Clinical Cerebrospinal Fluid Research Articles

Cerebrospinal Fluid Biomarkers of Synaptic Dysfunction are Altered in Parkinson's Disease and Related Disorders.

Synaptic dysfunction and degeneration are central contributors to the pathogenesis and progression of parkinsonian disorders. Therefore, identification and validation of biomarkers reflecting pathological synaptic alterations are greatly needed and could be used in prognostic assessment and to monitor treatment effects. To explore candidate biomarkers of synaptic dysfunction in Parkinson's disease (PD) and related disorders. Mass spectrometry was used to quantify 15 synaptic proteins in two clinical cerebrospinal fluid (CSF) cohorts, including PD (n1 =51, n2 =101), corticobasal degeneration (CBD) (n1 =11, n2 =3), progressive supranuclear palsy (PSP) (n1 =22, n2 =21), multiple system atrophy (MSA) (n1 =31, n2 =26), and healthy control (HC) (n1 =48, n2 =30) participants, as well as Alzheimer's disease (AD) (n2 =23) patients in the second cohort. Across both cohorts, lower levels of the neuronal pentraxins (NPTX; 1, 2, and receptor) were found in PD, MSA, and PSP, compared with HC. In MSA and PSP, lower neurogranin, AP2B1, and complexin-2 levels compared with HC were observed. In AD, levels of 14-3-3 zeta/delta, beta- and gamma-synuclein were higher compared with the parkinsonian disorders. Lower pentraxin levels in PD correlated with Mini-Mental State Exam scores and specific cognitive deficits (NPTX2; rho=0.25-0.32, P < 0.05) and reduced dopaminergic pre-synaptic integrity as measured by DaTSCAN (NPTX2; rho=0.29, P=0.023). Additionally, lower levels were associated with the progression of postural imbalance and gait difficulty symptoms (All NPTX; β-estimate=-0.025 to -0.038, P < 0.05) and cognitive decline (NPTX2; β-estimate=0.32, P=0.021). These novel findings show different alterations of synaptic proteins in parkinsonian disorders compared with AD and HC. The neuronal pentraxins may serve as prognostic CSF biomarkers for both cognitive and motor symptom progression in PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Nested PCR Assay for Eight Pathogens: A Rapid Tool for Diagnosis of Bacterial Meningitis.

Bacterial meningitis is a dreadful infectious disease with a high mortality and morbidity if remained undiagnosed. Traditional diagnostic methods for bacterial meningitis pose a challenge in accurate identification of pathogen, making prognosis difficult. The present study is therefore aimed to design and evaluate a specific and sensitive nested 16S rDNA genus-based polymerase chain reaction (PCR) assay using clinical cerebrospinal fluid (CSF) for rapid diagnosis of eight pathogens causing the disease. The present work was dedicated to development of an in-house genus specific 16S rDNA nested PCR covering pathogens of eight genera responsible for causing bacterial meningitis using newly designed as well as literature based primers for respective genus. A total 150 suspected meningitis CSF obtained from the patients admitted to Central India Institute of Medical Sciences (CIIMS), India during the period from August 2011 to May 2014, were used to evaluate clinical sensitivity and clinical specificity of optimized PCR assays. The analytical sensitivity and specificity of our newly designed genus-specific 16S rDNA PCR were found to be ≥92%. With such a high sensitivity and specificity, our in-house nested PCR was able to give 100% sensitivity in clinically confirmed positive cases and 100% specificity in clinically confirmed negative cases indicating its applicability in clinical diagnosis. Our in-house nested PCR system therefore can diagnose the accurate pathogen causing bacterial meningitis and therefore be useful in selecting a specific treatment line to minimize morbidity. Results are obtained within 24 h and high sensitivity makes this nested PCR assay a rapid and accurate diagnostic tool compared to traditional culture-based methods.

False-positive PCR detection of cyclovirus Malawi strain VS5700009 in human cerebrospinal fluid

BackgroundCyclovirus (CyCV) Malawi strain VS5700009 has recently been discovered and reported in clinical cerebrospinal fluid (CSF) samples. Further epidemiological and case-control studies are warranted. The availability of a highly sensitive and specific detection assay for this new virus is thus crucial. ObjectivesTo evaluate the performance of the first and the only available PCR assay for CyCV-VS5700009. Study designA total of 100 CSF samples collected during January–December 2010 were selected for PCR detection of CyCV-VS5700009. Positive PCR amplicons were subjected to sequencing confirmation and BLAST analysis. ResultsInitial PCR screening for CyCV-VS5700009 identified one sample, showing a PCR band of expected size (380 bp). Sequencing and BLAST analysis, however, indicated that the band was 364 bp in length and showed >99% nucleotide homology to a human gene known as nuclear receptor coactivator 6 (NCOA6). Pairwise sequence alignment confirmed that both the forward and reverse PCR primers used had significant homology (>70%) to NCOA6. None of the CSF samples tested were positive for CyCV-VS5700009. ConclusionsThe original PCR assay for CyCV-VS5700009 detection may have potential cross-reactivity with contaminating human genomic DNA. The assay may be of little diagnostic use on clinical specimens that are rich in host DNA such as biopsy tissues.

CSF and Brain Indices of Insulin Resistance, Oxidative Stress and Neuro-Inflammation in Early versus Late Alzheimer's Disease.

