Because this imaging method is not feasible in an acute clinical setting, the magnetic resonance imaging (MRI) mismatch between perfusion-weighted imaging and diffusion-weighted imaging, as well as computed tomography perfusion have been proposed as surrogate markers to identify the penumbra in acute ischemic stroke patients. Transversal studies comparing PET and MRI or using longitudinal assessment of a limited sample of patients have been used to define perfusion thresholds. However, in the era of mechanical thrombectomy, these thresholds are debatable. Using various MRI methods, the original penumbra definition has recently gained a significant interest. The aim of this review is to provide an overview of the evolution of the ischemic penumbra imaging methods, including their respective strengths and limitations, as well as to map the current intellectual structure of the field using bibliometric analysis and explore future directions.The ischemic penumbra is defined as the severely hypoperfused, functionally impaired, at-risk but not yet infarcted tissue that will be progressively recruited into the infarct core. Early reperfusion aims to save the ischemic penumbra by preventing infarct core expansion and is the mainstay of acute ischemic stroke therapy. Intravenous thrombolysis and mechanical thrombectomy for selected patients with large vessel occlusion has been shown to improve functional outcome. Given the varying speed of infarct core progression among individuals, a therapeutic window tailored to each patient has recently been proposed. Recent studies have demonstrated that reperfusion therapies are beneficial in patients with a persistent ischemic penumbra, beyond conventional time windows. As a result, mapping the penumbra has become crucial in emergency settings for guiding personalized therapy. The penumbra was first characterized as an area with a reduced cerebral blood flow, increased oxygen extraction fraction and preserved cerebral metabolic rate of oxygen using positron emission tomography (PET) with radiolabeled O Stroke is the second most common cause of death and a leading cause of disability in occidental countries, and the 20- to 30-year projection forecasts around 20–30% increase of its burden , 2.In case of acute ischemic stroke (AIS), occlusion of a cerebral blood vessel causes a variable decrease of blood flow in the downstream parenchyma. Three major zones have been identified: (i) the irreversibly damaged ischemic core; (ii) the ischemic penumbra, defined as the severely hypoperfused, electrically silent, at risk brain tissue; and (iii) the oligemia, a mildly hypoperfused area with preserved neuronal function , 4. As tTherefore, the primary target of AIS treatment is to restore brain perfusion as soon as possible in order to preserve the ischemic penumbra. Reperfusion therapies, such as intravenous (IV) thrombolysis with recombinant tissue plasminogen activator (rt-PA) and mechanical thrombectomy (MT) for selected patients with large vessel occlusion (LVO), have been shown to improve functional outcome within a time window of 4.5 and 6 h, respectively .Strict time windows, however, have been questioned as the infarct core progresses at different rates across individuals. The primary pathophysiological variable distinguishing “fast progressors” from “slow progressors” is the collateral status, and particularly the functionality of the leptomeningeal anastomoses . TherefoThe aim of this review is to provide an overview of the ischemic penumbra imaging methods from positron emission tomography (PET) imaging, the historical one, to multiparametric magnetic resonance imaging (MRI) and computed tomography (CT) that can be used as operational surrogate in AIS to guide therapeutic decisions. Using co-citation analysis, a bibliometric analysis of the field was carried out to observe the emergence of these methods. Additionally, the bibliographic coupling focusing on the MT era sketches the current state of research in order to position future contributions to the field.15O]-PET imaging on baboons validated the existence of the ischemic penumbra with metabolic parameters  and the cerebral metabolic rate of oxygen (CMRO2) is obtained through a series of PET acquisitions following inhalation and injection of molecules labeled with15O radioactive tracer. The steady state method was first implemented, and consists in maintaining the tracer's concentration in the blood to a constant level throughout the acquisition period. This method was replaced by a bolus injection and inhalation method that drastically reduced the radioactive doses required for image acquisition (2 parameter map (15O]H2O) bolus provides CBF mapping. The time activity curve following the injection enables the numerical calculation of the image-derived arterial input function (IDIF) and may prevent invasive procedures to obtain the arterial blood curves  with increased OEF and relatively preserved CMRO2 (CMRO2 ≥ 1.4 mL/100g/min)  . These t00g/min) .15O]-PET has been fundamental in demonstrating the existence of the ischemic penumbra in humans, thus driving the development of reperfusion therapies. However, it is impractical in the emergency setting and exposes patients to high doses of radioactivity  11C-flumazenil (FMZ) a ligand selective for the central benzodiazepine receptor, whose binding is reduced in irreversible tissue damage , 31; ii. AlthougAlternatively, single photon emission computed tomography (SPECT) using 99mTc-HMPAO (99m-Technetium hexamethylpropyleneamineoxime) or ECD (99mTc-ethyl-cysteinate-dimer) also provides data on perfusion in brain tissue. Although relative thresholds of CBF reduction have been proposed, the combination of SPECT and diffusion-weighted MRI seems more accurate to distinguish the core from the penumbra –36. HoweReperfusion therapies, such as IV thrombolysis with recombinant tissue plasminogen activator (rt-PA) and mechanical thrombectomy (MT) for selected patients with large vessel occlusion (LVO), have been shown to improve clinical outcome in AIS patients. While penumbra imaging is not formally required for patient's eligibility in early time windows, it has been proposed for the selection of patients who are the most amenable to reperfusion strategies in later time windows. Operational penumbra imaging methods, that are feasible in emergency setting, are required for this purpose. In this context, CT and MRI modalities have been introduced to estimate the penumbra in clinical routine , 38.15O]-PET. A parallel between these two penumbra can be found on Hemodynamic parameters maps can be computed using CT or MR perfusion imaging. Perfusion data processing requires the manual or automatic selection of an arterial input function, which is used for the deconvolution of each voxel's concentration time curve. Deconvolution can be done using a variety of techniques, ranging from the classical singular value decomposition (SVD)-based algorithms  to more To differentiate the penumbra from the oligemia, perfusion thresholds were initially defined using relative CBF and CBV parameters opposed to the contralateral control region (CCR) , 43. Wit−3mm2\s  thrombolysis  and up tThis historical association between imaging and AIS management was followed by a fast evolution of therapeutic and imaging methods in the field of AIS care. The scientific literature review of this field is a very challenging task as there are over 1000 articles on penumbra imaging in scientific databases, with about 50% of these articles published since 2015, in the era of MT. Therefore, in terms of data quantity, bibliometric analysis was a relevant approach to provide an overview of the field and an insight into the knowledge of the thrombectomy era. The bibliometric analysis was carried out in accordance with the methodology recommendations of Donthu et al.  and the The co-citation analysis is a bibliometric method that calculates how frequently two articles are cited together. It uncovers fundamental publications and enables a mapping of the field's foundation knowledge and evolutions. The analysis included the 75 most co-cited documents. This threshold increased the network's legibility and highlighted the most important contributions. The resulting network can be found in The applied mapping method retraces the timeline developed in 15O]-PET imaging. Several studies have compared MRI to [15O]-PET back-to-back in ischemic stroke patients and demonstrated that MRI overestimated the penumbra , it also includes articles about threshold and biomarkers of penumbral tissues using PET, CT, and MRI. These articles represent 10/27 documents within this cluster. This cluster also includes the first publication defining the ischemic penumbra . The detpenumbra , 53. Twopenumbra ; (ii) DiAll of the articles in the green cluster are focused on CT imaging. Perfusion CT and associated thresholds are covered in 10/16 publications. 3 articles elaborate on other CT imaging modalities  cluster contains 3 articles related to these biomarkers, however, 2/3 methods in this group are based on MRI modalities. Regarding the methods developed, 4/6 publications are based on variations of blood oxygen level dependent (BOLD) functional MRI principles . An exciting alternative consists in the fusion of the qBOLD approach with a new MRI framework called MR fingerprinting (MRF) where complex MR sequences are directly linked to advanced numerical simulations , the RHU MARVELOUS (ANR-16-RHUS-0009) of Lyon University, under the Investissements d'Avenir program of the French National Research Agency (ANR). The Ph.D. salary of LC  is co-funded by the French Ministry of Higher Education and Research (ANRT).This work was supported by ANR CYCLOPS and CMROLC, DR, and TB are employees of Olea Medical, a company developing the OleaSphere platform including pipelines for ischemic penumbra. