Presenilin (PS) with a genetic mutation generates abundant β-amyloid protein (Aβ) 43. Senile plaques are formed by Aβ43 in the cerebral parenchyma together with Aβ42 at middle ages. These brains cause the early onset of Alzheimer’s disease (AD), which is known as familial Alzheimer’s disease (FAD). Based on the stepwise processing model of Aβ generation by γ-secretase, we reassessed the levels of Aβs in the cerebrospinal fluid (CSF) of FAD participants. While low levels of Aβ38, Aβ40, and Aβ42 were generated in the CSF of FAD participants, the levels of Aβ43 were unchanged in some of them compared with other participants. We sought to investigate why the level of Aβ43 was unchanged in FAD participants. These characteristics of Aβ generation were observed in the γ-secretase assay in vitro using cells, which express FAD mutations in PS1. Aβ38 and Aβ40 generation from their precursors, Aβ42 and Aβ43, was decreased in PS1 mutants compared with wild-type (WT) PS1, as observed in the CSF. Both the ratios of Aβ38/Aβ42 and Aβ40/Aβ43 in PS1 mutants were lower than those in the WT. However, the ratio of Aβ43/amyloid precursor protein intracellular domain (AICD) increased in the PS1 mutants in an onset age dependency, while other Aβ/AICD ratios were decreased or unchanged. Importantly, liquid chromatography–mass spectrometry found that the generation of Aβ43 was stimulated from Aβ48 in PS1 mutants. This result indicates that PS1 mutants switched the Aβ43 generating line, which reflects the level of Aβ43 in the CSF and forming senile plaques. More thr brains . This par brains . Recentlr brains , 14. Theγ-Secretase generates each Aβ under two pathways. At first, Aβ49 and Aβ48 are cleaved from APP. Aβ49 is successively cleaved, mostly to Aβ40, via Aβ46 and Aβ43, while Aβ48 is similarly cleaved to Aβ38 via Aβ45 and Aβ42. Of note, the most abundant species, Aβ40, is derived not from Aβ42 but from Aβ43. Moreover, Aβ38 is derived mainly from Aβ42 and Aβ43 –20. ThesWe previously reported that (i) the levels of Aβ42 and Aβ43 are proportional, and the levels of Aβ38 and Aβ40 are also proportional in the cerebrospinal fluid (CSF) of sporadic AD participants, mild cognitive impairment (MCI) participants, and cognitively normal controls (NCs). The levels of Aβ concentration in the CSF might reflect lipid raft-associated γ-secretase activity from brain cortices , 22;  was collected in a polypropylene or polystyrene tube and gently inverted. After brief centrifugation, aliquots of CSF collected were transferred to polypropylene tubes (0.25–0.5 mL), which were kept at −80 °C until use. The CSF concentrations of Aβ38, Aβ40, Aβ42, and Aβ43 were quantified using commercially available enzyme-linked immunosorbent assay kits .A previously reported cell culture method was employed . BrieflyA previously reported assay method was employed with some modifications , 27. BriThe expected peptides were quantified using LC-MS/MS as previously described –20. A QuAll statistical analyses were conducted using GraphPad Prism version 8. Data transformation was required to achieve normal distributions in the CSF analysis. Data analyses were performed after logarithmic transformation of the data for Aβ38, Aβ40, Aβ42, and Aβ43 and compared with our previous results. In the case of Aβ generation in the in vitro assay, Pearson’s correlation coefficients were calculated to indicate the strength of the relationship between two variables.R = 0.9536). These Aβs are the set of major final products in the stepwise processing of Aβ. When Aβ38 generation from Aβ42 decreased in the CSF of FAD participants, Aβ40 generation from Aβ43 also decreased, as found in the CSF of other participants. This proportional plot indicates that both γ-secretase cleavages, from Aβ43 to Aβ40 and from Aβ42 to Aβ38, occurred simultaneously in FAD brains, as found in other participants. However, the plots of Aβ42 and Aβ43 in the FAD participants varied within those of other participants. In these plots, the levels of the FAD participants  digressed far from the regression line . The onset age of these mutations was 27 years for L85P, 49.6 years for G209R, and 29 years for L381V. These results indicate that the levels of Aβ38, Aβ40, and Aβ42 decreased in the CSF of FAD participants, while Aβ43 might be defective by those mutations . The highest levels of Aβ40 and Aβ38 were in the WT PS1 from both CHO and HEK293 cells . Although Aβ42 decreased in some PS1 mutants, the level of Aβ43 was unchanged, as observed in the CSF of three early-onset FAD mutants . These reductions in Aβ40 occurred in an onset age dependency, as previously reported [R = 0.6751). These results indicated that FAD mutations of the early-onset ages have inhibitory effects on both γ-secretase-mediated cleavage pathways, from Aβ43 to Aβ40 and from Aβ42 to Aβ38.Next, we compared the ratios of Aβ40/Aβ43 and Aβ38/Aβ42, which are Aβ product/precursor sets, with each AD onset age. Although Aβ43 generation was unchanged in the PS1 mutants Fig. , these Areported , 32. AnoR = −0.4466). The ratio of Aβ42/AICD was found to be almost constant . These findings demonstrate that the levels of Aβ42 generation depend on the value of AICD generation. However, the level of Aβ43 generation might be the independent manner of PS1 mutants. Other ratios, Aβ38/AICD and Aβ40/AICD, were reduced in an onset age dependency . Decreasing the values of Aβ38 and Aβ40 means increasing those of Aβ42 and Aβ43 . On the other hand, VIT and TVI were generated from Aβ48 to Aβ45 and from Aβ45 to Aβ42 . These clear correlations indicated that the levels of Aβ42 and Aβ43 generation depend on the levels of Aβ48 and Aβ49 generated by FAD mutation in PS1 . VIVIT was released from Aβ48 to Aβ43 by γ-secretase  the levels of Aβ38 and Aβ40 were on the regression line in the CSF; (ii) some levels of Aβ43 from FAD participants, but not those of Aβ42, diverged from the regression line; (iii) the in vitro γ-secretase assay followed these Aβ generations by PS1 mutations, as seen in the CSF; (iv) the levels of Aβ43/AICD reflected the onset age of FAD; and (v) Aβ43 was generated not only from Aβ46 but also from Aβ48 in the PS1 mutants. With these assumptions, the most important issue for the mechanism of Aβ43 generation in the PS1 mutant is that the Aβ43 generation line was switched from Aβ48. This is in contrast with the mechanism of γ-secretase activity in sporadic AD , 22.In the present study, we quantified the concentrations of four Aβs, Aβ38, Aβ40, Aβ42, and Aβ43, in the CSF from FAD participants for comparison with our previous study . Even ifNext, the CSF concentrations of both Aβ42 and Aβ43 were proportional except in some early-onset ages of PS1 mutants Fig. . In the Table S1Fig. S1Fig. S2Fig. S3Fig. S4Supplementary legends


Donor selection is an important factor influencing the engraftment and efficacy of fecal microbiota transplantation (FMT) for complex conditions associated with microbial dysbiosis. However, the degree, variation, and stability of strain engraftment have not yet been assessed in the context of multiple donors.We conducted a double-blinded randomized control trial of FMT in 87 adolescents with obesity. Participants were randomized to receive multi-donor FMT  or placebo . Following a bowel cleanse, participants ingested a total of 28 capsules over two consecutive days. Capsules from individual donors and participant stool samples collected at baseline, 6, 12, and 26 weeks post-treatment were analyzed by shotgun metagenomic sequencing allowing us to track bacterial strain engraftment and its functional implications on recipients’ gut microbiomes.Prevotella to Bacteroides (P/B) ratio. Engrafted strains led to enterotype-level shifts in community composition and provided genes that altered the metabolic potential of the community. Despite our attempts to standardize FMT dose and origin, FMT recipients varied widely in their engraftment of donor strains.Multi-donor FMT sustainably altered the structure and the function of the gut microbiome. In what was effectively a microbiome competition experiment, we discovered that two donor microbiomes  dominated strain engraftment and were characterized by high microbial diversity and a high Our study provides evidence for the existence of FMT super-donors whose microbiomes are highly effective at engrafting in the recipient gut. Dominant engrafting male and female donor microbiomes harbored diverse microbial species and genes and were characterized by a high P/B ratio. Yet, the high variability of strain engraftment among FMT recipients suggests the host environment also plays a critical role in mediating FMT receptivity.ACTRN12615001351505).The Gut Bugs trial was registered with the Australian New Zealand Clinical Trials Registry (https://bmjopen.bmj.com/content/9/4/e026174.The trial protocol is available at Video AbstractThe online version contains supplementary material available at 10.1186/s40168-021-01060-7. Fecal microbiota transplantation (FMT) is currently being investigated for its efficacy in treating a variety of chronic disorders in which the gut microbiome is presumably implicated. Among these, obesity and metabolic disorders have been a key focus, given their global health burden and lack of effective treatment options . PioneerEfficient engraftment of donor strains is likely a prerequisite for functional alteration of the host’s gut microbiome . SeveralReplacing a disease-associated microbiome with a one-off dose of donor microbiota is not trivial and presumably involves a variety of competitive interactions between the endogenous and exogenous communities, restrained within the niche environment supplied by the host. Strain-resolved metagenomics have been instrumental in improving our understanding of FMT engraftment, revealing that donor strains can stably coexist with recipient’s endogenous strains for months after FMT , 22. HowTo address these gaps, we metagenomically profiled stool samples from healthy, lean donors and adolescents with obesity participating in a double-blinded randomized-controlled trial for FMT. Fecal microbiota was harvested from multiple donors and administered over two consecutive days in a capsule form that enabled competition between the individual microbiomes. Recipients’ gut microbiomes were tracked for up to 6 months post-treatment. Specifically, we sought to quantify the degree of donor strain engraftment, uncover any donor-specific trends, track the persistence of donor strains, and identify any functional effects of strain engraftment on the host’s microbial community.2, total body fat 47.5 ± 5.6%). Nine donors  were selected following extensive health and pathogen screening as documented in the published trial protocol [The Gut Bugs trial was a double-blinded, randomized, placebo-controlled trial that tested the efficacy of FMT capsules to treat adolescents with obesity . This stprotocol .Recipients were stratified by sex and randomized 1:1 to receive 28 acid-resistant capsules over two consecutive days containing either the active treatment (multi-donor FMT) or placebo . The fecal microbiota used for FMT capsules were derived from four healthy same-sex lean donors. To standardize treatment, the same donors were used for the entire course of the study with the exception of one male donor, DM05, who was replaced after the first male treatment round with DM12 due to illness.The day before treatment, recipients ingested a GlycoPrep-C solution  to cleanse the bowel and reduce the endogenous microbial load. Participants were instructed to maintain their usual diet and physical activity patterns throughout the trial. Clinical assessments were conducted at baseline, 6 weeks, 12 weeks, and 26 weeks post-treatment .TM  which are specifically designed to release their contents in the proximal bowel. Each capsule contained 0.25 g of microbiota suspended in 0.5 ml cryoprotective saline solution . Placebo capsules were visually indistinguishable and contained 0.5 ml of sterile saline solution . Capsules were stored at −80°C for approximately 1 week before administration. The capsule bacterial load was assessed by quantitative PCR using universal 16S primers . We did not observe any significant differences in FMT capsule bacterial load between donors  and 12 capsules the following day (3 capsules/donor). Capsules were swallowed with a glass of water under clinical supervision.Fresh stool samples were collected on site from participants at baseline (prior to bowel cleansing