IL-8 and MCP-1 concentrations were measured with ELISA method. We demonstrated that CSF IL-8 concentration of UIA patients is significantly higher (p < 0.001) than that presented in the serum, which can indicate its local synthesis within central nervous system. CSF IL-8 concentration was also significantly related to aneurysm size, which may reflect the participation of IL-8 in the formation and development of brain aneurysms. IL-8 Quotient (CSF IL-8 divided by serum IL-8) in UIA patients was statistically higher compared to control individuals (p = 0.045). However, the diagnostic utility analysis did not equivocally indicate the diagnostic usefulness of the IL-8 Quotient evaluation in brain aneurysm patients. Nevertheless, this aspect requires further study.Only scarce data pertaining to interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human aneurysm can be found in the current literature. Therefore, the aim of this study was the evaluation of cerebrospinal fluid (CSF) and serum IL-8 and MCP-1 concentration in unruptured intracranial aneurysm (UIA) patients ( Intracranial cerebral aneurysms (IAs) occur in approximately 5% of the population ,2. Risk At present, both primary diagnosis of IAs and follow up of those treated conservatively rely exclusively on their visualization. Nevertheless, the imaging either with MRI or with CT is hampered by high costs, limited availability, and a degree of biohazard related to irradiation and the use of a contrast agent . TherefoChemokines are a heterogeneous group of soluble, short-acting proteins and peptides that are strong mediators of the inflammatory response ,16. AnimIn contrast to the animal models, there are only scarce data pertaining to aneurysms in humans ,28. It sTaking into consideration the above, the aim of the study was the evaluation of CSF and serum IL-8 and MCP-1 concentration in patients with unruptured intracranial aneurysm. To exclude influence of the blood–brain barrier and the blood–CSF barrier on mutual relationships of protein levels in these two compartments ,31,32, wThe study was conducted in agreement with the Helsinki-II-declaration and the protocol was approved by the Bioethics Human Research Committee of the Medical University of Bialystok (Permission No. R-I-002/383/2015). All subjects gave their informed consent for inclusion before they participated in the study.The study group consisted of 25 patients: five males/20 females, mean age 56 years, range 30–70 years. All of them were subjected to craniotomy and direct surgical clipping of unruptured intracranial aneurysm and were operated on by one surgeon (Z.M.). In all patients, their aneurysms were asymptomatic and located at the anterior part of the Willis circle. A decision regarding the need for intervention was undertaken after careful consideration of the risk of aneurysm rupture versus inherited risk of surgical procedure. The commonly accepted guidelines . The criteria of exclusion from the control group were exactly the same as observed in the study group.The comparative group was composed of 20 subjects , suffering from trigeminal neuralgia due to anatomical conflict between the trigeminal nerve and a cerebellar artery. All patients belonging to this group revealed refractory to conservative treatment and were qualified for posterior fossa craniotomy and microvascular decompression. The procedure involved exposition of the conflict at the cerebro-pontine angle, desinsertion of the compressing artery from the trigeminal nerve, displacement of the artery, and finally its fixation to the cerebellar tentorium with a sticking material (Tachosil2) were classified as obese.All patients before surgery had fasting basic laboratory tests done between 6:00 and 7:00 a.m. CSF was obtained during craniotomy from the subarachnoid space of the brain. Surgical procedures were performed in a standard manner: under general anesthesia and with the patient’s head fixed in a three-pin Mayfield headholder. Skin incision preceded the lifting of the bone flap and lancing of dura mater, which allowed visualization of the arachnoid membrane and subarachnoid space. With the aid of an operating microscope, the subarachnoid space was carefully opened and inflowing CSF aspirated with single-use, sterile syringe, and soft venous catheter. Particular care was taken to prevent any contamination of the CSF with blood and with warm saline solution used as irrigation. Therefore, all the aforementioned steps were taken in the very beginning of each procedure, before any bleeding may occur.g to obtain serum. CSF samples were centrifuged for 20 min at 1000× g. Obtained serum and CSF supernatant were aliquoted and stored at −75 °C until further analysis.Patients’ blood samples (2.7 mL) were collected into test tubes without anticoagulant . Within 0.5 h after the venipuncture, blood was centrifuged for 20 min at 1000× ® Human CXCL-8/IL-8 Immunoassay kit  and ELISA Quantikine® Human CCL2/MCP-1 Immunoassay kit , both from R&D Systems Europe Ltd., Abingdon, England. Experiment was conducted in accordance with the manufacturer’s instruction. CSF and serum samples for IL-8 evaluation were not diluted prior to analysis. The manufacturer of the