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vancomycin-resistant Enterococcus faecium (VRE) in particular has evolved as an important cause of hospital acquired infection, especially in immunocompromised hosts. Methods: We present a complex case of a patient with relapsed acute myeloid leukemia who underwent allogenic hematopoietic stem cell transplantation complicated by persistent VRE bacteremia and meningitis. To optimize therapy, various blood and cerebrospinal fluid (CSF) samples were sent to a research laboratory for extensive susceptibility testing, pharmacokinetic analyses, and time-kill experiments. Results: In vitro testing revealed resistance to all first-line treatment options and CSF sampling demonstrated sub-optimal central nervous system concentrations achieved by each antimicrobial agent administered in relation to their respective MIC value. Time-kill analyses at observed CSF concentrations confirmed the lack of bactericidal activity despite use of a four-drug combination regimen. Conclusions: This work is the first to report CSF concentrations of oritavancin and tedizolid in humans and adds to the limited data regarding in vitro susceptibility of new antimicrobial agents such as eravacycline, omadacycline, and lefamulin against VRE. Our study provides new insights into various aspects of treatment of extensively drug-resistant Enterococcus faecium meningitis and bacteremia and supports the continued pursuit of precision medicine for these challenging cases.Background:  Enterococcus spp. are now well-described and associated with significant morbidity, mortality, and excess healthcare costs [E. faecium are vancomycin-resistant (VREf), severely limiting the number of safe and effective treatment options [Although once considered an innocuous commensal organism, serious infections due to re costs . The dif options . In addi options . Further options . These difficulties are further augmented when attempting to treat VREf in less pervious body sites, such as the central nervous system (CNS) . EnterocThe patient was a 58-year-old man with relapsed acute myeloid leukemia (AML) who achieved remission with salvage chemotherapy and was admitted for a planned haplo-identical stem cell transplant (SCT). In addition to the AML, his previous medical history was significant for subdural hematoma status post craniectomy 9 months prior to this admission and numerous episodes of fever in the setting of neutropenia with associated bacteremia including previous VREf. The first episode of VREf bacteremia occurred 2.5 months prior to the current admission and was susceptible dose-dependent (MIC = 4 mg/L by Etest) to daptomycin and was therefore treated with daptomycin at a dose of 8 mg/kg every 24 h. Blood cultures initially cleared but bacteremia with a daptomycin-resistant VREf (MIC = 32 mg/L by broth microdilution), returned with fevers after 11 days of daptomycin therapy. The isolate remained susceptible to linezolid (MIC = 2 mg/L) and therapy was therefore changed to linezolid 600 mg every 12 h, wherein he successfully completed 29 days of therapy. 3 cells/uL throughout his hospitalization. On hospital day 22, he developed a fever, blood cultures grew VREf  and day 35 blood VREf isolates were sent to the research laboratory for more extensive susceptibility testing . On the Pseudomonas aeruginosa. Meropenem was immediately initiated but the patient quickly decompensated and died on hospital day 84. An autopsy revealed infection with Gram-negative bacilli in his lungs, gastrointestinal tract, and genitourinary tract but no obvious source of persistent VREf infection.On hospital day 52, the patient developed new fever and repeat blood cultures again grew VREf. A repeat LP on day 57 showed improved pleocytosis , although CSF cultures again grew VREf . This isInitial organism identification and susceptibility determination were performed via MALDI-TOF MS  and MicroScan Walkaway , respectively, in the clinical microbiology laboratory. Pure colonies of the VREf isolates cultured from the CSF on hospital days 33 and 57 and from blood on days 35 and 52 were sub-cultured to BD BBL chocolate II agar slants  and shipped immediately overnight to the research laboratory where all subsequent testing was performed. Identification and resistance marker detection was confirmed via the Verigene Gram-positive blood culture assay . Research use only (RUO) MTS strips for lefamulin, omadacycline, quinupristin-dalfopristin, eravacycline ; in vitro diagnostic (IVD) Etest strips for fosfomycin ; GPALL3F ; and FDANDPF  Sensititre AST plates  were utilized according to manufacturer’s instructions. Minimum inhibitory concentrations were determined in triplicate according to CLSI guidelines . Modal MfCmax) and the time of fCmax (Tmax) of each agent. Finally, these predictions were evaluated against published data, all of which were restricted to animal studies except for tigecycline [Throughout the duration of systemic gentamicin, serum trough concentrations were measured as part of routine therapeutic drug monitoring. Gentamicin concentrations in plasma were measured via commercially available, validated immunoassay kits (Abbott Laboratories) with a lower limit of quantitation of 0.5 mg/L. From the CSF sample obtained on hospital day 57 as part of standard medical care, two excess CSF tubes were scavenged for the measurement of gentamicin, oritavancin, tigecycline, tedizolid, quinupristin, and dalfopristin concentrations. CSF samples were immediately frozen at −80 °C and shipped overnight on dry ice. Concentrations of each agent in the CSF were quantified via liquid chromatography–tandem mass spectrometry by Keystone Bioanalytical, Inc.  with a lower limit of quantitation of 0.001 mg/L. All analytical runs met pre-specified acceptance criteria for standard curve and quality control samples with a coefficient of variation ≤15% and absolute mean bias within 15%. Results are presented as the mean (±SD) of the two measured CSF concentrations. Additionally, estimated corresponding unbound plasma concentrations at the time of CSF collection were extrapolated for each agent from published pharmacokinetic data in order to establish an estimate of the degree of CSF/plasma penetration. This % CSF/plasma penetration was then utilized to predict the maximum post-dose CSF concentration on the basis of the peak free plasma concentration  to 0.5 mg/L (susceptible). The Verigene platform confirmed the identification of E. faecium and presence of vanA in all four isolates. Phenotypic susceptibilities for each VREf isolate against the tested antimicrobial agents are displayed in Over the course of 40 days of systemic gentamicin therapy at a dose of 400 mg every 48 h, 18 serum trough concentrations were obtained and all but two were above the lower limit of quantitation. Excluding these two values, the mean (±SD) plasma trough concentration was 0.89 ± 0.5 mg/L. Even with more than 72 h elapsing between the time of the last dose and CSF collection, gentamicin displayed the highest mean CSF concentration of any drug by more than twofold at 0.410 ± 0.05 mg/L, despite undetectable plasma concentrations as evidenced by a trough concentration collected just 8 h prior to CSF collection which was below the lower limit of quantitation. max. Almost exactly 2 days had elapsed between the time of the patient’s last oritavancin dose and the time of CSF collection. The average (±SD) CSF concentration at this time was 0.013 ± 0.005 mg/L. On the basis of the fCmax after a 1200 mg dose of 20.7 mg/L and a half-life of 245 h, the estimated unbound plasma concentration at 48 h post-dose would be approximately 18.8 mg/L. This would result in a 48 h CSF/plasma ratio for this patient of 0.07%, leading to a projected maximal CSF concentration of 0.014 mg/L at plasma Tmax of 0.6 mg/L and 12 h half-life [max. Although this 50% ratio is notably higher than the 2.16% average observed in uninfected rats [The last doses of tedizolid and tigecycline were administered approximately 8.5 h prior to CSF collection and both demonstrated similar mean (±SD) CSF concentrations at 0.204 ± 0.006 mg/L and 0.172 ± 0.002 mg/L, respectively. Given the reported IV tedizolid steady-state plasma fCalf-life , the unbted rats ,19,20. Sted rats . In a seted rats . ConsideFinally, despite being administered just 2 h prior to CSF collection, concentrations of both quinupristin and dalfopristin were undetectable in all four samples analyzed. This is consistent with published rabbit pneumococcal meningitis data  and supp10 colony-forming units (CFU)/mL at 24 h compared to the starting concentration). Oritavancin was the second most active agent, allowing 0.69 log10 CFU/mL of growth by 24 h. Treatment with either gentamicin or tedizolid alone resulted in an increase of >1 log10 CFU/mL compared to the starting inoculum. When these four agents were combined at their respective measured CSF concentrations, >1.5 log10 CFU/mL of bacterial growth was observed over 24 h compared to the starting inoculum. Finally, when combined at the drugs’ respective estimated maximum CSF concentrations, the four-drug combination demonstrated the greatest antibacterial activity, resulting in a 1.62 log10 CFU/mL reduction at 24 h (Time-kill analyses were performed on the VREf isolate obtained from the CSF on hospital day 33  against  at 24 h . Despite the advent of new agents with in vitro activity against VREf, the morbidity and mortality associated with serious infections remains unacceptably high, due in large part to increasing antimicrobial resistance and the dearth of adequate data to inform treatment ,29. ThisHerein, we describe the case of an immunocompromised patient with persistent, extensively drug-resistant VREf bacteremia and meningitis who was unable to sustain a clinical or microbiological cure despite combination therapy with up to five agents guided by extensive in vitro susceptibility testing. This lack of efficacy is likely explained in part by the suboptimal CNS concentrations achieved by each agent , especiaLimitations to this work include the lack of repeated PK measurements, absence of concomitant plasma concentrations to correlate with CNS concentrations, and lack of whole genome sequencing analysis to identify genetic mechanisms of resistance. Moreover, the lack of inflammation present on the day 33 CSF sample likely contributed in part to the low drug concentrations observed in addition to the physiochemical properties of the agents tested such as the large molecular weight and high protein binding of oritavancin. Finally, despite that the results of our PK/PD analysis were not available in real time to assist with this patient’s care, they likely would not have changed their antimicrobial regimen as therapy was already optimized to the extent possible based on our in vitro susceptibility testing. The promise of precision antimicrobial therapy is that a deeper understanding of the PK/PD properties of these agents alone and in combination can guide us towards improved therapies and better patient outcomes. Although the collaboration utilized herein to achieve this goal is not currently universally applicable, it is a step towards improving the quality of care and combating antimicrobial resistance. We hope that these data are beneficial in helping future patients afflicted with this challenging pathogen.


Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with “sickness behaviors” and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the “sickness behaviors” of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.Rainbow trout ( Nature paper indicated that aquaculture might act as a solution for increased fishery production [Oncorhynchus mykiss) is one of the most important commercial and market fish with global production around 800 thousand tonnes . Compared to dramatically depleted wild fishery resources, cultured fish are making up an increasing proportion of the world’s fishery population [Fish serves as an important single source of high-quality protein . Based ooduction . For exaoduction ,4,5,6,7)pulation .Aeromonas salmonicida , which has been identified as one of the most infective and lethal pathogens for over 100 years, acts as the causative agent of furunculosis [A. salmonicida could enter the host cells of skin, gills, and intestines, and then rapidly infect the internal organs, thus resulting in severe symptoms and mortality of the infected individuals [A. salmonicida is reported to result in diseases in a wide range of freshwater fish, including salmonids, common carp (Cyprinus carpio), European catfish (Silurus glanis), Cottus gobio and fathead minnow , and the seawater species of turbot , and sea bream (Sparus aurata) [A. salmonicida exhibit high morbidity, severe symptoms, and high mortality, thus resulting in great economic loss [The cultured fish must cope with multiple environmental and pathogen challenges ,9. Moreonculosis ,13,14. Fnculosis . After iividuals ,17. The  aurata) ,19. Fishmic loss .A. salmonicida has been thoroughly investigated, as well as the symptoms, pathophysiology, and pathology [A. salmonicida exhibits the symptoms of anorexia, lethargy, abnormal swimming behavior, and haemorrhagic septicaemia with darkened skin pigmentation, skin ulcers and necrosis, and internal bleeding [A. salmonicida infection, including the expression profiles of cytokines, signaling pathways of toll-like receptors, and activation and migration of immune cells [A. salmonicida not only impairs the cell physiology and phagocytosis of trout, but also severely dysregulates gene transcription of immune mediators and disturbs the warning signals related to infection recognition and immunomodulation [A. salmonicida infection [A. salmonicida infection [A. salmonicida infection at early stages [A. salmonicida resistance [A. salmonicida infection will limit the better understanding of the immune mechanisms during A. salmonicida infection in trout .The characteristics, virulence, and genome of athology ,22,23,24bleeding ,23,25,26ne cells ,29,30,31dulation . In recenfection ,34,35,36y stages . Moreovesistance . Howeversistance . The infA. salmonicida infection resulted in homogeneously (or heterogeneously) immune effects on CNS and peripheral immune tissues in trout.Although early researchers generally studied the immune and central nervous system (CNS) as separate targets due to the concept of “immune privilege”, the last 30 years has witnessed the birth of brain (CNS)-immune communication and the following significant development ,41. PeriA. salmonicida infection. We selected the brain and kidney due to the following reasons: 1. Brain cells exert poor regenerative capacity and dysfunction in the brain (by immune cells and/or modulators) could lead to catastrophic effects on life. However, the immunomodulation status of trout brain in response to pathogen infection has been underestimated, so far. 2. Th kidney is a well-characterized immune tissue in teleost and serves as the major target of bacterial A. salmonicida infections [In our study, we investigated the transcriptional profiles and functional enrichment of brains (CNS) and kidneys  of trout in response to fections ,36. Furtfections ,47. TherA. salmonicida, RNA-seq analysis showed 855 (163 down and 692 up) and 603 (306 down and 297 up) DEGs in the brain and kidney, respectively  was involved in inflammatory regulation, cellular defenses, and cellular responses to cytokines in the brain and kidney (h2-q9 (with other genes) was associated with immune response and major histocompatibility complex (MHC) functions  were enriched in the pathways of phagosome, antigen processing and presentation, and natural killer cell mediated cytotoxicity . Trout and human CSF-1R showed relatively conserved amino acid sequences in juxtamembrane domains (Colony stimulating factor 1 receptor (CSF-1R) serves as the receptor of CSF-1 and interleukin-34 (IL-34). The CSF-1R signaling plays an important role in regulating cellular proliferation, differentiation, and survival ,50,51,52 domains A,B. Base domains C–F, incl domains C–F.il-34b, csf-1ra2, and csf-1rb2 showed up-regulation in infected trout while the kidney csf-1rb1 showed up-regulation in infected trout  gene was first identified in mouse macrophage cell line in response to lipopolysaccharide (LPS) stimulation [acod1 expressions in the brain and kidney, respectively (Cyprinus carpio carpio L.) exerts up-regulated acod1 expression in responses to LPS [acod1 was highly enriched in GO terms, such as immune response (GO:0006955), inflammatory response (GO:0006954), and cellular response to LPS (GO:0071222) (acod1 expression (~160-fold higher than brain), while the brain showed higher A. salmonicida-induced acod1 expression (~118 versus 34-fold increase), probably suggesting that CNS and peripheral tissues exerted distinct metabolic signatures during basal and activated immune states.We identified DEGs of dulation . The Acomulation . The recmulation ,55,56. Iectively , which is to LPS . Our enr0071222) , which sh2-q9 (LOC110496224) and ha2q (LOC110537685) were associated with biological functions of class I and class II histocompatibility and the up-regulated tapasin serves as a chaperone for MHC class I assembly [A. salmonicida or virus infection [A. salmonicida-resistant trout families due to the fact that MHC polymorphism is associated with resistance/susceptibility to A. salmonicida in Salmonids [MHC are polymorphic membrane glycoproteins that play an important role in regulating immune functions in response to self and invading antigens ,59. The assembly . Moreovenfection ,65. Thernfection . Our datalmonids .il-6 and il-6rα (il-6 receptor subunit α) expressions and up-regulated enrichments of cellular response to lipopolysaccharide (GO:0071222), cellular response to interleukin-1 (GO:0071347), TNF signaling pathway (ko04668) and Cytokine-cytokine receptor interaction  play important roles in regulating innate immune and the inflammatory responses ,79. Multed trout . Moreoveed trout . Our resnfection ,82. TakeA. salmonicida. After infection, trout exert defenses to foreign pathogens and initiate immune responses. TLR2 could detect the extracellular pathogen molecules while the intracellularly pathogen molecules were recognized by TLR7, TLR8, and TLR13. The activated TLRs trigger the downstream MyD88 and NF-κB signaling, enhancing transcriptional levels of genes involved in inflammatory regulation. Neuroinflammation further results in down-regulation of neural development and function , toll-like receptors and TNF signaling , and complement and coagulation cascades ko04610, . The kidcsf-1 gene is extensively expressed in multiple species including mammals, birds, and teleost [csf-1 gene is identified in most mammals, while teleost  exerts two csf-1 (csf-1a and csf-1b) copies due to the additional whole genome duplication  [csf-1 paralogs in trout, which is consistent with the fact that Salmonids’ ancestry exerts four rounds of genome duplication [c-fms proto-oncogene [csf-1r paralogs in rainbow trout. Phylogenetic analysis indicated orthologous relationships of csf-1 and csf-1r between mammalian and teleost equivalents  ,96. In tlication . The CSFoncogene , serves ivalents , potentiA. salmonicida infection resulted in significantly increased csf-1ra2 and csf-1rb2 expressions in the brain and decreased csf-1rb1 expression in the kidney, suggesting the functional diversity of csf-1r paralogs and the potential tissue-specific expressions. Further enrichment analyses showed up-regulated brain csf-1r genes were involved in up-regulated GO terms associated with innate immune response (GO:0045087) and inflammatory response , the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals . Our research did not involve endangered or protected species. In this study, trout juveniles were immature, and the effect of gender was not considered.A. salmonicida (1 × 108 CFU/mL), while trout in CT were injected by an equal volume (0.2 mL) of phosphate buffer solution (PBS). Furunculosis symptoms (Rainbow trout (~10 g and ~8 cm) were obtained from trout farm in Linqu, Shandong Province, and then acclimated for 7 days in individual aquariums at ~17 °C with the photoperiod of 12:12 (hours) of light and dark. During bacterial challenge, trout were randomly classified into two groups as control trout group (CT) and infected trout group (IT), respectively. IT contained two replications and each replication had 20 individuals (n = 20). Based on our previous study , trout osymptoms  were obsTM RNA Sample Prep Kit . The Illumina Hiseq X Ten platform  was used to generate 150 bp paired-end raw reads. Raw data were processed using the Trimmomatic [Total RNA of the brain and kidney was extracted using TRIzol reagent , and the RNA quality was evaluated by NanoDrop ND-1000  and 1% agarose gel electrophoresis. One transcriptome library was constructed by pooling equal quantities of RNA from two individuals. A total of 12 libraries (2 tissues x 3 replicated samples × 2 treatment groups) were constructed via TruSeqmmomatic . The clemmomatic . The seqp-value < 0.05 and |log2(fold change)| > 1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were evaluated, primarily focusing on functional/signaling pathways associated with immunomodulation and neural functions.The Fragments Per kb Per Million Reads (FPKM) value of each gene was calculated using cufflinks, and the read counts of each gene were obtained by htseq-count. Differentially expressed genes (DEGs) were identified by the DESeq2 R package ,104 and csf-1, also known as cfms) paralogs and their cognate receptors (csf-1r) were significantly involved in immune responses of trout in response to A. salmonicida challenges. To identify the novel paralogs of csf-1 and csf-1r, the whole genomic sequence database (GCA_013265735.3) of rainbow trout was researched using the TBLASTN program. The CSF-1 and CSF-1R full-length amino acid sequences of human and zebrafish were used as the queries with an E-value of 1 × 10−5. The TBLASTN and Clustal W were used to remove redundant sequence(s), thus generating initial candidate sequences for further analysis.The RNA-Seq data showed that macrophage colony stimulating factor 1 (Homo sapiens), mice (Mus musculus), zebrafish (Danio rerio), medaka (Oryzias latipes), channel catfish , Atlantic salmon  and other teleost were used for the construction of the phylogenetic tree. An alignment of multiple amino acid sequences was performed using ClustalW. The neighbor-joining (NJ) method and Jones–Taylor–Thornton (JTT) model were used for conducting phylogenetic and molecular evolution analysis by using MEGA 7.0 software. Bootstrap tests with 1000 replications were used to test the phylogenetic tree and gaps were removed via pairwise deletion. Comparison between trout and mammalian CSF-1R  were generated with the SWISS-MODEL  and PyMOL software package.Phylogenetic analysis was conducted to validate the annotation of the potential CSF-1 and CSF-1R paralogs in trout. The amino acid sequences of human ((−1/slopes) − 1) × 100. The β-actin was used as an internal reference gene [TM Real-time PCR system . The reaction volume was 10 μL, containing 1 μL cDNA, 5 μL SYBR® FAST qPCR Master Mix, 0.2 μL forward (reverse) primer, and 3.6 μL RNAase-free water. The qPCR was performed by following the following program: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and Tm for 30 s, followed by 72 °C for 30 s. The relative expression levels of mRNA were calculated using the comparative 2−ΔΔCT method [Thirteen DEGs were selected for qPCR analysis. They were shown in T method .https://www.xialab.ca/tools.xhtml, last accessed on 21 December 2021) for data processing and analyses [A. salonmicida challenge on gene expressions of trout in CT and IT. Means of gene expressions were further compared by Tukey’s multiple range tests when significant differences were observed by one-way ANOVA. The differences were considered statistically significant when the p < 0.05. Results were expressed as mean ± standard error (means ± S.E.). The correlation analysis of gene expression was investigated by MetaboAnalyst and GraphPad Prism 8.0 via Pearson’s correlation analysis.Based on published medical and fishery studies ,107 the analyses . The datA. salmonicida infections resulted in homogeneously immune effects on both central and peripheral tissues, rather than CNS immune privilege. In the brain, the up-regulated DEGs were associated with activation of the toll-like receptor signaling pathways, thus triggering neuroinflammation and dysregulated neuro functions. Therefore, the typical disease characteristics of infected trout, such as symptoms of anorexia and lethargy, could be partly attributed to the dyshomeostasis between immunomodulation and neuromodulation in brain. In addition, kidneys also showed up-regulated endocrine networks involved in estrogen, prolactin, and insulin signaling pathways and thyroid hormone synthesis, supporting the bi-directional crosstalk between endocrine and immune systems in response to pathogen infection.The pairwise comparison between trout of the CT and IT groups  showed t


