Collected Cerebrospinal Fluid Samples Research Articles

PATH-04. OPTIMIZING CELL-FREE DNA ASSAYS FOR LOW INPUT LIQUID BIOPSY SAMPLES FROM PEDIATRIC BRAIN TUMOR PATIENTS

Abstract BACKGROUND Genetic and epigenetic profiling have transformed biological understanding, diagnostics, and therapeutic approaches for pediatric central nervous system (CNS) tumors. Obtaining tumor tissue may be associated with neurosurgical risk, may not be feasible in some cases, and is not routinely performed during follow-up or at relapse. Liquid biopsies (LBs) have emerged as a minimally invasive, serially collectable source of tumor-derived DNA. Here, we collected cerebrospinal fluid (CSF) and serum LBs from a large, unselected, cross-entity pediatric CNS tumor cohort (n=55 patients) for method optimization. METHODS Cell-free DNA (cfDNA) isolated from CSF and serum samples was analyzed 1) by low-coverage whole genome sequencing for copy number variation (CNV)-based tumor detection and 2) by enzymatic conversion (EC)-based DNA methylation profiling using the EPIC DNA methylation array and the Heidelberg Brain Tumor Classifier. All library protocols were optimized for LB samples with low cfDNA amounts in the picogram range. Matched tumor samples were used as reference. RESULTS Our lcWGS-based cfDNA profiling protocol had a high technical success rate across the whole cohort (>95%), despite very low cfDNA amounts for most CSF samples. The majority of CSF LBs captured tumor-derived CNVs and displayed a high fraction of tumor-derived cfDNA (>5%, range 0-78%). For serum LBs, only few samples showed detectable CNVs. Proof-of-concept methylation analyses leveraging a new EC-based workflow allowed molecular classification using CSF, particularly for embryonal brain tumors (11/15 samples). Clinical workup of selected cases illustrates the potential of LBs to aid as diagnostic tool, monitor disease, and characterize tumor evolution. CONCLUSIONS Tackling a major roadblock of clinical translation of LBs, this study delineates new methods tailored to low input cfDNA samples. Results highlight the clinical utility of LBs for the management of brain tumor patients, providing a strong rationale for prospective validation within pediatric neuro-oncology trials.

Abstract PS11-03: Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD)

Abstract Introduction. Leptomeningeal disease (LMD) is a devastating complication of advanced malignancies and occurs in 5-15% of patients (pts) with metastatic breast cancer (MBC). Molecular characterization of the primary tumor to identify actionable mutations is standard of care, and targeted therapies have significantly improved survival. In contrast, metastatic tumors to the central nervous system (CNS) are not routinely assessed for the presence of actionable mutations, so the frequency and concordance of actionable mutations centrally vs. peripherally is not well characterized. In this single-center prospective study, we collected cerebrospinal fluid (CSF) in pts with known or suspected LMD and performed next generation sequencing (NGS) to evaluate the status of actionable mutations in the CSF and compared these results with NGS from matched peripheral blood and systemic metastatic tumor samples. Methods. We enrolled sixteen (16) pts with MBC and suspected or confirmed LMD at a single academic center from 2021-2023 in this non-therapeutic prospective study and collected CSF, peripheral blood, and archival systemic tumor samples from each pt. We used NGS to evaluate the status of actionable biomarkers in the CSF, blood, and tissue using the following NGS platforms: CNSide (Biocept) for CSF; Guardant 360, Foundation One CDx and CNSide for blood; and Caris and the UCSF CLIA-validated UCSF500 panel for the systemic metastatic tumor samples. We also evaluated CSF tumor cell enumeration and HER2 status in a subset of pts (CNSide). Results. Of the sixteen pts with MBC and suspected or confirmed LMD, 81% (13/16) were ultimately determined to be LMD-positive based on positive CSF cytology and/or convincing MRI imaging, including 6 pts with HR+/HER2- MBC, 6 pts with metastatic triple negative breast cancer (mTNBC), and 1 pt with HR+/HER2+ MBC; 3 pts were determined to be LMD-negative based on similar criteria. Of the 6 pts with HR+/HER2- MBC and LMD, NGS detected actionable mutations in the CSF in 67% (4/6), including two pts with PIK3CA E545E, one pt with both PIK3CA E542K and ERBB2 amplification, and 1 pt with ESR1 Y537S. Matched NGS analysis of the peripheral blood and/or systemic metastatic tissue showed concordance with PIK3CA E542K detected across tissue types in two pts, but discordance across tissue types in the other pts, including ESR1 detected systemically in two pts but not in the CSF. Of the 6 pts with mTNBC and LMD, CSF NGS detected actionable mutations in the CSF in 17% (1/6) of pts, including one pt with PIK3CA E542K; analysis of blood NGS for this pt is pending. 3 pts (50%) had mutations in TP53 in the CSF (1 pt with R273H and two pts with K132R), which were concordant across tissue types in all 3 pts. NGS did not detect any variants in the CSF of the one pt with HR+/HER2+ MBC and LMD. CSF tumor cell enumeration and central HER2 status will also be reported. Conclusion. This study demonstrates that NGS can detect actionable mutations in the CSF of pts with MBC and LMD. We observed concordance and heterogeneity in the status of actionable mutations between the CSF, peripheral blood, and systemic metastatic tumor tissue. Larger studies are needed to assess the clinical utility of these observations, particularly with the development of several novel targeted agents that are CNS-penetrant. Citation Format: Laura Huppert, Lindy Her, Christine Hodgdon, Susie Brain, Carol Simmons, Jo Chien, Melanie Majure, Hope Rugo, Mark Jesus Magbanua, Ron Balassanian, Michelle Melisko. Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-03.