assay kit referred to the intra-assay coefficient of variation (CV%) as 5.6% at IL-8 mean concentration of 168 pg/mL, SD = 9.4 pg/mL. CSF and serum samples for MCP-1 evaluation were diluted 2-fold prior analysis. The manufacturer of the assay kit referred to the intra-assay coefficient of variation (CV%) as 7.8% at MCP-1 mean concentration of 76.7 pg/mL, SD = 6.0 pg/mL.Cerebrospinal fluid (CSF) and serum IL-8 and MCP-1 concentrations were measured using enzyme-linked immunosorbent assay (ELISA) tests: ELISA QuantikineTo calculate IL-8 and MCP-1 Quotients, we divided CSF chemokine result by its concentration in serum, as described elsewhere ,31,32. S2-test), thus nonparametric statistical analyses were applied. The Mann–Whitney test was used in order to compare two independent samples. The X2-test with Yates correction was used to investigate whether patients were gender-matched and whether there were differences regarding risk factors present between them. Correlation coefficients were obtained by applying Spearman’s rank correlation. Values for continuous variables are presented as median with the 25th and 75th percentiles. For the variables CSF and serum chemokines concentration, chemokines Quotients, age, sex, and brain aneurysm risk factors, a logistic regression model was sought. The same covariables were used in the linear regression analysis of predictor variables influencing aneurysm size as the ones in the logistic regression analysis. Differences were considered statistically significant at p < 0.05. Receiver operator characteristic (ROC) curve was generated in order to determine the performance of cytokine evaluation in a task of discrimination between the study and control groups. The Youden index, a function of sensitivity and specificity, indicated an optimal trade-off between these two (cut-off point) for the parameters tested.The obtained results were analyzed with the use of the STATISTICA 12.0 PL software . The distribution of the studied concentrations did not follow the normal distribution (Xp < 0.001). In the control group, CSF IL-8 was 1.6-fold higher compared to median serum concentration, but the obtained difference was not significant (p = 0.212)  ., see IL-8 Quotient in UIA patients was statistically higher compared to control individuals   . The MCPctively) .For the variables CSF IL-8 concentration, serum IL-8 concentration, IL-8 Quotient, CSF MCP-1 concentration, serum MCP-1 concentration, MCP-1 Quotient, age, sex, and brain aneurysm risk factors  a multivariate logistic regression model was sought, but nothing of significance was discovered. Only a univariate linear regression model was obtained. We showed that if the IL-8 Quotient increases by 1, the chance of having unruptured brain aneurysm increases by 1.84 times (increases by 84%) .R = 0.41, p = 0.04), and positive correlation between both CSF and serum MCP-1 concentrations and the aneurysms number .In UIA individuals, correlation coefficient analysis revealed: positive correlation between CSF IL-8 concentration and aneurysm size  with an increase in IL-8 concentration in CSF by 10 pg/mL, the aneurysm size increases by 1.14 times (rises by 14%); (2) with an increase in BMI by 1, the aneurysm size increases by 1.035 times (rises by 3.5%) .2) for the created model equals 0.39, which indicates that this model explains 39% of the variance in dependent variable. Multivariate linear regression analysis results for brain aneurysm size revealed that: (1) with an increase in IL-8 concentration in CSF by 10 pg/mL, the aneurysm size increases by 1.15 times (rises by 15%); (2) with an increase in BMI by 1, the aneurysm size increases by 1.03 times (rises by 3%)  .p < 0.05). Interestingly, patients with multiple brain aneurysm had higher CSF MCP-1 levels compared to individuals with single aneurysm  had statistically higher CSF IL-8 levels compared to those with aneurysm size < median value  and to predict the number of aneurysms (single versus multiple) we performed a ROC curve analysis in the next step. The optimal cut-off point for CSF IL-8 concentration to predict a larger brain aneurysm size (≥5.4 mm) was 36.9 pg/mL . The optp < 0.01, see As to the “risk factors”, our results suggest that patients with arterial hypertension had significantly lower CSF MCP-1 concentration compared to individuals without hypertension . In the control group, we did not observe significant differences between these two compartments. These results indicate a local IL-8 synthesis in brain aneurysm patients ,34 and mMoreover, we found a weak but positive correlation between MCP-1 concentration in the CSF and the number of aneurysms. Our research shows that the assessment of these chemokines concentration in CSF and serum may be also useful in stratifying the risk of a “dangerous” brain aneurysm presence.Simultaneous evaluation of the chemokines both in CSF (which abuts the aneurysm), and serum is the advantage of our study. The calculation of protein Quotient eliminates the influence of biological factors like age, gender, infection, which can change the content of blood cytokines. Additionally, such approach minimizes the impact of methodological factors because chemokines are