and treatment), as well as at 6 weeks, 12 weeks, and 26 weeks post-treatment. A 200 mg aliquot was taken from the middle section of the stool and transferred to a 2-ml LoBind DNA tube for temporary storage at −80°C. In addition, FMT capsules from each batch of donations were reserved for microbiome assessment. Nucleic acid extraction was performed within 5 days of stool collection using a modified protocol of the AllPrep DNA/RNA Mini Kit  . Firstlyhttp://huttenhower.sph.harvard.edu/biobakery_workflows. Quality control and pre-processing steps involved removal of adaptor sequences using Trim Galore! followed by removal of low-quality reads and human sequences with KneadData. Post-processed metagenomic sequencing files and accompanying metadata are deposited in NCBI’s SRA database (BioProject PRJNA637785).A total of 381 stool samples from recipients and donors were analyzed by shotgun metagenomic sequencing. Metagenomic sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina  following the manufacturer’s protocol. In brief, DNA was fragmented by sonication to an average size of 300 bp, and resulting fragments were end-polished, A-tailed, and ligated with sequence adaptors. Following PCR amplification, DNA fragments were purified , assessed for size distribution , and quantified by real-time PCR. Clusters were generated using the cBot Cluster Generation System, and sequencing was performed on an Illumina NovaSeq6000 platform, generating an average of 23 million reads per sample (150 bp paired-end reads). Raw sequencing files were processed with bioBakery workflows using docker images available at Species-level taxonomic profiles were generated by MetaPhlAn2 v2.7.7 . Metaphlp-values from PERMANOVA were adjusted for multiple testing using Benjamini-Hochberg procedure to obtain q-values, and results with q < 0.1 were considered statistically significant.Differences in the overall structure of the gut microbiome, based on Bray-Curtis dissimilarities, were assessed by permutational multivariate analysis of variance (PERMANOVA) using the adonis2 function in the vegan R package . The effAssociations between individual species and metadata were examined using general linear models as implemented in the MaAsLin2 R package . SpeciesProfiling of the dominant strain of a given species was achieved by SNP-based haplotyping using StrainPhlAn , requiriTo systematically quantify strain engraftment events for all profiled species, we employed the same method used by Ferretti et al. . In brieFunctional profiling was performed by HUMAnN2 v0.11.2 , which iThe Gut Bugs trial was a double-blinded, randomized-controlled FMT trial that recruited 87 adolescents with obesity Fig. a. FMT req < 0.1, Suppl. Table p = 0.0043, Suppl. Fig. p = 0.014, Suppl. Fig. p < 0.005). This indicated that a one-off dose of FMT capsules was capable of inducing sustained changes in the gut microbiome for up to 26 weeks post-treatment, above the spontaneous drift observed over the same time period within placebo recipients.A total of 381 stool samples from recipients and donors were analyzed by shotgun metagenomic sequencing at a mean sequencing depth of 23 million reads/sample (mean 6.9 Gb/sample). To assess the impact of FMT on gut microbiome composition, we generated a Bray-Curtis dissimilarity matrix for all fecal metagenome samples and performed a series of cross-sectional PERMANOVA tests. After adjusting for known microbiome confounders , treatment group was found to have a significant effect on the gut microbiome composition at week 6 and week 12 accounting for 2.7% and 2.9% of the variance, respectively  on the species-level Bray-Curtis dissimilarities. Stratified by sex and time point, we observed distinct patterns of microbiome shift in response to FMT Fig. b. Strikip = 0.016; week 12, p = 0.27; week 26, p = 0.0031, Suppl. Table p = 0.059; week 26, p = 0.044, Suppl. Table After adjusting for baseline similarity, we confirmed that the gut microbiome of female FMT recipients became more similar to female donor DF16 .DF16 had the second highest alpha diversity among the female donors, while DM08 was the most diverse male donor  [Prevotella and others by high levels of Bacteroides . This shift from Bacteroides to Prevotella dominance was largely maintained by FMT recipients out to 26 weeks post-treatment  , 36. To B ratio) , 37, we des Fig. f. For plq < 0.1). Of these, 39 increased and 25 decreased in relative abundance , Bacteroides finegoldii, Bacteroides salyersiae, Bacteroides faecis, Bacteroides massiliensis, Prevotella copri, Desulfovibrio piger, and Barnesiella intestinihominis. Whereas in males, the most enriched species included Catenibacterium mitsuokai, Bacteroides finegoldii, Prevotella copri, Collinsella aerofaciens, and Ruminococcus lactaris. For some species, enrichment could be traced back to one specific donor, predominantly DF16. In fact, many of the species that were unique to, or abundant within DF16, were found to be elevated in female recipients post-FMT . This observation is consistent with donor microbiomes providing recipients with novel taxa that fill available niches, while also signaling for wider changes in the endogenous community structure.To assess whether the shift towards donors was driven by the transfer of novel donor taxa, we removed donor species that were not present in FMT recipients at baseline. Repeating our dissimilarity assessments, we observed that the gut microbiomes of female FMT recipients still shifted in similarity towards DF16 despite the absence of her unique species  were located in close genetic proximity to donor strains and were clearly separated from their pre-FMT strain  haplotype representing the dominant strain of a given bacterial species within a sample. The SNP haplotypes were used to construct phylogenetic trees and identify donor strain engraftment events. For example, ain Fig. a.Fig. 3p < 2.2e−16). Collectively, these observation imply that FMT leads to greater fluctuations in strain dominance, which is not solely influenced by donor-engrafting strains, but may also include (a) a higher intrinsic rate of strain fluctuation within the recipient’s endogenous microbiome, (b) an influx of novel donor diversity that facilitates greater engraftment of environmentally acquired strains due to ecosystem rearrangement , under which strains were considered to be similar enough to constitute a strain match Fig. b. On avety model ), or (c)By tracking strain dominance over time, we identified a variety of ecological scenarios which included no replacement, replace with novel, replace and retain, temporary replacement, gain and retain, temporary gain, and loss of strain Fig. d. CombinBacteroides stercoris, whereas recipient strains from Ruminococcus bromii, Bacteroides vulgatus, Eubacterium ventriosum, and Eubacterium rectale remained stable, despite also being present in donors. Recipient strains that were present at lower relative abundance were more likely to be replaced than strains that were at higher relative abundance . Clinical parameters assessed included anthropometric measures, lipid profile, and markers of glucose metabolism. Thus, in the context of obesity, higher levels of donor engraftment were not associated with improvements in any of the clinical outcomes measured.DF16’s p < 0.005, males p = 0.022). Donors with higher microbial diversity tended to have higher levels of strain engraftment. Among female donors, DF16 exhibited the highest engraftment efficiency with 4.7% of her strains engrafting in the average FMT recipient . Consistent with the observed variation in alpha diversity  and week 26 . This effect was particularly notable within females , with concomitant reductions in the potential of a number of pathways, particularly pantothenate and coenzyme A biosynthesis. Subsequent associations at weeks 12 and 26 revealed that the majority of the treatment-associated pathways identified at week 6 were not differentially abundant at later time points. Instead, we identified a further 14 pathways with reduced potential among FMT recipients , Ruminococcus obeum, and Sutterella wadsworthensis.We hypothesized that these sex-specific differences were due to differences in the FMT donor material. To test this, we firstly identified the bacterial species that were responsible for pathway enrichment. Across the three female FMT-enriched pathways, we identified 19 contributing species, four of which were shown to be elevated post-FMT: Prevotella copri, and various species from genus Megamonas. Importantly, the gain of these genes occurred specifically within FMT recipients, ruling out the possibility that they were naturally acquired from environmental sources. Our ability to track functionally relevant gene transfer from donor to recipient suggests that FMT-related augmentation is largely dependent on which strains the donor provides and which strains engraft within the recipient. Thus, the observed differences in pathway enrichment between males and females suggest that functional alterations induced by FMT are not universal but, rather, specific to the genes present on engrafting strains.To determine individual donor contributions to enriched pathways, we tested for genes that had been gained post-FMT that were also present in any of the contributing donors. Across all enriched pathways, we found FMT recipients had gained a higher proportion of these genes compared to placebo recipients, suggesting these had likely been acquired by donor-engrafting species. The majority of the gained genes were traced back to species present within one specific donor. When multiple donors contained the same gene family from the same bacteria, we leveraged dominant strain data to assign the likely donor source. Consistent with these donors having a higher proportion of strain engraftment, we identified the same dominant donors (DF16 and DM08) as contributing proportionately more gene families within the FMT-enriched pathways. For example, female donor DF16 was responsible for the transfer of multiple genes involved in the NAD biosynthesis from aspartate pathway to 18 FMT recipients Fig. b. These Prevotella to Bacteroides ratio. FMT with these dominant microbiomes resulted in a stable transition towards a Prevotella-dominant enterotype in recipients. We unraveled a variety of strain competition dynamics, including replacement of endogenous strains with conspecific donor strains that persisted within their new hosts for at least 6 months. Finally, we tracked the transfer of genes from donor-engrafting strains that led to the enrichment of various metabolic pathways within the recipients’ microbial communities.Administration of FMT capsules from multiple donors led to sustained alterations in the structure and function of the gut microbiome in adolescents with obesity. In what was in practice a competition experiment between donor microbiomes, we showed that higher levels of strain engraftment occurred for specific bacterial strains from certain donors. The dominant engrafting female and male microbiomes were characterized by high microbial diversity, high gene richness, and a high Previous studies, based on single donor approaches, have shown that bacterial strains from donors engraft with variable success rates , 22. A lMegamonas species. Notably, when we removed DF16’s novel taxa from our analysis, we observed that the recipients’ microbiomes were still more similar to hers than those of the other donors. This suggests that, in addition to providing recipients with novel species, engraftment from DF16’s microbiome was also able to alter recipients’ endogenous microbial population structure.We contend that female donor DF16 is an example of a super-donor because of her gut microbiomes’ ability to engraft and augment recipients’ local community structure and function. Remarkably, the microbiome composition of all female FMT recipients shifted towards DF16’s and retained this structure throughout the 6-month trial. Similar donor-polarizing shifts were not observed in male recipients, despite DM08 contributing towards similar levels of strain engraftment and outcompeting the other male donors. We initially hypothesized that the prominent effect of DF16 was due to the transfer of novel taxa that were not present in recipients’ microbiomes at baseline. DF16 had a number of novel species that efficiently transferred, including various Although we could not assess the clinical efficacy of each individual