Alzheimer’s disease (AD) is characterized by progressive impairments in cognitive and behavioral functions with deficits in learning, memory and executive reasoning. Growing evidence points toward brain insulin and insulin-like growth factor (IGF) resistance-mediated metabolic derangements as critical etiologic factors in AD. This suggests that indices of insulin/IGF resistance and their consequences, i.e. oxidative stress, neuro-inflammation, and reduced neuronal plasticity, should be included in biomarker panels for AD. Herein, we examine a range of metabolic, inflammatory, stress, and neuronal plasticity related proteins in early AD, late AD, and aged control postmortem brain, postmortem ventricular fluid (VF), and clinical cerebrospinal fluid (CSF) samples. In AD brain, VF, and CSF samples the trends with respect to alterations in metabolic, neurotrophin, and stress indices were similar, but for pro-inflammatory cytokines, the patterns were discordant. With the greater severities of dementia and neurodegeneration, the differences from control were more pronounced for late AD (VF and brain) than early or moderate AD (brain, VF and CSF). The findings suggest that the inclusion of metabolic, neurotrophin, stress biomarkers in AβPP-Aβ+pTau CSF-based panels could provide more information about the status and progression of neurodegeneration, as well as aid in predicting progression from early- to late-stage AD. Furthermore, standardized multi-targeted molecular assays of neurodegeneration could help streamline postmortem diagnoses, including assessments of AD severity and pathology.

A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara

This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in “”127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples.

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%–3.67% and 0.79%–4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample.

Evaluation of MeningoFinder, a Novel Multiplex Ligation-Dependent Probe Amplification Assay for Simultaneous Detection of Six Virus Species Causing Central Nervous System Infections

A multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species was developed and tested on clinical cerebrospinal fluid (CSF) samples. The assay, termed MeningoFinder, showed an accordance of 97%, concordance of 96%, interlaboratory sensitivity of 90%, and interlaboratory specificity of 94% compared to PCRs.

Infezioni del Sistema Nervoso centrale da Coxsackievirus ed Echovirus - Risultati di 5 anni di osservazione nel territorio Piemontese

The report is an overview of enterovirus epidemiology in Piemonte during a 5-year period from 2000 to 2004 with an investigative protocol recording epidemiologic, clinical, and laboratory data. A total of 1232 clinical cerebrospinal fluid (CSF) were collected from patients having clinical manifestations of aseptic meningitis (AAM) or encephalitis. Enterovirus detection was performed by isolation on cell culture (MRC5, BGM, Hep 2 e VERO) according to World Health Organization recommended protocols, and molecular methods based on reverse transcription (RT)-PCR. Isolates were identificated by indirect immunofluorescence staining with commercially available monoclonal antibodies and serotype identification was performed by seroneutralization (Lim and Benyesh-Melnick) of the cytopathic effect using pools of specific antisera. Twenty-six patients (2.1%), 15 males and 21 females, were found positive for Enterovirus with at least one of the test used.The average age was 23 (6-43).A total of 26 Non-Polio Enterovirus (NPEV) strains were isolated (11 Echovirus, 5 Coxsackievirus, 10 not identified); the dominant strain of the outbreak was identified as a human Echovirus 6 (4 cases) followed by Echo 31 (3), Coxsackie B5 (3), Echo 17 (2) Echo 9, 11, (1) and Coxsackie A16 (1). The yearly distribution of positive cases was homogeneous; a seasonal variation was noted with a predominance in summer-autumn; the higher transmission period starts in May and peaks in June-July; sporadic cases were also observed in winter and spring.

INFEZIONI DEL SISTEMA NERVOSO CENTRALE DA COXSACKIEVIRUS ED ECHOVIRUS – RISULTATI DI 5 ANNI DI OSSERVAZIONE NEL TERRITORIO PIEMONTESE

The report is an overview of enterovirus epidemiology in Piemonte during a 5-year period from 2000 to 2004 with an investigative protocol recording epidemiologic, clinical, and laboratory data. A total of 1232 clinical cerebrospinal fluid (CSF) were collected from patients having clinical manifestations of aseptic meningitis (AAM) or encephalitis. Enterovirus detection was performed by isolation on cell culture (MRC5, BGM, Hep 2 e VERO) according to World Health Organization recommended protocols, and molecular methods based on reverse transcription (RT)-PCR. Isolates were identificated by indirect immunofluorescence staining with commercially available monoclonal antibodies and serotype identification was performed by seroneutralization (Lim and Benyesh-Melnick) of the cytopathic effect using pools of specific antisera. Twenty-six patients (2.1%), 15 males and 21 females, were found positive for Enterovirus with at least one of the test used.The average age was 23 (6-43).A total of 26 Non-Polio Enterovirus (NPEV) strains were isolated (11 Echovirus, 5 Coxsackievirus, 10 not identified); the dominant strain of the outbreak was identified as a human Echovirus 6 (4 cases) followed by Echo 31 (3), Coxsackie B5 (3), Echo 17 (2) Echo 9, 11, (1) and Coxsackie A16 (1). The yearly distribution of positive cases was homogeneous; a seasonal variation was noted with a predominance in summer-autumn; the higher transmission period starts in May and peaks in June-July; sporadic cases were also observed in winter and spring.