Clusterin is a multifunctional protein, which is associated with the pathogenesis and the development of Alzheimer’s disease (AD). Compared with normal controls, inconsistent results have yielded in previous studies for concentration of cerebrospinal fluid (CSF) clusterin in AD patients. We explored CSF clusterin levels in different pathological processes of AD.42(A), phosphorylated-Tau (T), and total-tau (N). Based on previously published cutoffs and the close correlation between CSF p-tau and t-tau, 276 participants from the publicly available ADNI database with CSF biomarkers were divided into four groups: A-(TN)- , A+(TN)- , A+(TN) + , A-(TN) + . To assess CSF clusterin levels in AD continuum, intergroup differences in four groups were compared. Pairwise comparisons were conducted as appropriate followed by Bonferroni post hoc analyses. To further study the relationships between CSF clusterin levels and AD core pathological biomarkers, we employed multiple linear regression method in subgroups.Following the National Institute on Aging-Alzheimer’s Association (NIA-AA) criteria, we employed on the levels of CSF AβP = 0.002 after Bonferroni correction), but increased in the A+(TN) + group and the A-(TN) + group (both P <  0.001 after Bonferroni correction). Moreover, we found CSF clusterin levels are positively associated with CSF Aβ42 , CSF p-tau  and CSF t-tau .Compared with the A-(TN)- group, CSF clusterin levels were decreased in A+ (TN)- group (Our results indicated that there are differences levels of CSF clusterin in different stages of AD pathology. The CSF clusterin level decreased at the early stage are related to abnormal Aβ pathology; and the increased levels are associated with tau pathology and neurodegeneration.The online version contains supplementary material available at 10.1186/s12883-022-03038-w. Alzheimer’s disease (AD) is a devastating neurodegenerative disorder affecting in the world widely . At the CLU gene which contains single nucleotide polymorphisms (SNP’s) associated with the risk of late-onset Alzheimer’s disease (LOAD) [Clusterin  is a multifunction protein widely expressed in vivo, attributing functions obtaining immune modulation, regulation Aβ metabolism, Aβ clearance in AD . Primarye (LOAD) –7. Accume (LOAD) –10.Howeve (LOAD) , 12. Rece (LOAD) , 14. We Studies have shown clusterin involved in AD pathology. Previous studies have shown that clusterin exerts protective effects in AD, including markedly improving Aβ clearance by binding to Aβ across the blood-brain barrier (BBB) , preventWe employed the ATN classification system based on the CSF levels of amyloid-β (1–42) (A), phosphorylated-Tau (T), and total-tau (N) , which iThis study was based on publicly available data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. ADNI aims to examine whether clinical, imaging and genetic assessments as well as CSF biomarkers can be integrated to detect and track AD as soon as possible. Any neurological disease patients were excluded from the database, except suspected AD. The ages of participants are in 55 to 90. On the ADNI website, more detailed information about inclusive or exclusive criteria can be found. In accordance with the Declaration of Helsinki, written informed consent was acquired from all the participants or their representatives. ADNI was approved by the institutional review boards of all the participating institutions. For latest information, see ADNI website. , 23.42), phosphorylated-Tau (p-tau), and total-tau (t-tau) levels from the ADNI database. Nine participants with values outside the mean ± 3 standard deviations (SD) were excluded, leaving 276 participants for further analysis.We included 285 participants with baseline data on CSF clusterin, CSF amyloid-β (1–42) . At the same time, these CSF core biomarkers were analyzed by the fully automated Elecsys immune assay platform® at the University of Pennsylvania. More detailed information about Elecsys method measuring AD biomarkers in ADNI is provided elsewhere [http://adni.loni.usc.edu/.CSF Aβlsewhere . All CSFlsewhere . The who42 or amyloid PET); “T” refers to aggregated tau (CSF p-tau or tau PET); and “N” refers to neurodegeneration  [42 levels (A); CSF p-tau levels (T); CSF t-tau (N). To reduce comparison groups numbers, the tau pathology group (T) and neurodegeneration group (N) were merged, given that CSF p-tau and t-tau are closely correlated. ‘(TN)-’ profile was defined as both normal range of CSF p-tau and t-tau and ‘(TN)+’ profile was defined as p-tau or t-tau with abnormal range. We dichotomized biomarkers according to use previously published cutoffs as normal (negative) or abnormal (positive). CSF Aβ42 < 976.6 pg/mL, CSF p-tau > 21.8 pg/mL, CSF t-tau > 245 pg/ml were respectively defined as A positive (A+), T positive (T+) and N positive (N+) [Following 2018 NIA-AA criteria, each ADNI participant was assigned into groups by the ATN framework . At pres or MRI) , 27. In ive (N+) . Then, w42, CSF p-tau and t-tau levels were log-transformed to obtain normal distributions. We excluded values outside the mean ± 3 standard deviations (SD) to eliminate the influence of extreme values. Totally, nine individuals were excluded in this step. To explore the differences in CSF biomarkers and sociodemographic data, we employed one-way analyses of variance (ANOVA) for continuous variables and chi square for dichotomous variables (such as APOE ε4 status and gender). Subsequently, to test the levels of CSF clusterin in different ATN groups, we employed an analysis of covariance (ANCOVA) followed by Bonferroni post hoc analyses. Thus, the threshold P values of groups differences for statistical significance was set at 0.008 (= 0.05/6). Finally, in the whole cohort, the biomarker normal group and the AD continuum group, a multiple linear regression was performed to study the associations of the concentration of CSF clusterin with CSF core pathological biomarkers, adjusting for age, diagnosis, gender, education, and APOE ε4 status. P <  0.05 was defined as statistical significance the correlation analysis. We used R software version 4.1.0 and IBM SPSS Statistics to perform all statistical analyses.The data downloaded from the ADNI database showed that the concentration of CSF clusterin displayed an approximate normal distribution, which was presented in the Q-Q plot -, n = 39; A+(TN)+, n = 147; A-(TN)+, n = 40). The study population had a female population of 39.5%, an APOE ε4 positive percentage of 47.8%, an average age of 75.11 ± 6.86 years, and 15.72 ± 2.99 years of education. Four groups showed statistically significant differences in the distribution of APOE ε4 status and MMSE score (both P <  0.001) rather than age, education level, and gender. The proportion of MMSE scores and APOE ε4 are dependent on the ATN framework, of which the highest percentage of APOE ε4 and lowest MMSE scores are in A+ profiles. Compared with the other three groups, A+(TN)- group showed the lowest levels of CSF clusterin.The demographics and clinical features of participants at baseline are summarized in the Table P <  0.001 after Bonferroni correction). Considering age, gender, APOE ε4, education level all influence AD, we conducted another ANCOVA after adjusting for all these factors, which yielded the same results as previous studies.To assess the levels of CSF clusterin across the AD continuum, we employed ATN classification framework. Significant differences were shown by one-way ANCOVA after Bonferroni correction among the four groups Fig. . As mentWe also repeated ANCOVA in groups classified by Aβ pathology and tau pathology status or in groups classified by Aβ pathology and neurodegeneration status. The results of the two analyses are presented in the Supplementary Fig. Our results suggest that during AD progression, CSF clusterin levels are different, which may be associated with the pathological changes in ATN biomarkers.P <  0.001 after Bonferroni correction), the A+(TN)- group , and the A+(TN) + group  after adjusting for age, diagnosis, education, gender, and APOE ε4 status. This result suggests that the elevation of CSF clusterin level may be associated with tau pathology/neurodegeneration.Participants who had abnormal tau pathology or neurodegeneration without amyloidosis(A-(TN) + group)were considered as suspected AD. The results of the Bonferroni post hoc test showed CSF clusterin levels were different among groups. As shown in Fig. APOE ε4 status. We excluded participants in the A-(TN) + group in this analysis. These results were presented in Supplementary Table n = 237), CSF clusterin was positively associated with CSF Aβ42    . In AD continuum groups, there was a positive relationship between CSF clusterin and CSF Aβ42 in Fig. 42 among A-TN- and A + TN- subjects. There was a positive relationship between CSF clusterin and CSF Aβ42 . We repeated the analysis after excluding the outliers, which yielded the similar results Finally, to study the associations between CSF clusterin and the core AD biomarkers based on ATN system, we employed the linear regression methods adjusting for age, diagnosis, education, gender, and In our study, we assessed the CSF clusterin levels across AD continuum using the ATN classification system. We further explored the associations of CSF clusterin with AD core pathological biomarkers in different subgroups. Our results indicated that CSF clusterin levels were different across AD continuum influenced AD pathologies: the decreased levels of CSF clusterin at early stages are related to abnormal Aβ pathology; and the elevated levels of CSF clusterin are related to tau pathology and neurodegeneration.Our data proved our hypothesis that the levels of CSF clusterin have different levels across the AD continuum rather than a unidirectional increase or decrease as shown by previous studies. The different levels of clusterin during different AD stages have not been observed by previous studies due to their adoption of clinical staging. Compared to a previous ADNI study with clusterin increasingly in AD patients , our stuIn the present study, compared to the A-(TN)- group, higher levels of CSF clusterin were observed in both A+(TN) + and A-(TN) + groups, suggesting that increases in CSF clusterin levels were related to tau pathology or neurodegeneration. These results are consistent with that of a recent study which indicated that increased clusterin levels occurred not only in the presence of amyloid plaques but also the primary tauopathy . The cau42, CSF p-tau, and CSF t-tau, which was consistent with most previous studies. Clusterin and p-tau/t-tau might be linked via neuronal damage [We further investigated the associations between CSF clusterin and CSF core biomarkers separately in total individuals, biomarker normal, and AD continuum group. Both in total participants and AD continuum group, CSF clusterin levels were positively related to CSF Aβl damage . Interesl damage –39.Previous studies did not focus on levels of CSF clusterin in different stages of AD. The highlight of the current research is that it is the first to study the CSF clusterin levels in different AD pathological stages. And an AD biomarker classification system we used based on the diagnostic guidelines of the NIA-AA study. There is a temporal sequence on the models of AD pathophysiology theorize. Extracellular Aβ deposition(A) initiates a biological cascade, followed by phosphorylated tau aggregation (T) that leads to neurodegeneration (N) , 40. BasIn the current research, we assessed CSF clusterin levels in the Alzheimer’s continuum based on ATN system classification. The application of this system made us unravel that CSF clusterin levels are different in the development of the pathological stage in AD. To sum up, the decreased levels of CSF clusterin at an early stage are related to abnormal Aβ pathology; and the enhanced levels of CSF clusterin are related to tau pathology and neurodegeneration. Our study provided underlying simultaneous processes in AD progression. Future studies should use longitudinal data and explore the underlying mechanisms in the dynamic CSF clusterin.Additional file 1: Supplementary Fig. 1. CSF clusterin normal Quantile-Quantile Plot, Supplementary Fig. 2. Association of CSF clusterin with age, Supplementary Fig. 3. CSF clusterin in groups defined only by CSF Aβ42 and p-tau, Supplementary Fig. 4. CSF clusterin in groups defined only by CSF Aβ42 and t-tau, Supplementary Table 1. Association of CSF clusterin and CSF core biomarkers, Supplementary Table 2. Association of CSF clusterin and CSF core biomarkers containing outliers.