donor, the use of a single super-donor that induces greater metabolic shifts in recipients’ gut microbiomes may, in theory, lead to greater therapeutic benefits . Using mRecipients were frequently colonized by strains from multiple donors that were largely retained long-term. These results are particularly encouraging given recipients were not asked to change their diet or lifestyle during the course of the study . We did,Prevotella copri was found to consistently transfer and stably colonize FMT recipients. Although formal testing of enterotypes was not performed, engraftment of Prevotella copri increased the P/B ratio of FMT recipients, effectively switching the recipients’ microbial enterotype. A Prevotella-type signature has previously been shown to be more favorable for weight loss in the context of a high-fiber diet [Bacteroides to a Prevotella-type signature. A similar transition from Bacteroides to Prevotella dominance has been reported before in a multi-donor FMT study for ulcerative colitis which involved regular enema-based administration of pooled fecal material from multiple donors [Prevotella species to any one particular donor. In our study population, this transition was attributed to the presence of just one Prevotella-dominant donor within the multi-donor pool. This suggests that Prevotella strains from these donors were able to outcompete the Bacteroides strains from the recipients as well as those from the three other donors.ber diet , 44. Howber diet . Our rese donors . Unlike Prevotella-dominant donors we identified were also the most effective donors for overall strain engraftment. Whether this association was causative or not remains unclear. A similar observation was recently reported by a small FMT pilot trial for obesity where the most effective donor for engraftment was also characterized by a high P/B ratio and consistently transferred Prevotella species shifting recipient enterotypes [Interestingly, the erotypes . Based oWe demonstrated functional shifts in the metabolic potential of the microbial community following FMT. Importantly, we showed that alterations in microbial metabolic pathways were linked to strain engraftment and newly obtained genes from donor microbiomes, particularly those from the dominant donors. Among the altered microbial pathways, we observed an increased biosynthesis potential for a number of bioactive molecules, including NAD, polyamines, and vitamin precursors, which have previously been implicated in metabolic disorders , 46. NADIn conclusion, our study provides further evidence for the existence of FMT super-donors. Dominant engrafting male and female donor microbiomes harbored diverse microbial species and genes, and were characterized by a high P/B ratio. Pre-screening donor microbiomes for these characteristics may help improve donor strain engraftment and elicit greater change in microbial community function. However, donor selection is just one piece of the puzzle. The high variability of strain engraftment among FMT recipients suggests that undetermined host factors still play a significant role in mediating FMT receptivity. Future experiments should focus on identifying the host components that moderate strain engraftment and their interactions with phenotype.Additional file 1. Supplementary FiguresAdditional file 2. Supplementary Table 1Additional file 3. Supplementary Table 2Additional file 4. Supplementary Table 3


Comprehensive data on the cerebrospinal fluid (CSF) profile in patients with COVID-19 and neurological involvement from large-scale multicenter studies are missing so far.To analyze systematically the CSF profile in COVID-19.Retrospective analysis of 150 lumbar punctures in 127 patients with PCR-proven COVID-19 and neurological symptoms seen at 17 European university centersl-lactate was increased in 26/109 . CSF-IgG was elevated in 50/100 (50%), but was of peripheral origin, since QIgG was normal in almost all cases, as were QIgA and QIgM. In 58/103 samples (56%) pattern 4 oligoclonal bands (OCB) compatible with systemic inflammation were present, while CSF-restricted OCB were found in only 2/103 (1.9%). SARS-CoV-2-CSF-PCR was negative in 76/76 samples. Routine CSF findings were normal in 35%. Cytokine levels were frequently elevated in the CSF (often associated with BCB dysfunction) and serum, partly remaining positive at high levels for weeks/months (939 tests). Of note, a positive SARS-CoV-2-IgG-antibody index (AI) was found in 2/19 (10.5%) patients which was associated with unusually high WCC in both of them and a strongly increased interleukin-6 (IL-6) index in one (not tested in the other). Anti-neuronal/anti-glial autoantibodies were mostly absent in the CSF and serum (1509 tests). In samples from patients with pre-/coexisting CNS disorders , CSF findings were mostly representative of the respective disease.The most frequent pathological finding was blood-CSF barrier (BCB) dysfunction (median QAlb 11.4 [6.72–50.8]), which was present in 58/116 (50%) samples from patients without pre-/coexisting CNS diseases (group I). QAlb remained elevated > 14d (47.6%) and even > 30d (55.6%) after neurological onset. CSF total protein was elevated in 54/118 (45.8%) samples (median 65.35 mg/dl [45.3–240.4]) and strongly correlated with QAlb. The CSF white cell count (WCC) was increased in 14/128 (11%) samples . An albuminocytological dissociation (ACD) was found in 43/115 (37.4%) samples. CSF The CSF profile in COVID-19 with neurological symptoms is mainly characterized by BCB disruption in the absence of intrathecal inflammation, compatible with cerebrospinal endotheliopathy. Persistent BCB dysfunction and elevated cytokine levels may contribute to both acute symptoms and ‘long COVID’. Direct infection of the CNS with SARS-CoV-2, if occurring at all, seems to be rare. Broad differential diagnostic considerations are recommended to avoid misinterpretation of treatable coexisting neurological disorders as complications of COVID-19.The online version contains supplementary material available at 10.1186/s12974-021-02339-0. COVID-19, first described in December 2019, is an infectious disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Acute COVID-19 has been reported to affect—directly or indirectly—the nervous system in a substantial number of cases . A broadl-lactate, glucose, and SARS-CoV-2 CSF polymerase chain reaction (PCR), antibody indices (AI), autoantibody findings, and cytokine levels, are widely missing so far. On behalf of the German Society for CSF Diagnostics and Clinical Neurochemistry  (DGLN) we conducted a systematic analysis of CSF findings from 150 lumbar punctures in 127 patients with PCR-proven COVID-19 and neurological symptoms. Patients were stratified according to the type and severity of the neurological symptoms, acuity, co-/preexisting neurological conditions, and treatment status.Cerebrospinal fluid (CSF) analysis is a diagnostic mainstay in neurology. However, limited information on CSF findings in patients with COVID-19 is currently available. Many of the studies published so far are restricted in terms of the number of patients included , 8 and/oThe results of 150 lumbar punctures (LPs) in 127 adult patients with COVID-19 and neurological symptoms were obtained from the patient records and analyzed retrospectively. LPs were performed between 03/2020 and 01/2021. In all cases, SARS-CoV-2 infection was confirmed by PCR . All patients were diagnosed with COVID-19 at 17 German , Austrian , Italian  and Swiss (Zurich) university hospitals. See Additional file Eleven patients (19 CSF samples) were excluded from primary analysis due to pre-/coexisting inflammatory , neoplastic , or cerebrovascular (severe subarachnoid hemorrhage [SAH] resulting in severe blood contamination of the CSF sample in 2) CNS conditions known to cause CSF alterations and considered by the treating physicians to be most likely not related to COVID-19; these patients were analyzed separately as ‘group II’ .N = 7); in the remaining patients, either serum antibody testing was not done because of the short time interval between COVID-19 onset and LP or data were not available retrospectively.Of the remaining patients , 90 were male (f:m ratio = 1:3.5). Eighty-eight percent were of Caucasian, 5% of African, 4% of Turkish or Middle-Eastern, and 3% of Asian origin. Overall, 131 LPs were performed in these 116 patients following onset of COVID-19 . The median age at LP was 65 years (range 19–89). The median time between first SARS-CoV-2-PCR-positive swab and first LP was 12 days (percentile range [0.1–0.9] 0–44), and the median time between LP and onset of the first neurological symptoms that prompted it was 5 days (percentile range 1–26). The median time between the first SARS-CoV-2-PCR-positive swab and the onset of neurological symptoms was 7 days. Neurological manifestations at the time of LP included encephalopathy, disturbed consciousness, or delayed wake-up reaction (63 LPs), seizures or epilepsy-like EEG changes (27 LPs), cerebral ischemia or bleeding (14 LPs), myelitis (3 LPs), other CNS manifestations , peripheral neuropathy  [at least 6 LPs]), cranial nerve symptoms , and headache  (multiple manifestations per patient possible). The presence of neurological disease was supported by paraclinical evidence from magnetic resonance imaging (MRI) or computed tomography (CT) at the time of LP in 94 cases, by EEG in 35, and by ENG/EMG in 18. At the time of LP, most patients had symptoms attributable to brain or spinal cord involvement , while some had exclusively hyposmia, dysgeusia, isolated cranial nerve involvement, peripheral nerve damage, and/or headache  (no exact classification possible in 1 case). The neurological symptoms present at the time of LP were coarsely classified by the treating neurologists as ‘severe’ for 57.9% of LPs, ‘moderate’ for 24.8%, and ‘mild’ for 17.4%, and samples were stratified accordingly for further analysis; at least 57% of the samples were taken during or within 1 week before or after ICU treatment and 57% during or within 1 week before or after ventilation. Serum IgG antibodies against SARS-CoV-2 were determined by enzyme-linked immunosorbent assay (ELISA) in 58 samples from 56 patients in group I  and were positive in 51 (88%) samples from 49 (87.5%) patients  and negative in 7 samples from 6 patients . To avoid bias due to differences in acuity, some subgroup analyses were restricted to ‘acute’ samples . The study was approved by the review boards of the participating centers and patients or their legal representatives gave written informed consent. LPs were performed for diagnostic purposes in all cases; no samples were obtained for use in this study.Due to the retrospective nature of this study, not all data of interest were available for all patients and samples. Accordingly, absolute patient numbers and/or sample numbers differed among subanalyses . No data were excluded from analysis unless explicitly stated .Methods were adopted from our previous studies on CSF findings in inflammatory CNS disorders –18 adherCSF[mg/l]/Igserum[g/l]. The upper limits of the respective reference ranges, Qlim(IgG), Qlim(IgM), and Qlim(IgA), were calculated against QAlb according to Reiber’s revised hyperbolic function [lim(Ig) were considered to indicate intrathecal immunoglobulin synthesis [lim(Ig)] × Igserum × 100 and Ig-loc[mg/L] = [QIg−Qlim(Ig)] × Igserum, respectively /Qlim(Ig), if QIg[total] > Qlim(Ig) with QIg[spec] = IgG[spec]CSF/IgG[spec]serum and QIgG[total] = IgG[total]CSF/IgG[total]serum)/QIg[total], if QIg[total] < Qal]serum). The uppformulas . AI valuformulas .CSF[mg/l]/Albserum[g/l], was used to assess BCB function. As the upper reference limit of QAlb is age dependent, Qlim(Alb) was calculated as (4 + ( a/15)) × 10–3 with a representing patient´s age according to Reiber et al.  vs. 7.7 [N = 16] if all samples with available data were considered, and 13 [N = 36] vs. 10.9 [N = 9] if only those with elevated QAlb were considered), but the differences did not reach statistical significance (Table The frequency of BCB dysfunction was slightly higher in the ‘acute B/SC subgroup’ (56.9% [37/65]) than in the ‘acute PN/CN/H subgroup’ (50% 8/16]), as was median QAlb (8.6  patients tested), with a median concentration of 3.04 mmol/l (range 2.2–4) ; however, the difference did not reach statistical significance . Median CSF l-lactate concentrations did not differ between the two subgroups (2.1 vs. 2 