measured in one analytical process ,34.We are the first to calculate Quotients for the IL-8 and MCP-1 chemokines in patients with unruptured brain aneurysm. We observed that the Quotient for IL-8 was significantly higher in patients with an unruptured intracranial aneurysm compared to those without vascular lesions in the brain. It is also worth mentioning that IL-8 Quotient tended to increase in patients with larger, polycyclic aneurysms and in patients over 60 years of age, smokers and obese. Univariate logistic regression analysis showed that if the IL-8 Quotient increases by 1, the chance of having unruptured brain aneurysm increases by 84%. Unfortunately, we did not obtain a significant multivariate logistic regression model for IL-8 Quotient.To assess diagnostic utility of the IL-8 Quotient calculation, we constructed an ROC curve for ex post discrimination between our patients with and without cerebral aneurysm. The area under the curve (amounting to 0.72) corresponds to a moderate overall diagnostic accuracy of the test . The posAn intriguing question pertains to a possible mechanism by which cytokines leak to CSF. Obviously, our results gave no unequivocal basis for concluding on this matter, whereas the number of sources in the literature is scarce. Chalouhi et al.  evaluateHowever, some presumptive evidence indicates also the astroglia and microglia as a local source of chemokines present in the CSF. Both astrocytes and microglia were shown to release IL-8 and MCP-1 when triggered by immune activation, stress and nociceptive input ,40. SimiChalouhi et al.  found inThough general conclusions from the work of Chalouhi et al. and Zhang et al. are rather convergent with ours, some differences in the set-up must also be indicated. First, the two groups of authors conducted their study in mixed populations of patients, containing both ruptured and unruptured aneurysms. This seems important to indicate, as the SAH is a separate pathological entity with serious consequences, also including an aspect of cytokine release, such as cerebral vasospasm ,42,43. AChalouhi et al. and Zhang et al. analyzed plasma IL-8 and MCP-1 levels by means of the multiplex method; in our study, the concentration of CSF and serum chemokines was analyzed with the use of the ELISA method. Both the ELISA and multiplex technique use an immobilized antibody to capture the soluble ligand and then detect the captured ligand by a second “reporter” antibody. However, there are significant differences between these two techniques, which include a reporter system, solid phase, or suspension in which the reaction occurs. Determination of the concentration of many ligands simultaneously in the same sample can lead to a cross-reaction , while ELISA only tests one analyte at a time, thus avoiding such a situation. Moreover, artifacts related to the analysis of many analytes simultaneously can result from: antibodies that can cross-react with other proteins, interspecies antibodies and the presence of other interfering substances . The difThe average size of aneurysms was in our patients was smaller than those in the hitherto published studies. It must be also admitted that patients belonging to our “control group” were not entirely healthy people, as they suffered from persistent facial pain caused by trigeminal nerve irritation. Nevertheless, in spite of these limitations, we still were able to demonstrate statistically significant differences.CSF samples were not obtained via the lumbar puncture but during craniotomy, so it is not clear if these results are reproducible with CSF lumbar puncture samples. This aspect could also be recognized as a study limitation. However, both unruptured intracranial aneurysm as well as trigeminal neuralgia are not indications for CSF collection for diagnostics purposes, thus the local Bioethics Committee did not give permission for CSF collection via the lumbar puncture. The available literature indicates that the total protein concentration in CSF obtained by lumbar puncture is higher than in CSF obtained from lateral ventricles of the brain. In the case of specific proteins, it is difficult to predict the concentration difference between different space of CSF collection ,46,47. TFinally, the setup of our study does not enable us to address the problem of the cellular sources of chemokines elevation in CSF and these specific mechanisms still need to be determined.We demonstrated that IL-8 concentration in the cerebrospinal fluid of patients with unruptured intracranial aneurysm is significantly higher than that presented in serum, which can indicate its local synthesis within the central nervous system. CSF IL-8 concentration was also significantly related to aneurysm size, which may reflect the participation of IL-8 in the formation and development of brain aneurysm. We also observed that the Quotient for IL-8 was significantly higher in patients with unruptured intracranial aneurysm compared to those without vascular lesions in the brain. However, further statistical analysis did not clearly indicate the diagnostic usefulness of the IL-8 Quotient evaluation in brain aneurysm patients. Nevertheless, this aspect requires further study.


Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01 to 0.66 μg/ml) and micafungin levels (<0.01 to 0.16 μg/ml) were lower than those in plasma concentrations  drawn simultaneously. In cerebral cortex, anidulafungin and micafungin levels were 0.21 to 2.34 and 0.18 to 2.88 μg/g, respectively. Candida strains exceed concentrations in CSF and in brain.Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01 to 0.66 μg/ml) and micafungin levels (<0.01 to 0.16 μg/ml) were lower than those in plasma concentrations  drawn simultaneously. In cerebral cortex, anidulafungin and micafungin levels were 0.21 to 2.34 and 0.18 to 2.88 μg/g, respectively. Thus, MIC values of several pathogenic  The echinocandins anidulafungin and micafungin are recommended for treatment of invasive candidiasis , 2. CandPost hoc consent was obtained from patients who were incompetent at the time of enrollment. Autopsy samples were taken from deceased patients who, on admission, had permitted scientific use of residual specimens taken for clinical laboratory tests.The study was approved by the local ethics committee and performed in accordance with the Declaration of Helsinki and Austrian law. Written informed consent for scientific use of CSF and blood samples was granted by competent patients. CSF was taken during diagnostic lumbar puncture (LP) or via external ventricular drain (EVD) from critically ill adults treated with anidulafungin or micafungin. Simultaneously, we took 2-ml arterial blood samples. CSF and plasma were stored at –80°C.0–24) was computed with the log-linear method when the concentration in a trapezoid decreased or with the trapezoidal method when the concentration increased.Anidulafungin and micafungin concentrations were quantified by high-performance liquid chromatography and UV detection (HPLC-UV) as described previously . Noncompg. For calibration, we used porcine brain samples spiked with anidulafungin or micafungin and homogenized and proceeded in the same manner. Both echinocandins were quantified by HPLC-UV at 306 nm , homogenization with a Precellys homogenizer  at 4,500 rpm , and centrifugation for 5 min at 8°C and 4,665 × U test using IBM SPSS statistics 24.0. PR was the ratio between the AUC0–24 in CSF and that in plasma over 24 h (AUC0–24 CSF/AUC0–24 plasma), when multiple samples were drawn via EVD. For single samples drawn by LP, PR was the ratio between the concentration in CSF and that in plasma (CCSF/Cplasma).The significance of the difference between CSF and plasma concentrations was assessed by Wilcoxon matched-pairs test, and the difference between the penetration ratio (PR) of anidulafungin and PR of micafungin was calculated by Mann-Whitney P < 0.05 and P < 0.01, respectively) (P = 0.40). In the cerebral cortex of four deceased patients, anidulafungin had reached concentrations of 0.21 to 2.34 μg/g. Brain concentrations of micafungin were <0.10 to 2.88 μg/g (n = 6). Anidulafungin and micafungin were measurable even 13 and 10 days, respectively, after treatment (CSF samples were obtained from three patients on anidulafungin and three patients on micafungin. One patient on anidulafungin (patient 2) and one patient on micafungin (patient 5) had undergone EVD ; Fig. 1.ctively) ; Fig. 1.reatment .Candida species   . In the  strains , 7. Howects MICs . HoweverDuring cerebral aspergillosis, the 2-fold maximum dose of micafungin  achieved CSF concentrations of <0.02 μg/ml . After i3H]caspofungin brain concentrations were ≤0.16 μg eq/g


Surface electromyography (sEMG) is a non-invasive method, which may be used in France by health practitioners without medical degree, such as physiotherapists, who are taught in Institutes of physiotherapy. However, very few hours are devoted to sEMG teaching in physiotherapist educational programs, especially in a form of practical work. In order to motivate using sEMG in physiotherapy to the students, we propose an example of sEMG practical work, applied to muscle stretching. Passive stretching exercises are often used by physiotherapists to maintain or improve range of motion. During a passive stretching session, subjects are given specific instructions to relax and not to activate their muscles during the procedure. In the proposed practical work, the sEMG is used to study the plantar flexor activation level during passive stretching. Therefore, this work may provide students with deeper understanding of physiology and biomechanics, trigger an interest in sEMG as a tool, and give knowledge about good sEMG practice, according to SENIAM and other recommendations. The integration of Institutes of physiotherapy in the University system may provide an opportunity to revisit the physiotherapist educational program and to provide students with more practical courses on sEMG application. The clinical use of EMG in France for neuromuscular diseases diagnosis is performed by medical doctors using needle EMG only. However, according to French Common Classification of Medical Acts  nomenclStarting from 2015, physiotherapist educational program duration in France is 4 years in Institutes of physiotherapy, preceded by 1 year of either medical study, sport-science