Abnormal spindle microtubule assembly (ASPM) is a centrosomal protein and that is related to a poor clinical prognosis and recurrence. However, the relationship between ASPM expression, tumor immunity, and the prognosis of different cancers remains unclear.ASPM expression and its influence on tumor prognosis were analyzed using the Tumor Immune Estimation Resource (TIMER), UALCAN, OncoLnc, and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The relationship between ASPM expression and tumor immunity was analyzed using the TIMER and GEPIA databases, and the results were further verified using qPCR, western blot, and multiplex quantitative immuno fluorescence.+ T cells, and M2 macrophages in KIRC and LIHC.The results showed that ASPM expression was significantly higher in most cancer tissues than in corresponding normal tissues, including kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), and breast invasive carcinoma (BRCA). ASPM expression was significantly higher in late-stage cancers than in early-stages cancers , demonstrating a possible role of ASPM in cancer progression and invasion. Moreover, our data showed that high ASPM expression was associated with poor overall survival, and disease-specific survival in KIRC and LIHC (p < 0.05). Besides, Cox hazard regression analysis results showed that ASPM may be an independent prognostic factor for KIRC and LIHC. ASPM expression showed a strong correlation with tumor-infiltrating B cells, CD8These findings demonstrate that the high expression of ASPM indicates poor prognosis as well as increased levels of immune cell infiltration in KIRC and LIHC. ASPM expression may serve as a novel prognostic biomarker for both the clinical outcome and immune cell infiltration in KIRC and LIHC. Immune ls (DCs) . Studiesls (DCs) , includils (DCs) , triple-ls (DCs) , colorecls (DCs) , esophagls (DCs) , and hepls (DCs) . Kashiwals (DCs) . In addi therapy . HoweverAbnormal spindle microtubule assembly (ASPM) is ubiquitously expressed in the ventricular region of the posterior cranial fossa and plays a vital role in the regulation of the mitotic spindle, neurogenesis, and brain size –14. In rIn the present study, we investigated the expression pattern of ASPM by using the Tumor Immune Estimation Resource (TIMER) and UALCAN databases. The functional protein association network and functional enrichment were analyzed using the STRING and Enrichr databases. The correlation between the expression of ASPM and patient survival was calculated by the OncoLnc and Gene Expression Profiling Interactive Analysis (GEPIA) databases. To determine any potential correlation of ASPM expression with immune cell infiltration in cancers, Spearman’s correlation was used to evaluate samples from the TIMER and GEPIA databases. The results of this report reveal the important role of ASPM in cancer and provide an underlying mechanism and potential relationship between ASPM and tumor immunity.This study was performed on archived tissues from 20 diagnosed cases of LIHC and 20 control samples , as well as 20 diagnosed cases of LIHC and 20 control samples .These samples were obtained from Shenzhen Second People’s Hospital. This study was approved by the Ethics Committee of Shenzhen Second People’s Hospital in accordance with the principles of the Declaration of Helsinki.ASPM in several cancers were determined from the TIMER database (https://cistrome.shinyapps.io/timer/), It includes 10,897 samples for 32 cancer types from The Cancer Genome Atlas (TCGA) database to estimate the role of immune infiltration , which is an effective online analysis and mining website for an in-depth analysis of gene expression data using TCGA levels 3 RNA-seq and clinical data from 31 cancer types (ASPM from various cancers and investigated ASPM expression on the basis of tumor grade.The expression levels of ltration . TIMER ultration . Besideser types . In thishttps://string-db.org/) , which is a comprehensive tool for gene enrichment analysis  . Proteinectively . Subsequanalysis .ASPM and survival of patients with KIRC, KIRP, LIHC, LUAD, PAAD, and BRCA was assessed by OncoLnc (http://www.oncolnc.org/), which provides an online database of TCGA survival data related to mRNA, miRNA, and lncRNA expression levels and was used to investigate their prognostic values , an online database used to evaluate the correlation between ASPM expression and clinicopathologic information in cancers . We explored the expression of ASPM and the genetic markers of immune cell subsets through the correlation module in the TIMER database, calculated Spearman’s correlation coefficient, and estimated the statistical significance , and M2 macrophages (marker: CD163), we performed immunohistochemistry to assess ASPM, CD19, CD8A, and CD163. The concentration of the four antibodies was optimized; the following primary antibodies were used: rabbit anti-ASPM , anti-CD19 , anti-CD8A , and anti-CD163 . All of the above fresh tissue was under cryopreservation and processed into frozen sections. The slides were deparaffinized and rehydrated through graded alcohols and were then subjected to an antigen retrieval procedure . After treatment with 3% H2O2 for 10 min to quench endogenous peroxidase activity. Then the tissues were incubated by primary antibodies and incubated at 4 ˚C overnight and incubated by secondary antibodies. We defined an appropriate scoring system based on the immune response scoring method proposed by Remmele and Stegner, using the product of staining intensity and the percentage of positive cells. The staining intensity is divided into 4 levels, that is, level 0 is negative if no positive cells are seen, level 1 is weakly positive, level 2 is moderately positive, and level 3 is strong positive. The percentage of positive cells is divided into 5 levels, that is, level 0 is negative, level 1 ≤ 10%, level 2 is 11%-50%, level 3 is 51%-80%, and level 4 > 80%. The expression density of ASPM, CD8A, CD19, and CD163 was quantitated by scoring staining intensity through automated analysis, including negative (–), weak (+), moderate (++), and strong (+++) staining, respectively. IHC scores were graded by manual interpretation by three statisticians and three pathology experts.We used archived tissues from 20 diagnosed cases of LIHC and 20 control samples , as well as 20 diagnosed cases of LIHC and 20 control samples  for immunohistochemistry. To validate the relationship between ASPM expression and B cells (marker: CD19), CD8+ T cells, and M2 macrophages. The following primary antibodies were used: rabbit anti-ASPM , anti-CD19 , anti-CD8A , and anti-CD163 . Different primary antibodies were applied in turn, and then incubated with horseradish peroxidase bound secondary antibodies and tyramine signal amplification (TSA) technology for high-resolution subcellular localization of proteins. After TSA, the slides were subjected to microwave heat treatment. All human antigens were labeled and stained with 4’-6’-diamino-2-phenylindole . Stained slides were scanned using a mantra system , which captured fluorescence spectra at 20 nm wavelength intervals at the same exposure time; the scans are combined to form a single laminated image.We reused previously collected 20 diagnosed cases of KIRC and 20 diagnosed cases of LIHC samples for ultiplex quantitative immunofluorescence staining was obtained using pano 7-plex IHC kit, cat 000410100 . We performed multiplex quantitative immunofluorescence to assess the relationship between ASPM expression and B cells, CD8In this study, we reused previously collected 10 diagnosed cases of KIRC and 10 diagnosed cases of LIHC archived tissues for western blot and RT-qPCR analysis, these tissues which do have ethics. This study was approved by the Ethics Committee of Shenzhen Second People’s Hospital in accordance with the principles of the Declaration of Helsinki. We extracted proteins with phosphate-safe extraction reagent.The protein was separated using 10% SDS-PAGE and was then transferred to a polyvinylidene fluoride (PVDF) membrane and blocked using 5% skimmed milk in triethanolamine-buffered saline solution (TBS) at 25°C for 1 h, incubated with specific primary ASPM antibodie  overnight at 4°C and then incubated with secondary antibodies for 2 h at 37°C. Images of protein bands were captured by ECL substrate and ImageQuant RT ECL System . The images were analyzed using Quantity One software.® Green PCR Kit (QIAGEN). The primers for detection of ASPM  and GAPDH  were synthesized by Sangon Biotech .We performed RNA extraction from the above-collected tissue . The total RNA was extracted by using the RNeasy Mini Kit (QIAGEN). Total RNA was then reverse-transcribed using Script II RT Kit (QIAGEN). Samples were quantified following the instructions of SYBRSurvival curves were generated through analysis on OncoLnc online tool and GEPIA database. The data were processed using a one-way analysis of variance (ANOVA). Data were presented as the means of the results from at least three independent experiments. Overall survival (OS) was calculated using Kaplan-Meier analysis and log-rank test. Use univariate Cox analysis to screen potential prognostic factors, and multivariate Cox analysis to verify the effect of ASPM expression on survival along with other clinical variables. A value of p < 0.05 was considered statistically significant.ASPM in cancer progression, the mRNA levels of ASPM in various types of cancer tissues and their corresponding normal tissues were analyzed using the TIMER database. As shown in ASPM was upregulated in BLCA , BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA , HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRC, KIRP, LIHC, LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), SKCM (skin cutaneous melanoma), STAD (stomach adenocarcinoma), THCA (thyroid carcinoma), and UCEC . Additionally, to confirm the differential expression of ASPM between tumor and normal tissues, ASPM expression was analyzed using the UALCAN database, and we found that ASPM mRNA expression was significantly higher in KIRC (p < 0.05), KIRP (p < 0.05), LIHC (p < 0.05), LUAD (p < 0.05), PAAD , and BRCA (p < 0.05) tissues than in normal tissues  (p < 10−8) and clear cell type B (ccB) (p < 10−13) subtype than that in the normal subtype, ASPM expression in ccB subtype was significant higher than in ccA subtype (p < 10−4). In KIRP tumors, the increase was significant for the CpG island methylator phenotype (CIMP). ASPM overexpression was more significant for type 2 papillary renal cell carcinoma (RCC) than for type1 papillary RCC (p < 10−3). The expression of ASPM was higher in all LIHC tumor tissues than in normal tissues, with that in grade 3 tissues being statistically significant. We found that ASPM expression in grade 3 was significant high in grade 1 and grade 2 (p < 10−4), but the change in ASPM expression between grade 3 and grade 4 for LIHC was not significant. Regarding lung cancers, all histological subtypes of LUAD tumors showed a notable increase in ASPM increase, which was more statistically significant for lung adenocarcinoma-not otherwise specified and lung adenocarcinoma mixed subtype (Mixed) than lung bronchioloalveolar carcinoma mucinous (LBC-Mucinous). Regarding patients’ smoking habits, all histological subtypes of LUAD tumor showed increased ASPM