Long-term kidney transplant survival at the population level is consistently favorable, but this survival varies widely at an individual level due to both recipient and donor factors. The distinct contribution of recipient and donor factors to individual post kidney transplant outcome remains unclear. Comparing outcomes in deceased donor (DD) recipients with potential but non-actualized living donors (DD1) to those recipients with actualized living donors (LD), and to DD recipients without potential living donors (DD0) may provide transplant candidates with more information about their own post-transplant prognosis.We conducted an observational retrospective cohort study of kidney transplant candidates presenting to our centre for evaluation between 01/01/06 and 31/12/18, and who also received a transplant during that time. Patients were followed to 31/08/2019. Candidates were classified as DD0, DD1, or LD based on whether they had an identified living donor at the time of initial pre-transplant assessment, and if the donor actualized or not. Primary outcome was 5-year death-censored graft survival, adjusted for common pre- and post-transplant donor and recipient risk factors. Secondary outcomes analyzed included patient survival and graft function.p = 0.19). DD0 graft survival was inferior to LD (p = 0.005), but also trended inferior to DD1 (p = 0.052). By multivariate Cox regression analysis, LD demonstrated similar 5-year graft survival to DD1  but LD graft survival was superior to DD0 . The 5-year patient survival in DD1 was similar to LD (p = 0.26) but was superior to DD0 (p = 0.01).There were 453 kidney transplant recipients  who received a transplant during the study period. DD0 and DD1 did not differ in key donor organ characteristics. The 5-year death censored graft survival of DD1 was similar to LD (DD recipients with potential but non-actualized living donors exhibit similar mid-term graft and patient survival compared to LD recipients. Having an identified living donor at the time of pre-transplant assessment portends a favorable prognosis for the recipient.The online version contains supplementary material available at 10.1186/s12882-022-02718-6. Successful kidney transplantation (KT) in patients with end-stage kidney disease (ESKD) improves survival and quality of life compared to chronic dialysis .AlthoughDonor factors including the organ source are considered variables that are fixed at the time of implantation; post-transplant organ performance then depends on acute perioperative and postoperative immune and load factors . Graft sIf discriminating a living donor from a deceased donor organ source defined at the time of transplantation does not sufficiently predict individual post-transplant graft survival, then examining the intended donor organ source as determined at the time of pre-transplant assessment rather than the actual donor organ source at time of transplantation might add useful information to predicting post-transplant graft survival. Greater explication of the donor source-post-transplant outcome relationship may also help guide public awareness strategies about organ donation, and assist decisions in particular cases about proceeding with living donation or not.St. Michael’s Hospital (SMH) is an urban tertiary medical-surgical centre in Toronto, Canada that performs approximately 120 adult single-organ kidney transplants annually and provides post-transplant care to about 1800 kidney transplant recipients (KTR) as of January 1, 2021. KT candidates referred to SMH are assessed by interview and investigations to conform to Canadian guidelines . WheneveIn keeping with the current paradigm, we hypothesized that graft outcomes in deceased donor KTR either with or without a previously identified potential living donor will be similar, but the outcomes in both will be inferior to those KTR with actualized living donors. To this end, we performed a retrospective cohort observational study of single-organ KT candidates who were either pre-dialysis or on dialysis and interviewed at SMH between January 1, 2006 and December 31, 2018, and who also subsequently underwent KT at SMH with at least 6 months of post-transplant follow-up. Patients with graft loss within 6 months were included. Data were collected from the SMH transplant program’s electronic database to August 31, 2019. Potential donor source was collected at interview and recorded in the pre-transplant chart, while the ultimate donor source, either living or deceased, was recorded along with demographic variables, early post-transplant events, and post-transplant outcomes from the post-transplant database. KTR with type 1 diabetes mellitus were excluded since they are referred to another centre for the possibility of a subsequent pancreas transplant. Similarly, recipients of a double-kidney or multi-organ transplant were excluded, along with candidates not transplanted during the study period or who were transplanted at another centre.We compared the graft and patient outcomes of actualized living donor KTR (LD), to both KTR with a prospective living donor but who subsequently received a deceased donor organ because their living donor did not actualize , and KTR without a prospective living donor at initial evaluation who then received a deceased donor organ (DD0). We also compared DD1 and DD0 recipients. This KTR classification was independently verified by two investigators. The primary outcome was 5-year death-censored graft survival, adjusted for donor and recipient age and other characteristics, race, cause of end-stage kidney disease (ESKD), peak panel-reactive antibody (PRA), dialysis duration, acute rejection (AR), and delayed graft function (DGF). Preemptive transplants were assigned a dialysis duration of 0. If a patient received more than one transplant, each transplant was counted as discrete. Secondary outcomes included graft survival at 1 and 3 years, as well as graft function and patient survival at 1-, 3- and 5-years. Graft function based on the estimated glomerular function rate was derived from the CKD-Epi Equation  .p < 0.05. The study was approved by the SMH Research Ethics Board .Within-group and between-group comparisons were made using paired or unpaired student t-testing, chi-square testing, or Fisher’s exact testing as appropriate. Graft and patient outcomes were compared using Kaplan–Meier methodology and the log-rank test. Independence of predictor variables considered most clinically relevant was verified by multivariable Cox proportional hazards analysis, while avoiding model overfitting. Since this was a population-based study of all KTR transplanted during a pre-defined period, a formal sample size calculation was not performed. Significance was taken as N = 18) were excluded from further study either due to type 1 diabetes mellitus or because they were transplanted at another centre. Reasons for non-actualization of DD1 donors included rejection for medical reasons in 58%, rejection for non-medical  reasons in 23%, donor withdrawal in 3%, and loss to follow-up for unknown reasons in 16%. Among all evaluated donors, 46% were biologically related to their recipient, 44% were emotionally related, and 10% had no pre-existing relationship to the recipient .There were 471 KTR who met entry criteria for the study . A small number of candidates . There were several important differences between the DD0 and DD1 groups, with the DD1 group being younger, on dialysis for a shorter duration, and being more sensitized to human leukocyte antigens (HLA). However, the DD0 and DD1 groups did not differ in important organ characteristics including donor age, terminal serum creatinine; neurological determination of death (NDD) versus donation after cardio-circulatory death (DCD); and standard criteria donor (SCD) versus expanded criteria donor (ECD) status. More DD0 recipients had diabetes and diabetes-related ESKD compared to the other two groups. None of the DD1 and DD0 transplants were preemptive.The most common immunosuppressive regimen in recipients was basiliximab induction followed by tacrolimus, mycophenolic acid, and prednisone as maintenance immunosuppression. Table p = 0.052) and was statistically inferior to LD (p = 0.005). However, DD1 survival was not inferior to that of LD (p = 0.19). LD displayed a higher 5-year eGFR (59.7 ml/min/1.73m2) compared to DD1 (53.5 ml/min/1.73m2) and DD0 (52.0 ml/min/1.73m2) (p = 0.009)  and DD0 alone  but not DD1 in isolation (p = 0.72). AR, DGF, and diabetic ESKD were also significant predictors of graft survival across models. Adding peak PRA and dialysis duration to each model rendered DD type insignificant (data not shown).The 5-year death censored graft survival is shown in Fig. In a post-hoc analysis, we compared the DD1 and DD0 recipients in three additional multivariate models to determine the relative importance of NDD/DCD, SCD/ECD, and cold ischemia time (CIT): these variables plus donor age, terminal serum creatinine and the other variables shown in Table N = 8) subsequently received an LD transplant when a living donor came forward after the initial recipient pre-transplant assessment. However, adding these patients to either the DD0 or LD groups in sensitivity analyses did not significantly affect any results. There was no difference in donor age or recipient age between the DD0 and DD1 groups. There were no DD0 patients known to identify a potential living donor subsequent to their initial assessment (and who could therefore have been assigned to DD1 instead), who then did not actualize.A small number of DD0 recipients (This study demonstrates important variation in graft survival within deceased donor KTR populations defined at initial evaluation. Graft survival in KTR with a non-actualized living donor did not significantly differ from KTR with an actualized living donor, while both these groups demonstrated outcomes superior to KTR who never had a potential living donor. This difference in graft survival occurred regardless of whether the deceased donor kidney type fulfilled standard criteria or expanded criteria, or occurred from donation after cardio-circulatory or neurological death. These findings suggest that the ultimate source of the donor organ whether living or deceased, while important for graft function and survival, carries with it an important caveat. The heterogeneity in outcome within the deceased donor KTR population likely relates to differences in recipient characteristics, since DD organ quality reassuringly did not differ between the DD0 and DD1 groups. The smaller sample size of DD1 may however have precluded detecting a statistically significant difference between the DD1 and DD0, as well as between the DD1 and LD groups.KTR with a prospective but non-actualized living donor may share greater similarity to KTR with an actualized living donor than to recipients who never had a prospective living donor at the beginning of their evaluation. The study result therefore raises the interesting hypothesis that potential but non-actualized living donors themselves favorably influence the post-transplant course of their intended recipient. In other words, intended donor source may be a recipient characteristic, while actual donor source is a donor characteristic. It is also possible that candidates with a potential living donor may opt to accept a deceased donor kidney only if considered good quality by the clinical judgment of the clinical team and patient, factors not easily measurable in a retrospective analysis. Medium-term post-transplant outcomes are similar amongst standard criteria deceased and living donor recipients . A risk Differences in DD recipient outcomes may reflect social support, medication adherence, and other recipient characteristics. While important biological factors pertaining to the organ influence post-transplant outcome, the present study shows that post-transplant outcomes differ among deceased donor recipient groups despite equivalent donor biological quality. Therefore, non-biological factors may be just as important. For example, a previous study demonstrated that longer-term graft survival is worse in recipients of living donor kidneys obtained through transplant tourism compared to domestic deceased donor transplants, and this inferiority of living donor transplants could not be explained by early post-transplant events . TherefoThe simple binary discriminatory variable of having a potential living donor identified many years before the actual transplant takes place may be a useful tool to predict medium-term graft survival. Large registry studies do not provide the required level of data granularity for pre-transplant information. Outcomes are typically captured based on actualized donors, living or deceased. Similarly, a randomized control trial of actualizing or non-actualizing a potential living donor is neither practical nor ethical. Studies to capture sociological variables are intense to perform and limited to small patient numbers. Granular single-centre level data about potential living donors on the other hand may be the best source for information that links social characteristics to graft survival.Predictors of living donor dropout may include female recipient, systemic disease as cause of kidney failure in the recipient, or relationship as a friend . Social The present study has several strengths and limitations. To our knowledge, this is the first study to identify the potentiality, not just actuality of a living kidney donor as a predictor variable for post-KT graft survival. The study splits the deceased donor recipient population into two sub-populations, who may be at different risk for adverse post-transplant events. The study also highlights the critical importance of transferring pre-transplant social variables to the post-transplant record. The variable is simple and inexpensive to collect, and can be routinely collected by large registries in the future. The study is limited by its single-centre and retrospective nature, and must therefore be considered hypothesis-generating. We were unable to collect detailed information on donor-recipient relationships beyond biological relationship, reasons for donor drop-out, or post-transplant social factors. Since the number of events was small, we could not dissect their nature further or control for more than only a limited number of potentially confounding variables. We did not have information on human leukocyte antigen mismatch or pre-transplant donor specific antibodies. Future studies can help elucidate social factors and the effect of corresponding interventions, but in the meantime reproducing these findings at other transplant centres and through registries is needed. While larger sample sizes and longer follow-up will be valuable to confirming these findings, there remains sufficient reason for transplant centres to henceforth collect this simple binary pre-transplant variable. KT candidates whose living donors do not actualize may also obtain some reassurance that their post-transplant outcomes will not necessarily be inferior to LD transplant recipients if they accept a DD organ. Since the potential living donor’s organ still lives outside the recipient’s bodily confines, an identified potential living donor may be serving as a surrogate for numerous impossible-to-collect post-transplant recipient factors affecting graft survival.Deceased donor kidney transplant recipients with a potential but not actualized living donor demonstrate mid-term graft survival comparable to living donor kidney transplant recipients. Deceased donor transplant recipients without a potential living donor demonstrate outcomes inferior to both these groups, and may benefit from more targeted study of assessments and interventions to improve their post-transplant graft survival.Additional file 1.