studies or natural/formal science bachelor programs, i.e., overall education time is 5 years after high school , 4. The The recommended educational program for Institutes of physiotherapy contains a course about the theories, models, methods, and tools in physiotherapy with a total volume of 40 h of lectures and 40 h of tutorials. This course is conducted during four first semesters. EMG is cited among the other methods, like motion capture, force plate and gauge, inertial sensors as the methods recommended to be presented in this course.To our knowledge based on the inquiry among French Institutes of physiotherapy, the time devoted to teaching musculoskeletal EMG is little .One of the main parts of physiotherapist practice is to recover or maintain the maximal range of motion which may be performed by passive mobilization or stretching, performed by a physiotherapist or a patient under the supervision of the former. The ability to move efficiently through a large range of motion (ROM) is essential for the successful completion of numerous tasks in daily living, work, and sports. Stretching exercises are often advocated to maintain and improve ROM . OptimalThere is a number of studies, aimed to quantify the muscle activation level during stretching, when the subjects were asked to maintain a relaxed state –9. For tgastrocnemius medialis, gastrocnemius lateralis, and soleus, respectively), when the participants activated their muscles at 5% activation during slow dorsiflexions of the ankle. Nevertheless, even at a lower activation level of sEMG amplitude (2%) the change in joint torque was significant (+14%).The recent study  has quanOne of the main stretching methods commonly used in clinical practice is called “wall stretching” . Wall stWe propose to integrate the practical work on sEMG into the physiotherapist educational program in order to demonstrate the interest on sEMG application in practice related to force application on passive structures.gastrocnemius medialis, gastrocnemius lateralis, and soleus muscles , which are plantar flexors, forming tripces surae muscles, and on tibialis anterior muscle (TA) according to SENIAM recommendations  might give an opportunity to develop the educational programs in scientific areas. Due to this educational reform, relevant sEMG application courses may be integrated into the French physiotherapist academic programs. Currently , French At the same time, further development of sEMG equipment could improve its usability, such as textile-based electrode arrays, which do not require precise electrode placement; capacitive electrodes, which do not require skin treatment; wireless sensors, which do not restrain movement. We believe that improving the usability of sEMG, together with available professional training program will lead practitioners to use of sEMG in their practice.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


We aimed to derive an algorithm to define the optimal proportion of patients with mild cognitive impairment (MCI) in whom cerebrospinal fluid (CSF) testing is of added prognostic value.n = 402). Three-year progression probabilities to dementia were predicted using previously published models with and without CSF data (amyloid-beta1-42 (Abeta), phosphorylated tau (p-tau)). We incrementally augmented the proportion of patients undergoing CSF, starting with the 10% patients with prognostic probabilities based on clinical data around the median (percentile 45–55), until all patients received CSF. The optimal proportion was defined as the proportion where the stepwise algorithm showed similar prognostic discrimination (Harrell’s C) and accuracy (three-year Brier scores) compared to CSF testing of all patients. We used the BioFINDER study (n = 221) for validation.MCI patients were selected from the Amsterdam Dementia Cohort  to Harrell’s C = 0.67 and Brier = 0.190, and performed similarly to a model in which all patients received CSF testing. Applying the stepwise approach in the BioFINDER study would again select half of the MCI patients and yielded robust results with respect to prognostic performance.CSF biomarker testing adds prognostic value in half of the MCI patients. As such, we achieve a CSF saving recommendation while simultaneously retaining optimal prognostic accuracy.The online version contains supplementary material available at 10.1186/s13195-020-00763-7. Biomarkers such as amyloid beta1-42 (Abeta) and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) provide evidence on the neuropathological process underlying a patient’s cognitive decline , 4. The In a former study, we constructed personalized prognostic models that enable estimation of prognosis in terms of dementia conversion for an individual MCI patient, based on available biomarkers , 10. We We aimed to derive an algorithm to select MCI patients for CSF testing and to provide an estimate of the optimal proportion of patients to undergo CSF biomarker testing.n = 402 patients with a baseline diagnosis of MCI from the Amsterdam Dementia Cohort . HerWe report Harrell’s C statistics  and 3-yeWe reasoned that patients with high or low progression probabilities