mmol/l) , and the frequency of CSF l-lactate elevation did not differ significantly between samples with and without neutrophil granulocytes (40% [4/10] vs. 35% [13/37]), widely ruling out neutrophil pleocytosis as a major cause of CSF l-lactate elevation in our patients. However, a weak yet significant positive correlation of l-lactate with the CSF WCC was found . l-Lactate levels were also significantly higher in patients who required ventilation at the time of LP or within 1 week before or after LP . Finally, CSF l-lactate levels correlated with QAlb  and, accordingly, also with CSF total protein . The correlation of L-lactate with QAlb was much stronger after exclusion of samples with unusually high QAlb values ≥ 30 × 10–3   and CSF l-lactate elevation significantly more frequent  in patients with elevated QAlb than in those with normal QAlb.CSF lim[IgG] 5.6) in just 1/115 (1%) samples or 1/106 (0.9%) patients tested, namely in a patient with CSF-restricted OCB and multiple brain infarctions (Table lim(IgG). However, low amounts of intrathecal IgG may in fact be detectable only by isoelectric focusing but not by QIgG determination; of note this patient was one of only two with elevated QIgM (see below). In the first patient, no pre- or coexisting condition known to cause OCB positivity has been detected as of last follow-up; the second patient had been previously diagnosed with mild dementia of unknown cause. Pattern 5, indicating monoclonal gammopathy, was present in 1/103 samples (1%), obtained from a patient with punctate multiple ischemia on brain MRI, bilateral visual impairment, decreased vigilance, and capillary leak syndrome. Altogether, intrathecal IgG synthesis was very rare and even below the rate reported in healthy central European volunteers  1–25). However, 26 samples were taken 7 or more days and 15 of these 14 or more days after neurological onset. PCR was negative in 69 initial CSF samples and 6 CSF samples obtained at follow-up LP (in 6 different patients). CNS tissue PCR from biopsy or autopsy samples was performed in none of the patients.Results from 113 further PCR tests in 32 samples from 30 patients were available for analysis  (Table Measles virus (M), rubella virus (R), and varicella zoster virus (Z) IgG AI results were available for only a small number of samples (N = 22) in group I. Moreover, all three AI were tested only in 1 sample and two AI in 0 samples. A positive Z-AI was found in 1/20 (5%) samples , a positive M-AI in 0/1 (0%), and a positive R-AI in 0/1 (0%). A positive MRZ reaction, as defined by the presence of a positive IgG AI for at least two of its three constituents M, R and Z , which is detectable in around 63% of cases in MS , was absBorrelia burgdorferi (BB), CMV, or EBV; one patient exhibited a borderline positive  IgM-AIfor BB  for herpes simplex virus (HSV) was found in 1/22 (4.5%) samples tested. While symptoms were generally compatible with HSVE in this patient , HSV-PCR was negative and no CSF pleocytosis present; in consequence, no diagnosis of HSVE was made by the then treating physicians. SARS-CoV-2-AI testing was not done in this case. None of the patients tested had a positive IgG-AI for N-methyl-D-aspartate receptor), anti-AMPA1/2-R (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor), anti-GABA-B1/2-R (G-protein coupled receptors B1/2 for gamma-aminobutyric acid), anti-LG1 , anti-CASPR (contactin-associated protein-like 2), anti-DPPX (dipeptidyl-peptidase-like protein-6), anti-mGluR5 (metabotropic glutamate receptor 5), anti-glycine receptor, anti-IgLON-5 (IgLON family member 5), anti-dopamin-2 receptor, anti-Hu, anti-Yo, anti-Ri, anti-Ma2/Ta, anti-Ma1, anti-Tr/DNER , anti-recoverin, anti-GAD65 (glutamic acid decarboxylase 65), anti-amphiphysin, anti-Zic4 (Zic family member 4), anti-SOX1 (SRY-box transcription factor 1), anti-nicotinergic (i.e. ganglionic) acetylcholine receptors, anti-MOG (myelin oligodendrocyte glycoprotein), anti-AQP4 (aquaporin-4)  1–27.4) and 18 days (percentile range 4–46), respectively.Free kappa light chains (FLC-kappa) and free lambda light chains (FLC-lambda) , which are produced by B cells during antibody synthesis in excess and subsequently secreted, are determined by some laboratories as a supportive quantitative marker of  IgG synthesis. FLC-kappa, and FLC-lambda , 44, 45 FLC-kappa concentrations were elevated in 5/6 serum samples  and in 1/4 CSF samples , but in none of the four patients tested was a positive K index  found  according to the manufacturer's cut-off (6.35). However, when applying the hyperbolic reference ranges proposed by Reiber et al. (2019)  instead Increased FLC-lambda concentrations were present in the serum in 4/6 patients . The L index  was normal (cut-off 5.51) in three patients. In the fourth patient, serum and CSF FLC-lambda concentrations did not exceed the upper reference limit, but the L index was elevated . However, this patient had developed peripheral neuropathy during COVID-19 with signs neither of CNS disease nor of intrathecal total IgG synthesis  and QAlb was unusually low, rendering a false-positive result at least conceivable.Interleukin-6 (IL-6) CSF levels were determined in 14 samples from 14 patients (CNS involvement in 13) in group I and were found to be elevated in 11  according to the manufacturers’ cut-off. The median CSF IL-6 concentration was 10.05 pg/ml , with particularly high concentrations (> 20 pg/ml) in 5 samples . In a patient with an unusually high WCC  and a positive SARS-CoV-2-AI, IL-6 was determined in both the CSF (> 300 pg/ml) and serum (17.3 pg/ml), revealing a very high IL-6 CSF/serum ratio of > 17 . IL-6 wIncreased blood  IL-6 levels  were noted in 27  of 29 samples (from 29 patients) tested, and BCB dysfunction, as indicated by elevated QAlb, was present in 8/10 (80%) samples with elevated CSF IL-6, rendering it possible that IL-6 was partly of peripheral origin in some of these cases.To take into account a possible effect of BCB dysfunction, we also calculated the IL-6 index . Here, the marked difference between the SARS-CoV-2-IgG-AI-positive sample with pleocytosis (247 cells/µl) and the SARS-CoV-2-IgG-AI-negative patients with a normal or almost normal WCC was still present (IL-6 index 0.5 vs. median index of 0.03 in the remainder)  around the date of the LP. In total, 723 (96.7%) of these samples exhibited elevated IL-6 levels  .CSF interleukin-8 (IL-8) was elevated in 7/7  CSF samples from 7 patients in group I, based on a cut-off of 40 pg/ml derived from a German cohort of patients with normal pressure hydrocephalus (21.40 ± 7.96 pg/ml)  . Regrettably, none of the 4 samples with high WCC > 100/µl  was tested for CSF IL-8.Elevated QAlb, indicating BCB dysfunction, was found in 4/6 (67%) samples with elevated CSF IL-8, and plasma IL-8 levels were elevated in 5/8  samples tested, suggesting a possible contribution of extrathecally produced IL-8 to CSF IL-8 levels. Whether intrathecal IL-8 synthesis occurred, remains unknown since no well-established cut-off exists. 6/6 samples exhibited a CSF/plasma IL-8 ratio > 1 . Based on an upper reference limit of 2.7 found in another Central European control cohort that comprised patients with trigeminal neuralgia , 3/6 wouIn 6/7 (86%) samples with elevated CSF IL-8 levels and available data also increased CSF IL-6 levels were found.Overall, 32 plasma samples from 19 patients in group I were tested for IL-8 during COVID-19 hospitalization. Of these, 29 (90.6%) samples exhibited elevated plasma IL-8 levels , and 18/19 (94.7%) patients showed elevated IL-8 concentrations at least once.The normal range for tumor necrosis factor alpha  in the CSF is not well defined  came from the abovementioned patient with an unusually high WCC (247 cells/µl), an unusually high IL-6 ratio and index, an elevated SARS-CoV-2-IgG AI, compatible with possible direct CNS infection with SARS-CoV-2, and severe clinical CNS involvement. The corresponding serum sample was positive for TNF-alpha at a concentration of 9.4 pg/ml, resulting in a CSF/serum ratio of 1.14. By contrast, the TNF-alpha CSF/serum ratio was markedly below 1 and thus at least 10 times lower in all (N = 7) further cases with available data , 6 of them without pleocytosis, and also not beyond the mean TNF-alpha CSF/serum ratio (0.29) reported in patients with low backpain .A marked difference was also found for the TNF-alpha index , which was 0.032 in the SARS-CoV-2-IgG-AI-positive sample with marked pleocytosis and a positive IL-6 index and 0.0038  in the remainder with normal or almost normal WCC  in 51 (81%) of them. Serum TNF-alpha concentrations were elevated in 21/26 (80.8%) patients at least once.Interferon-gamma (IFN-gamma) was detectable in only 1 CSF sample (from the B/SC subgroup) out of 7 samples from 7 patients tested (6 × B/SC subgroup), at a concentration of 2.6 pg/ml. While a generally accepted reference range for CSF IFN-gamma has not been defined, that level is lower than the mean CSF IFN-gamma concentrations in a recent cohort of control patients with non-inflammatory neurological diseases (mean 7.99 ± 2.75 pg/ml) , in patiInterleukin-1beta (IL-1beta) was determined in 10 plasma samples in group I, and was positive in 3 (30%) samples from 3/9 (33.3%) patients . IL-1beta secretion by monocytes after lipopolysaccharide (LPS) stimulation for 24 h was measured in 4 patients and was elevated in all of them .Interleukin-10 (IL-10) was elevated  in 21/22 (95.5%) serum samples (from 12 patients) obtained during hospitalization for COVID-19 in group I. Overall, 11/12 (91.7%) patients showed increased serum levels at least once. Of note, IL-10 levels were also increased in aqueous humor in one patient (177 pg/ml). Serum concentrations of the anti-inflammatory IL-1 receptor antagonist (IL-1RA), which is induced by IL-10, were markedly increased in 5/5 (100%) serum samples from 2 patients tested . No CSF samples were tested for IL-10 or IL-1RA.Staphylococcus aureus enterotoxin B (SEB) for 24 h, a marker of lymphocyte function/immunodeficiency, was assessed in 10 samples from 10 patients in group I. Interestingly, decreased IL-17 secretion was found in 4 samples from 4 patients , none of whom was treated with immunosuppressants or steroids at the time of testing.CSF concentration of the chemokine CXCL-13 (C-X-C motif chemokine ligand 13) was tested in a single patient in group I  and was below the detection limit (< 4 pg/ml). By contrast, CXCL-13 CSF levels were extremely high  in a patient from group II; however, this patient suffered from primary CNS lymphoma at the time he acquired COVID-19. Serum concentrations of the soluble interleukin-2 receptor (sIL2R), a marker of active cellular immunity, was determined in 4 patients in group I and was elevated in all of them; in total, 13/13 (100%) samples were positive . Relatively high levels were also found in the aqueous humor in one patient (1068 IU/ml), in whom CSF was not tested. Serum neopterin was tested in two patients in group I and was elevated in both . Interleukin-17 (IL-17), an important mediator of the mucosal immune response, was not directly assessed in CSF or serum, but IL-17 secretion after whole blood stimulation with In 9 patients, blood levels of 3 cytokines and cytokine receptors  were assessed; in 8 of these, increased levels of all three were observed at least once and in 1 patient increased levels of two markers. In another 11 patients, blood levels of 5 markers from the same panel were determined; in 4 of these, levels of all 5 markers were elevated; in 5 samples those of four markers, and in 2 samples those of at least two markers. In a further patient, 6 of the beforementioned cytokine markers were assessed in the blood and all were elevated.Not only IL-6 blood levels remained detectable over weeks or months but also those of other cytokines. IL-6, IL-8, TNF-alpha, IL-10 and sIL2 were still elevated in 12/13 (92.3%), 4/4, 5/6, 4/4 and 1/1 samples taken > 14 days after onset of the patients' neurological symptoms, respectively, and in 20/21 (95.2%), 6/7, 8/11, 3/3 and 3/3 samples obtained ≥30 days  after onset of the first non-neurological symptoms of COVID-19. CSF IL-6 and IL-8 were also still elevated in the repeat sample with the longest follow-up tested, which was obtained 16 days after neurological and 35 days after non-neurological COVID-19 onset.l-lactate elevation did not differ significantly between the first LP and follow-up LP  in group I  over time was noted. Similarly, 