Lumbar puncture increases Alzheimer’s disease biomarker levels in cerebrospinal fluid of rhesus monkeys

Cerebrospinal fluid (CSF) samples are commonly collected via lumbar puncture (LP) in both clinical and research settings for measurement of biomarkers of Alzheimer's disease (AD). To determine the effects of LP on CSF AD biomarkers, we collected CSF samples at seven different time points after an LP in rhesus monkeys. We find that amyloid-beta (Aβ) and Tau levels increased significantly on day 1, peaked on day 3, and returned to baseline on day 10 after LP. The NFL levels increased significantly on day 5, peaked on day 10, and returned to baseline after day 30. The increased AD biomarker levels were mainly due to CSF outflow and deep intrathecal invasion during LP. Therefore, if LPs are repeated within a short period of time, prior LP can affect Aβ and Tau levels within 10days and NFL levels within 30days, which may lead to clinical misdiagnosis or incorrect scientific conclusions.

EGFR amplification indicated poor prognosis in EGFR‐mutated lung cancer with leptomeningeal metastases

AbstractBackgroundLeptomeningeal metastasis (LM) is a lethal complication of advanced lung cancer, and due to limited access to the leptomeningeal lesion, we explored the potential role of cerebrospinal fluid (CSF) as a source for liquid biopsy in LM patients of lung cancer with EGFR mutation.Materials and MethodsFrom August 2018 to June 2021, we collected CSF samples of lung cancer patients at First Affiliated Hospital of Zhengzhou University. Next‐generation sequencing was performed to detect the mutations in EGFR genes, and 38 patients detected with EGFR mutations were finally enrolled in further clinical analyses.ResultsTP53 missense mutation (50%) was the most frequently detected concurrent gene in CSF. In those 10 patients whose CSF was obtained upon resistance to first TKI, TP53 missense mutation (40%, n = 4) and EGFR copy number amplification (40%, n = 4) was detected with high frequency; meanwhile, T790M mutation was found only in two patients. Known mechanisms of acquired resistance to third EGFR‐TKIs were found in 31.8% of cases. C797S or C797G mutation was identified in three patients. Possible EGFR‐independent resistant mechanism included MET amplification (4.5%), RET gene fusion (4.5%), PIK3CA missense mutation (4.5%) and CDK4 amplification (4.5%). The median OS was 55 months, the median OSLM was 38 months. EGFR amplification was associated with shortened OS in these EGFR‐mutated lung cancer with leptomeningeal metastases.ConclusionEGFR amplification indicated poor prognosis in EGFR‐mutated lung cancer with leptomeningeal metastases, providing a novel pathogenesis and treatment direction of these patients.