Unbiased metagenomic next-generation sequencing (mNGS) detects pathogens in a target-independent manner. It is not well-understood whether mNGS has comparable sensitivity to target-dependent nucleic acid test for pathogen identification.This study included 31 patients with chickenpox and neurological symptoms for screening of possible varicella-zoster virus (VZV) central nervous system (CNS) infection. Microbiological diagnosing of VZV cerebrospinal fluid (CSF) infection was performed on stored CSF samples using mNGS, quantitative and qualitative VZV-specific PCR assays, and VZV IgM antibodies test.6/L). VZV IgM antibodies presented in 16.1% of the CSF samples, and nucleic acids were detectable in 16.1 and 9.7% using two different VZV-specific real-time PCR protocols. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS , with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples. All VZV PCR positive samples were positive when analyzed by mNGS. Of note, human betaherpesvirus 6A with clinical significance was unexpectedly detected in one CSF sample.The median age was 30.0  years. 51.6% of the patients were men. About 80.6% of the patients had normal CSF white blood cell counts (≤ 5 × 10Our study suggests that CSF mNGS may have higher sensitivity for VZV detection than CSF VZV PCR and antibody tests, and has the advantage of identifying unexpected pathogens. Central nervous system (CNS) infections have led to a substantial morbidity and mortality in clinical practice; however, the etiologic agents remain undetermined in around half of the cases , 2. The Mycobacterium tuberculosis (MTB) infection, studies have shown that mNGS has higher sensitivity as compared with MTB-specific PCR  identifies pathogens in a target-independent way . It has ific PCR , 13. WheThe sensitivity of CSF mNGS for pathogen detection may be greatly influenced by the CSF pleocytosis which leads to a substantial human DNA contamination . Since vThis study included 31 patients with suspected VZV CNS infection admitted to the second hospital of Nanjing, China, from August 2018 to November 2020. In Nanjing, the second hospital of Nanjing is the only designated hospital for managing patients with primary VZV infection. Patients with characteristic skin rashes suggestive of varicella and concomitant neurological symptoms were hospitalized for screening of possible VZV CNS involvement. Diagnostic procedures in routine clinical care for those patients included a cerebral MRI and analysis of CSF cell counts, chemistry, and bacterial culture. After obtained written informed consent, additional CSF samples were collected and stored at −80°C until analysis for VZV IgM antibodies, VZV PCR, and unbiased mNGS.The medical records, such as epidemiological and demographic data, medical history, underlying comorbidities, clinical manifestations, laboratory tests, cerebral imaging findings, treatments, and outcomes, were retrieved from an electronic health record system. This study was approved by the ethics committee of the second hospital of Nanjing (reference number: 2020-LY-kt061).ftp://ftp.ncbi.nlm.nih.gov/genomes/) which contained 4,945 whole genome sequences of viral taxa, 6,350 bacterial genomes or scaffolds, 1,064 pathogenic fungi genomes, and sequences of 234 parasites associated with human diseases.Cerebrospinal fluid samples were sent for PMSeq-DNA, a commercially available service from BGI-Shenzhen, which detects pathogenic microorganisms through mNGS. In this study, 600 μl CSF sample was mixed with enzyme and glass beads, and was vortexed vigorously at 2,800–3,200 rpm for 30 min. Then, total DNA was extracted from 300 μl each CSF sample using TIANamp Micro DNA Kit  according to the recommendation of manufacturer. DNA libraries were constructed through DNA-fragmentation, end-repair, adapter-ligation, and PCR amplification. After quality control with Agilent 2100 , libraries were sequenced by BGISEQ-50 platform. Low-quality sequence reads and sequence reads mapped to the human reference genome (hg19) were removed. The remaining data were classified by aligning to four Microbial Genome Databases containing bacteria, fungi, viruses, and parasites. The reference databases were downloaded from NCBI  was used to measure VZV-IgM antibodies in CSF samples. The ELISA procedure was done following SERION ELISA classic CSF Diagnostics instructions with a modification that the dilution factor of CSF samples was changed from 1:20 to 1:10. As a CSF standard was not included in the kit, the interpretation of ELISA results was based on average optical density (OD) 450 values. The ELISA test was considered negative if the average OD value from duplicate sample wells is <0.1 or was <2-fold of that in the negative control. In our study, CSF samples from nine patients with syphilis-seropositive were used as negative control. Those nine patients were excluded from neurosyphilis based on normal CSF cell counts and chemistry tests, and negative CSF The VZV nucleic acid was detected by real-time PCR assay. For the qualitative assay, 30 μl of total DNA was extracted from 200 μl CSF sample using TIANamp Micro DNA Kit . Then, the presence of VZV DNA was detected using a commercially available VZV nucleic acid detection kit following the instruction of manufacturer . Each sample was testes two times. The following PCR program was run in the applied biosystems (ABI) 7500 Thermal Cycler : 50°C for 2 min; 95°C for 15 min; 45 cycles of 94°C for 15 s; and 55°C for 45 s. A cycle threshold (Ct) value of 38 or less was considered positive.In addition, the CSF samples were sent for absolute quantification of the VZV viral load using an in-house real-time quantification PCR test at a third-party diagnostic company . Then, 100 μl of total DNA was extracted from 200 μl CSF sample using a DNA/RNA extraction Kit . Furthermore, 5 μl DNA sample was added to the reaction mix prepared following instruction of QuantiTect Probe PCR Kit . The primers and probe set for VZV DNA amplification and detection were as follows: forward primer: 5′-TGCAGGGCATGGCTCAGT-3′; reverse primer: 5′-CCCAAGAACCACATGTCCAAC-3′; and probe: 5′-FAM-TCCTCAGTGCGGTGGTTGCCCA-BHQ1-3′ . The folp < 0.05 is considered statistically significant.Continuous variables were expressed as the medians and interquartile ranges (IQRs). Categorical variables were shown as the counts and percentages in each category. Comparison between groups was done using McNemar's test for categorical variables, when appropriate. Statistical analysis was done by using SPSS version 22.0 . A 6 cells/L.The characteristics of the 31 patients admitted for screening of the VZV CNS infection were summarized in 6 cells/L). The diagnosis of VZV CNS infection in the remaining 7 patients with normal CSF white blood cell counts was made by expert penal after thoroughly evaluating the clinical course and imaging findings of those patients. To explore the microbiological evidence of VZV CNS infection, stored CSF samples were tested for VZV IgM antibodies, VZV PCR, and unbiased mNGS. VZV IgM antibodies were presented in 16.1% (5/31) of the CSF samples.At the time of discharge, 41.9% (13/31) of the patients were clinically diagnosed with VZV CNS infection. Of the 13 patients with a clinical diagnosis of VZV CNS infection, 6 had CSF pleocytosis , with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples (Varicella-zoster virus nucleic acids were detected in 16.1% (5/31) and 9.7% (3/31) of the CSF samples, respectively, when different real-time PCR reagents were used. VZV nucleic acids were detected in three CSF samples by both qualitative and quantitative VZV PCR. Two CSF samples, with negative quantitative VZV PCR results, tested weak positive for VZV by using qualitative VZV PCR with cycle threshold of 37.88 and 37.93, respectively. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS  ; VZV was6 cells/L. A cerebral MRI showed multiple abnormalities in left thalamus, cerebellar vermis, and left cerebral peduncles. At that time, she was clinically diagnosed with VZV CNS infection, and was treated with acyclovir, intravenous immunoglobulin, and dexamethasone. The clinical condition was gradually improved. After hospitalization for 26 days, the patient was discharged without obvious symptoms. The presence of HHV-6A in the stored CSF sample, which was unexpectedly identified by mNGS, was further confirmed by real-time PCR following the protocol described by Gautheret-Dejean et al.  and low sequence reads of VZV . The patient was admitted to the hospital because of fever and itchy blisters for 6 days and difficulty urinating for 1 day. She had a close contact with her son who was recently diagnosed with chickenpox. CSF analysis demonstrated pleocytosis with leukocytes of 120 × 10n et al. .Viral encephalitis accounts for 20–50% of microbiologically confirmed cases of encephalitis, with herpes simplex virus being the most common etiologic agent followed by VZV . DetectiFor the above-mentioned reasons, a negative CSF VZV IgM test or nucleic acid test does not necessarily rule out VZV CNS infection. Therefore, in the present study, we did not arbitrarily define “true positive” or “true negative” cases of VZV CNS infection. In this context, the exact sensitivity of each microbiological test for diagnosing of CNS infection was not calculated. Instead, the magnitude of positive result was compared. Our study showed that, compared with CSF VZV IgM antibody test or CSF VZV PCR test, CSF mNGS identified much higher number of cases with clues of VZV CNS infection. Of note, all the CSF samples with positive VZV PCR results tested positive for VZV sequence reads by using mNGS. Those findings suggest that CSF VZV-specific PCR may not improve the sensitivity of diagnosing VZV CNS infection when mNGS has already been performed on CSF samples.The mNGS focuses on the whole genome sequence of the pathogen which is broken into small sequence segments that are unbiasedly amplified and measured. Therefore, sequences of a given pathogen are more likely to be detected by mNGS targeting the whole genome of the pathogen, as compared with pathogen-specific PCR which generally targets single or two regions of the genome sequence . MoreoveThe main advantage of mNGS is unbiased sampling which enables broad identification of pathogens in a hypothesis-free manner . Using tThe mNGS is not a perfect diagnostic strategy in clinical microbiology. The unbiased sampling is not just an advantage but also, to some extent, could be a drawback. First, clinical samples are dominated by human host background. Unwanted human DNA occupies the majority of sequence reads of the mNGS which would limit the sensitivity of this strategy for pathogen identification , 34. SecAs shown in In conclusion, our study found that CSF mNGS was the most sensitive microbiological test for diagnosing VZV CNS infection, and could unexpectedly identify the causative agent that were not recognized based on traditional diagnostic tests. Perhaps, for the diagnoses of viral encephalitis caused by DNA virus, with continuing improving the methodology of mNGS, pathogen-specific PCR may not be needed when mNGS has already been used for pathogen detection. Nevertheless, pathogen-specific antibody detection may still be performed because it provides different biological information. Finally, although the sensitivity of mNGS can be further increased by technical innovation, the specificity will continue to be a great concern. Clinical judgement of the treating physicians is very important for interpretating the results.PRJNA784638.