based on clinical data only are unlikely to benefit from additional biomarker testing, in terms of improving the prognostic accuracy for dementia conversion. On the other hand, in patients that have an initial progression probability in the center of all patients’ prognostic probabilities, additional biomarker testing could improve the prognosis. Therefore, in our MCI group, we defined the median 3-year progression probability according to the demographic and/or MRI information as most uncertain since it is the predicted prevalence of 3-year progression.Subsequently, we used a stepwise approach and added additional CSF biomarker data  to refine prognosis in the 10% (between percentile 45–55) of patients surrounding the median 3-year progression probabilities. Of note, due to the high correlation of p-tau and total tau (t-tau), t-tau concentrations are not included in the models. Details on the selection of variables in the models are described elsewhere , 10. MeaWe plotted cross-validated Harrell’s C and 3-year Brier scores of stepwise models with increasing proportion of patients receiving biomarker testing against the models with clinical data only (demographics only/demographics and MRI) and the model with additional CSF testing for all patients. This allowed us to identify the optimal proportion of patients where the stepwise approach performed better than the model with clinical data only and equally good as the additional CSF biomarker model in terms of prognostic discrimination (Harrell’s C) and prognostic accuracy (3-year Brier scores).As we used percentiles of the calculated prognostic probabilities with demographic and/or MRI data, the optimal proportion that is selected corresponds with certain demographic or MRI-model derived probabilities  and patients above the upper probability threshold (not selected).Table In Fig. Figure Subsequently, we evaluated the identified probability thresholds in the BioFINDER study , sex , and MMSE score (28), the 3-year progression probability was estimated to be 49.7%. This probability falls within the identified probability of the 50% of patients surrounding the median, and therefore additional CSF testing would be recommended. Adding CSF information  resulted in a far lower progression probability of 17.8%.3), the 3-year progression probability was estimated to be 14.0%. This probability falls outside the identified probabilities of the 50% of patients surrounding the median based on demographic information and MRI. As the progression probability was already low, the algorithm does not recommend to add CSF testing. The progression probability of the ATN model  for this patient was 9.2% and showed that CSF indeed did not meaningfully alter the estimated prognosis of this patient.For patient B, both demographic and imaging information were available. Based on age (54 years), sex , MMSE (29), and HCV  pathology, predict clinical progression, change disease management, and/or improve well-being. The BIOMARKAPD project, a multidisciplinary working group, ranked these clinical questions on importance, and showed that CSF biomarkers are particularly useful to identify AD pathology and to predict progression to AD dementia in MCI patients . Their rThe current study provides clinicians with an easy-to-use algorithm that uses readily available information  to identify MCI patients for CSF biomarker measurement. We took as a starting point progression probabilities based on basic clinical information only. By identifying the range of progression probabilities close to the progression prevalence in the population, where CSF is likely to add prognostic value, we allow the clinician to make an informed decision on performing biomarker testing. The clinician could also use this information to inform the patient before embarking on biomarker testing and manage expectations about potential outcomes. The communication of considerations to perform or not perform a diagnostic test was given high priority in a recent Delphi consensus study among clinicians, patients, and caregivers . The BIOIn the search for practical guidelines on which patient to test, several previous studies developed prediction models for amyloid positivity. Although these studies differ in their methodological details, they all focus on only one of the pathological hallmarks of Alzheimer’s disease as tauopathy and neurodegeneration are not considered , 37. MorOne of the strengths of the current study is that our algorithms make use of validated prognostic models to estimate the prognosis of each patient using available clinical information . Moreover, we used measures that are easily available to the clinician, i.e., patient characteristics, the widely used MMSE score, and hippocampal volume. Although we described this stepwise approach for the decision to perform CSF testing in MCI patients, our approach has general applicability to investigate a stepwise approach from any two prognostic models. The novelty in our study is that we used a data-driven approach to define the proportion of patients that would benefit from additional biomarker testing, i.e., the performance of the stepwise