Safety of spinal anesthesia and analysis of cerebrospinal fluid in SARS-CoV-2 pregnant women undergoing cesarean section: an observational prospective study

BackgroundSystemic infection has always been considered a relative contraindication to neuraxial anesthesia, despite the fact that infectious complications are relatively uncommon. Pregnancy-related physiological changes and coronavirus disease (COVID-19) neurotropic features may facilitate the virus’ entry into the central nervous system. The principal aim of this study was to test the safety of spinal anesthesia in “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2)-positive pregnant women and to examine cerebrospinal fluid (CSF) characteristics.MethodsWe conducted a prospective observational single-center study in asymptomatic or paucisymptomatic consecutive pregnant SARS-CoV-2 patients who underwent spinal anesthesia for cesarean section. Women with severe infection were excluded because they underwent general anesthesia. At the time of spinal anesthesia, we collected CSF samples, and then we performed a chemical-physical analysis to look for signs of inflammation and for SARS-CoV-2 genome.ResultsWe included 26 women. No spinal anesthesia complications were reported in the perioperative period and after 2 months. All CSF samples were crystal clear, and all physical-chemical values were within physiological ranges: the median concentration of CSF/plasma glucose ratio was 0.66, IQR 0.5500 (0.6000–0.7100), and the average CSF protein concentration value was 23.2 mg/dl (SD 4.87). In all samples, genomes of SARS-CoV-2 and other neurotropic viruses were not detected.ConclusionsSpinal anesthesia was safe in SARS-CoV-2 pregnant women with mild disease; no clinical maternal complications were detected, and no CSF changes indicative of inflammatory or infectious diseases that would compromise the safety of the procedure were found.

The Novel Biomarkers for Predicting Prognosis and Recurrence in Primary Central Nervous System Lymphoma: Exosomal Protein and Exosomal microRNA

Abstract Purpose Primary central nervous system lymphoma (PCNSL) is a highly heterogeneous tumor. Therefore, it is crucial to identify biomarkers that can dynamically monitor disease progression for individualized treatment and to improve prognosis. Exosomes carry a large amount of genetic and proteomic information and are stable and easy to obtain. They play important roles in disease diagnosis, treatment, and prognosis. This study aimed to evaluate the use of cerebrospinal fluid (CSF) exosomes in patients with PCNSL as biomarkers of tumor evolution. Methods From October 2021 to October 2022, patients diagnosed with PCNSL via pathology were selected from Beijing Tiantan Hospital affiliated to Capital Medical University. None of the patients had received any cancer-related treatment before diagnosis. Primary intraocular lymphoma and HIV-positive patients were excluded. Clinical information and laboratory findings of patients were recorded in detail. The responses were evaluated according to the response criteria formulated by the International Primary Nervous System Lymphoma Cooperation Group in 2005. According to the time of collecting CSF samples, the patients were divided into diagnosis group (before treatment, n=12), treatment group (after 2-4 treatment cycles, n=11) and complete remission (CR) group (achieved CR after 6 treatment cycles, n=11). We analyzed and compared the exosomal protein concentrations and microRNA expression in the CSF of patients in different groups using western blotting and quantitative reverse transcription PCR. Results There are CD20-positive exosomes in the CSF of patients with PCNSL (Figure 1). In the diagnosis group, the exosomal protein concentration of patients who achieved CR after 2 cycles induction treatment (early remission) was significantly lower than that of patients who did not achieve early remission (p=0.03). Higher exosomal protein concentrations (p=0.022) and exosomal miR-3681-3p expression (p=0.036) at diagnosis were significantly associated with shorter progression-free survival (Figure 2). The expression levels of miR-21-5p (p=0.001), miR-29b-3p (p=0.043), and miR-101-3p (p=0.007) in exosomes were significantly higher in the diagnosis group than in the CR group. Higher exosomal protein concentrations (p=0.012) and exosomal miR-101-3p expression (p=0.013) after CR were significantly associated with recurrence and shorter disease-free survival (Figure 2). The predictive efficacy of exosomal protein concentration and exosomal has-miR-101-3p in PCNSL was analyzed. The area under the receiver operator characteristic curve (AUC) was 0.833 (95% confidence interval 0.524-1.000; p<0.05) and 0.917 (95% confidence interval 0.731-1.000; p=0.033), respectively. Conclusions Exosomes in CSF may have the potential to evaluate prognosis and predict recurrence in PCNSL. It provides clinicians with selectable biomarkers for dynamic evaluation of disease progression.

Detection of cell-free histones in the cerebrospinal fluid of pediatric central nervous system malignancies by imaging flow cytometry.

