis, and incoordination. Brain MRI revealed a large right subcortical frontal-parietal FLAIR hyperintense and peripherally enhancing lesion with leading-edge diffusion restriction. CSF studies revealed elevated protein (385 mg/dL) and negative JCV PCR. A brain biopsy was performed with tissue staining positive for SV40, consistent with PML. Her CD4 count was 88 cells/mm3. A repeat CSF sample one month later showed near normalization CSF protein (47 mg/dL) and negative CSF JCV PCR. MRI of the brain one month later demonstrated complete enhancement resolution and decreased FLAIR abnormality, which correlated with subjective and objective clinical improvement. Her serum CD4 also increased to 134 cells/mm3. This highlights a rare case of PML related to rituximab with spontaneous improvement, which may help guide clinicians in counseling patients who may also develop this complication. Furthermore, this supports the biological rationale for continued investigation of T-cell therapies to achieve immune reconstitution.

Phenotypic, Molecular Identification and Virulence Assessment of Cronobacter spp Isolated from Clinical Samples of Children Under Two Years in Mosul City/Iraq

Cronobacter spp. is known to be a foodborne causative agent for a variety of diseases in both humans and animals. This study focused on isolating Cronobacter species from 150 clinical samples of children under two years (60 from blood and stool samples, 30 from CSF samples) collected from Ibn al Atheer and Al Khanssa Hospitals. The phenotypic identification of bacterial isolates were performed through culture, microscopic, and biochemical examinations and vitek-2 system. The results revealed a mixture of bacteria in 6.5% of the clinical specimens. The identity of isolates was 99% using the vitek-2 system for identifying Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter pulveris. Out of 150 specimens there were 8 (5.33 %) of specimens gave positive for Cronobacter spp which included: C. sakazaki 2/60 blood specimens (3.3%), C. sakazaki 2/60 stool specimens (3.3%), C. malonaticus 2/60 stool specimens (3.3%), 1/60 C. muytjensii and C. pulveris 1/60 (3.3%). The eight isolates phenotypically identified as Cronobacter were confirmed at the molecular level through 16S rRNA sequencing and submitted to NCBI under the accession numbers (OR825874, OR825875, PP126443, PP126444, PP126445, PP126455). The virulence profile of these isolates showed that 7/8 (87.5%) of Cronobacter strains exhibited haemolysin activity, 5/8 strains (62.5%) were able to produce the protease enzyme and 2/8 (25%) of Cronobacter strains were positive for lipase and lecithinase. All strains lacked the ability to produce a slime layer. The results also showed the ability of Cronobacter strains to produce biofilm by using the tube method and microtiter plate method but with different levels.

α-Synuclein plastic antibody applied to monitor monomeric structures and discriminate aggregated forms in human CSF

Aggregation of alpha-synuclein (aSyn) occurs in presynaptic neurons and constitutes a key factor for the progression of Parkinson's disease, emphasising the urgency of early detection to support effective treatment. Unfortunately, a reliable, sensitive and cost-effective diagnostic tool has so far been lacking.Thus, this work presents a novel biosensor for detecting aSyn using plastic antibodies coupled to electrochemical detection. This biosensor was designed for portability and compatibility with point-of-care devices and exploits the electropolymerization of methylene blue (MB) together with aSyn on the carbon working electrode of screen-printed electrodes (SPEs). By electrochemical impedance spectroscopy (EIS) measurements, the sensor showed exceptional analytical performance in detecting aSyn monomers in human CSF samples. It showed a linear trend of response from 1 fM to 10 pM with an impressively low limit of detection of 69 aM. Selectivity tests confirmed the predominant response to aSyn monomers, a less intense response to oligomers and insensitivity to fibrils.Overall, this plastic antibody-based electrochemical sensor represents a significant breakthrough as it is the first of its kind to accurately, sensitively and selectively detect aSyn monomers with a partial response to oligomers. Its simplicity and reproducibility promise to contribute to the early and effective diagnosis of Parkinson's disease.