expression compared with normal tissues, we found that ASPM expression was higher in patients who smoked longer (p < 10-6). Concerning PAAD tumors, on the basis of tumor grade, ASPM expression was higher in all tumor tissues than in normal tissues  and HER2 positive (p < 10-4) breast cancer. ASPM expression showed the most significant increase in infiltrating ductal carcinoma and infiltrating lobular carcinoma. The expression of ASPM was higher in pre-menopause, peri-menopausal, and post-menopause women than in normal women , stage 4 than stage 2 (p < 10-3), stage 4 than stage 3 (p < 10-2) in KIRC. For KIRP, the expression levels of ASPM were significantly higher in stage 3/4 than stage 1/2. The expression levels of ASPM were significantly higher in stage 2 than stage 1 for LIHC (p < 10-2) and BRCA (p < 10-3). However, there is no association in PAAD. These results demonstrate a possible role of ASPM in cancer progression and invasion.We investigated  tissues Table 1.To determine the biological interaction network of ASPM, we analyzed the functional protein association network generated by the STRING database  of the identified genes ASPM, we compared the overall survival rate of patients with a high expression level of ASPM (50%) to those with a low expression level of ASPM (50%) using the OncoLnc online tool , KIRP (p = 1.10e-10), LIHC (p = 1.10e-03), LUAD (p = 2.30e-05), and PAAD (p = 5.70e-04). However, there was no significant correlation between ASPM expression and the survival rates in BRCA patients (p = 1.20e-01), which were used as negative controls in subsequent analyses.To investigate whether the survival rate of KIRC, KIRP, LIHC, LUAD, PAAD, and BRCA patients is related to the expression level of ine tool Figure 4ASPM and cancer prognosis , but positively correlated with tumor purity in KIRP , LIHC , and BRCA . There was no significant correlation between ASPM expression and tumor purity in LUAD and PAAD (p > 0.05). Interestingly, high ASPM expression in KIRC tissues markedly increased the infiltration of immune cells, such as B cells , CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and DCs . In addition, we found that the expression of ASPM had a positive correlation with B cells , neutrophils , and DCs , but a negative correlation with macrophages . There was no significant correlation between ASPM expression and CD8+ T cells or CD4+ T cells (p > 0.05) in KIRP. For LIHC, the expression of ASPM was positively correlated with B cells , CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and DCs . These findings implicate that ASPM plays a specific role in immune cell infiltration in KIRC, KIRP, and LIHC.Previous studies have shown that the number and activity of tumor infiltrating lymphocytes determine the survival time of several tumor patients , 35. The samples . FigureASPM and the state of immune cells based on the expression levels of immune marker genes using the TIMER database and immunohistochemistry in KIRC, KIRP, and LIHC tissues, including B cells, T cells, CD8+ T cells, monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages, neutrophils, NK cells, and DCs , pathologic stage (p < 0.001), TNM stages (p < 0.001) were significant parameters that affect the survival time of KIRC patients (ASPM expression was significant related with pathologic stage (p < 0.001) and T stage (p < 0.001) in LIHC , CD8+ T cells (marker: CD8A), and M2 macrophages (marker: CD163) infiltration. The results are consistent with our hypothesis. Macrophages are a group of differentiated immune cells and classify as M1 macrophages and M2 macrophages, they play an important role in development, homeostasis, and immunity ; UALCAN database ; STRING (https://string-db.org/); Enrichr database (http://amp.pharm.mssm.edu/Enrichr); OncoLnc (http://www.oncolnc.org/); GEPIA (http://gepia.cancer-pku.cn/index.html).Publicly available datasets were analyzed in this study. This data can be found here: TIMER database  for the publication of any potentially identifiable images or data included in this article.TD and YL conceived the project and wrote the manuscript. JZ performed part of the experiments. QH participated in data analysis and participated in discussion and language editing. YT reviewed the manuscript and supervised this project. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China (82002936); Guangdong Natural Science Foundation (2020A1515010347); Shenzhen Science and Technology Innovation Committee ; the Major Scientific and Technological Project of Guangdong Province (2020A1515110419); Shenzhen Municipal Government of China ; China Postdoctoral Science Foundation Funded Project (2021T140478).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Borrelia burgdorferi sensu lato bacteria in patient samples for diagnosis of Lyme neuroborreliosis (LNB) is hampered by low diagnostic sensitivity, due to few bacteria in cerebrospinal fluids (CSF) samples. Evaluation of novel molecular methods, including digital PCR (dPCR), as future tools in diagnostics of LNB is desirable. This study aimed to establish a dPCR assay and validate pre-PCR procedures for detection of Borrelia in CSF. Synthetic DNA fragments and cultured Borrelia reference strains were used during optimisation experiments. In addition, 59 CSF specimens from patients examined for LNB were included for clinical validation. The results showed that the pre-PCR parameters with the highest impact on Borrelia-specific dPCR method performance were incubation of the PCR-plate at 4 °C for stabilization of droplets, centrifugation for target concentration, quick-spin for dPCR rain reduction, and PCR inhibition by matrix components. Borrelia DNA in CSF was detected in one out of nine patients with LNB. Diagnostic sensitivity was determined to be 11.1% and specificity 100%. In conclusion, this study reports an optimized Borrelia-specific dPCR method for direct detection of Borrelia in CSF samples. The present study does not support the use of Borrelia-specific dPCR as a routine method for diagnosing LNB.Direct detection of  Borrelia burgdorferi sensu lato complex. The Borrelia bacteria (Bb) may disseminate from a tick bite and establish infection in the central nervous system (CNS) causing neurological disease manifestations2. The most common neurological manifestation of early LNB in Europe is lymphocytic meningoradiculitis, which is defined by painful radicular neuritis, cranial nerve abnormalities such as facial nerve palsy, and mononuclear cell CNS inflammation4. Various Bb can cause LNB including B. burgdorferi sensu stricto, B. garinii, B. spielmanii, B. bavariensis, and B. afzelii5.Lyme neuroborreliosis (LNB) is a nervous system disorder caused by bacteria belonging to the 6. The polymerase chain reaction (PCR) technique has, due to its turnaround time and high sensitivity and specificity, revolutionized the diagnostics of diseases with microbiological aetiology. However, studies using PCR in the diagnostics of LNB have shown variable results with clinical sensitivities ranging between 12 and 46%10. Hence, PCR is not recommended as a routine test for diagnosis of LNB11. Therefore, the diagnosis of LNB is based on neurological symptoms suggestive of LNB together with CSF pleocytosis and detection of intrathecally produced Borrelia-specific antibodies 11. The AI result can be negative in patients with a short duration of neurologic symptoms, due to limited synthesis of intrathecal antibodies12. In these early LNB cases, PCR-based technologies may serve as supplemental diagnostic tools, because the bacterial load in CSF is relatively higher early in the disease course13. To detect these early LNB cases by PCR, the analysis still needs to be highly sensitive and specific.Direct detection of Bb in cerebrospinal fluid (CSF) samples is challenging because the Bb are present in extremely low numbers and Bb culturing is laborious and may take up to 12 weeks14. The detection principle of the dPCR technique relies on target molecules that are randomly distributed across many small and independent partitions. In each partition with a target molecule, PCR is performed, resulting in partitions with high amounts of fluorescent signal that can be separated from the empty negative partitions with baseline signal only, thus resulting in a digital output. An evaluation of the dPCR technology as a future tool in diagnostics of LNB is desirable as dPCR has several advantages over conventional PCR, including the ability to detect DNA in samples with minimal target numbers using the concentration effect of the partitions and by enrichment of the target molecules from interfering compounds or background signals17.During the last decade, novel digital PCR (dPCR) platforms have become commercially available and affordable leading to rapid application in fields of clinical oncology, prenatal diagnostics, and infectious diseases19, but the dPCR technique has not yet been applied in the context of LNB.Digital PCR assays for detection of Bb have been established in relation to ticks and blood samples20. Therefore, it is critical that the pre-analytical steps (concentration and purification) are evaluated and adjusted as well to optimize the entire PCR method. We hypothesise that a crude sample preparation in combination with a sensitive dPCR assay may be used for detection of Bb DNA in CSF specimens.In addition, suboptimal treatment of samples before the PCR analysis is performed  may have adverse effects on the PCR performance, and thus result in low diagnostic sensitivityA.Borrelia dPCR assay.Establishment of B.without DNA extraction.Optimization of “direct” pre-PCR method C.Compare the “direct” method to the automated Maxwell DNA extraction method.D.Test of patient samples with optimized pre-analytical method and dPCR assay.The objective of this study was to establish an optimized dPCR method for Bb-specific detection in cerebrospinal fluid samples. This was done by systematically evaluating different analytical steps with a potential effect on overall dPCR method performance:Borrelia dPCR method are outlined in Fig. A detailed description of the experimental setups is presented in Supplementary. Our approach to establish and optimise a highly specific and sensitive Borrelia species  known to cause LNB, we chose to use the Borrelia-specific primer and probe sequences described by Ornstein and Barbour in 2006. We performed an in-silico verification of the primers and probe in two stages. First, primers and probes were entered into the NCBI Nucleotide BLAST web application and checked against the nucleotide collection database (nr/nt) to look for sequence homology with different Borrelia strains as well as potential non-specific matching of particularly human or microbial origin. BLAST default settings were applied. Second, the primers and probes were checked for oligo-dimerization using the IDT OligoAnalyzer™ Tool . All primers and probes fulfilled our acceptance criteria, e.g., “Expect value”21 for sequence homology below 0.01.For detection of the five During the initial dPCR experiments we observed that it was important to incubate the dPCR plates on the PCR thermal cycler at 4 °C, overnight, prior to reading to obtain a minimum of 15,000 droplets per well. When comparing the total droplet counts from plates that were read directly