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

P14.05.B 2-HYDROXYGLUTARATE IN CEREBROSPINAL FLUID AS A MONITORING BIOMARKER FOR IDH-MUTANT GLIOMAS

Abstract BACKGROUND D-2-hydroxyglutarate (D-2-HG) is a well-established oncometabolite of isocitrate dehydrogenase (IDH) mutant gliomas. While prior studies have demonstrated that D-2-HG is elevated in the cerebrospinal fluid (CSF) of patients with IDH-mutant gliomas, no study has evaluated the ability of CSF D-2-HG to quantitatively monitor tumor burden and therapeutic response. Toward this goal, we are routinely obtaining CSF samples from patients prior to, during, and after treatment via intra-operative collection and CSF-access devices. MATERIAL AND METHODS Patients were enrolled into our Ommaya reservoir study (NCT04692337), which enables longitudinal access to CSF. The catheter for the implanted reservoir was placed within the surgical resection cavity following a standard-of-care resection. Intra-operatively collected CSF samples were also biobanked through Mayo Clinic’s Neuro-Oncology biorepository. CSF samples were also acquired via clinically indicated ventriculoperitoneal shunts (VP shunts) or external ventricular drains via our ongoing CSF biomarkers study (NCT04692324). D-and-L-2-HG were assayed from each CSF sample via gas chromatography-mass spectrometry (GC-MS) at Mayo Clinic’s Biochemical Genetics Laboratory. RESULTS CSF was collected prior to and after resection in patients via Ommaya reservoirs or VP shunts, all of whom underwent near gross total resections (>95% of tumor volume). D-2-HG and the D/L ratio fell on average by 57.9% (range: 15.1%-98.6%) and 68.8% (range: 49.3%-98.9%), respectively, following resection. In one patient with a largely intraventricular grade 4 IDH-mutant astrocytoma, D-and-L-2-HG fell by 99.1% and 99.3% by post-operative day 2, consistent with the impact of ventricular contact on baseline CSF D-2-HG levels. In patients for whom CSF was collected throughout chemoradiation, with or without immunotherapy, D-2-HG and the D/L ratio continued to decrease over time, despite concern for pseudoprogression versus progression on imaging. CONCLUSION Our early experience with this strategy demonstrates the potential for intracranial CSF D-2-HG as a monitoring biomarker for IDH-mutant gliomas. Work is ongoing to evaluate the reproducibility of these findings in a larger cohort of patients with low or high-grade gliomas, in addition to the impact of tumor recurrence on 2-HG. Future studies will evaluate the impact of IDH inhibitors and other experimental therapies on CSF 2-HG levels for pharmacodynamic monitoring of treatment response.

Lower levels of Th1 and Th2 cytokines in cerebrospinal fluid (CSF) at the time of initial CSF shunt placement in children are associated with subsequent shunt revision surgeries

ObjectiveWe compare cytokine profiles at the time of initial CSF shunt placement between children who required no subsequent shunt revision surgeries and children requiring repeated CSF shunt revision surgeries for CSF shunt failure. We also describe the cytokine profiles across surgical episodes for children who undergo multiple subsequent revision surgeries. MethodsThis pilot study was nested within an ongoing prospective multicenter study collecting CSF samples and clinical data at the time of CSF shunt surgeries since August 2014. We selected cases where CSF was available for children who underwent an initial CSF shunt placement and had no subsequent shunt revision surgeries during >=24 months of follow-up (n = 7); as well as children who underwent an initial CSF shunt placement and then required repeated CSF shunt revision surgeries (n = 3). Levels of 92 human cytokines were measured using the Olink immunoassay and 41 human cytokines were measured using Luminex based bead array on CSF obtained at the time of each child’s initial CSF shunt placement and were displayed in heat maps. ResultsQualitatively similar profiles for the majority of cytokines were observed among the patients in each group in both Olink and Luminex assays. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Pro- and anti-inflammatory cytokines were selected a priori and shown across subsequent revision surgeries for the 3 patients. Cytokine patterns differed between patients, but within a given patient pro-inflammatory and anti-inflammatory cytokines acted in a parallel fashion, with the exception of IL-4. ConclusionsHeat maps of cytokine levels at the time of initial CSF shunt placement for each child undergoing only a single initial CSF shunt placement and for each child undergoing repeat CSF shunt revision surgeries demonstrated qualitatively similar profiles for the majority of cytokines. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Better stratification by patient age, etiology, and mechanism of failure is needed to develop a deeper understanding of the mechanism of inflammation in the development of hydrocephalus and response to shunting in children.

Longitudinal ventricular cerebrospinal fluid profile in patients with spontaneous subarachnoid hemorrhage.