Autoimmune Encephalitis and Paraneoplastic Neurologic Syndromes: A Nationwide Study on Epidemiology and Antibody Testing Performance.

Autoimmune encephalitis (AIE) and paraneoplastic neurologic syndromes (PNSs) encompass a heterogeneous group of antibody-associated disorders. Both the number of syndromes and commercially available antibody tests have increased considerably over the past decade. High-quality population-based data on epidemiology of these disorders and real-world performance of antibody tests are needed. In this nationwide retrospective cohort study, we identified all serum and CSF samples tested for antibodies against intracellular antigens (IAs: Hu [ANNA1], Yo [PCA1], CV2 [CRMP5], Ri [ANNA2], Ma1, Ma2 [Ta], amphiphysin, GAD65, GFAP, KLHL11, CARP VIII) or extracellular antigens (EAs: NMDAR, LGI1, Caspr2, GABA-B-R, GABA-A-R, AMPAR, DPPX, GlyR, mGluR1, VGCC, IgLON5, Tr [DNER]) between January 2016 and December 2021 in the Netherlands. Nationwide coverage was guaranteed for all antibodies except anti-GAD65 and anti-VGCC. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV); obtained clinical information about patients who tested positive; assigned diagnosis of AIE/PNS according to diagnostic criteria; and calculated incidence rates for IA, EA, and individual antibody-associated syndromes. In the study period, 2,877 (9.5%) of 30,246 samples, belonging to 1,228 patients, tested positive. Sensitivity and specificity were high (>95%) to very high (>99%) for most tests in both serum and CSF. PPVs for several tests were moderate to poor, especially for serum testing of IA antibodies (25%-80%). Clinical data were available for 940 (76.5%) of 1,228 patients. A total of 578 AIE/PNS diagnoses were made. The incidence rate for AIE/PNS (per million person-years) increased from 4.70 (95% CI 3.72-5.85) in 2016 to 5.76 (4.69-7.00) in 2021. Overall, the incidence rate was 5.57 (5.13-6.05), 2.96 (2.64-3.31) for the EA and 2.61 (2.31-2.94) for the IA subgroup. The 4 most common AIE/PNS types were anti-NMDAR, anti-LGI1, anti-Hu, and anti-GAD65, together comprising almost two-thirds of all diagnoses (364/578, 63.0%). Most commercial antibody tests perform well overall, but important pitfalls remain. Although almost all tests had high specificity, PPV was only modest in the setting of these rare diseases and mass testing. We observe trends toward increasing incidence of antibody-associated AIE/PNS.

West Nile virus encephalitis: Clinical characteristics and a comparison to other infectious encephalitides

PurposeTo compare functional outcomes and help differentiate between important causative agents of acute infectious encephalitis in adults, focusing on West Nile virus encephalitis (WNVE). MethodsThe electronic database of the Tel Aviv Medical Center was screened for patients admitted during 2010–2020 with acute encephalitis. Additionally, patient laboratory results during the same period were screened for CSF samples positive for common pathogens causing encephalitis. The main patient groups were compared in terms of clinical characteristics and functional outcomes. ResultsOne hundred and five infectious encephalitis patients were identified. WNVE patients (n = 31) and VZV encephalitis (VZVE) patients (n = 31) were older than HSV1 encephalitis (HSV1E) patients (n = 15) (median ages 73, 76, 51, respectively). WNVE patients had a more prominent inflammatory profile. CSF characteristics significantly differed between groups, with an extreme mononuclear white blood cell predominance in VZVE patients (median 98 %).Functional outcomes at discharge were significantly worse in WNVE patients (median modified Rankin Scale score 4 at hospital discharge, 2.5 at last follow-up) when compared with HSV1E patients (2.5, 1, respectively) and VZVE patients (1.5, 1, respectively). ConclusionIn odds with previous reports, WNVE and VZVE in this study were far more prevalent than HSV1E. Differences in clinical characteristics could prove clinically useful early in encephalitis, including an association of WNVE with a relatively prominent inflammatory profile (somewhat resembling a bacterial infection) and an extreme mononuclear white blood cell predominance in VZVE. The detrimental outcome of WNVE emphasizes the need to advance research on WNV infection.