after PCR amplification  to the total droplet counts from incubated plates , the difference was statistically significant (p < 0.001).Borrelia dPCR assay was optimised using a stepwise approach to evaluate how the different parameters  with four reverse primer concentrations (300–1200 nM) in a primer matrix. The resulting data , medium (50 copies) and low (5 copies) of Borrelia gBlocks per PCR reaction  was used as target for the internal positive PCR control (IPC). First, the IPC assay was tested alone to assess the separation of the IPC positive cluster from the IPC negative cluster (data not shown). Then the Taq errors. Therefore, we assessed the frequency of false-positive partitions by testing samples containing only PCR-grade water, the “negative CSF matrix”, or Maxwell elution buffer . The analytical sensitivity of the Borrelia assay was determined by a twofold dilution series using known copies of Borrelia gBlock in water , together with an assessment of limit of detection (sensitivity). The analytical specificity was evaluated using a collection of purified bacterial and viral DNA from either laboratory culture strains or QCMD past panels, representing various important differential diagnostic CNS infections  calculated by the QuantaSoft software . Bb were spiked into either “negative CSF matrix” or the “CSF matrix with pleocytosis” and run in parallel using the optimised direct method or the Maxwell DNA extraction method as detailed in Supplementary Borrelia and the IPC dPCR assays in the direct pre-PCR method compared to the Maxwell method, as indicated by the increase in amount of rain. The analytical sensitivity of the Maxwell method was evaluated to be approximately 10 Borrelia targets per mL sample, when using the lowest concentration (0.14 copies/µL) calculated by the QuantaSoft software  results and fifty patients with negative AI results and no CSF pleocytosis, respectively. All patient samples were centrifuged, and DNA was extracted using the Maxwell instrument, before dPCR analysis.The clinical validation of the Borrelia DNA in the dPCR analysis. Thus, the clinical sensitivity was calculated to be 11.1% (1/9) and the clinical specificity was 100% (50/50). The patient with the positive Borrelia dPCR test had a short duration of symptoms (< 7 days) prior to lumbar puncture. The initial Borrelia dPCR result was confirmed by a repeat run on the same sample eluate. Both dPCR runs demonstrated very low amounts of Borrelia DNA in the patient sample  some droplets may shrink during PCR due to the evaporation of water and this is overturned by the cooling of the droplets. The size of the droplets is detected during the reading of the droplets and fluorescence signals from tiny droplets (assessed by a detector) will be dismissed by the Quantasoft software resulting in a reduced number of accepted droplets. A high number of accepted droplets is essential when detecting rare events, e.g., very few Borrelia bacteria, and therefore necessary for our experimental setup. However, a cooling step before droplet reading is not required in all dPCR applications.In order to facilitate replication of our experiments, we have described the optimization process of our Borrelia DNA targets in CSF.By including an incubation step at 4 °C over-night of the PCR-plate prior to data acquisition, the overall method time is extended. This may be a disadvantage, when aiming to use the method for diagnosing LNB in the clinical laboratory. However, we consider that the benefit of maximizing the total droplet counts will greatly outweigh the disadvantage of increased turnaround time because more droplets read increases the chance of not missing the extremely rare Borrelia DNA by dPCR without the often-applied DNA extraction procedure prior to PCR analysis. This was done to avoid the theoretically loss of DNA material during purification procedures, and to exploit the ability of the dPCR analysis to amplify target molecules in reactions with PCR interfering components. To release the Borrelia DNA for detection, the spiked bacteria samples were subjected to simple boiling lysis. Our results confirm that Borrelia bacteria can be broken by boiling only, and that concentration of Borrelia targets by centrifugation is a central condition that improves the analytical sensitivity  and bacteria in clinical samples are evident, thus we also analysed patient CSF samples. We included fifty-nine patients examined for LNB, with samples stored at − 20 °C containing at least 0.5 mL CSF sample material. We detected Borrelia DNA in one clinical sample from a patient diagnosed with LNB resulting in a clinical sensitivity of 11% (1/9), which is within range of previous reports25. Positive PCR results for detection of Borrelia targets in CSF samples often have high cycle threshold values (> 38), indicating low amounts of targets in the samples26. The dPCR analysis of our positive patient sample resulted in similar weak signal with < 4 positive partitions per duplicate in each run 25. Consequently, it has been suggested that larger CSF sample volumes (> 1 mL) in combination with centrifugation may improve the possibility of detecting the Borrelia target by PCR23. A different approach enabling enrichment of Borrelia DNA prior to PCR could be to culture the clinical CSF samples. The turnaround time for Borrelia PCR results will increase significantly with this approach, however, the isolation and direct detection of the etiological agent from CSF samples will substantiate a clinical diagnosis of early LNB given patients with short duration of illness and initial negative Borrelia-specific AI results. Notably, we found one patient positive of the two patients with a duration of symptoms of less than a week indicating the importance of an early clinical suspicion of neuroborreliosis in patients with relevant symptoms.In general, the analytical sensitivity and specificity of PCR assays detecting Borrelia-specific dPCR assay performance and direct detection of Borrelia infection in CNS. The present study does not support the use of Borrelia-specific dPCR as a routine diagnostic method for diagnosing LNB. Future studies with focus on CSF pre-culture enrichment of Borrelia targets prior to PCR need to be performed during the earliest stage of a LNB possible.In conclusion, our study has evaluated parameters important for An overview of the experimental workflow in this study is presented in Fig. Borrelia gBlock) was initially used as positive PCR control during optimization of the in-house dPCR assay. Additionally, a gBlock containing a gene fragment of the Phocid herpesvirus (PhHV)28 was used as internal positive PCR control (IPC). Further details are provided in Supplementary Double-stranded DNA gBlock  containing the target amplification sequence of the Bb-specific 16S rRNA gene (hereafter Borrelia burgdorferi sensu lato reference strains (Supplementary Borrelia strains were cultured in 8 mL modified Kelly–Pettenkofer (MKP) medium at 35 °C under microaerophilic conditions29. The viability of the Borrelia spirochetes was assessed visually by dark-field microscopy  with 20× and 40× objectives. The Bb were counted using a Petroff–Hausser counting chamber .Borrelia PCR assay was originally designed by Ornstein and Barbour to target a Borrelia burgdorferi sensu lato homogen-specific region of the 16S rRNA gene30. For the PhHV PCR assay, in-house validated forward and reverse primers were used together with a probe from the Wisselink study28. Table The primers and probe for the dPCR was performed on the QX100 Droplet Digital PCR System (Bio-Rad) throughout this study as per the manufacturer’s instructions, as described below and in the “Borrelia and IPC oligonucleotides, ddPCR Supermix for probes (No dUTP) (Bio-Rad 186-3023), and DNA template to a final volume of 22.0 μL (10% excess) and transferred them to a 96-well plate. Droplets were produced on the Automated Droplet Generator (Bio-Rad) from 20 μL of the prepared reaction. The PCR plate was sealed (Bio-Rad) and PCR amplification was performed with the Veriti thermal cycler (Applied Biosystems). The PCR cycling conditions are given in Table The preparation of the digital droplet PCR assay is given in details in Supplementary Borrelia burgdorferi sensu lato strains were used for inclusivity/exclusivity experiments. The content of QCMD CNS 2017 + 2018 and ME 2017 past panels and the Bb reference strains are presented in Supplementary Quality Control for Molecular Diagnostics (QCMD) past panels (Qnostics) and cultured without CSF pleocytosis (< 5 cells/µL) and non-infectious CNS diseases, were pooled and used as CSF spike matrix for optimization experiments, hereafter designated “negative CSF matrix”. To ensure that the CSF spike matrix was negative for CNS disease causing microorganisms, the negative CSF matrix was examined by the FilmArray Meningitis/Encephalitis (ME) PCR Panel .Anonymised CSF specimens from 150 patients with CSF pleocytosis (> 4 cells/µL) were pooled and diluted to contain approximately 100 cells/µL and designated “CSF pleocytosis matrix” hereafter.For evaluation of the effect of CSF pleocytosis on dPCR interference, anonymised CSF specimens from patients The Maxwell® 16 Cell LEV DNA Purification Kit  was used with the automated Maxwell® 16 Instrument (Promega) configured with the low elution volume (LEV) hardware according to the manufacturer’s instructions. Samples (100 µL) was subjected to the purification kit together with 100 µL elution buffer.Borrelia dPCR method if they contained ≥ 0.5 mL CSF. The need for patient approval and informed consent was waived by the ethics committee of the institutional review board at Lillebaelt Hospital.CSF sample aliquots from patients examined for LNB at the Department of Clinical Microbiology Lillebaelt Hospital in the Region of Southern Denmark were included in this study. All samples were sent to the laboratory from October 2019 to March 2021 as part of routine LNB diagnosis and CSF aliquots were stored at − 20 °C until dPCR analysis. Patient aliquots were only analysed by the There were nine CSF samples from patients diagnosed with definite LNB, defined by a positive CSF/serum Bb-specific antibody index (AI) , neurological symptoms consistent with LNB, and concurrent CSF pleocytosis. Patient information provided by the electronic medical record and the local electronic laboratory data management systems for clinical biochemistry (BCC) and microbiology (MADS) included duration of illness, antibiotic treatment prior to lumbar puncture, CSF leukocyte counts, CSF/serum Bb-specific AI results and CSF CXCL13 quantities. As controls we used CSF samples that tested negative in the CSF/serum Bb-specific AI, designated the non-LNB patients.All data analysis was done using the QuantaSoft™ v.1.7.4.0917 and the QuantaSoft™ Analysis Pro 1.0.596 software. Figures and tables were made in Word and Excel v. 2102 .Collection and use of data for this study were conducted in accordance with the declaration of Helsinki and was approved as a quality development project by the ethics committee of the institutional review board at Lillebaelt Hospital (18/62866). This study was registered as public research of the Region of Southern Denmark according to the General Data Protection Regulation (18/63046).Supplementary Information.