8/8 (100%) patients who were tested more than once had a normal IgG CSF/serum ratio both at first LP and at follow-up. An intrathecal ‘IgM to IgG switch’ was observed in 0/8 patients in whom QIgM and QIgG were determined more than once .l-lactate levels in those with and those without elevated l-lactate, and the frequency of samples with elevated l-lactate , median QIgG and QAlb levels, the frequency of BCB dysfunction, median TP levels in those with elevated TP, the frequency of TP values > 100 mg/dl, median te Table . As pleol-lactate concentrations in those with elevated values). However, the differences did not reach statistical significance. The steroids used included methylprednisolone, prednisone and dexamethasone (N = 26); the immunosuppressive or immunomodulatory treatments used included intravenous immunoglobulins, sulfasalazine, tacrolimus, mycophenolate mofetil, and ciclosporin A and were given either alone (N = 3) or in combination with steroids (N = 3).Several CSF parameters were either pathologically altered only in samples from patients untreated at the time of LP (N = 89) or more severely pathologically altered in these samples than in samples from patients treated with steroids, immunosuppressants or immunomodulatory drugs at the time of LP (N = 29) in group I  were found with equal or even higher frequency in samples from treated patients versus untreated patients. This could partly reflect the fact that the subgroup of treated patients included more patients with severe disease and/or a delayed or insufficient effect of the treatments applied on these parameters. Of note, WCC was rarely elevated in general in this cohort, including in untreated patients, which would in any case render it highly difficult to detect treatment effects on the rate of samples with ACD. To explain the apparent lack of effect on BCB function and related findings, detailed analysis would be required of all individual cases taking account of steroid timing, dosage, and the severity of BCB dysfunction, which was not part of the study protocol.A substantial number of CSF samples in group I exhibited no pathological changes. If CSF WCC, OCB, QIgG, Link index, QIgM, QIgA, QAlb, CSF TP, and CSF L-lactate are taken into account, 28 (35%) of 80 samples in group I in which all of these 9 parameters were assessed showed exclusively normal values. Of these 28 samples, 17 were obtained ≤14 d and 7 > 14 days after onset of neurological symptoms (no data in 4). If only a basic panel consisting of CSF WCC, CSF TP, and CSF L-lactate is considered , 49 (46.2%) of 106 samples in which all of these 3 parameters were determined samples would have been classified as 'normal'. Of those, 42.9% (21/49) would have been false-negatives (with the more extensive panel of 9 CSF parameters serving as gold standard).lim, of the respective immunoglobulin class), are shown for group I in Fig. Plots of QIgG, QIgA, and QIgM , 23, resCSF findings in patients with pre- or co-existing CNS disorders did not substantially differ from those typically present in patients with the respective disorders and no COVID-19.–3; typically < 12 × 10–3 in MS) in one case, probably due to additional JC virus-associated (and possibly SARS-CoV-2-associated) BCB dysfunction.In group II, positive pattern 2 or 3 OCB, which were extremely rare in group I (~ 1%), pointed to the presence of coexisting MS  or viral meningoencephalitis . A bispecific MRZ reaction was noted in none of the patients in group I but was present in the only MS patient in group II tested for this marker. However, QAlb values were higher than expected  than observed in 97% of all patients in group I, CSF-restricted OCB in 2/3 patients, and BCB dysfunction in 3/3 with QAlb values (higher than those in 90% of patients in group II). In an immunosuppressed patient (after kidney transplantation), CSF PCR for CMV and HSV1/2 as well as serum PCR for CMV, HSV1/2 and EBV were all positive, further underlining the need for extensive PCR diagnostics in patients presenting with COVID-19 and neurological symptoms. Neutrophilic (70%) pleocytosis (32 cells/µl) was also noted in this patient, probably because septic brain infarction has occurred as a result of acute bacterial endocarditis rather than reflecting early viral infection. Neutrophils were found in only 3% of patients with available cytology data in group I and should thus be considered a red flag. In a further patient with a history of chronic viral meningitis of unknown cause , COVID-19 and headache, WCC, OCB, QIgG/IgA/IgM and QAlb were all normal; only CSF TP was elevated, albeit markedly so .In patients with additional SAH, high CSF erythrocyte counts , siderophages, and xanthochrome CSF were noted as well as high TP level (up to 300 mg/dl).l-lactate levels and marked BCB dysfunction . In a patient with non-small cell-lung cancer, meningeal carcinomatosis and cerebral metastases, mild pleocytosis (10 cells/µl), and, elevated CSF l-lactate and TP levels were found.Primary CNS lymphoma, present in one patient, was associated with a higher WCC (21 and 88 cells/µl) than observed in most patients in group I, elevated Finally, in a patient with delayed wake-up reaction after sedation, encephalopathy, and myoclonus, a PET-CT-supported diagnosis of anti-Yo-associated autoimmune encephalitis (with positive antibodies in both CSF and serum) was made. CSF analysis revealed severe BCB damage (QAlb 26.7) and, of note, intrathecal synthesis of IgA and IgM , a feature not seen in any of the patients in group I; WCC could be determined only at follow-up and was then mildly elevated (17 cells/µl). SARS-CoV-2 PCR was negative in 8/8 CSF samples tested (from 6 patients) in group II.A substantial proportion of patients with COVID-19 develop neurological symptoms. However, the pathophysiology of such complications is not fully understood. CSF analysis may aid in addressing this question. Furthermore, CSF analysis may be clinically important by virtue of guiding diagnostic and differential diagnostic decisions.This study, which is, to the best of our knowledge, the largest and most comprehensive on CSF findings in COVID-19 conducted to date, demonstrates that in the majority of cases neurological involvement in COVID-19 is not associated with signs of intrathecal inflammation, as indicated by a normal WCC and a lack of quantitative  or qualitative (CSF-restricted OCB) evidence of total or SARS-CoV-2-specific IgG, IgA, or IgM synthesis within the CNS. Importantly, the remarkable absence of intrathecal inflammation was not restricted to patients with mild disease or those with peripheral symptoms but was found also in almost all patients with severe disease and in the B/SC subgroup.The median interval between onset of the non-neurological COVID-19 symptoms or the first positive nasopharyngeal SARS-CoV-2 PCR and LP was just 19 and 12 days, respectively. Moreover, and, most importantly, the neurological symptoms had started only 5 days (median) before LP. It is theoretically possible that intrathecal antibody synthesis began later and was thus not detected. In fact, 10.9% of patients tested had not yet developed SARS-CoV-2 serum antibodies at the time of LP. On the other hand, OCB were missing also in all 21 samples obtained > 14 days after neurological onset and even in all 8 samples taken after more than 30 days (range 31–82), suggesting that the observed lack of intrathecal immunoglobulin synthesis was not simply due to timing. Rather, our findings favor the notion that the neurological symptoms observed in patients with COVID-19 are not caused by direct CNS infection with SARS-CoV-2 in most cases.This said, it should not go unmentioned that in two patients in group I direct infection of the CNS with SARS-CoV-2 cannot be ruled out. These were the almost only patients with a strongly elevated WCC  and both exhibited at the same time a positive SARS-CoV-2-IgG-AI. Further support in favor of infection of the CNS comes from the finding of a strongly elevated IL-6 CSF/serum ratio in one of them —no IL-6 ratio (and index) as high was present in any of the SARS-CoV-2-IgG-AI-negative samples tested (which showed a median ratio of just 0.35) –, of a positive TNF-alpha ratio (and high index), and of a positive SARS-CoV-2-IgM AI and SARS-CoV-2-IgA AI in the same patient (not tested in the second patient). In both cases, severe BCB dysfunction was noted. The capability of SARS-CoV-2 to infect neurons has been suggested by a recent study, and neuropathological autopsy findings suggesting CNS infection in some patients with COVID-19 have indeed been published [As a limitation, SARS-CoV-2-CSF-PCR was negative in both cases. PCR was also negative in 82 further samples tested in this study. These findings from a large cohort confirm results from small case series and case reports also describing negative SARS-CoV-2-CSF-PCR results in patients with COVID-19 and neurological symptoms (summarized in ) and froA negative PCR result does not per se rule out infection of the nervous system. In a recent study, 96/96 Danish CSF samples from patients with encephalitis due to infection with tick-borne encephalitis virus, all of whom met the European Centre for Disease Prevention and Control (ECDC) criteria, were found to be reverse transcriptase (RT)-PCR-negative. In HSVE, a positive CSF PCR result is sometimes obtained only in sequential LP –51, probSimilarly, the lack of CSF-restricted total IgG OCB and of quantitative evidence of intrathecal total IgG synthesis in those two patients with a positive SARS-CoV-2-AI and pleocytosis does not formally rule out CNS infection with SARS-CoV-2 either. Total IgG OCB  are rather markers of chronic inflammation and may be negative at onset. Higher sensitivity of antigen-specific IgG AI than of markers of total IgG synthesis such as OCBs and immunoglobulin ratios has been previously shown –62. For We recommend that future studies on patients with COVID-19 and neurological symptoms should generally consider not only WCC, PCR, and markers of total IgG synthesis such as OCB and QIgG but also the SARS-CoV-2-IgG-AI , which might have diagnostic and prognostic implications, and, provided sequential LP are performed for clinical purposes, should examine the temporal dynamics of the intrathecal antiviral immune response in more detail. As the median interval between onset of neurological symptoms and AI determination was relatively short in the present cohort , results from follow-up LPs may be of particular interest. Based on the finding of high IL-6 and TNF-alpha CSF/serum ratios and indices in the only SARS-CoV-2-IgG-AI-positive patient tested but not in any other patients, such studies should evaluate also IL-6 and TNF-alpha as supplementary markers of acute intrathecal inflammation in COVID-19. Finally, also potential cross-reactivity between anti-SARS-CoV-2 antibodies and neuronal antigens (as recently discussed ) could eA striking feature in this cohort was the high frequency of BCB dysfunction. Disturbed BCB integrity, indicated by an increased albumin CSF/serum ratio, was present not only at onset but also in samples taken more than 30 days later. The presence of BCB dysfunction sufficiently explains the elevated CSF immunoglobulin and free kappa and lambda light chain concentrations and the high total TP concentrations found in some patients, as indicated by a highly significant correlation of both CSF IgG and CSF TP with QAlb and the lack of QIg elevation.Most importantly, in most cases BCB dysfunction was not associated with signs of cellular or humoral inflammation. Accordingly, a high rate of ACD was found . It is thus likely that BCB dysfunction was not primarily a result of intrathecal inflammation in our patients. The generally high rate of ACD needs also to be considered as a caveat when diagnosing patients presenting with COVID-19 and signs and symptoms of peripheral nerve disease. While ACD is an important marker of GBS, its predictive value for that disease may be lower in patients with COVID-19.Little is known about the exact mechanisms leading to impaired BCB function in COVID-19. These may include direct infection (and subsequent apoptosis) of endothelial cells and other cells constituting the blood–brain barrier (BBB) and the anatomic BCB with SARS-CoV-2 as well as nonspecific inflammatory bystander damage to these structures –67, SARSN = 3) or brain infarction/bleeding (N = 7), conditions known to cause BCB dysfunction, in a relatively