Free Kappa light chain in a sample of Egyptian multiple sclerosis patients (a pilot study)

BackgroundMultiple sclerosis is a chronic autoimmune-mediated demyelinating disease of the central nervous system that is usually associated with varying degrees of progressive disability. Free Kappa light chain (FKLC) has attracted growing attention as a significant diagnostic marker of MS. Our aim was to study the diagnostic utility of cerebrospinal fluid free Kappa light chain and related indices in a sample of Egyptian MS patients vs. CSF IgG oligoclonal bands. It was a prospective case–control study of MS patients carried in our hospital during the period from January 2021 till January 2022. Our study carried on 30 patients with multiple sclerosis and other inflammatory neurologic diseases and 20 age and sex matched controls. The study measured FKLC in the CSF and serum sample pairs of patients and control group. Indices calculated using FKLC measured in CSF and serum included; FKLC index, FKLC intrathecal fraction and quotient of FKLC. Indices were used to assess intrathecal synthesis of FKLC considering blood–CSF barrier function. Receiver operating characteristic curve analysis was used to determine diagnostic performance of FKLC and related indices in comparison to CSF–OCB testing.ResultsMeasured FKLC levels as well as its calculated indices have shown statistically significant higher values among MS patients against OIND patients and healthy control group. Both FKLC index and FKLC IF were similarly showing 100% diagnostic sensitivity and 100% diagnostic specificity for MS diagnosis.ConclusionsFKLC biomarkers are proposed to be highly sensitive and easy to detect first-line markers of intrathecal immunoglobulin synthesis with accurate performance and low cost that might prove to be promising diagnostic markers of MS.

Molecular Biology for Diagnosis of Congenital and Neonatal Infections in the Cerebrospinal Fluid of Newborns from a Brazilian Tertiary Hospital

The risk of infection transmission from mother to fetus depends on the pathogen. TORCH agents cause some neuroinfections, including Toxoplasmosis, rubella, Cytomegalovirus, herpes simplex 1 and 2, and others (Varicella Zoster, Parvovirus B-19, Epstein–Barr virus, and Zika virus). The consequences can be stillbirth, prematurity, uterine growth restriction, and congenital malformations. The detection of DNA/RNA from CSF by molecular methods is a marker of the involvement of congenital infection in the central nervous system. This study aimed to identify the frequency of these pathogens in CSF samples from newborns (1 to 28 days old) at a tertiary hospital, using PCR, and determine the clinical consequences. Methods: This was a prospective descriptive study involving the molecular analysis of 151 CSF samples from neonates, collected for cytological and biochemical diagnosis from 2017 to 2021. After the results and consent from the participants’ caregivers were obtained, the leftover material was sent to the University’s Virology Laboratory and submitted for DNA/RNA extraction and Nested-PCR/RT-PCR. A review of the patients’ medical records and descriptive statistics was performed. This work was approved by the Ethics Committee (CAAE: 86760218.3.0000.5404). Results: A total of 151 CSF samples were obtained, 16 of which were positive (10.6% [95% CI%: 6.18–16.63%]). Two of these were PCR-positive for HSV-1 (1.3%), four for VZV (2.6%), one for CMV (0.67%), two for Toxoplasmosis (1.3%), four for Parvovirus B-19 (2.6%), and four for Zika (2.6%). The proportion of positive PCR results was higher in the group that presented with malformations (25.0% vs. 8.4%, p = 0.040). Conclusions: The pathogens identified by PCR were mostly Zika virus, VZV, and B-19, and these were mainly found in newborns with malformations.

CSF and Plasma Biomarkers in Patients With Iatrogenic Cerebral Amyloid Angiopathy.