This cross-sectional study of 2 observational cohorts investigates if phosphorylated tau biomarkers are more associated with the presence of cerebral β-amyloidosis or tau neurofibrillary tangle aggregation. Do soluble phosphorylated tau (p-tau) biomarkers preferentially reflect the presence of cerebral β-amyloidosis or tau neurofibrillary tangle aggregation?181, p-tau217, p-tau231, p-tau235) were significantly more closely associated with amyloid PET (positron emission tomography) than with tau PET. These results were replicated in an independent group of individuals with plasma p-tau181, p-tau217, and p-tau231 and in another independent cohort with CSF p-tau181.In this cross-sectional study of 2 observational cohorts, 4 p-tau biomarkers in the cerebrospinal fluid , framework. The recent proliferation of phosphorylated tau (p-tau) biomarkers has raised questions about their preferential association with the hallmark pathologies of Alzheimer disease (AD): amyloid-β plaques and tau neurofibrillary tangles.To determine whether cerebrospinal fluid (CSF) and plasma p-tau biomarkers preferentially reflect cerebral β-amyloidosis or neurofibrillary tangle aggregation measured with positron emission tomography (PET).181, p-tau217, p-tau231, p-tau235), [18F]AZD4694 amyloid PET, and [18F]MK6240 tau PET. The second TRIAD subsample included individuals assessed with plasma p-tau , [18F]AZD4694 amyloid PET, and [18F]MK6240 tau PET. An independent cohort from ADNI comprised individuals assessed with CSF p-tau181, [18F]florbetapir PET, and [18F]flortaucipir PET. Participants were included based on the availability of p-tau and PET biomarker assessments collected within 9 months of each other. Exclusion criteria were a history of head trauma or magnetic resonance imaging/PET safety contraindications. No participants who met eligibility criteria were excluded.This was a cross-sectional study of 2 observational cohorts: the Translational Biomarkers in Aging and Dementia (TRIAD) study, with data collected between October 2017 and August 2021, and the Alzheimer’s Disease Neuroimaging Initiative (ADNI), with data collected between September 2015 and November 2019. TRIAD was a single-center study, and ADNI was a multicenter study. Two independent subsamples were derived from TRIAD. The first TRIAD subsample comprised individuals assessed with CSF p-tau  assay or enzyme-linked immunosorbent assay.Associations between p-tau biomarkers with amyloid PET and tau PET.181 difference, 13%; 95% CI, 3%-22%; P = .006; p-tau217 difference, 11%; 95% CI, 3%-20%; P = .003; p-tau231 difference, 15%; 95% CI, 5%-22%; P < .001; p-tau235 difference, 9%; 95% CI, 1%-19%; P = .02) . These results were replicated with plasma p-tau181 , p-tau217 , p-tau231 , and CSF p-tau181  in independent cohorts.A total of 609 participants  were included in the study. For all 4 phosphorylation sites assessed in CSF, p-tau was significantly more closely associated with amyloid-PET values than tau-PET values , framework. Tau biomarkers include quantification of insoluble neurofibrillary tangles using PET, as well as soluble phosphorylated tau (p-tau) in the CSF and plasma. Because of their specificity, amyloid-β and tau biomarkers are increasingly used in AD diagnosis5 and as inclusion criteria for disease-modifying clinical trials.7Alzheimer disease (AD) is defined by the presence of cerebral amyloid-β plaques and tau neurofibrillary tangles.14 A longitudinal study in autosomal dominant AD provides evidence that concentrations of soluble p-tau biomarkers begin to rise in conjunction with amyloid-β aggregation, several years before neocortical tau abnormality.15 Furthermore, longitudinal biomarker studies in sporadic AD report that soluble p-tau217 mediates the association between amyloid-β and tau-PET change.16 Correspondingly, recent biomarker models of AD suggest that p-tau reflects a state between amyloid-β plaques and tau aggregation.18 However, it is unclear to what extent biofluid measurements of p-tau are preferentially associated with the presence of amyloid-β or tau neurofibrillary tangles in the brain.Although soluble p-tau biomarkers are interpreted as biomarkers of tau pathology, several recent observational studies provide evidence that concentrations of p-tau are closely correlated with amyloid-β deposition.181, p-tau217, p-tau231, p-tau235) and 3 in plasma  with amyloid-β and tau aggregation assessed with PET in the Translational Biomarkers in Aging and Dementia (TRIAD) study cohort. In addition, we evaluated the association between CSF p-tau181 with amyloid PET and tau PET in the Alzheimer Disease Neuroimaging Initiative (ADNI) cohort.The objective of the current study was to determine whether soluble p-tau biomarkers are preferentially associated with cerebral amyloid-β plaques or tau neurofibrillary tangles. We evaluated the association between 4 p-tau biomarkers in the CSF (p-tau19 cohort: a CSF p-tau subsample (n = 181) and a plasma p-tau subsample (n = 171). Participants included in the CSF subsample had measures of CSF p-tau , amyloid PET with [18F]AZD4694, tau PET with [18F]MK6240, and magnetic resonance imaging (MRI). The median (IQR) time difference between CSF and PET data collection was 53 (86) days. In the second subsample (n = 171), individuals had measures of plasma p-tau , amyloid PET with [18F]AZD4694, tau PET with [18F]MK6240, and MRI. The median (IQR) time difference between plasma and PET data collection was 70 (112) days. The individuals in the second TRIAD subsample did not have CSF measures of p-tau and thus represent an independent sample of individuals. Participants had paired fluid p-tau and PET biomarker assessments available within a 9-month interval. All individuals were included between October 2017 and August 2021. Individuals from the following race and ethnicity categories were included: Asian, Black, Hawaiian/Pacific Islander, Hispanic/Latinx, non-Hispanic/Latinx, multiracial, White, and unknown/not reported. Race and ethnicity were identified using official National Institutes of Health classifications. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines.This study was approved by the Montreal Neurological Institute PET working committee and the Douglas Mental Health University Institute Research Ethics Board. Written informed consent was obtained for all participants. For this cross-sectional study, we assessed 2 independent subsamples of participants in the TRIAD20 Individuals with dementia had a CDR score of 1 or 2.21 Participants were excluded from this study if they had systemic conditions that were not adequately controlled through a stable medication regimen. Other exclusion criteria were active substance abuse, recent head trauma, recent major surgery, or MRI/PET safety contraindications. PET acquisition and processing are described in eMethods 1 of the 181 and p-tau231, were quantified in the Clinical Neurochemistry Laboratory, University of Gothenburg, Mölndal, Sweden, by scientists blinded to participant clinical and PET information; this information is described in detail in eMethods 2 of 217 was quantified by scientists at Janssen Research & Development blinded to clinical and PET information.Cognitively unimpaired (CU) individuals had no objective cognitive impairment and a Clinical Dementia Rating (CDR) score of 0. Individuals with mild cognitive impairment had subjective and/or objective cognitive impairment and a CDR score of 0.5.18F]florbetapir, tau-PET with [18F]flortaucipir, and CSF p-tau181. ADNI PET acquisition and processing data are described in eMethods 1 of the 181 quantification is described in eMethods 2 of the 22 Plasma p-tau181 was not investigated in ADNI due to the small number of individuals with plasma p-tau evaluations and tau PET at the same visit.The ADNI study was approved by the institutional review boards of all participating institutions. All participants provided informed written consent. We examined the open-access ADNI cohort, a North American multisite cohort launched in 2003. All participants had amyloid PET with [18F]AZD4694 PET and [18F]MK6240 PET were investigated using the Spearman nonparametric test. Statistical evaluation of whether correlations were significantly different was performed in R using the cocor package,23 a statistical framework for comparing associations between intercorrelated measurements. As secondary confirmatory analyses, we conducted partial correlation analyses to determine the extent to which p-tau biomarker concentrations were associated with amyloid PET when correcting for tau PET and tau PET correcting for amyloid PET using the ppcor package. We also conducted analyses correcting for age and sex. P values were 2-sided, and statistical significance was defined as P <.05.Statistical analyses were performed in R, version 4.1.1  and Matlab, version 2015a (MathWorks). Assumptions of normality were tested using the D’Agastino-Pearson normality test. Associations between p-tau biomarkers with ) for their target. However, because [18F]MK6240 has a higher affinity for tau tangles than [18F]AZD4694 does for amyloid-β plaques, the stronger association of p-tau with amyloid PET is unlikely to be driven by sensitivity issues. Replication in an independent cohort (ADNI) with different PET imaging agents helps further attenuate these concerns. Similar to PET biomarkers, biofluid assay performance can affect interpretation of the results in this study. For example, it is unclear to what degree the stronger associations of p-tau217 and p-tau231 with PET biomarkers are driven by biological properties of phosphorylation of a specific amino acid compared with differences in the assays used to detect them . Other limitations of this study include the lack of availability of plasma p-tau235 in the TRIAD cohort and the lack of availability of plasma p-tau181 at the same time point as tau-PET in ADNI. Furthermore, the TRIAD and ADNI cohorts consist of individuals motivated to participate in a study of AD, which may limit generalizability. Finally, the cohorts are not demographically representative of the populations at risk for dementia in North America.Results of this study should be interpreted in the context of several limitations. First, tau (hyper)phosphorylation is a dynamic process, the understanding of which is anticipated to evolve with respect to the availability of more biomarkers. Because tau can be phosphorylated at over 80 sites, some of which are hypothesized to have site-specific associations with disease stage, it is unknown whether all future p-tau biomarkers will exhibit the preferential association with amyloid PET reported in this study. Another limitation is that PET biomarker signals (used in this study as measurements of amyloid-β plaques and tau tangles) are influenced by their affinities (1 / equilibrium dissociation constant [K7In conclusion, results of this cross-sectional study of 2 observational cohorts suggest that p-tau biomarkers better reflect the concentration of amyloid-β plaques than cerebral tau pathology quantified with PET. Our findings contribute to the growing understanding of the role of tau phosphorylation in the amyloid-β cascade and highlight the need for careful interpretation of p-tau biomarkers in CU individuals and as outcomes in disease-modifying clinical trials.