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The raw sequences of mNGS were deposited to the NCBI database under accession numbers The studies involving human participants were reviewed and approved by the Second Hospital of Nanjing. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.ZH designed this study. YZ, CD, and ZH collected the data. MX, WW, and ZH completed the experiment. MX, ZP, WC, and ZH analyzed the data. BS, JC, and CC were involved in the clinical management of the patients and analyzed the clinical data. This manuscript was initially drafted by YZ, CD, and ZH and then revised by MX, ZP, WW, BS, JC, CC, WC, HW, and ZH. All authors approved the final manuscript.This study was funded in part by the project of Jiangsu province medical youth talent (QNRC2016059) and the Nanjing medical science and technique development foundation (YKK18153). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Growth differentiation factor 15 (GDF15) increases in serum during pregnancy to levels not seen in any other physiological state and is suggested to be involved in pregnancy-induced nausea, weight regulation and glucose metabolism. The main action of GDF15 is regulated through a receptor of the brainstem, i.e., through exposure of GDF15 in both blood and cerebrospinal fluid (CSF). The aim of the current study was to measure GDF15 in both CSF and serum during pregnancy, and to compare it longitudinally to non-pregnant levels.Women were sampled at elective caesarean section  and were followed up 5 years after pregnancy (n = 25). GDF15, insulin and leptin were measured in CSF and serum. Additional measurements included plasma glucose, and serum adiponectin and Hs-CRP.P<0.001) and serum . CSF levels correlated with serum levels during pregnancy (P<0.001), but not in the non-pregnant state (P = 0.98). Both CSF and serum GDF15 were highest in women carrying a female fetus (P<0.001). Serum GDF15 correlated with the homeostatic model assessment for beta-cell function and placental weight, and CSF GDF15 correlated inversely with CSF insulin levels.GDF15 levels were higher during pregnancy compared with follow-up in both CSF (385±128 vs. 115±32 ng/l, This, the first study to measure CSF GDF15 during pregnancy, demonstrated increased GDF15 levels in both serum and CSF during pregnancy. The results suggest that effects of GDF15 during pregnancy can be mediated by increases in both CSF and serum levels. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta family, was first shown to be involved in inflammation and stress pathways but has also emerged as a potentially important metabolic regulator . GDF15 hResearch interest has recently grown regarding GDF15 during pregnancy as substantial and progressive increases in serum GDF15 have been shown from early to late pregnancy, ending with serum levels much higher than in any other physiological or pathophysiological state –12. PregFood intake and energy expenditure are primarily controlled by the central nervous system, with the arcuate nucleus of the hypothalamus identified as a key area. Several appetite-suppressing/stimulating neuropeptides have been shown to change both in the circulation and in the cerebrospinal fluid (CSF) during pregnancy , 20. GDFLocal hypothalamic expression of GDF15 or intracerebroventricular injections of recombinant human GDF15 in mice resulted in a direct central action which induced anorexia and weight loss . AlthougThe study was approved by the ethical committee at the University of Gothenburg (dnr 402-08/dnr 750–15). All women received oral and written information about the study and gave informed written consent before enrolment.Women were recruited at admission for elective cesarean section as previously described , and folThe pregnant subjects underwent elective cesarean section the morning after an overnight fast. A 10-ml venous blood sample was taken by venipuncture before infusion of Ringer-acetate solution. Before spinal anesthesia, an introducer needle was inserted into the interspinous ligament at L3-4, and a 25-gauge Whitacre needle or a 25-gauge Pajunk Pencil Point Spinal Needle was inserted through the introducer into the subarachnoid space. Ten milliliters of CSF were removed with a 10-ml syringe. Hemorrhagic samples were excluded. The first 0.5 ml of CSF was discarded. Study samples were transferred to polyethylene tubes, placed on ice, centrifuged, aliquoted, and stored (–80°C). Serum samples were similarly centrifuged, aliquoted, and stored. At the 5-year follow-up, women came to the laboratory after an over-night fast and CSF and blood samples were taken and processed in the same way as at the cesarean section.Dietary intake was assessed with a self-administered questionnaire for the three previous months. The questionnaire had a semi-quantitative food frequency design and was validated in Swedish men and non-pregnant women against a 4-day food record and assessments of 24-h energy expenditure and nitrogen excretion. From these comparisons, valid estimates of energy intake were obtained in normal weight, overweight, and obese subjects .Blood glucose and insulin were analysed with a Cobas Modular system  at the Clinical Chemistry Laboratory, Sahlgrenska University Hospital . CSF insulin was analyzed with a double-antibody radioimmunoassay (Linco Research) at the Department of Clinical Science, Lund University. Leptin and adiponectin were measured with ELISA kits (R&D Systems) in the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital Mölndal. ELISA plates were read on a Vmax plate reader, and concentrations were determined with Softmax software (Molecular Devices). Insulin was measured in undiluted samples and adiponectin in 100-fold diluted samples. For leptin analysis, CSF samples were diluted 2-fold and serum samples 100-fold. HOMA-IR was calculated as (fasting glucose × fasting insulin)/22.5 and HOMA-B as (20 × fasting insulin)/(fasting glucose − 3.5) . GDF15 cVariables are expressed as mean ± standard deviation (SD). Differences between pregnancy and follow-up were assessed using paired t-tests; between-group differences for fetal sex were assessed using independent t-tests and univariate tests. Associations were analyzed using Pearson correlations and linear regression models. For analysis during pregnancy, adjustments were made for gestational age of pregnancy ss GDF15 levels increase steeply and progressively during pregnancy . Further2, P = 0.01; glucose, 4.5 ± 1.3 vs 3.9 ± 0.6 mmol/l, P = 0.04; insulin, 13.9 ± 8.0 vs9.0 ± 5.4 mU/l, P = 0.02). There were no differences in age, gestational weight gain, placental weight or birth weight (P > 0.3), and importantly, there was no difference in GDF15 levels in serum or CSF at pregnancy between women that attended one or two visits (P > 0.5).The characteristics and blood/CSF measurements for the women in the study are presented in During pregnancy, GDF15 was almost 200 times higher in serum and more than 3 times higher in CSF compared with levels five years after pregnancy . The GDFPregnancy GDF15 levels did not correlate with post-pregnancy GDF15 levels in either serum or CSF. However, a strongly significant correlation was observed for the ratio CSF:serum between the two time points .P<0.001) between GDF15 serum and CSF levels .Women carrying a female fetus had significantly higher GDF15 levels both in serum and in CSF during pregnancy compared with women carrying a male fetus , whetherP = 0.18) or gestational weight gain (P = 0.92). There was a positive association for serum GDF15 with HOMA-B . In agreement with our previous study of serum GDF15 in pregnancy, we did not see a correlation with gestational weight gain. We also did not observe any association with self-reported dietary intake, although it should be noted that only approximately half of the women returned a completed dietary questionnaire.We also confirmed in this new cohort of women our previously shown association between serum GDF15 and HOMA-B in serum. Additionally, we observed a negative association of CSF GDF15 with CSF insulin, but no association with CSF leptin. Both insulin and leptin are believed to be involved in the central regulation of energy balance and peripheral glucose metabolism during pregnancy . Both hoAs most research on GDF15 in the CNS has been performed in animals, this study adds important new knowledge in the human. The design of the study with women acting as their own controls is a strength of the study. However, limitations include the shortfall in number of women attending the follow-up CSF sampling. This reduces power and could open for potential bias. The women attending both visits had lower BMI, glucose and insulin compared with the women only sampled at pregnancy. Importantly, however, there were no differences in GDF15 levels, and with only performing paired longitudinal analysis for changes between the two time points the bias should not influence the results of the present study. Also, we did not have information about pregnancy induced nausea, which would have been valuable to evaluate the role played by CSF and serum GDF15 in this respect.In conclusion, we have measured GDF15 in CSF of pregnant women and compared the levels longitudinally to the non-pregnant state. We showed increased levels of GDF15 in both CSF and serum during pregnancy compared with follow-up, and that both CSF and serum levels were highest in women carrying a female fetus. We propose that effects of GDF15 during pregnancy can be mediated by changes in both CSF and serum levels. These types of human studies are important to start elucidating how GDF15 might be centrally regulated in its proposed actions involving appetite, nausea and glucose metabolism.