Recently, a subset of patients affected by cerebral amyloid angiopathy (CAA) distinguished by atypical juvenile onset and a hypothesized iatrogenic origin (iatrogenic CAA, iCAA) has emerged. β-Amyloid (Aβ) accumulation evidenced by amyloid PET positivity or CSF Aβ decrease was included in the iCAA diagnostic criteria. Conversely, diagnostic criteria for sporadic CAA (sCAA) do not involve biomarker analysis. The aim of this study was to assess CSF and plasma levels of Aβ and tau in iCAA and sCAA cohorts. Patients affected by probable or possible CAA according to established criteria (Boston 2.0) were prospectively recruited at Fondazione IRCCS Carlo Besta and San Gerardo dei Tintori from May 2021 to January 2024. Patients with probable and possible iCAA or sCAA with available plasma and/or CSF samples were included. Clinical and neurologic data were collected, and levels of Aβ40, Aβ42, total tau, and phospho-tau (p-tau) were assessed in CSF and plasma by SiMoA and Lumipulse. 21 patients with iCAA (72% male, mean age at symptom onset 50 years [36-74]) and 32 patients with sCAA (44% male, mean age at symptom onset 68 years [52-80]) were identified. Cognitive impairment and cardiovascular risk factors in the sCAA cohort were more common compared with the iCAA cohort. Patients with sCAA and iCAA showed similar CSF levels for Aβ40 (p = 0.5 [sCAA, 95% CI 2,604-4,228; iCAA, 95% CI 1,958-3,736]), Aβ42 (p = 0.7 [sCAA, 95% CI 88-157; iCAA, 95% CI 83-155]), and total tau (p = 0.08 [sCAA, 95% CI 80-134; iCAA, 95% CI 37-99]). Plasma levels of Aβ40 (p = 0.08, 95% CI 181-222), Aβ42 (p = 0.3, 95% CI 6-8), and total tau (p = 0.4, 95% CI 3-6) were not statistically different in patients with sCAA compared with iCAA ones (Aβ40, 95% CI 153-193; Aβ42, 95% CI 6-7 and total tau, 95% CI 2-4). Despite presenting with a younger age at onset, fewer cardiovascular risk factors, and lower cognitive impairment, patients with iCAA demonstrated Aβ and tau levels comparable with elderly patients with sCAA, supporting a common molecular paradigm between the 2 CAA forms.

Torque teno virus (TTV) Infection in Patients with Encephalitis

Torque teno virus (TTV) is a ssDNA orphan virus belonging to the Anelloviridae family, but some recent studies suggested its possible involvement in central nervous system (CNS) pathology. We analyzed serum and cerebrospinal fluid samples (CSF) from 109 patients with encephalitis for TTV infection using serological and molecular testing, virus quantitative measurement, and next-generation sequencing-based (NGS) phylogenetic analysis. TTV noncoding region (UTR) and/or open reading frame 1 (ORF-1) sequences were detected in serum of 86 (79%) patients and in nine (8%) patients in CSF. Five of the latter patients were coinfected with various entero- and herpesviruses. Anti-TTV-IgG were detected in 80 (73.4%) sera and in two (1.8%) CSF samples, while anti-TTV-IgM were present in three (2.8%) sera and in none of the CSFs. Phylogenic analysis of CSF-derived TTV ORF-1 sequences revealed the presence of three unique variants in one patient. TTV was quantified in five CSF-serum pairs: in two patients viral loads were similar, and in three serum TTV loads were approximately one log higher. Our results suggest at least an occasional replication of TTV in CNS. However, whether TTV could be the cause of encephalitis requires further studies.

Accuracy of urinary dipstick for glucose and protein determination in canine cerebrospinal fluid.

To evaluate the precision of urinary dipstick (UD) to assess protein and glucose concentrations in canine CSF samples compared to the standard methods. Cerebrospinal fluid protein and glucose were measured in 22 samples from dogs with neurological diseases affecting the CNS using UD and biochemistry (pyrogallol red and glucose oxidase reaction, respectively). Results were converted into scores to allow comparison between methods. The proportion of divergence between methods and its CI were calculated. The sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and accuracy (Ac) of UD were determined for 2 cutoff levels of CSF protein (15 and 30 mg/dL) and glucose (40 and 100 mg/dL). The proportion of divergence between methods was 64% (95% CI, 44% to 84%) for CSF protein (representing 14 of 22 erroneous samples), of which 92.9% (13 of 14) had a UD score lower than biochemistry. For CSF glucose, 73% (16 of 22 erroneous samples; 95% CI, 54% to 91%) had divergence between methods, of which 87.5% (14 of 16) had a UD score higher than biochemistry. Urinary dipstick had better results when the cutoff level was 15 mg/dL for protein (Se, 78.9%; Sp, 66.7%; PPV, 93.7%; NPV, 33.3%; Ac, 77.3%) and 40 mg/dL for glucose (Se, 89.5%; Sp, 33.3%; PPV, 89.5%; NPV, 33.3%; Ac, 81.8%) concentrations. Urinary dipstick was unreliable in estimating canine CSF protein and glucose concentrations. The UD underestimated protein and overestimated glucose levels in the canine CSF, which could have a significant diagnostic impact and should discourage its use as a bedside test.

Modified rat pup cerebrospinal fluid collection method

BackgroundCerebrospinal fluid (CSF) reflects biochemical changes in the brain due to its direct contact with brain interstitial fluid, making it a valuable tool for diagnosing and monitoring disease progression and therapeutic effectiveness in clinical practice. However, collecting CSF in animal studies, particularly from small animals like rat pups or mice, poses significant challenges. New methodAfter attempting various reported protocols, we encountered difficulties in consistently obtaining sufficient CSF from rat pups (P7-P42). Consequently, we modified these methods and developed a protocol with controllable and precise parameters for each step, enhancing reproducibility across different researchers. ResultsThe newly developed method enables rapid, single-operator, and reproducible CSF extraction while ensuring high-quality (the absorbance of the “quality control solution” at 415 nm < 0.05 AU, an indicator of oxyhemoglobin contamination for the collected CSF samples) and high-yield samples (33 ± 2.128 μL for P7 pups, 34.10 ± 2.747 μL for P8 pups, 36.67 ± 3.997 μL for P9 pups, 36.90 ± 1.946 μL for P10 pups, 35.11 ± 3.285 μL for P10 hypoxic-ischemic brain damage (HIBD) pups and 51.70 ± 5.256 μL for P42 pups, respectively). Comparison with existing methodsUnlike existing methods of CSF extraction in rat pups, our protocol has reproducible capillary pipette pulling parameters, controllable CSF quality indexes, and can be operated by a single person with high yield in a short time. ConclusionsThis paper provides a step-by-step comparison and discussion of the CSF collection process, establishing a method that enables a single operator to collect CSF rapidly, consistently, sufficiently, and with controlled quality.

Long-Term Trends in Antibiotic Use in a Low-Resource Medical Setting: Amman, Jordan (2010–2023)