Traumatic brain injury and aneurysmal subarachnoid haemorrhage are a major cause of morbidity and mortality worldwide. Treatment options remain limited and are hampered by our understanding of the cellular and molecular mechanisms, including the inflammatory response observed in the brain. Mitochondrial DNA (mtDNA) has been shown to activate an innate inflammatory response by acting as a damage-associated molecular pattern (DAMP). Here, we show raised circulating cell-free (ccf) mtDNA levels in both cerebrospinal fluid (CSF) and serum within 48 h of brain injury. CSF ccf-mtDNA levels correlated with clinical severity and the interleukin-6 cytokine response. These findings support the use of ccf-mtDNA as a biomarker after acute brain injury linked to the inflammatory disease mechanism. There are estimated to be ∼50 million cases of TBI and 5 million cases of aSAH  occurring annually worldwide.5 An early neuroinflammatory response is evident after acute brain injury, demonstrated by the release of cytokine mediators, such as Interleukin-6 (IL-6), within hours of injury.6 However, the links between serum and cerebrospinal fluid (CSF) levels of ccf-mtDNA after acute brain injury, and their relationship to an early inflammatory response, have not been systematically examined. We hypothesized that levels of ccf-mtDNA in both CSF and serum would be raised after acute brain injury and correlate with the neuroinflammatory response.Mitochondria contain their own genome , which codes for 13 essential proteins involved in oxidative energy metabolism. When released from organelles and cells, mtDNA contributes to damage-associated molecular patterns (DAMPs), and raised plasma levels of circulating cell-free mtDNA (ccf-mtDNA) are associated with organ dysfunction after systemic trauma.Patients with severe TBI or aSAH requiring admission to the Neuro-Critical Care Unit at Cambridge University Hospitals (CUH) were recruited within 48 h of injury. Serum was extracted from blood samples taken from an arterial line. Next, 5 mL of CSF was extracted as a fresh, clean sample from the external ventricular drain when present. All samples of serum and CSF (when available) were obtained within 48 h of injury. This study was approved by the NHS Health Research Authority and the CUH institutional ethics board.Control CSF and serum samples were obtained from patients undergoing investigation for normal pressure hydrocephalus or idiopathic intracranial hypertension. These patients underwent lumbar drain or lumbar infusion studies, requiring a lumbar puncture and removal of CSF as part of standard clinical investigations. All control patients consented to use of their CSF and serum for research purposes as part of the CUH Neurosurgical Biobank.g for 10 min at 4°C with the supernatant stored in cryovials at −80 oC. Blood samples were collected into tubes , gently inverted three times and left to clot for 30 min. Tubes were then centrifuged at 2000g for 10 min at 4°C. The supernatant was then stored at −80°C.All CSF samples were centrifuged within 15 min of collection at 2000Demographic and clinical data were extracted from the electronic health record system, with the initial Glasgow Coma Scale (GCS) as the best responses at the point of first presentation to a clinician.MTND1 and MTND4, using the Bio-Rad QX100 Digital Droplet PCR platform . For serum samples, DNA was first extracted using the DNeasy Blood and Tissue kit . CSF samples were loaded directly, without DNA extraction. The absolute mtDNA copy number is expressed in copies per microliter, derived as the mean of ND1 and ND4 values. Cases and control samples were randomly assigned to each run. IL-6 concentration was measured in duplicate, directly from both serum and CSF samples, using the Human IL-6 ELISA kit .Quantification of ccf-mtDNA was performed in triplicate by droplet digital polymerase chain reaction amplification of the mitochondrial genes, p < 0.05.Data were analyzed using GraphPad Prism  and R statistical language software . Statistical significance testing was carried out using data appropriate tests (detailed in the text), with significance level set at p = 0.067). Median initial GCS score was lower in the TBI compared with aSAH cohort , which did not reach significance . Demographic data for the cohort are summarized in Twenty-five acute brain injury (15 TBI and 10 aSAH) and 10 control patients were recruited. Mean age (±SD [standard deviation]) in the cohorts was 52.6 (±16.8), 64.8 (±9.3), and 66.9 (±14.6) years for TBI, aSAH, and control groups, respectively .Serum samples were collected from all brain injury and control patients. CSF was available and collected from all aSAH patients, 5 TBI, and all control patients. Median time of sampling (± interquartile range) was 24 (±26) and 26 h (±19) after injury for the TBI and aSAH cohorts, respectively  and CSF (p = 0.0007). Similarly, in the aSAH cohort, ccf-mtDNA copy number was raised compared to controls in both serum (p = 0.0015) and CSF (p < 0.0001). Between the two acute brain injury cohorts, there was a higher ccf-mtDNA mean copy number in TBI compared with SAH in both serum (p = 0.0009) and CSF , but not serum  or serum .Initial GCS score correlated with ccf-mtDNA copy number in CSF  and aSAH  cohorts (p = 0.08). CSF concentration of IL-6 correlated with CSF ccf-mtDNA copy number  characterized by mitochondrial dysfunction and impaired mitophagy.14In either scenario, the presence of raised ccf-mtDNA in the central nervous system in the context of injury is of interest. mtDNA, unlike nuclear DNA, contains unmethylated CpG sequences—a pattern common to bacterial DNA—which act as a DAMP.15 However, the mechanism for this response is poorly understood, and this is highlighted by the failure of numerous clinical trials for broad “anti-inflammatory” agents  in these cohorts.15 In concordance with previous studies, we demonstrate an acute IL-6 cytokine response in brain injury patients that is isolated to the CSF and reflects the severity of injury.6 We found that this response is highly correlated to the ccf-mtDNA copy number in CSF. This finding, alongside existing in vitro evidence of mtDNA inducing IL-6 production, suggests a role for ccf-mtDNA as an initiator of neuroinflammation after acute brain injury.14 mtDNA thus presents as both a potential biomarker for the process of neuroinflammation and a target for investigating how this process is initiated. mtDNA, or its specific downstream inflammatory effectors, may also present new avenues for therapeutic intervention, as has already been explored pre-clinically, using nucleic acid scavenging polymers.5After acute brain injury, there is ample evidence of a significant neuroinflammatory response, with IL-6 implicated as a prototypical cytokine released within hours of injury.17One limitation of this study is the relatively small sample size, which was insufficiently powered to stratify patients by demographic parameters, such as age or sex, or pre-morbid conditions. As such, we have not attempted to draw any conclusions with regard to prognostication or association to long-term outcome, but these warrant future exploration in larger longitudinal studies, with more frequent sampling, alongside further measures of injury severity such as imaging scores. We highlight also that our control cohort was drawn from patient groups undergoing investigation for neurological diseases, but we found that both serum and CSF copy number values of ccf-mtDNA of control subjects in this study resemble previous healthy subject findings.In summary, we demonstrate ccf-mtDNA as a potential molecular marker of acute brain injury with CSF levels reflecting severity of brain injury and correlating with an inflammatory cytokine response. These findings open a new avenue for investigating the mechanism and treatment of neuroinflammation after acute brain insults.


Cholesterol pools in the central nervous system (CNS) are largely separate from those in blood, and CNS cholesterol excess may promote neurodegeneration. CEC of cerebrospinal fluid (CSF) may be a useful measure of CNS cholesterol trafficking. We hypothesized that subjects with AD and mild cognitive impairment (MCI) would have reduced CSF CEC compared with Cognitively Normal (CN) and that CSF apolipoproteins apoA-I, apoJ, and apoE might have associations with CSF CEC.Alzheimer’s disease (AD) shares risk factors with cardiovascular disease (CVD) and dysregulated cholesterol metabolism is a mechanism common to both diseases. Cholesterol efflux capacity (CEC) is an ex vivo metric of plasma high-density lipoprotein (HDL) function and inversely predicts incident CVD independently of other risk factors3H]-cholesterol for 24 h, had ABCA1 expression upregulated for 6 h, were exposed to 33 μl of CSF, and then were incubated for 2.5 h. CEC was quantified as percent [3H]-cholesterol counts in medium of total counts medium+cells, normalized to a pool sample. ApoA-I, ApoJ, ApoE, and cholesterol were also measured in CSF.We retrieved CSF and same-day ethylenediaminetetraacetic acid (EDTA) plasma from 108 subjects  from the Center for Neurodegenerative Disease Research biobank at the Perelman School of Medicine, University of Pennsylvania. For CSF CEC assays, we used N9 mouse microglial cells and SH-SY5Y human neuroblastoma cells, and the corresponding plasma assay used J774 cells. Cells were labeled with [We found that CSF CEC was significantly lower in MCI compared with controls and was poorly correlated with plasma CEC. CSF levels of ApoJ/Clusterin were also significantly lower in MCI and were significantly associated with CSF CEC. While CSF ApoA-I was also associated with CSF CEC, CSF ApoE had no association with CSF CEC. CSF CEC is significantly and positively associated with CSF Aβ. Taken together, ApoJ/Clusterin may be an important determinant of CSF CEC, which in turn could mitigate risk of MCI and AD risk by promoting cellular efflux of cholesterol or other lipids. In contrast, CSF ApoE does not appear to play a role in determining CSF CEC.The online version contains supplementary material available at 10.1186/s13195-022-01119-z. Alzheimer’s disease (AD), the most common cause of dementia, is characterized by the abnormal accumulation of amyloid-beta (Aβ) and hyperphosphorylated tau proteins, synaptic and neuronal dysfunction, neuroinflammation, and brain degeneration. Importantly, the brain is the most lipid-rich organ in the body and depends on tight regulation of lipid metabolism and transport to maintain proper neural signaling and cognitive function. This is particularly pertinent to AD, because increased cellular cholesterol content can increase Aβ production  and chanCholesterol biosynthesis occurs in all cells within the central nervous system (CNS) and some cell types can actively take up cholesterol from the extracellular space , 7. ExpeCerebrospinal fluid (CSF) contains at least three apolipoproteins that are known to be lipid transport proteins and could serve as extracellular acceptors of cellular cholesterol and other lipids in the brain. ApoA-I, the major protein in plasma high density lipoproteins (HDL), is not synthesized in the brain, and CSF ApoA-I is derived from the plasma and is in concentrations in CSF approximately 1% of those in plasma  (and SupThe cholesterol efflux capacity (CEC) assay was first developed by the Rader lab, using plasma  in orderWe hypothesized that CSF CEC may be inversely associated with AD: high CSF CEC may facilitate cellular efflux of cholesterol (or other lipids) in a manner that is protective against AD, while low CSF CEC may have an opposite effect, potentially contributing to Aβ accumulation and the development of amyloid plaques in AD. We adapted our plasma CEC assay for CSF and showed that it is reproducible using several cell lines relevant to the CNS . In thatCSF and plasma samples were obtained from the Biosample Repository of the Center for Neurodegenerative Disease Research (CNDR) at the Perelman School of Medicine (PSOM), University of Pennsylvania . This case-control study examined participants enrolled in a prospective longitudinal study under the auspices of the National Institute on Aging (NIA)-funded Alzheimer’s Disease Clinical Center (ADCC) at PSOM. The participants were recruited at the Penn Memory Center, PSOM, and the Maria de los Santos Health Center , following written informed consent under approval of the University of Pennsylvania Institutional Review Board.Cases were classified as AD or MCI based on standard diagnostic criteria according to procedures established for ADCCs and the National Alzheimer’s Coordinating Center (NACC) –21. From1-42), total tau (TTau), and phosphorylated tau (pTau) at the threonine 181 are available in previous publications  cholesterol overnight. On day 2, SH-SY5Y were treated with 2 μM T0901317, and N9 and J774 cells were treated with 0.3 mM 8-CPT-cAMP to upregulate ABCA1. Cells were exposed to 33 μl of individual CSF and cholesterol efflux was allowed to proceed for 2.5 h. Cell medium was counted upon removal of floating cells and cell lipids were extracted with isopropanol read in a scintillation counter. Cell cholesterol efflux was