approach should be significantly better than the clinical model and similar to the full  model. Similar approaches have been proposed for the classification of cancer samples by means of high-dimensional genomic markers  With ourAmong the limitations is that we were unable to construct a stepwise approach from the demographic model to the MRI model, as the demographic and MRI model performed similarly in our sample (data not shown). Although the addition of MRI does not result in a more accurate prognosis in MCI patients, performing MRI or CT is still valuable to exclude other (reversible) causes for cognitive impairment. Other diagnostic tests, like amyloid-PET, were not part of the current study. In a future study, we aim to apply the same approach to amyloid-PET. Based on previous research on prognostic models in amyloid-PET, we expect similar results as reported here . AnotherIn conclusion, we showed that by performing CSF testing in 50% of the MCI patients the same prognostic accuracy is reached compared to testing all patients. Our algorithm uses prognostic models without CSF data to identify those patients most likely to benefit from CSF testing. This has important implications with respect to cost-efficient use of CSF testing. Furthermore, this approach also aids clinicians to set appropriate expectations before diagnostic testing.Additional file 1: Supplemental Table 1. Probability thresholds for proportion of patients receiving additional CSF testing. Supplemental Table 2. BioFINDER patient characteristics. Supplemental Table 3. Prognostic discrimination and prognostic accuracy in the BioFINDER study.


Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients. Is it ok to order a genetic test or do I need to wait?” is a commonly encountered question in the clinical genetics laboratory due to concerns about the possible presence of donor DNA in the transfused blood product(s).Microchimerism, the presence of two genetically distinct populations within an individual, can occur as a result of natural or iatrogenic processes. Naturally acquired microchimerism arises from twin to twin transfusions in utero or more commonly, as a result of bi-directional cellular exchange during pregnancy between a mother and fetus and is often still present even decades after the pregnancy . Microch6 in 95% of units tested after filtration; and the degree of leukoreduction within the acceptable range can vary among centers depending on the process. A number of filter iterations have been tried in order to achieve these endpoints and the current third and fourth generation pre-storage filtration systems are able to achieve a 3–4 log reduction (99.99%) in the WBC counts, which far surpasses the mandated thresholds set forth by the FDA and The Council of Europe. In addition, while most medical centers currently transfuse leukoreduced red blood cell products for non-trauma patients, it is not a requirement; approximately 70% of RBC and platelet products are pre-storage leukoreduced in the United States [Transfusion-associated microchimerism has been well documented in trauma patients receiving both whole and leukoreduced blood products; however, study results have been mixed regarding the presence of donor DNA from the transfusion of leukoreduced (LR) blood components in non-trauma patients ,7,8,9,10d States . Whole bMolecular genetic testing has drastically increased over the past decade for a wide spectrum of conditions. As of 2017, there were 75,000 commercially available genetic tests with an additional 10 entering the market daily, the majority of which were prenatal with hereditary following closely behind . In addi® WB System  in order to establish both an LR  and a PLR unit. The latter was used to simulate the transfusion of units with white counts just outside the AABB criteria for LR, since the degree of LR can vary among institutions, and the content of leukocytes in our institution’s LR units may not reflect that elsewhere. This PLR unit may also mimic an instance of inadequate filtration due to a faulty filter or process given that the AABB standard requires confirmation in only 95% of its’ products and served as another data point from which we could determine when detectable, whether any levels of donor DNA are observed. An NLR unit was used because not all centers provide LR products and NLR whole blood products are also routinely used in the trauma setting. We performed an in vitro spiking study utilizing four whole blood units collected from anonymous donors . All uni6 and 17.0 × 106, respectively. A third unit was processed similarly, but not filtered and was labeled as non-leukoreduced with a WBC count of 2630 × 106. All three units  were subsequently treated as our in vitro donors (IVD).WBC counts were measured using the NanoEnTek Adam  per our standard practice. The WBC counts for the LR and PLR units were 0.34 × 106/unit (corresponding to 0.48 × 109/L in vivo), which is below the threshold of clinical leukopenia (3.4 × 109/L) in vivo. The second half remained un-manipulated and served to represent our immunocompetent normocellular recipient (ICR) , one of these halves was leukoreduced