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Due to shortage of donor, kidney transplants (KTs) from donors with acute kidney injury (AKI) are expanding. Although previous studies comparing clinical outcomes between AKI and non-AKI donors in KTs have shown comparable results, data on high-volume analysis of KTs outcomes with AKI donors are limited. This study aimed to analyze the selection trends of AKI donors and investigate the impact of AKI on graft failure using the United states cohort data. We analyzed a total 52,757 KTs collected in the Scientific Registry of Transplant Recipient (SRTR) from 2010 to 2015. The sample included 4,962 (9.4%) cases of KTs with AKI donors (creatinine ≥ 2 mg/dL). Clinical characteristics of AKI and non-AKI donors were analyzed and outcomes of both groups were compared. We also analyzed risk factors for graft failure in AKI donor KTs. Although the incidence of delayed graft function was higher in recipients of AKI donors compared to non-AKI donors, graft and patient survival were not significantly different between the two groups. We found donor hypertension, cold ischemic time, the proportion of African American donors, and high KDPI were risk factors for graft failure in AKI donor KTs. KTs from deceased donor with AKI showed comparable outcomes. Thus, donors with AKI need to be considered more actively to expand donor pool. Caution is still needed when donors have additional risk factors of graft failure. Kidney transplantation is the most effective treatment to improve the survival and quality of life for patients with end-stage renal disease –3. HowevThis study used data obtained by the SRTR for kidney transplant recipients from 2010 to 2015. The SRTR data system includes data on all donors, waitlisted candidates, and transplant recipients in the US submitted by members of OPTN and has been previously described in detail . Health A total of 87,272 adult recipients who underwent KT between January 1st, 2010 and December 31st, 2015 were evaluated for inclusion in this study. We excluded living donors , those with missing donor creatinine (n = 643), incompatible blood groups (n = 5), and those with implausible values . A total of 52,757 recipients were included in the main analysis. The sample was further divided into two groups based on donor serum creatinine at allocation: 1) Non-AKI donor serum creatinine < 2 mg/dL; and 2) AKI donor serum creatinine ≥ 2 mg/dL.2, Kruskal-Wallis rank-sum, and Student’s t-tests were used to compare group differences in recipient, donor, and transplant characteristics. Due to large number of patients in AKI and non-AKI groups, a more conservative p-value of p < 0.0001 were considered significant. For comparisons within the AKI group among those with and without graft failure, p < 0.05 was considered significant. Kidney Donor Profile Index (KDPI) was calculated based on OPTN guidelines  for normally distributed variables, or median and interquartile range [IQR] for skewed distributions. χidelines . The 201P-values < 0.05 were considered significant predictors of graft failure. Additionally, we evaluated KDPI both continuously and as a categorical variable (KDPI>85 vs ≤84) in separate models. For models including KDPI, variables included in the calculation of KDPI were excluded to avoid collinearity. All analyses for this paper were completed using SAS 9.4 .Logistic regression with backward selection was used to model the odds of graft loss among the AKI group. We entered significant predictors from Table 3 into the model, including donor gender, donor age, mechanism of death , glomerular sclerosis, donor creatinine, donor hypertension status, cold ischemia time (hours), donor AA race, recipient AA race, and recipient age. This study cohort consisted of 52,757 adult deceased donor kidney transplants (DDKTs) in the U.S. alone between January 1, 2010 and December 31, 2015. Four thousand nine hundred sixty-two (9.4%) KTs were performed using donors with AKI. The control group included 47,795 non-AKI donor KTs.p < 0.0001. In addition, proportions of extended criteria donors (ECDs) and those with hypertension (HTN) history over 10 years were lower in the AKI group compared to those in the non-AKI group   (P < 0.0001) and roughly 34% of AKI kidneys were from non-local donors. Consequently, AKI group had a higher odds of pump use . Finally, cold ischemic time (CIT) was longer in AKI group   . Fewer rctively) . This ma 0.0001) .p < 0.0001). However, acute rejection rate, BKV, and CMV infection rate were similar between the two groups . Although creatinine at the time of discharge was higher in the AKI group compared to that in the non-AKI group , the last follow-up creatinine was not significantly different between the two groups (The incidence of delayed graft function (DGF) was significantly higher in the AKI group  months .p < 0.02). The proportion of African-American (AA) donors and ECDs was also higher in the graft failure group . KDPI and HTN rate were both higher in the graft failure group . The proportion of brain death from cardiovascular event was higher and mean CIT was longer in the graft failure group  (p < 0.0001)  . Acute r 0.0001) .Logistic regression was performed to identify potential risk factors for graft failure among the AKI group while adjusting for confounding factors. We found that donor creatinine, donor HTN, CIT, AA donor, and acute rejection were significant predictors for graft failure in the AKI group . A higher donor creatinine was associated with lower odds of graft failure . While donor HTN, CIT and AA donor were all associated with a higher odds of graft failure . In a seIn the era of donor shortage, efforts to expand the donor pool has continued. Increasing awareness in the potential use of kidneys from marginal donors, including those with AKI, has been increased accordingly , 7, 16. Lower graft survival in the AKI group has been previously found in a study of 1,869 kidney with AKI in the UK . ConsistOur study intends to provide clinicians ample information to assist in deciding whether to use AKI kidneys. This data is based on a registration generalizable to the US population. In logistic regression analysis, risk factors for graft failure in AKI donors were AA donors and donors with HTN, CIT, and acute rejection episode. Among risk factors for graft failure in the AKI group, donor factors included AA race, HTN, and CIT. When we examined KDPI in separate models: AA recipient, KDPI, and acute rejection were found to be risk factors for graft failure in AKI donors.The incidence of ESRD in AA is four times higher than in whites . PreviouP = 0.03) in multivariate analysis [Long CIT was one risk factor for predicting graft failure. In an analysis of SRTR data from 2005 to 2015, Dube et al. found there was an increased risk of graft failure when the CIT exceeded 36 hours. It was predictive of death-censored graft survival (DCGS)  for deceased donor kidneys to quantify graft failure risk based on donor and transplant characteristics . Since KThis study has potential limitations. First, registry data were used. Therefore, we are limited to exploring only the factors collected and there may be selection bias. Selection bias might have occurred from the moment of donor allocation for AKI donor according to the selection policy of each institution. Such favourable graft outcomes and survival in the AKI group in this study may, in part, reflect a careful selection of AKI kidney. One of results possibly driven from this selection bias was that the mean creatinine level was statistically higher in the non-graft fail group. We attempted to address confounding by adjusting for other donor factors using statistical techniques. We found some relations with creatinine and CIT with the scatter plot. It showed that high creatinine donor kidneys were correlated with lower CIT. Besides, there might be some other unmeasured confounders. Second, we could not adopt AKI definition or stage in this study because SRTR data provided only a single pretransplant creatinine level. Consequently, we defined AKI as a terminal creatinine greater than or equaled 2 mg/dL instead of using the recent AKI definition such as AKIN criteria. However, as shown in the biopsy rate (92.3% in the AKI group), we can expect that most donors with high creatinine caused by chronic kidney disease were excluded depending on the biopsy findings. Third, SRTR does not provide sequential follow-up creatinine values. Thus, we could not measure or compare graft function after KTs. Follow-up creatinine shown in Nevertheless, our study suggested that KTs from deceased donor with AKI showed comparable outcomes with good long-term allograft survival. Graft failure in AKI donor KT was higher in AA donor and donor with HTN and long CIT. Creatinine was not a weighty factor in predicting graft failure in the AKI group. Thus, the use from donors with AKI needs to be considered more actively to expand the donor pool. Caution is needed when donor is AA or has HTN, long CIT, and obviously high KDPI.