quantified as the percent of [3H]-cholesterol counts in the medium relative to the total counts in medium and cells. A reference CSF sample was included on each plate to decrease inter-assay variability, and CEC activity was calculated as a unitless ratio of cholesterol efflux to sample CSF normalized to the reference CSF.The CSF CEC assay using N9 microglial cells and SH-SY5Y neuroblastoma cells has been described previously . BrieflyA similar assay with J774 mouse macrophages was used to determine plasma CEC using 1% ApoB depleted plasma in place of CSF and is described in Khera et al.  and HoriP < 0.05 were considered significant. CEC values were log-transformed, and the N9 and SH-SY5Y CEC were not significant for the Shapiro-Wilk normality test (P = 0.58 for N9 and P = 0.66 for SH-SY5Y). For the association analysis, linear regression models were used to predict CEC, including lipoproteins and CSF biomarkers. Age, sex, race, and CSF (or plasma) storage time were incorporated as covariates in all analyses. We assessed the contribution of predictors in multinomial logistic regression, and calculated β, the logistic regression coefficient that shows the direction and size of the relationship between predictor and diagnosis. Odds ratio (OR) is the ratio of the odds that are calculated as the exponent of β with 95% confidence interval. The association between plasma CEC and biomarkers were analyzed using the same model. All analysis was carried out in the R application (v 3.6.0).For demographics, categorical variables are in percentages, and continuous variables as means with standard deviations or medians with range. Demographic statistics were computed using ANOVA and chi-square test for the continuous variable and categorical variables, respectively. For the post hoc multiple comparisons, we used the Tukey honestly significant difference (HSD) test. The correlation of efflux capacity between different cell lines was assessed with Spearman’s correlation coefficients. Correlations with 1-42 levels were significantly reduced in AD, whereas total Tau (TTau) and phosphorylated Tau (pTau) were significantly increased in AD. MCI subjects were intermediate between CN and AD in all cases  . We further investigated the relationship between groups using multinomial logistic regression and observed a significant difference between MCI and CN for CECs from both cell types  and from N9 microglial cells  , and phosphorylated-Tau (pTau) in the same CSF samples. Aβ1-42 had a modestly significant association with CSF CEC in SH-SY5Y cells but not in N9 cells  identified several genes involved in cholesterol movement as risk factors, including ApoE, ApoJ, ABCA1, and ABCA7. In this study, we used CSF from subjects with AD and MCI as well as cognitively normal controls to measure CSF cholesterol efflux capacity from two different cell types as well as lipids, apolipoproteins, and AD biomarkers. We studied whether these data were associated with AD/MCI diagnosis as well as with each other. We observed that both AD and MCI patients had significantly lower CSF AβOur major findings are that reduced CSF CEC is a feature of MCI and early-stage AD, and the CSF CEC is strongly correlated with CSF ApoA-I and ApoJ, but not with CSF ApoE. CSF ApoA-I and ApoJ were significantly lower in MCI subjects than in controls, while ApoE is significantly lower in AD subjects compared to controls. Importantly, neither CSF CEC nor CSF ApoA-I were correlated with plasma CEC and ApoA-I; thus, plasma levels of ApoA-I and CEC cannot be used as surrogates for CSF.Importantly, we measured CSF CEC with a novel approach using two different biologically relevant cell lines—the human microglial N9 cell line and the human neuronal SH-SY5Y cell line—by labeling the cells with radiolabeled cholesterol and then testing the capacity of the CSF samples to promote efflux. While previous studies have suggested decreased levels of CSF CEC in MCI and AD subjects , 29, theIn our studies, the two independent CEC assays correlated well with each other. The most significant association with CSF CEC was with CSF ApoA-I and ApoJ, both of which are highly plausible acceptors of cellular cholesterol efflux. Indeed, ApoA-I and ApoJ are known to occur on the same lipoprotein particles in plasma . One mig1-42, and the complex is transported across the blood brain barrier (BBB) into the circulation through low-density lipoprotein receptor related protein 2 (LRP2) to promote clearance of Aβ peptides from the brain [The association between CSF ApoA-I and AD is not consistent in the literature –36. Our he brain , 41. Mor1-42, similar to a previous study [We previously reported that ApoJ/Clusterin predicts CSF CEC from microglia and astrocytes . In our us study . Our resAPOE ε4 allele in reducing CSF ApoE levels. Despite the fact that ApoE can act as an acceptor of cellular lipid efflux, we found no correlation of CSF CEC with CSF ApoE; we did, however, note a significant positive correlation between CSF ApoE and CSF Aβ1-42. In a previous study, the concentration of CSF ApoE was not associated with Aβ1-42 or clinical dementia diagnosis [1-42 [ApoE has a well-established role in AD , though iagnosis . Anotheris [1-42 . Our stuIn summary, we found that CSF CEC measured using two different brain-relevant cell types was significantly lower in MCI patients than in cognitively normal controls. Both CSF ApoA-I and ApoJ (but not ApoE) were significantly associated with CEC. In addition, CSF ApoJ was significantly associated with MCI (lower) and trending in AD. Our major conclusions are that impaired CSF CEC, determined in part by CSF ApoJ concentrations, is a risk factor for MCI and AD. We speculate that factors regulating the synthesis and secretion of ApoJ in the brain influence the ability to promote efflux of cholesterol (and possibly other toxic lipids) from brain cells and therefore influence the risk of AD.Additional file 1: Supplemental Figure 1. Multinomial logistic regression analyses for CSF AD biomarkers and diagnosis. (A) Aβ1-42 is significantly lower in MCI and AD  (B) Total Tau is significantly higher in AD only (***P = 1.58e-04) (C) Phosphorylated Tau is significantly higher in MCI and AD .Additional file 2: Supplemental Figure 2. Multivariate linear regression between CSF CEC from N9, SH-SY5Y, and J774 efflux. (A) Spearman correlation of N9 human microglial cell efflux and SH-SY5Y human neuroblastoma cell efflux. The two CSF CEC are strongly correlated . (B) Spearman correlation of N9 human microglial cell efflux and plasma efflux is not significant . (C) Spearman correlation of SH-SY5Y human neuroblastoma cell efflux and plasma efflux is also not significant .Additional file 3: Supplemental Figure 3. Multivariate linear regression between CSF cholesterol and phospholipids with CSF CEC. (A) Phospholipids measured in CSF (μM) are not associated with SH-SY5Y CEC or N9. (B) Cholesterol measured in CSF (μg/mL) showed strong association with both N9 human microglial cell efflux and SH-SY5Y human neuroblastoma cell efflux .Additional file 4: Supplemental Figure 4. Multivariate linear regression between CSF apolipoproteins. (A-C) CSF apolipoproteins are not significantly associated with each other. (D) ApoE is positively associated with Aβ1-42 (***P<0.001).Additional file 5: Supplemental Figure 5. (A) Association between ApoE genotype and AopE concentration in CSF. The x-axis presents the ApoE genotype dosage. Each additional APOE ε4 allele are assigned as a positive integer, while each ε2 allele acts as a negative integer. Ε3 allele does not have any effect on the equation. This genotype model shows that the APOE genotype has a significant effect on ApoE concentration in CSF (***P = 2.45e-11). (B) The occurrence of ε4 allele carriers is significantly higher in the AD group .Additional file 6: Supplemental Figure 6. Multivariate linear regression of AD biomarkers with CEC in CSF. (A) SH-SY5Y human neuroblastoma cell CEC significantly associated with increased Aβ1-42 (*P = 0.0226) but not N9 microglial cell CEC. (B) TTau is not significantly associated with either SH-SY5Y human neuroblastoma cell or N9 microglial cell CEC. (C) PTau is not significantly associated with either SH-SY5Y human neuroblastoma cell or N9 microglial cell CEC. (D) PTau/Aβ1-42 ratio is not significantly associated with either SH-SY5Y human neuroblastoma cell or N9 microglial cell CEC.Additional file 7: Supplemental Table 1. Concentrations of apolipoproteins in plasma.


The manufacturer's instructions for the venereal disease research laboratory (VDRL) antigen test for diagnosing neurosyphilis describe testing of serum samples and do not include procedures for cerebrospinal fluid (CSF) testing. This study compared the CSF-VDRL test with 10 μL of antigen (CSF-VDRL-10) according to the American Public Health Association to the CSF-VDRL test with 17 μL of antigen (CSF-VDRL-17) according to the VDRL serum procedure. A total of 121 neurosyphilis patients and 86 syphilis/non-neurosyphilis patients were included. The sensitivities of the CSF-VDRL-10 and CSF-VDRL-17 tests were comparable for neurosyphilis diagnosis. The positive rate of the CSF-VDRL-17 test was higher than that of the CSF-VDRL-10 test. In all, 78.3% of the quantitative CSF-VDRL-17 results were consistent with those of the CSF-VDRL-10 test, 18.4% exhibited one-titer higher results than those of the CSF-VDRL-10 test, and 3.4% had positive CSF-VDRL-17 results but negative CSF-VDRL-10 results. The CSF-VDRL test with 17 μL of antigen was more sensitive, and it is worth performing longitudinal studies to understand its practical implications. The cerebrospinal fluid venereal disease research laboratory (CSF-VDRL) test remains the current standard for the laboratory diagnosis of neurosyphilis –3. HowevBetween August 2017 and January 2021, 207 patients at Zhongshan Hospital, School of Medicine, Xiamen University, were suspected of having neurosyphilis and received non-treponemal tests and treponemal tests of both serum and CSF samples. This study was approved by the Ethics Committee of the School of Medicine, Xiamen University, and it was conducted in compliance with national legislation of China and the Declaration of Helsinki guidelines. Written informed consent was obtained from all the study participants.Treponema pallidum particle agglutination (TPPA) assay result based on a CSF sample and a combination of the following findings: (1) reactive VDRL in a CSF sample, (2) clinical symptoms or signs consistent with neurosyphilis without other known causes for these clinical abnormalities, and (3) elevated CSF protein concentration (>500 mg/L) or leukocyte count (>5 cells/μL) in the absence of other known causes of these abnormalities. Verified neurosyphilis was defined as meeting conditions (1) and (2); likely neurosyphilis was defined as meeting conditions (2) and (3); and possible neurosyphilis was defined as meeting condition (3). The above three classifications were all treated as neurosyphilis.The diagnostic criteria for neurosyphilis complied with the guidelines of the Centers for Disease Control in the United States and Europe , 7. BrieSerological tests for syphilis were performed on all participants using a toluidine red unheated serum (TRUST) test  and TPPA tests  according to the manufacturer's instructions and as previously reported . The VDR2 test was used to test the difference between paired proportions. All statistical analyses were performed using SPSS 25.0 for Windows . A two-sided P value of <0.05 was considered statistically significant.The diagnostic performance of the CSF-VDRL-10 and CSF-VDRL-17 tests was determined and compared to the results of diagnostic criteria for neurosyphilis, according to the Yerushalmy model. McNemar's χP = 0.125) (2 test between the two tests in terms of specificity could not be performed because the number of rows and columns of the crosstab was <2 (P = 0.016). There were seven patients who had negative CSF-VDRL-10 results but positive CSF-VDRL-17 results. The agreement rate between the two tests was 96.6% (200/207).A total of 207 participants were included in the study, namely, 121 neurosyphilis patients and 86 syphilis/non-neurosyphilis patients. The clinical characteristics of the study participants are shown in = 0.125) . The speb was <2 . The posP < 0.001). Compared with that of the quantitative CSF-VDRL-10 test, 78.3% (162/207) of the quantitative CSF-VDRL-17 results were in complete agreement, including 114 patients having negative results and 48 patients having the same titers  to lipoidal antigens  released from the treponemal cell surface and damaged host cells through a flocculation reaction . To dateOur findings showed that the sensitivity of the CSF-VDRL-17 test for neurosyphilis diagnosis was comparable to that of the CSF-VDRL-10 test. Moreover, the positive rate of the CSF-VDRL-17 test was significantly higher than that of the routine CSF-VDRL-10 test. Four patients with positive CSF-VDRL-17 but negative CSF-VDRL-10 results were diagnosed with neurosyphilis. A larger concentration of antigen may promote antigen-antibody lattice formation, indicating a better sensitivity of the CSF-VDRL-17 test for patients with very low concentrations of antibodies.Moreover, three patients with discordant results were not diagnosed with neurosyphilis, because a reactive CSF-VDRL-10 test was the only diagnostic criterion for neurosyphilis for syphilis patients without neurological symptoms or other CSF abnormalities. The discordant results may be due to that the probability of false positives increased as the concentration of antigen in the CSF-VDRL-17 test increased. Antilipoidal antibodies are produced not only as a consequence of syphilis and other treponemal diseases but also in response to non-treponemal diseases of an acute and chronic nature in which tissue damage occurs , some stIn conclusion, our study demonstrated that the CSF-VDRL test with 10 or 17 μL of antigen had comparable diagnostic performance for neurosyphilis diagnosis. The CSF-VDRL test with 17 μL of antigen was more sensitive than that with 10 μL, and it is worth performing longitudinal studies to understand its practical implications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of School of Medicine, Xiamen University. The patients/participants provided their written informed consent to participate in this study.YX and WL wrote the main manuscript text. Q-LL and Q-YX contributed to sample collection and laboratory tests. YY and T-CY performed the statistical analysis. L-LL and W-MG conceived the study and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , the Key Projects for Province Science and Technology Program of Fujian Province, China (Grant No. 2018D0014), and the Natural Science Foundation of Fujian Province, China (Grant No. 2021J02055).