small subset of patients in this cohort.Given the existing reports on severe peripheral inflammation in COVID-19 and a cytokine storm in the blood of severely affected patients, inflammatory bystander damage of peripheral origin to the BCB seems at least conceivable. Blood leukocytosis is present in many patients with acute COVID-19, and a ‘mirror OCB pattern’ , which may indicate peripheral, polyclonal acute inflammation, was present in 56% of our patients. Moreover, inflammatory cytokines, including markers indicating cellular inflammation, were frequently and persistently elevated in many patients in this cohort at the time of LP, with very high values in some. The spectrum of increased soluble markers of inflammation observed in our patients was broad, including both proinflammatory  and anti-inflammatory (IL-10 and IL-1RA) cytokines and cytokine receptors. In some patients extremely high serum IL-6 levels were noted, suggesting severe systemic inflammation, and IL-6 remained persistently positive over time in > 95% of more than 700 samples taken over a median period of around 1 month during COVID-19-related hospitalization. This broad and strong activation of inflammatory pathways renders it likely that other mediators of inflammation implicated in BBB disruption not investigated in this cohort such as ROS (reactive oxygen species), NETs (neutrophil extracellular traps), MMPs  and complement may have been elevated as well. In addition, hypoxia- or acidosis-related damage to the BCB in ICU-treated patients , who accAn alternative  explanation for QAlb elevation is a change in CSF flow rate , which cl-lactate levels were elevated in a quarter of our patients, based on age-dependent reference ranges, with a trend towards higher levels in acute samples. Physiologically, CSF l-lactate levels are relatively independent from serum l-lactate levels due to saturated lactate transporters. CSF l-lactate correlated significantly with QAlb, however, and the median l-lactate CSF level was significantly higher and CSF-lactate elevation significantly more common in patients with BCB dysfunction, suggesting a possible contribution of serum l-lactate to CSF level. Regrettably, l-lactate levels were not determined in the serum. However, CSF levels are usually higher than serum levels, rendering it more likely that CSF l-lactate and QAlb independently reflect the extent of CNS damage.CSF l-lactate levels were also weakly correlated with the CSF WCC. Among CSF white cells, granulocytes are a known source of CSF l-lactate [l-lactate elevation did not differ between samples with and without granulocytes and no significant correlation between CSF granulocyte counts and CSF l-lactate levels was found in the present cohort. CSF l-lactate is thought to be produced also by astrocytes, which form an essential part of the BBB, following glutamate stimulation [CSF -lactate –82. Howemulation , 84.l-lactate levels may thus reflect cerebral hypoxia due to COVID-19-induced pneumonia or cerebral ischemia [l-lactate levels were indeed significantly higher in the subgroup of patients requiring ventilation at the time of LP or within 1 week before or after LP. Further studies are needed to better characterize the sources of intrathecal l-lactate in COVID-19.Finally, neurons may switch to glycolysis, in particular when their capacity to metabolize anaerobically the lactate of astrocytic origin is exhausted . The eleischemia , 86. CSFThe relatively high frequency of normal CSF findings means that altered CSF parameters in patients presenting with COVID-19 and neurological symptoms, in particular pleocytosis, intrathecal immunoglobulin synthesis and unusually high QAlb values, should always prompt clinicians to consider the presence of pre- or coexisting conditions, which may require specific treatment and must thus not be overlooked or mistaken for direct effects of COVID-19. In fact, a broad panel of coexisting neurological diseases was present in this cohort, which is hardly astonishing given the currently high prevalence of COVID-19. Importantly, these patients were analyzed separately (group II). The CSF findings in this subgroup were mainly representative of the respective comorbidity.l-lactate), as is often practiced in non-tertiary care or emergency settings, was associated with a relatively high rate of false-negative reports in our cohort. We strongly recommend applying a more complete panel including QAlb and, to rule out differential diagnoses, also QIgG, QIgA and QIgM, whenever possible.From a clinical point of view, it is also important to keep in mind that restricting CSF analysis to a basic panel  suggests that timing issues were not a general confounder, it cannot be fully ruled out that the humoral immune response had simply not yet developed at the time of LP at least in some cases. In HSVE, CSF is normal in 10–20% of cases during the early phase of disease, with a delay of around 1 week in the onset of the antibody response , 87, 88.A lack of inflammatory pleocytosis and intrathecal total IgG synthesis does also per se not exclude CNS inflammation of autoimmune etiology. For example, in myelin oligodendrocyte glycoprotein-IgG-associated encephalomyelitis (MOG-EM) , a normaIt is of interest in this context, that antibodies to neural antigens have been reported in some patients with COVID-19 , 95, renThe fact that BCB dysfunction and elevated levels of inflammatory cytokine levels  persisted over weeks and months, respectively, is of interest in light of the many reports of patients with ‘non-specific’ long-term sequelae of COVID-19 such as malaise, fatigue, or cognitive impairment. Studies addressing the relevance of cytokines and BCB disruption in ‘long COVID’ are currently underway.Of note, HSVE and VZV meningoencephalitis, respectively, were present in two patients. Whether infection with SARS-CoV-2 may promote reactivation of latent herpes virus infection remains to be investigated. While a number of reports on COVID-19-associated VZV  reactivWe consider the high number of samples included and the availability of follow-up samples as particular strengths of this study, together with the high number of parameters assessed. A further strength is the stratification according to the presence of pre- or coexisting disorders, which helped to identify CSF alterations attributable to conditions other than SARS-CoV-2 infection.The retrospective and multicenter design of this study is a potential limitation. However, LP is an invasive and potentially harmful procedure that widely precludes prospective studies. The multicenter design can also be viewed as a strength, since it helped to reduce potential center-specific selection bias. Moreover, CSF diagnosis is highly standardized in central European university CSF laboratories, with centers participating in regular round-robin tests required for obtaining quality certification. Finally, given the relatively low prevalence of neurological involvement severe enough to justify LP in COVID-19, a multicenter approach was necessary for inclusion of a sufficiently large number of patients in a short time, which was important given the current pandemic situation.It is a limitation that some parameters, such as anti-neural autoantibodies, interleukins, interleukin receptors, chemokines, and kappa and lambda light chains, are not routinely assessed at some centers and, accordingly, data were available only for a subset of patients. It is therefore unknown whether the results reported here are representative for the total cohort. While we provide a large dataset for some of these parameters , more studies investigating these parameters are certainly warranted, for which our results may provide a rationale. Similarly, no predefined criteria for performing LP were used. Instead, the decision for LP was made in a clinical ‘real-life’ context based on the patient’s clinical and paraclinical presentation. While this could theoretically have introduced referral bias , this is unlikely given that all LPs were performed in the setting of academic tertiary care hospitals with similar standards of care. CSF analysis is part of the general routine workup of patients with neurological symptoms of unknown cause in Germany, Austria, Switzerland, and Italy.A trend towards more frequent and more severe CSF alterations in patients classified as having ‘severe’ or ‘moderate’ neurological deficits at the time of LP by the treating centers than in those with ‘mild’ deficits was noted. However, no standardized criteria for disease severity could be applied due to the retrospective nature of this study and the plethora of manifestations present. Further studies are needed to better define the relationship of disease severity and CSF findings in patients with COVID-19 and neurological involvement.Finally, as this study was performed at European centers, most patients were of ‘Caucasian’ origin. It is unknown if our results are valid also for other ethnicities.In summary, our study demonstrates (i.) that BCB dysfunction, as indicated by elevated QAlb, is the most common pathological CSF feature in patients with COVID-19 and neurological symptoms and may remain present for several weeks; (ii.) that BCB dysfunction in COVID-19 is not typically caused by intrathecal inflammation, as indicated by a lack of pleocytosis  and intrathecal IgG synthesis in most patients with elevated QAlb, favoring a peripheral origin ; (iii.) that classical signs of intrathecal CSF inflammation  are generally rare in patients with COVID-19 and neurological symptoms, both in samples obtained in the acute phase and in samples obtained later and irrespective of clinical disease severity; (iv.) that the SARS-CoV-2-IgG antibody index, which was elevated in 2 of the 3 only patients with marked pleocytosis (and in 2/20 patients tested), may be a marker of potential interest  that may indicate either rare SARS-CoV-2 infection of the CNS in COVID-19 or cross reactivity of SARS-CoV-2-IgG with CNS antigens and which may be more sensitive than markers of total IgG synthesis, although more studies on the frequency, diagnostic and prognostic implications, and temporal dynamics of SARS-CoV-2 AI elevation in COVID-19 are needed; (v.) that proinflammatory cytokine levels are frequently elevated in the CSF in patients with COVID-19 and that this often reflects BCB dysfunction rather than intrathecal cytokine synthesis, as indicated by CSF/serum cytokine ratios and indices; (vi.) that the IL-6 and TNF-alpha CSF/serum ratios and indices may be worthwhile being investigated in future studies on CSF findings in COVID-19 as potential markers of CNS infection with SARS-CoV-2, given that the only patient positive also exhibited increased SARS-CoV-2-IgG, -IgM and -IgA AI as well as marked CSF pleocytosis; (vii.) that elevated cytokine levels, like BCB dysfunction, may remain detectable over weeks and months, suggesting a potential role in ‘long COVID’ (or ‘post-COVID’), all the more since some of these cytokines are known to exert also non-inflammation-related undesirable effects in the CNS; (viii.) that anti-neuronal and anti-glial autoantibodies as detectable by routine methodology are rare in patients with COVID-19 and neurological symptoms ; (ix.) that SARS-CoV-2-PCR is typically negative in CSF samples from patients with COVID-19 and neurological symptoms  and should always be accompanied by PCR testing for other viral diseases of the CNS, which can coexist with COVID-19, as was the case in several patients of this cohort; (x.) that ‘normal’ CSF findings are not rare ; and, last but not least, (xi.) the importance of considering differential diagnoses in all patients presenting with neurological symptoms and a positive SARS-CoV-2 swab. Broad CSF analysis, including extensive screening for viruses by PCR as well as for pathological AIs and autoantibodies, is essential to avoid misinterpretation of treatable coexisting neurological disorders as complications of COVID-19. Given the relative rarity of CSF pathology observed in group I, we recommend that markedly altered CSF findings, in particular an increased WCC, should not only prompt SARS-CoV-2-IgG AI and SARS-CoV-2 PCR testing to detect cases of possible CNS infection with that virus but also broad differential diagnostic endeavors to identify or rule out co-existing CNS disorders in patients with COVID-19 and neurological symptoms.Additional file 1. Table S1. Patient and subgroup characteristics.