Abstract Background Antimicrobial resistance (AMR) contributes to substantial morbidity and mortality. The misuse and overuse of antibiotics are major drivers of AMR. Lack of point-of-care testing to discern viral from bacterial infections complicates appropriate management in low-resource settings. This study examines antibiotic prescription practices in Amman, Jordan. Methods We conducted three prospective viral surveillance studies at the largest public hospital in Amman Jordan (2010–2013, 2020, and 2023). Our target population for this study was children &amp;lt;2 years old hospitalized with fever or respiratory symptoms. Through parental interviews and chart abstractions, we collected demographic and clinical data, including antibiotic use, as well as results of any blood, urine, and cerebrospinal fluid cultures performed at providers’ discretion. We classified antibiotics according to the 2023 World Health Organization AWaRe (Access, Watch, Reserve) antibiotic classification system, focusing on the utilization of Watch group antibiotics, which are recommended when first-line Access group antibiotics are ineffective or inappropriate. Research personnel also collected nasal or throat swabs from enrolled children, and these samples were tested in a research laboratory for common respiratory viruses using RT-PCR. Providers were unaware of viral testing results. We described categorical variables using absolute and relative frequencies and summarized continuous variables using the median and interquartile range (IQR). Results Of 4,950 children enrolled, we included 4,703. The median age was 3.5 months (IQR, 1.6–8.3), and 59.4% (n=2,793) were male. Most children were enrolled from the general ward (n=4,312 [91.9%]), with the remaining 380 (8.1%) being enrolled from the pediatric intensive care unit. Only 8 children (0.2%) among those eligible for seasonal influenza vaccination (n=1,624) had been vaccinated at presentation. The five most common admission diagnoses were bronchopneumonia (n=1,255 [26.7%]), rule-out sepsis (n=1,217 [25.9%]), bronchiolitis (n=866 [18.4%]), pneumonia (n=606 [12.9%]), and paroxysmal coughing or pertussis-like cough (n=273 [5.8%]). Blood, urine, or CSF samples were collected from 2,556 children (54.5%), 355 (13.9%) of whom tested positive for at least one bacterial species (Figure 1A). During hospitalization, 4,356 children (92.6%) received at least one antibiotic. Of those, 4,227 (97.0%) received at least one antibiotic from the Watch group (Figure 1B). The three most common Access group antibiotics prescribed were ampicillin (n=830 [17.7%]), gentamicin (n=96 [2.0%]), and amikacin (n=85 [1.8%]), whereas the three most common Watch group antibiotics prescribed were ceftriaxone (n=1,956 [41.7%]), cefuroxime (n=1,628 [34.7%]), and azithromycin (n=754 [16.1%]) Overall, 3,898 children (82.9%) were positive for at least one respiratory virus (Figure 1C). Providers ordered cultures most often for children 0–2 months old (n=1,575 [73.5%]) and those with an admission diagnosis of rule-out sepsis (n=1,161 [95.4%]). Despite seasonal variation, prescription and testing practices were consistent across study years. Conclusion Inappropriate antibiotic use remains a significant healthcare challenge in Jordan. Incorporating point-of-care viral testing for suspected respiratory infections and improving diagnostic testing for bacterial infections is crucial. Preventive strategies including influenza vaccination and the use of emerging therapies for RSV or other pathogens associated with high morbidity could indirectly limit inappropriate antibiotic use. Development of context-specific management guidelines and antimicrobial stewardship programs are urgently required to guide prescription practices.

B-076 Detection of KLHL11 Autoantibodies Using a New Cell-based Indirect Immunofluorescence Assay

Abstract Background Autoantibodies against kelch-like protein 11 (KLHL11) were first described in 2019 as markers of paraneoplastic encephalitis. Early diagnosis and treatment are imperative to improve prognosis and outcome of affected patients. Rat brain immunohistochemistry was reported to be not useful in routine screening for KLHL11 antibodies. This study evaluates the performance of a new recombinant cell-based assay for the standardized detection of KLHL11-specific IgG and reports the characteristics of the examined patients. Methods Serum (n=9), CSF (n=5) and/or plasmapheresis (n=1) samples from 10 patients with clinical presentations compatible with anti-KLHL11 encephalitis as well as sera from 100 healthy blood donors were analyzed using a prototype indirect immunofluorescence assay (IFA; EUROIMMUN) based on recombinant HEK293 cells expressing human KLHL11. Results KLHL11 autoantibodies were detected at high titers in all samples (serum &amp;gt;1:1,000; CSF &amp;gt;1:320; plasma &amp;gt;1:100,000) from all (10/10) patients but in none (0/100) of the blood donors, indicating 100% sensitivity and specificity. Anti-KLHL11-positive patients had a median age of 52 years (range 27-71) and 80% were male. The most common reported clinical presentations were cerebellar syndrome (ataxia), brainstem diencephalic encephalitis, rhombencephalitis and limbic encephalitis. Testicular or ovarian tumors were found in 87.5% (7/8) of the cases with available results from malignancy screening. Conclusions The new cell-based IFA enables the sensitive and specific detection of KLHL11 autoantibodies, thus supporting the diagnosis of patients with predominantly paraneoplastic brainstem cerebellar syndrome. Future studies will evaluate assay performance in larger patient cohorts to address the reported association of KLHL11 autoimmunity with a wider spectrum of syndromes and tumors.