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nasal liquorrhea indicates a cerebrospinal fluid fistula, an open communication between the intracranial cerebrospinal fluid and the nasal cavity. It can be traumatic and spontaneous.The aim of this study was to assess the outcome of endoscopic repair of cerebrospinal fluid fistula using fluorescein.This retrospective study included 30 patients of both sexes, with a mean age of 48.7 years, treated in the period from 2007 to 2015. All patients underwent lumbar administration of 5% sodium fluorescein solution preoperatively. Fistula was closed using three-layer graft and fibrin glue.Cerebrospinal fluid fistulas were commonly located in the ethmoid (37%) and sphenoid sinus (33%). Most patients presented with traumatic cerebrospinal fluid fistulas (2/3 of patients). The reported success rate for the first repair attempt was 97%. Complications occurred in three patients: one patient presented with acute hydrocephalus, one with reversible encephalopathy syndrome on the fifth postoperative day with bilateral loss of vision, and one patient was diagnosed with hydrocephalus two years after the repair of cerebrospinal fluid fistula.Endoscopic diagnosis and repair of cerebrospinal fluid fistulas using fluorescein intrathecally has high success rate and low complication rate. Nasal liquorrhea is the leakage of cerebrospinal fluid (CSF) into the nasal cavity. It may be either spontaneous or traumatic. Traumatic CSF leakage usually occurs following basilar skull fractures, or it can appear as iatrogenic after surgical interventions. Spontaneous leakage can be with or without elevated intracranial pressure.3Although basilar skull fractures are often associated with leakage of the cerebrospinal fluid, Miller initially described spontaneous CSF leakage in a child with hydrocephalus in 1826.Endoscopic CSF fistula repair is an extracranial extradural approach. It has been accepted worldwide as the method of choice because of excellent visualization, precise graft placement, minimal damage to surrounding tissue, preservation of olfactory function in case of fistula leak through the cribriform plate, shortened operating time and faster recovery time.12Intrathecal administration of a 5% fluorescein solution is highly effective in the detection of CSF fistulas. The use of fluorescein gives no false positive results. False negative findings may be due to temporary formation of granulation tissue closing the CSF fistula and preventing the leakage to be visualized at the moment of examination.The aim of this study was to analyze the success rate of endoscopic repair of cerebrospinal fluid fistulas using intrathecal administration of fluorescein for intraoperative visualization.This retrospective study included 30 patients of both sexes surgically treated for CSF leakage during the period from 2007 to 2015. There were 19 male and 11 female patients with a mean age of 48.7 years. Age ranged between 19 and 68 years. None of the patients had previous CSF fistula repair operation.All patients underwent clinical examination, computed tomography (CT) scanning and beta-trace protein test for the diagnosis of nasal liquorrhea preoperatively. Magnetic resonance imaging (MRI) was not routinely performed preoperatively in all examinees; it was indicated in patients with spontaneous CSF leakage, in patients with suspected hydrocephalus and in case of postoperative complications. Lumbar puncture was performed in all patients on the day of surgery by the neurosurgeon. The samples of CSF were taken for biochemical and microbiological analysis. Intrathecal administration of 5% sodium fluorescein solution was done through the performed lumbar puncture one hour prior surgery. Solution of 5% sodium fluorescein was diluted in 5 mL of CSF and gradually injected intrathecally for five minutes with the close monitoring of the patient. During the next one hour prior surgery, patients remained in supine position, lying down on their back and received intravenous antibiotic prophylaxis with 2 g ceftriaxone. The dosage of fluorescein solution was measured according to the body weight (0.1 mL/10 kg of body weight), but the highest dose of intrathecal fluorescein was 1 mL, regardless of the amount of body weight over 100 kg. In order to enhance the visualization of fluorescein, blue light filter (Storz) was used intraoperatively. The appearance of cerebrospinal fluid fistula without using a blue light filter, and with blue light filter is shown in Having localized the defect, it was closed using three layers – two layers of fascia lata graft and one mucosal layer  with fibrin glue. The endoscopic repair was performed by the otorhinolaryngologist under general anesthesia. After CSF fistula repair, lumbar drain was kept for five postoperative days with complete bed rest. Antibiotics were administered intravenously during this period.The investigation was done with approval of the Ethics Committee of the institution where the study was carried out (protocol number of Ethics Committee is 00-81/110).Retrospective analysis of the etiologic factors of CSF leaks showed that the largest number of patients had previous head trauma – 17 patients (57%), followed by spontaneous CSF leaks in 10 patients (33%) while 3 (10%) patients previously underwent a surgical procedure . Distribution or the patients depending on the etiology of CSF leakage is shown in Cerebrospinal fluid leaks were most commonly located in the area of the ethmoid sinus – in 11 patients (37%), followed by sphenoid sinus and cribriform plate as shown in Success rate of endoscopic repair was high (97%) since only one patient had recurrent CSF leakage postoperatively. It developed on his eighth postoperative day. This patient had the CSF leak as a result of previous olfactory meningioma surgery. Due to the development of recurrent CSF leaks and considering that the patient had loss of sense of smell, it was decided, together with neurosurgeons, to perform a transcranial repair of CSF fistula.In examined group of patients, postoperative complications occurred in three of them; two presented with early, and one with late complications. One male patient, 40 years of age, developed acute postoperative hydrocephalus eight hours after repair of the lateral wall of the sphenoid sinus, and required an urgent neurosurgical intervention .On the fifth postoperative day, one female patient, 49 years of age, developed a Posterior Reversible Encephalopathy Syndrome (PRES), manifesting with bilateral loss of vision. Magnetic resonance imaging showed edema in the occipital region. After conservative antiedema therapy using mannitol and corticosteroids, all symptoms resolved within two days.Two years after the repair of CSF fistula, one male patient, was diagnosed with hydrocephalus, which was managed with a ventriculoperitoneal shunt.There were no adverse reactions related to sodium fluorescein administration, whatsoever.Nasal liquorrhea is a serious condition that may lead to bacterial meningitis by spread of infection from the nasal cavity to the endocranium. During a 10 year period, almost 80% of patients present with at least one attack of purulent meningitis, some even more. Meningitis is more common in patients with traumatic nasal liquorrhea.14The authors reported no significant difference related to sex distribution, whereas in our sample 2/3 of patients were male, male patients outnumbered female ones in the group of traumatic CSF leaks, while most of the patients with spontaneous CSF leaks were female ones. This may be due to the fact that traffic injuries are more common among males, as well as occupational injuries of male workers whose job includes hard physical labor. Virk et al.More than half of studied patients had a traumatic CSF leakage, and together with those with iatrogenic nasal liquorrhea, it accounts for 2/3 of patients. Kapitanov and associatesThe predominance of traumatic CSF leaks in our sample may be due to the fact that our Clinic for ear, nose and throat diseases is a tertiary health institution with close cooperation with the Clinic of Neurosurgery; this is where patients with severe injuries are referred to when they cannot be treated at the secondary level.Cerebrospinal fluid fistulas are commonly located in the ethmoid (35%), and sphenoid sinus (32%). CSF leaks in the ethmoid sinus may be caused by trauma, either as a part of head trauma or iatrogenic, or they are spontaneous. A highly pneumatized sphenoid sinus may also cause CSF leaks. Extreme pneumatization of the sphenoid sinus may cause bone resorption in the skull base and, sometimes, a prolapse and tearing of the dura. A small meningocele may originate from bone dehiscence in the overpneumatized areas and this pathogenic mechanism should be considered an important etiological factor for spontaneous CSF leak development. Spontaneous CSF leaks are commonly located in the lateral wall of the sphenoid sinus.Endoscopic endonasal repair of CSF leaks had a high success rate of 97%. This can be attributed to the use of sodium fluorescein solution and blue light filter for detection of cerebrospinal fluid fistula. Only one patient had recurrent CSF leakage which developed on his eighth postoperative day. This patient had the CSF leak as a result of previous olfactory meningioma surgery. Otorhinolaryngologist suggested endoscopic reoperation, but since the patient already had no sense of smell, it was decided, together with neurosurgeons, to perform a transcranial repair of CSF fistula.Nyquist and associatesWe believe that use of lumbar drains for 5 days has positive effects on the success of CSF repair, because it reduces the pressure at the point of defect closure; however, some authors do not share this opinion and believe that lumbar drains are necessary only in patients with increased intracranial pressure.Early postoperative complications were present in 2 patients (6%); one male patient presented with acute hydrocephalus eight hours following surgery, which was probably the reason for spontaneously nasal liquorrhea, although preoperative MRI in this patient showed no signs of hydrocephalus and/or altered CSF flow. The second female patient developed a Posterior Reversible Encephalopathy Syndrome (PRES), manifesting with bilateral loss of vision. She was examined by the ophthalmologist, the ocular fundus appeared normal. Endocranial CT was done with no pathology detected, but MRI showed edema in the occipital region. Anti-edematous therapy successfully resolved the symptoms. PRES was described by Hinchey and associates in 1996.One male patient developed late complication, two years following surgery, in the form of hydrocephalus which was managed with ventriculoperitoneal shunt. He also underwent preoperative endocranial MRI showing no abnormalities in CSF flow. MRI is indicated in all patients with non-traumatic CSF leaks in order to detect alteration in CSF flow and in cases of developed postoperative complications.There were no complications or side effects related to sodium fluorescein.Nasal liquorrhea usually occurs in male patients as a result of previous trauma.Endoscopic detection and repair of cerebrospinal fluid fistulas with three-layer graft and fibrin glue has high success rate and low complication rate.The use of 5% sodium fluorescein solution for intrathecal administration, in the appropriate dosage, is a safe procedure for detection of cerebrospinal fluid fistulas during endoscopic surgery and causes no adverse effects.The authors declare no conflicts of interest.