BackgroundSpontaneous subarachnoid hemorrhage (SAH) is a severe neurological disease that frequently requires placement of external ventricular drainage (EVD). Cerebrospinal fluid (CSF) obtained via the drain is used to detect potential complications of SAH.ObjectiveThis study aimed to describe the longitudinal profile of routine CSF parameters in patients with SAH and to identify associations with neurological complications.MethodsA total of thirty-three patients with spontaneous SAH who required an EVD and had at least three consecutive CSF samples collected over a period of more than 7 days were included in this study.ResultsA median of 6 longitudinally collected CSF samples per patient were available within 1–22 days after SAH onset. Overall, red blood cells (RBC) steadily decreased over time, whereas white blood cells (WBC) and total protein (TP) increased until days 6 and 13, respectively, and decreased thereafter. The estimated decay rates of RBC, WBC, and TP were 28, 22, and 6% per day. Distinct CSF patterns over time were linked to known complications after SAH. Patients with rebleeding showed increased RBC, TP, and phagocytosing cells compared to patients without re-bleeding. For ventriculitis, an elevated cell index with a higher proportion of granulocytes was characteristic. CSF of patients with delayed cerebral ischemia showed increased RBC and WBC compared to patients without DCI. Early CSF WBC and cell index were predictive for the occurrence of DCI and ventriculitis later during the disease course. The amount of daily CSF drainage via EVD had no impact on routine CSF parameters.ConclusionLongitudinal CSF characteristics are associated with SAH-related complications.

Evaluation of human bocavirus as well as other well-known viral etiologic agents by PCR in the cerebrospinal fluid of children with clinical impression of viral meningoencephalitis referred to the Children's Medical Center in Tehran, Iran, from 2019 to 2020.

Viral meningoencephalitis is one of the most important diseases that most commonly affect children. In many cases of viral meningoencephalitis, the underlying cause of the disease is not identified, raising the possibility of a variety of pathogens that are not routinely tested. Bocaviruses belong to a newly identified class of viruses that have been reported in some studies to be associated with viral encephalitis. In the present study, we investigated the prevalence of bocaviruses and other viruses in the patients suspected of having viral encephalitis and their associations with various demographic and clinical variables. Two hundred patients with suspected viral meningoencephalitis referred to Children's Medical Center were studied from 2019 to 2020. Age, sex, length of hospitalization, and course of the disease were gathered. Cerebrospinal fluid (CSF) samples were taken from the patients and subjected to biochemical examinations and PCR to identify the underlying cause. Bocaviruses were detected in none of the DNA samples extracted from the CSF specimens. The most identified organisms were mumps and enteroviruses. In 92% of cases, the underlying cause was not identified. PCR-based identification of the underlying causes of viral meningoencephalitis in CSF specimens was not successful in most cases. Bocavirus was not found in any of the collected CSF samples. Further studies are required for drawing more accurate conclusions.

Learnings from a Cutibacterium acnes pseudo-outbreak in pediatric neurosurgical patients

Background:Cutibacterium acnes is normal skin flora as well as a common culture contaminant. It can cause infections in the setting of sterile implants, although clinical presentations can be subtle. Differentiating true infection from sample contamination is challenging and has implications for patient care. We describe an investigation of a cluster of 7 hospitalized pediatric patients with C. acnes isolated from anaerobic cultures of cerebrospinal fluid (CSF) over 3 weeks at a quaternary-care children’s hospital. Methods: An outbreak response was coordinated between the infection prevention and control (IPC), microbiology, and neurosurgery teams. We defined a case as a hospitalized patient with C. acnes isolated from a CSF culture beginning in November 2020. We reviewed charts of all cases and CSF culture collection on all case units, transport to and processing at the microbiology laboratory, and the IPC team measured adherence for all processes. Results: There were 8 positive cultures in 7 cases from November 10 to 27, 2020. The median case age was 2 months (range, 0–119). Cases occurred on 4 different units. All positive patients had at least 1 implanted neurosurgical device used for CSF drainage. There were no clear commonalities in surgeon responsible for device placement, hardware type placed, or staff collecting CSF samples. A standard protocol for CSF collection was followed for all cases. Overall, 3 patients cleared cultures without intervention, 2 received oral antibiotics, and 2 underwent surgical removal of their device. Specimen processing was unchanged, although due to supply issues, an alternative anaerobic culture media (Anaerobic Systems, Morgan Hills, CA) was used for 6 weeks, during which all cases were identified. Compared to routine media, the alternative is known to enhance organism detection. The company reported no concerns for media contamination or C. acnes outbreaks. Once routine media became available, CSF culture positivity for C. acnes returned to baseline (late November or early December) (Fig. 1). Conclusions: We identified a likely pseudo-outbreak related to temporary use of a more sensitive culture media. No direct patient harm was identified, although many had increased risk of harm by surgical intervention or prolonged length of stay. Technological advances may enhance organism identification but challenge existing paradigms of care. More studies are needed to better delineate the intersection of diagnostic advancements with patient care standards.Funding: NoneDisclosures: None