ABCA7, CR1, etc. A few studies have explored the association between the common variants, mainly in the non-coding regions of these genes, and cerebrospinal fluid (CSF) biomarkers. Fewer studies target the variants in the coding regions.The previous studies have identified several genes in relation to Alzheimer’s disease (AD), such as To illustrate the association between the common variants within or adjacent to the coding regions of AD susceptible genes and CSF biomarkers in AD patients.42, 181p-tau, and t-tau, and next-generation DNA sequencing. Sixty-nine common single nucleotide polymorphisms (SNPs)  within or near the coding region of 13 AD susceptible genes were included in the analysis.75 sporadic probable AD patients were extracted from the dementia cohort of Peking Union Medical College Hospital. They all had history inquiry, physical examination, blood test, cognitive assessment, brain MRI, CSF testing of AβAPOE) genotype showed lower CSF Aβ42 level and higher p-tau/Aβ42 ratio than the rs7412-CT genotype. The rs3752246-C (ABCA7) allele correlated with lower CSF Aβ42 level. The alternate alleles of six ABCA7 SNPs were related to lower CSF p-tau, including rs3745842, rs3764648, rs3764652, rs4147930, rs4147934 and rs881768. The rs11609582-TT (A2M) genotype showed higher CSF p-tau than the rs11609582-TA genotype. The p-tau/Aβ42 ratio was higher in the rs490460-TT (BACE1) genotype relative to the rs490460-GT genotype.The rs7412-CC (APOE, ABCA7, A2M, and BACE1 are associated with CSF Aβ42, p-tau. or p-tau/Aβ42.Some common variants within or near the coding regions of  APP), the presenilin 1 (PSEN1), and the presenilin 2 (PSEN2) genes can explain less than 1% of the AD population [APOE) ɛ4 haplotype is a well-known genetic risk factor for AD. It is composed of two single nucleotide polymorphisms (SNPs), rs429358-C and rs7412-C. However, more than half of AD patients do not carry the APOEɛ4 allele [Alzheimer’s disease (AD) is the most prevalent neurodegenerative dementia. In 2010, there were about 3.71 million people in China who lived with AD [42 and elevated p-tau in cerebrospinal fluid (CSF) are biomarkers of Aβ plaques and fibrillar tau, respectively [AD is pathologically characterized by extracellular deposition of amyloid-β (Aβ) plaques and intracellular aggregation of highly phosphorylated tau (p-tau)-containing neurofibrillary tangles . Based oectively . In thisA2M), the ATP-Binding Cassette Subfamily A Member 7 (ABCA7), the beta-Site amyloid precursor protein-cleaving enzyme 1 (BACE1), the bridging integrator 1 (BIN1), the Clusterin (CLU), the complement component (3b/4b) receptor 1 (CR1), the membrane spanning 4-domains A6A (MS4A6A), the membrane spanning 4-domains A6E (MS4A6E), the NME/NM23 family member 8 (NME8), the phosphatidylinositol binding clathrin assembly protein (PICALM), the plasminogen activator urokinase (PLAU), and the sortilin related receptor 1 (SORL1) genes [Genome-wide association studies and meta-analyses have identified several genes associated with AD risk, such as the alpha 2 Macroglobulin (1) genes . They ar1) genes . Most suABCA7 and 83 CR1 SNPs and CSF biomarkers. However, most of the SNPs are in the non-coding regions, such as intron and untranslated region [A few studies have been devoted to exploring the association between susceptibility loci and CSF biomarkers. With the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database, previous studies have investigated the correlation between 15 d region . Fewer sWe hypothesized that the common SNPs within or near the coding regions of AD risk genes were potential functional loci for AD. The mutations close to the splicing junction might affect mRNA splicing. Missense mutations in the coding region could alter amino acid products. And synonymous mutations might influence promoter activity, pre-mRNAs conformation, stability, protein folding, or function . The altAPOE, A2M, ABCA7, BACE1, BIN1, CLU, CR1, MS4A6A, MS4A6E, NME8, PICALM, PLAU, and SORL1). We would illustrate the association between these SNPs and CSF Aβ42, p-tau, and total tau (t-tau).In present study, we focused on the common SNPs within or near the coding regions of 13 known AD susceptible genes , activities of daily living (ADL), etc. 315 cases received whole exon sequencing, and 1,144 cases had targeted exon sequencing of 278 dementia-related genes. CSF testing of Aβ42, 181p-tau, and t-tau; and 4) informed consent was obtained. This study was approved by ethics committee of PUMCH (No. JS-1836).75 Chinese Han patients were included in the study. The inclusion criteria were as follows: 1) met the diagnostic criteria for probable AD based on 2011 diagnostic guidelines for AD from National Institute on Aging-Alzheimer’s Association workgroups ; 2) no fIDT xGen Lockdown Probes were used in whole exon sequencing with 50 bp flanking intron length. The targeted exon sequencing covered 3927 exons with 20 bp flanking intron regions. The DNA libraries were sequenced on Illumina HiSeq X Ten Analyzers . “Clean reads” were generated with AfterQC . All reaAPOE, A2M, ABCA7, BACE1, BIN1, CLU, CR1, MS4A6A, MS4A6E, NME8, PICALM, PLAU, and SORL1. Finally, 69 SNPs were included in the analysis. The inclusion criteria were as follows: 1) minor allele frequency > 0.01, according to ExAC, Genomes databases, etc.; 2) located in the exon region or within 10 bp of splicing junction.This report focused on 13 known AD susceptible genes, including 42, 181p-tau, and t-tau were measured using ELISA method ; Fujirebio, Ghent, Belgium).CSF samples was collected using low protein binding tube . The tubes were filled more than 75%. Within one day, the samples were centrifuged at 1800 rpm, 4°C for 10 min. After that, the supernatant was moved to new tubes and stored in freezers at –80°C. Within two weeks, CSF Aβhttps://doi.org/10.1016/j.jalz.2019.06.790). With the increasing sample size, the current CSF biomarker cut-offs for clinical AD have been set as follows: Aβ42 <570.9 pg/ml, p-tau > 56.49 pg/ml, t-tau > 241.6 pg/ml, p-tau/Aβ42 > 0.08465, and t-tau/Aβ42 > 0.5290. The sensitivities of these cut-offs are 56.6%, 88.7%, 81.1%, 77.4%, and 84.9%, respectively. The specificities are 84.6%, 84.6%, 90.4%, 90.4%, and 90.4%, respectively. These cut-offs are unpublished data.The CSF biomarker cut-off values for clinical AD in our laboratory were submitted to Alzheimer’s Association International Conference in 2019  were compared by analysis of covariate (ANCOVA). Sex, age, disease course, and APOE status (ɛ4 carrier or non-carrier) were included in the model as fixed factor or covariate. Unadjusted means were displayed in The CSF biomarker data (AβAs shown in As shown in APOE) (24.0% versus 8.6%) and lower rs7412-T (APOE) allele frequencies (2.0% versus 10.0%). Additionally, the cohort showed lower rs2274567-G (CR1) (15.3% versus 32.7%), rs3811381-G (CR1) (15.3% versus 32.2%), rs6691117-G (CR1) (17.3% versus 34.3%), and higher rs12364988-C (SORL1) (46.7% versus 31.2%) allele frequencies. As for the other SNPs, the alternate allele frequencies in this AD cohort were comparable to those from 1000 Genomes database.Compared with the allele frequency in east Asian populations from 1000 Genomes database, the cohort had higher rs429358-C , the rs7412-CC (APOE) genotype showed lower Aβ42 level and higher p-tau/Aβ42 ratio than the rs7412-CT genotype . Among the rs429358-TT, rs429358-TC, and rs429358-CC genotypes, CSF Aβ42 level showed a declining trend . The rs429358-CC genotype showed higher p-tau/Aβ42 ratio than the rs429358-TT and rs429358-TC genotypes . However, some of these differences did not reach statistical significance.After adjustment by the rs429358 (ABCA7) allele correlated with lower Aβ42 level . The alternate alleles of seven ABCA7 SNPs were related to lower p-tau, including rs3745842, rs3752246, rs3764648, rs3764652, rs4147930, rs4147934, and rs881768. Of them, the alternate alleles of rs3745842, rs3764652, rs4147930, and rs881768 were also relevant to lower p-tau/Aβ42 ratio. In addition, the rs4147914-GA genotype showed higher p-tau level than the rs4147914-AA and rs4147914-GG genotypes .The rs3752246-C (A2M) genotype showed higher p-tau level than the rs11609582-TA genotype . Also, the reference allele homozygote of rs1799759 had higher p-tau than the rs1799759 heterozygote .The rs11609582-TT (BACE1) genotype showed lower Aβ42 and higher p-tau levels than the rs490460-GT genotype . These differences were not statistically significant. However, the p-tau/Aβ42 ratio was significantly higher in the rs490460-TT genotype relative to the rs490460-GT genotype .The rs490460-TT   and the rs7232 (MS4A6A) . Two NME8 SNPs showed sex*SNPs interactions on p-tau/Aβ42, including the rs2598044  and the rs2722372 .As shown in 42 level than females. Among the participants with the rs2598044-CT and the rs2722372-GA genotypes, the p-tau/Aβ42 ratio was higher in males relative to females. However, among those with rs3752240-AG, rs7232-AA, rs2598044-CC, and rs2722372-GG genotypes, Aβ42 or p-tau/Aβ42 ratio did not differ between males and females.Among the subjects with the rs3752240-AA and the rs7232-AT genotypes, males had lower AβAPOEɛ4 allele is a well-known genetic risk factor for AD. Conversely, the ɛ2 allele confers a reduced AD risk [ɛ2 and ɛ4 allele frequencies in the cohort are 2.0% and 24.0%, respectively, which are lower and higher than those in Chinese general population  [42 level, and the rs429358-C allele shows a trend associated with decreased Aβ42. The correlation between these APOE SNPs and CSF Aβ42 level is related to the role of APOE in Aβ metabolism. The APOEɛ4 can increase Aβ production by improving the activity of γ-secretase and inhibit Aβ clearance by impairing its lysosomal and proteolytic degradation [APOEɛ2 in AD is less clear. Compared with the APOEɛ4, the APOEɛ2 allele is more efficient at promoting the degradation of soluble Aβ and its transport across vessel walls [The  AD risk . These a%, 7.1%) . Besidesradation . ComparaABCA7. Six novel SNPs  show effects on CSF p-tau level. Two known SNPs  correlate with Aβ42 and p-tau burden, respectively. Like the APOE, the ABCA7 is also located on chromosome 19q13.3. It has 47 exons. It might influence Aβ production, aggregation by mediating β-secretase cleavage, microglial endocytosis, or macrophage phagocytosis [Among the 13 potential functional SNPs, 61.5% (8/13) SNPs are from the ocytosis . Aβ depoocytosis .A2M SNPs  are related to CSF p-tau burden. The A2M might affect tau pathogenesis by its interaction with calcineurin and regulator of calcineurin 1, which are the enzyme and regulator of tau phosphorylation, respectively [Two novel ectively .BACE1 SNP (rs490460) is relevant to p-tau/Aβ42 ratio. This might be due to the role of the BACE1 in Aβ metabolism. The BACE1 protease is implicated in the β-site cleaving of amyloid precursor protein, which can eventually lead to the synthesis of Aβ peptide [One novel  peptide .CR1), rs3811381 (CR1), rs6691117 (CR1), and rs12364988-C (SORL1) in this cohort are far from those in the East Asian population from 1000 Genomes database. However, these allele frequency data in this cohort are close to the data from some previous studies. In Chinese late-onset AD patients, the minor allele frequencies of rs2274567, rs3811381, and rs6691117 are 12%, 14%, and 11%, respectively. In Chinese cognitively normal controls, the minor allele frequencies are 19%, 18%, and 19%, respectively [The alternate allele frequencies of rs2274567 , rs7232-AT (MS4A6A), rs2598044-CT (NME8), and rs2722372-GA (NME8) genotypes, males have lower Aβ42 or higher p-tau/Aβ42 than females. The sex differences in the effect of these SNPs on CSF biomarkers might be somewhat responsible for the sex differences in the amyloid load. Cavedo et al. has found that there is higher anterior cingulate cortex amyloid burden in men relative to women [This is the first time that we demonstrate the interactions between sex and to women .42. This might be partly due to the dynamic evolution of CSF biomarkers at different stages of cognitive impairment. The CSF Aβ42 level decreases mainly in the preclinical stage and reaches a plateau in the dementia stage, whereas the CSF tau level increases gradually in the mild dementia stage [In this paper, ten SNPs show effects on CSF p-tau. Comparatively, there is less association between these SNPs and CSF Aβia stage . T-tau iia stage . This miAPOE, ABCA7, A2M, and BACE1 SNPs show associations with CSF Aβ42, p-tau, and p-tau/Aβ42, including four known and nine novel SNPs. ABCA7, MS4A6A, and NME8 have sex*SNPs interactions on CSF Aβ42 and p-tau/Aβ42.This study involves 69 common SNPs within or near coding regions of 13 AD risk genes. 42 > 570.9 pg/ml, and 25.3% (19/75) subjects have CSF p-tau <56.49 pg/ml. The atypical CSF profile might be attributed to individual differences in CSF dynamics. Grothe studied the CSF biomarkers in pathology-confirmed AD patients from ADNI database. They reported a considerably higher CSF Aβ42 cutoff  for differentiating high and low Thal phases, as well as a lower p-tau cutoff (19 pg/ml) for discriminating high and low neuritic plaque scores [The main limitation of this study is the small sample size and absence of pathological evidence. All the subjects in this study are clinically diagnosed with probable AD. None of them have pathological evidence. According to the CSF biomarker cut-offs for AD in our laboratory, only 50.7% (38/75) subjects reach the cutoff values of all the five variables. 29.3% (22/75) subjects have CSF Aβe scores . In thisClick here for additional data file.


Encephalitis in children may lead to adverse outcomes and long-term neurodevelopmental sequelae. The prompt identification of the causative agent is important to guide proper management in cases with encephalitis; however, the etiology often remains undetermined. The use of polymerase chain reaction (PCR) analysis in the cerebrospinal fluid (CSF) has increased the diagnostic yield in encephalitis cases; however, it may be occasionally misleading. In this article, we describe the case of a male immunocompetent child with encephalitis in which human herpesvirus-7 (HHV-7) was detected in CSF by PCR. As the detection of HHV-7 DNA in the CSF alone is insufficient to prove an etiologic association of severe encephalitis in immunocompetent children, alternative diagnoses were pursued. Enterovirus (E-11) was detected by PCR analysis of the nasopharyngeal and rectal swabs of the male patient. The final diagnosis was facilitated by the findings in his sibling, which presented concurrently with enteroviral encephalitis. Failure to detect enterovirus in the CSF by PCR does not exclude enteroviral encephalitis; screening of other samples, from other body sites, may be necessary to identify the virus, and physicians should take into consideration all evidence, including history, clinical presentation, and sick contacts’ clinical status. Encephalitis is a severe infection of the central nervous system (CNS) that may lead to adverse outcomes and long-term neurodevelopmental sequelae ,2. The lA previously healthy 3-year-old male was admitted during the winter season with fever, vomiting, and lethargy. The history started six days before with fever (38.6 °C) for three days, followed by two days of apyrexia. Twelve hours prior to admission, fever relapsed, and the patient developed drowsiness, vomiting, and episodes of jaw trismus. The patient is the first child of a four-member family, with an uneventful medical history, adequately vaccinated for age, with no history of travel or animal contact.At presentation, he was confused and disoriented. He soon became intermittently lethargic, non-verbal, and irritable upon waking with intervals when he retained eye contact and responded to his mother. His walking was unstable. He was hemodynamically stable with a heart rate of 98 beats/min, blood pressure of 93/61 mmHg, temperature of 38.7 °C, and oxygen saturation of 99%. There was no nuchal rigidity or other meningeal signs. Physical examination was otherwise unremarkable. Soon after admission, his general condition deteriorated; he was more lethargic, unresponsive to vocal stimulation, the episodes of starring gaze and jaw trismus became more frequent, and he also developed focal tonic seizures of the right hand.3, neutrophil—2900/mm3, hemoglobin—12.1 g/dL, and platelets—159,000/mm3) and elevation of erythrocyte sedimentation rate (61 mm/hour) and C-reactive protein . Blood serum and urine biochemical analyses were unremarkable. Blood and throat cultures were obtained. The chest X-ray was normal. Brain computed tomography (CT) was urgently requested and was unremarkable. CSF analysis revealed 55 cells (52% neutrophils), with protein levels of 45 mg/dL glucose levels of 80 mg/dL with normal CSF to serum glucose ratio. Further serological analyses and PCR analyses for viruses and bacteria were performed in the serum, CSF, and nasopharyngeal samples  revealed an area of T2 hyperintensity at the left parietal cortex and enlarged hyperintense thalami, while signal intensity at the posterior portion of the pons was borderline hyperintense . With a Blood and CSF cultures were sterile and the CSF PCR for herpes simplex virus (HSV)-1,2 came out negative. From the analyses depicted in The patient’s sibling was a previously healthy, 30-day-old female, breast-fed, born at term after an uneventful pregnancy and delivery. The infant developed fever (39 °C) one day prior to admission and an episode of loss of consciousness with starring gaze and lip cyanosis. She had a mild cough the previous two days. Upon examination, the infant was lethargic and tachycardic, had mottled skin and a capillary refill time of 3 sec. The complete blood count, as well as the blood biochemical and urine analyses were unremarkable apart from mildly elevated troponin. CSF analysis showed 22 cells (62% lymphocytes), with normal glucose and protein. Empiric treatment with cefotaxime, ampicillin, and acyclovir was initiated. Within the following three days, CSF gram stain, culture, and CSF PCR for bacterial pathogens came out negative and antibiotics were discontinued. CSF analysis of the sibling was positive for enterovirus, while PCR for other pathogens, including HHV-7, was negative.The biphasic pattern of fever and the concomitant presentation of the sibling with enteroviral encephalitis raised the suspicion of enteroviral infection in the older child as well. Nasopharyngeal and rectal swabs of both siblings were sent for PCR for enteroviruses, and all came out positive. Serotyping revealed enterovirus E-11. The presence of E-11 in two distinct body sites in the male sibling, the detection of E-11 in the CSF of the female sibling and the relevant clinical presentation led us to the diagnosis of enteroviral infection in these children .The female patient exhibited sepsis-like presentation, encephalitis, and myocarditis. She received no specific treatment and recovered well within seven days of presentation. The male patient received intravenous immunoglobulin (IVIG) (500 mg/kg/day for 5 days). Fever subsequently subsided, and the patient gradually regained consciousness and was able to speak, communicate, and perform normally within ten days of presentation. Mild irritability and fine tremor were evident at discharge but improved and gradually resolved. Repeated brain MRI 6 months later showed complete elimination of findings .Mycoplasma spp., and others, may be performed, depending on the local epidemiology and clinical presentation [Encephalitis is defined by the presence of brain inflammation in association with clinical evidence of neurologic dysfunction . In patientation . Howeverentation ,3,4,5.We present herein the case of an immunocompetent child with encephalitis that posed a diagnostic challenge, as extensive investigations were inconclusive and potentially misleading. CSF PCR was negative for common pathogens, apart from HHV-7. HHV-7 causes primary infection during the first 5 years of life, which may be asymptomatic or present with roseola infantum or simple febrile seizures, though the latter is rare . HHV-7 hEnteroviruses are a leading cause of encephalitis in young children worldwide ,2. The sThe diagnosis of enteroviral encephalitis is mainly based on positive PCR of the CSF, the sensitivity of which is reported to be 75–95% . HoweverThe presence of enteroviruses in at least two different body sites, such as rectal/stool swabs, nasopharyngeal specimen, and/or blood, is required for the diagnosis of enteroviral encephalitis, especially in cases with late presentation, where the window of opportunity to detect enterovirus in the CSF is missed, which was the case in our male patient ,12. AddiMRI in enterovirus encephalitis may disclose findings that range from normal to hyperintensities at the posterior brainstem, medulla oblongata, and thalami . BrainstFor enteroviral (EV) infections, there are currently no specific therapies with a proven benefit . For infThese cases highlight the diagnostic challenges that physicians may confront during evaluation of encephalitis in children. Extensive CSF PCR testing contributes a lot to diagnosis but may lead to detecting pathogens with unknown clinical significance. Furthermore, failure to detect enterovirus in the CSF does not necessary preclude enteroviral encephalitis; screening of other sample types may be necessary to identify the etiological agent, and physicians should take into consideration all evidence, including contacts’ clinical status.