Postoperative delirium (POD) is a common complication of older people undergoing hip fracture surgery, which negatively affects clinical- and healthcare-related outcomes. Unfortunately, POD pathophysiology is still largely unknown, despite previous studies showing that neuroinflammation, neuroendocrine dysfunction, increased reactive oxidative stress (ROS), and endothelial dysfunctions may be involved. There is also evidence that many of the pathophysiological mechanisms which are involved in delirium are involved in sarcopenia too.This article describes the protocol of a pilot study to evaluate the feasibility of a larger one that will explore the pathophysiological mechanisms correlating POD with sarcopenia. We will analyse whether various biomarkers reflecting neuroinflammation, ROS, neuroendocrine disorders, and microvasculature lesions will be simultaneously expressed in in the blood, cerebrospinal fluid (CSF), and muscles of patients developing POD.Two centres will be involved in this study, each recruiting a convenient sample of ten older patients with hip fracture. All of them will undergo a baseline Comprehensive Geriatric Assessment, which will be used to construct a Rockwood-based Frailty Index (FI). Blood samples will be collected for each patient on the day of surgery and 1 day before. Additionally, CSF and muscle fragments will be taken and given to a biologist for subsequent analyses. The presence of POD will be assessed in each patient every morning until hospital discharge using the 4AT. Delirium subtypes and severity will be assessed using the Delirium Motor Subtype Scale-4 and the Delirium-O-Meter, respectively. We will also evaluate the patient’s functional status at discharge, using the Cumulated Ambulation Score.This study will be the first to correlate biomarkers of blood, CSF, and muscle in older patients with hip fracture.The online version contains supplementary material available at 10.1186/s12877-021-02584-1. Delirium is a neuropsychiatric disorder characterized by impairment of many cognitive functions (mainly attention and executive functions) that develops as the result of one or more acute clinical problems or because of drugs or due to mixed aetiology, It tends to fluctuate, with patient’s cognition and awareness rapidly changing from normal to either hyperactive or hypoactive states .The term postoperative delirium (POD) refers to a condition of delirium that develops after surgery. Up to 40% of older people are affected by POD after hip fracture surgery , 3. It iThe pathophysiology of POD remains largely unknown. However, an increasing body of evidence suggests that neuroinflammation and a damage of blood-brain barrier (BBB) are involved: endothelium lesions at the BBB level can result in an increased permeability, allowing immune cells, cytokines and other neuroinflammatory products to enter brain parenchyma. This mechanism is particularly likely to occur in old and demented subjects with hip fracture, as elevated serum levels of pro-inflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and IL-8 have been reported in these patients . After cInflammatory mediators promote the infiltration of myeloid immune system cells in the central nervous system, such as peripheral blood mononuclear cells (PBMCs), which have been reported to have a role in neuroinflammation in mouse models of Alzheimer’s disease . Animal A second pathophysiological mechanism has been proposed to explain delirium, relying on the oxidative stress hypothesis . Under sA third pathophysiological hypothesis proposes that delirium represents a response to acute or chronic stress, mediated by abnormally high glucocorticoid (GC) levels . GCs exePrevious studies have shown that patients who experience POD have higher postoperative cortisol levels with enhanced postoperative elevation in relation to baseline . Over tiAll these hypotheses should not be considered as mutually exclusive pathophysiological mechanisms for delirium occurrence but, on the contrary, they should be regarded as complementary, with many areas of intersection and reciprocal influence .Sarcopenia is defined as a condition of reduced skeletal muscle mass and strength, which is associated with several negative outcomes . It is aThis is a feasibility study for the prospective collection of blood, CSF, and muscular samples in older patients undergoing surgery for hip fracture. The study will be conducted at the Orthogeriatric Units (OGUs) of the San Gerardo hospital, Monza and of the Azienda Ospedaliero-Universitaria S. Anna, Ferrara, Italy from October 2021 to October 2022.Ten patients per centre will be recruited. All patients admitted to the participating orthogeriatric units (OGUs) will be screened for participation in the study, after their arrival to the hospital and before surgery. Each patient will be asked to give informed consent for the participation in the study; if he/she is able to give the consent because of severe cognitive impairment, the consent will be asked to patients’ next of kin.Inclusion criteria for this study will be: age 65 years and above; a femoral neck fracture (FNF) caused by a low energy trauma  needing surgical repair; suitability for subarachnoid anaesthesia; willingness to participate in the study and ability to understand and speak Italian.> 4 at Hoehn and Yahr Scale) [> 6 at Global Deterioration Scale) [Exclusion criteria will be: a pathologic FNF; ongoing anti-inflammatory or corticosteroid treatment; aphasia, advanced Parkinson’s disease or Parkinsonism  ; advancen Scale)  and; ascn Scale) . The 4ATn Scale) , 29. A sn Scale) . If deliTable All eligible patients will undergo a Comprehensive Geriatric Assessment (CGA), a globally approved method used to evaluate the older patients . InformaBefore surgery, the anaesthesiologist will assess the ASA score, a commonly used tool to evaluate a patient’s risk of dying, and the Sequential Organ Failure Assessment (SOFA) score . On the After surgery, each patient will be evaluated every morning until hospital discharge to assess the development of POD. This assessment will include the same procedures as for evaluating the presence of pre-surgical delirium, according to a previous protocol . This inBefore discharge, change in haemoglobin serum levels from admission to discharge and the number of units of blood transfused will be recorded. At discharge we will assess the patient’s calf circumference, the grip strength with the hand dynamometer, and the functional status with the Cumulated Ambulation Score (CAS) . The CASAll patients giving informed consent to the study will be eligible for the collection of blood, CSF, and muscle samples. Patients who die before or during surgical procedure will be considered drop-outs and therefore their samples will not be analysed.The first blood sample (4–5 mL obtained by venous puncture) will be collected on the day after OGU admission. On the day of surgery, CSF will be collected during subarachnoid anaesthesia. After dura mater puncture, 1.5 mL of CSF will be collected in sterile conditions, before injection of the local anaesthetic. Thus, the patient will not undergo any additional invasive procedure. Another sample of blood will be collected by venous puncture right after CSF collection (4–5 mL per patient). Polypropylene tubes with EDTA  will be used for the collection of blood and CSF.Muscle tissue will be collected by local biopsy during surgery, once the patient is anaesthetized, so that no pain or damage will be caused. A compact longitudinal cylinder  will be taken with a biopsy needle from the vastus lateralis muscle, carefully keeping fibres in a longitudinal orientation. The sample will be inserted in a polypropylene tube containing RNA Later Solution (Sigma-Aldrich), which allows to preserve sample and RNA integrity. All samples will be delivered to the researcher of reference as soon as possible.The procedure will be as follows: the patient will be admitted in the operating room carrying a sealable bag containing the tubes for the collection of blood, CSF, and the muscle samples. Inside the bag, written information to remind anaesthesiologists and surgeons about the procedures will be reported. Finally, another blood sample will be collected the day after surgery.Once the collection is complete, all samples will be given to a biologist. Blood and CSF will be centrifuged at 2000 x g for 10 min; centrifuged CSF will be aliquoted in cryotubes (0.5–1.0 ml per cryotube), while plasma will be collected from blood after centrifugation and will be in turn aliquoted in cryotubes (0.5–1.0 ml per cryotube). All the samples will be stored at − 80 °C in dedicated freezers. The list of the biomarkers which we have planned to assess are shown in Table Part of the muscle will be embedded, stored at − 80 °C and cut at the cryostat. We will perform immunohistochemistry and immunofluorescence techniques to evaluate muscle composition in terms of proportion of type I and type II fibers and the degree of intra- and intermuscular adipose tissue. This analysis will be done by the laboratory directed by two coauthors (RG and CD).The primary outcome measure of the pilot study will be the ability to collect the pre-planned amount of blood, CSF, and muscle samples in the centres participating in the study. We will measure the number of biological samples collected, the number of those suitable for further processing, and the number of analyses performed. Finally, the reasons for failure in the collection or storage of the biological samples will be considered. Additionally, we will compare the characteristics of patients developing POD in terms of clinical features and difference in blood, CSF, and muscle biomarkers. Overall, we plan to screen no more than 20 patients per centre to achieve the expected sample size. Every centre admits an average number of 300 hip fractured patients per year.The data will be analysed at the Biostatistics Centre for Clinical Epidemiology of the Milano-Bicocca University. The study variables will be described as mean, standard deviation or median and interquartile range, if continuous, or as frequencies and percentages if categorical. The characteristics of patients who will develop POD or not will be compared using the chi-squared or Fisher test for the categorical variables, and with the t-test for mean comparison or the Mann-Whitney U test for the continuous ones. The number of patients needed to detect a significant difference for some specific biomarkers  in the blood and CSF of hip fractured patients was calculated. However, we do not know whether this expected sample size would be informative to detect a significant difference with the same biomarkers in the muscle too. Therefore, we opted for a convenience sample. The biomarker levels in all the biological samples  will be used to estimate the difference and variability, which will constitute the premises to conduct a larger multicentre study.The study has been approved by the Brianza Ethics Committee and will be conducted in accordance with the European Union (EU) 2016/679 and 2016/680 directive and guidelines. Information will be collected on the Research Electronic Data Capture platform . Site acDelirium, and in particular POD, is common and associated with several negative outcomes. However, its pathophysiology is still largely unknown, which is an important barrier to the identification of patients at risk, as well as of those who may respond to specific treatments.Previous studies have tried to assess the mechanisms potentially involved in delirium pathophysiology, by measuring biomarkers of inflammation, neuroendocrine dysfunction, increased oxidative stress at the blood and CSF levels. However, most of these studies were carried out by separately assessing the putative biomarkers either in the blood or CSF, whereas only a minority assessed these biomarkers simultaneously in the blood and CSF.The most studied markers are the ones related to the inflammatory response since the well-known relationship between systemic and brain inflammation. A recent systematic review has shown that IL-1β, IL-6, IL-8, IL-10, and IFN-γ are significantly associated with delirium onset, displaying higher levels, both in plasma and CSF, in relation to baseline . Not onlGiven that many of the described pathophysiologic mechanisms of delirium have been reported to be potentially involved in sarcopenia, it appears crucial to put in practice a multicentre study aimed at exploring the existence of shared biomarkers between the two. Furthermore, focusing on the biomarker’s kinetics rather than on single measurements, would be helpful in determining their potential role in the pathogenesis of POD. However, no study has assessed the potential cross-talk and interaction between the blood and CSF districts and the muscle of hip fracture patients, despite preliminary evidence reporting that delirium is associated with sarcopenia . This stIn conclusion, this pilot study aims to pave the way for a larger multicentre study which will explore the in-depth pathophysiological mechanisms linking POD development and muscle abnormalities. It will also potentially inform future studies on POD, as well as on sarcopenia, aiming at early identification of patients at risk for the two conditions.Additional file 1.