Risk factors for neurological complications in children with Flavivirus infection.

This study aims to characterize the acute neurological manifestations caused by DENV, ZIKV, and YFV during hospitalization; identify the risk factors associated with persistent neurological complications after discharge; and evaluate the time to resolution during clinical follow-up. A prospective study evaluated 505 children, between March 2014 and July 2019, hospitalized with neurological manifestations and that doctors suspected infection of the central nervous system (CNS). Viral infection of collected cerebrospinal fluid (CSF) was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Patients were clinically followed up after hospital discharge. Analysis of predictive factors and survival curves was performed. This study identified clinical symptoms and changes in the CSF laboratory, electroencephalogram (EEG), and CNS image as predictors of complications in children with confirmed infection in the CNS by DENV, ZIKV, or YFV. No statistical difference was found (p value 0.574) in the time to the resolution of complications in children after hospital discharge between the three types of flaviviruses. Children with YFV, detected in CSF samples, had a 53% higher risk of developing neurological complications. Performing etiological diagnosis by RT-PCR of CSF samples of children with neurological manifestations, especially during Flavivirus outbreaks, is an essential tool for improving the prognosis and clinical follow-up of these patients.

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.

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

The Role of Hemoglobin Oxidation Products in Triggering Inflammatory Response Upon Intraventricular Hemorrhage in Premature Infants.

Intraventricular hemorrhage (IVH) is a frequent complication of prematurity that is associated with high neonatal mortality and morbidity. IVH is accompanied by red blood cell (RBC) lysis, hemoglobin (Hb) oxidation, and sterile inflammation. Here we investigated whether extracellular Hb, metHb, ferrylHb, and heme contribute to the inflammatory response after IVH. We collected cerebrospinal fluid (CSF) (n = 20) from premature infants with grade III IVH at different time points after the onset of IVH. Levels of Hb, metHb, total heme, and free heme were the highest in CSF samples obtained between days 0 and 20 after the onset of IVH and were mostly non-detectable in CSF collected between days 41 and 60 of post-IVH. Besides Hb monomers, we detected cross-linked Hb dimers and tetramers in post-IVH CSF samples obtained in days 0–20 and 21–40, but only Hb tetramers were present in CSF samples obtained after 41–60 days. Vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) levels were higher in CSF samples obtained between days 0 and 20 than in CSF collected between days 41 and 60 of post-IVH. Concentrations of VCAM-1, intercellular adhesion molecule-1 (ICAM-1), and IL-8 strongly correlated with total heme levels in CSF. Applying the identified heme sources on human brain microvascular endothelial cells revealed that Hb oxidation products and free heme contribute to the inflammatory response. We concluded that RBC lysis, Hb oxidation, and heme release are important components of the inflammatory response in IVH. Pharmacological interventions targeting cell-free Hb, Hb oxidation products, and free heme could have potential to limit the neuroinflammatory response following IVH.

Tumor-Specific Cell-Free DNA (cfDNA) in the Cerebrospinal Fluid (CSF) Detects Cytologically Occult Central Nervous System (CNS) Involvement in Aggressive Lymphomas

Background: CNS involvement in aggressive lymphomas (including diffuse large B-cell lymphoma, DLBCL) is associated with poor survival, requiring aggressive CNS-directed therapy to avert dismal outcomes. Early detection of clinically occult CNS invasion is thus critical. Clinical risk factors have low specificity to select patients for CNS prophylaxis, and routine evaluations of the CSF by cytology or flow cytometry have low sensitivity (Doolittle et al., Blood 2008). Moreover, most CNS recurrences of DLBCL occur in the brain parenchyma and remain occult on CSF examination. There is a high unmet need for an accurate method to identify subclinical CNS invasion and help non-invasively diagnose parenchymal disease. Clonotype-specific cell-free DNA (cfDNA) can be detected in the plasma of patients with DLBCL using high-throughput sequencing (HTS) combined with multiplex PCR, and cfDNA reappearance after therapy precedes systemic recurrence (Roschewski et al., Lancet Oncol. 2015). However, clonotypic cfDNA has not been systematically evaluated in the CSF compartment. Because of the blood-brain barrier that isolates CSF from plasma, clonotypic cfDNA might hold promise as a sensitive method of CNS lymphoma detection. Therefore, we sought to evaluate the ability of HTS-based cfDNA assay in the CSF to detect CNS invasion. Methods: We collected CSF samples from consenting patients with aggressive lymphomas who either had clinically overt CNS disease, or who were newly diagnosed and without evident CNS disease, but at high clinical risk. Genomic DNA from primary tumors (formalin-fixed paraffin-embedded [FFPE] tissue; Fig. A) was analyzed for tumor-specific clonotype using a quantitative HTS of rearranged IGK, IGH (VJ or DJ), or IGL loci, as previously described (Carlson CS et al., Nat Commun. 2013). Clonotypes in each primary tumor were selected for subsequent tracking in the CSF based on frequency relative to other B cells and all nucleated cells, as well as distribution. HTS of B-cell receptor (BCR) was performed on 5 mL CSF centrifuged at 1000x for 5 min at 4°C and separated into acellular portion for cfDNA, and a cell pellet suspension. Clonotype copy numbers are expressed per mL for acellular fluid, and per 106 cells for cell pellets. Results: The HTS assay identified median 3 (range, 2-6) dominant immunoglobulin sequences in each primary FFPE lymphoma sample (N=9), with median dominant clonotype frequency 39.0% (range, 22.0-97.9%) and median copy count 753,618 per 106 cells (range 47,455-1,407,748). In the CSF, the HTS/ASO-PCR assay detected the dominant clonotype in 5 out of 9 samples (Table), including all cases (N=3) with overt CNS disease, and 2 without known CNS invasion (sensitivity=100% and specificity=67% for clinically overt disease). Of note, the cfDNA assay detected tumor-specific clonotype in the CSF of a patient with primary CNS lymphoma (PCNSL) who had negative CSF evaluations by flow cytometry and IGH PCR. In the cell-free CSF, median detectable cfDNA clonotype copy count was 1,649/mL (range, 2-5,620), and in the CSF cell suspension it was 389,558 per 106 cells (range, 10,373-1,181,171). Copy counts were significantly higher (P=.004 for cfDNA in CSF fluid, P=.004 for cell suspension) in patients with overt leptomeningeal involvement (positive CSF cytology) than those with parenchymal or clinically occult disease (Fig. B). The 2 cases with positive CSF cfDNA but negative clinical staging and CSF cytology were notable for high risk of CNS dissemination due to ruptured epidural tumor with spinal cord compression, or double-hit histology (MYC-IGH and BCL2-IGH rearrangements) with extensive extranodal invasion of kidneys, pleura, and liver. Both patients were recommended CNS-directed prophylaxis based on clinical risk. Conclusions: HTS of BCR loci in the CSF of patients with aggressive lymphomas can identify intraparenchymal CNS or leptomeningeal disease with high sensitivity. More importantly, it can detect intraparenchymal involvement not identifiable using traditional means of CSF analysis (flow cytometry, standard IGH PCR). Detection of cfDNA in CSF of some high-risk patients without overt CNS disease will be further explored in a larger sample. It suggests that pre-treatment HTS of BCR loci could be prospectively tested to predict the risk of CNS recurrence, with a future potential to enable more sensitive and specific selection of patients for CNS prophylaxis therapy. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech: Research Funding. Reagan:Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding. Mullins:Adaptive Biotechnologies: Employment.

Aetiology of encephalitis and meningitis in children aged 1-59 months admitted to the Children's Hospital, Lusaka, Zambia

Background: Meningitis and encephalitis are important causes of admissions and mortality in Zambia. Apart from bacterial causes, no data is available on viral agents that cause disease at the Children's Hospital, Lusaka, Zambia. We conducted a prospective descriptive study to determine the viral and bacterial causes of encephalitis and meningitis in children aged 1-59 months.
 Methods: From November 2016 to February 2018, we collected cerebrospinal fluid (CSF) samples and clinical details from children admitted to the inpatient wards with encephalitis and meningitis. Macroscopic examination, microscopy, bacterialculture and real-time (Multiplex) PCR were performed on the CSF samples.
 Results: A total of 106 patients were enrolled. The median age was 10 months and 81 (76.4%) had meningitis while 25 (23.6%) had encephalitis. One (0.9%) participant had Haemophilus influenzae detected by both culture and PCR. Two (1.9%) had Neisseria meningitidis while 5 (4.7%) had Streptococcus pneumoniae detected only by PCR. Viruses were detected in 26.4% (28/106) and 64% had meningitis. The viral agents detected were: EBV (10%); Parvovirus B19, Human herpes virus type 6, Human herpes virus type 7 and CMV at 2.8% each. A raised CSF WBC was associated with the case definition (P=0.01) of meningitis. Patients with meningitis were more likely to be alive at discharge than those with encephalitis (OR = 3.6, CI = 1.96 – 6.68, P-value <0.001).
 Conclusions: Viral infections of the central nervous system (CNS) are the commonest causes of both encephalitis and meningitis at the Children's Hospital, Lusaka, Zambia.

Aetiology of Encephalitis and Meningitis in Children Aged 1-59 Months Admitted to the Children's Hospital, Lusaka, Zambia

Background: Meningitis and encephalitis are important causes of admissions and mortality in Zambia. Apart from bacterial causes, no data is available on viral agents that cause disease at the Children's Hospital, Lusaka, Zambia. We conducted a prospective descriptive study to determine the viral and bacterial causes of encephalitis andmeningitis in children aged 1-59 months.
 Methods: From November 2016 to February 2018, we collected cerebrospinal fluid (CSF) samples and clinical details from children admitted to the inpatient wards with encephalitis and meningitis. Macroscopic examination, microscopy, bacterial culture and real-time (Multiplex) PCR were performed on the CSF samples.
 Results: A total of 106 patients were enrolled. The median age was 10 months and 81 (76.4%) had meningitis while 25 (23.6%) had encephalitis. One (0.9%) participant had Haemophilus influenzae detected by both culture and PCR. Two (1.9%) had Neisseria meningitidis while 5 (4.7%) had Streptococcus pneumoniae detected only by PCR. Viruses were detected in 26.4% (28/106) and 64% had meningitis. The viral agents detected were: EBV (10%); Parvovirus B19, Human herpes virus type 6, Human herpes virus type 7 and CMV at 2.8% each. A raised CSF WBC was associated with the case definition (P=0.01) of meningitis. Patients with meningitis were more likely to be alive at discharge than those with encephalitis (OR = 3.6, CI = 1.96 – 6.68, P-value <0.001).
 Conclusions: Viral infections of the central nervous system (CNS) are the commonest causes of both encephalitis and meningitis at the Children's Hospital, Lusaka, Zambia.
 Disclosure: The authors declare no conflict of interest.

Evaluation of Next-Generation Sequencing for the Diagnosis of Infections of the Central Nervous System Caused by the Neurotropic Herpes Viruses: A Pilot Study

Background: There are sparse and limited studies on small sample size reporting the application of next-generation sequencing (NGS) in the detection of central nervous system (CNS) viral infections. We assessed the diagnostic performance of NGS of cerebrospinal fluid (CSF) for predicting viral infections of the CNS caused by the neurotropic herpes viruses in a pilot population. Materials and Methods: We prospectively collected CSF samples from 24 patients with CNS viral infection from April 2017 to October 2018. Of the 24 patients, 19 patients were infected with herpes simplex virus 1 (HSV-1), 1 patient with HSV-2, and 4 patients with varicella-zoster virus (VZV). All CSF samples were screened for viral DNA using NGS technologies to detect viral CNS infections. Results: Of the 24 patients with confirmed viral CNS infection caused by the neurotropic herpes viruses, 10 (10/24, 41.67%) patients exhibited positive NGS results. With the help of NGS, HSV-1 DNA was detected in the CSF of 6 patients (6/19; 31.58%). HSV-2 DNA was detected in 1 patient (1/1; 100%) and VZV DNA was detected in 3 patients (3/4; 75%). The positive rate of virus detected by NGS decreased with time. The positive rates of NGS of CSF in the first, second, and third weeks were 54.5% (6/11), 44.4% (4/9), and 0% (0/4), respectively. Conclusions: NGS method is a promising pathogen detection tool for identifying viral CNS infections. It should be recommended to sequence viral DNA of CSF in the early stage of CNS viral infections.