to the level of 59.32 × 10nt (ICR) .For the in vitro experiments, 10 mL was set to represent the pre-transfusion total blood volume (TBV) for each recipient, assumed to be a 70 kg patient. From this assumption, we then derived the volume of blood in mL that would represent a transfusion of 2, 5, or 16 units. In our model, 1.2 mL of blood equals approximately 2 units and corresponds clinically to an apheresis double RBC product, which is the maximum possible dose of white blood cells from a single donor that could be realistically transfused into a recipient within a 16 week period due to regulations on blood donation. Ten milliliters is equivalent to sixteen units, which is the volume of each of our recipient aliquots. These were intended to represent the combination of blood from two genetically different individuals in a 1:1 ratio, which would clinically model neonatal transfusion situations where one transfused aliquot (from a single donor) could result in a 1:1 ratio as a result of a neonate’s minimal starting TBV. For example, a 3.5 kg infant  would have an approximate TBV of 298 mL; one unit of RBCs can range anywhere from 300 to 550 mL. The five-unit combination was an arbitrary midpoint intended to serve as a point of reference should our initial results necessitate further refinement of our volumes or aliquots. The three aliquots, representing 2, 5, and 16 mL, were then removed from each of our 3 IVD units  and combined with a fixed 10 mL aliquot from each of our two recipients for our total of (18) combinations. Donor and recipient samples were mixed and incubated overnight at room temperature, without agitation. An additional aliquot of 3 mL was removed from the three donor units and single recipient unit as well for a baseline DNA signature . AllelicDNA extraction was performed using the QIAgen EZ1  instrument and chemistry and concentrations were determined using the Promega Quantus Fluorometer . To assess the degree of detectable donor DNA vs. recipient DNA, chimerism detection was performed by analyzing STR using the GlobalFiler PCR amplification kit  followed by ChimerMarker analysis software . Twenty-four STR markers corresponding to donors and/or recipients were evaluated. The area under each signal peak for informative markers (those that differed between the donor and recipient) was used to calculate the percentage donor. The area under the peak corresponding to the donor was divided by the total area of the donor plus recipient peak. This is a clinically validated and commercially available assay thorough Mayo Clinic Laboratories (Test ID: CHIMU) and has a clinically validated lower limit of detection of 5%, but typically detects chimerism (or donor contamination in this study) at levels as low as 1%. This means that it will likely be able to detect donor DNA in concentrations of ≥1% and always >5%. As additional confirmation, we also calculated the expected values for each donor/recipient combination based on the DNA concentrations after extraction and then compared those results to what we observed by STR analysis . The perA literature search was performed to determine the approximate analytic sensitivity of commonly used molecular techniques. Search terms included the name of each individual technique and “sensitivity” or “analytic sensitivity”. Additionally, this information was obtained by reviewing the validated limits of detection for assays performed at our institution. In the LR product, 0.013 ng/uL of DNA was detected, whereas 0.836 ng/uL was detected in the PLR product, and 47.0 ng/uL in the NLR product prior to mixing. No donor DNA was detected when any level of transfusion with the LR product was simulated with either the immunocompetent or leukopenic recipients . Given tWhen transfusions of PLR product, which had a WBC count above that required to be labeled LR by the AABB, were simulated, donor DNA was undetectable after two units in the immunocompetent recipient. At 5 and 16 units, donor DNA comprised 0.1 and 1.5% of the total DNA, respectively, in the immunocompetent recipient, which is below the limit of detection for most clinical assays evaluating germline genetic variation . TransfuThe transfusion of two or more units of NLR products resulted in detectable donor DNA in both the immunocompetent and leukopenic recipients. Donor DNA accounted for 13.3% of total DNA when two units were transfused to the immunocompetent recipient, 24.1% when five units were transfused, and 45.1% when 16 units were transfused . When thThis in vitro transfusion model reliably confirmed that donor DNA was not detectable after a simulated transfusion with any volume of the leukoreduced product, while the partially leukoreduced product displayed a very low level range of 0.1–1.5% chimerism in the immunocompetent recipient and a clinically significant (from a testing standpoint) range of 6.3–27.8% in the leukopenic recipient. Donor DNA was observed in each simulated transfusion scenario with the NLR product and would be expected to have a significant impact on testing. Donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients, but depending on the degree and quality of the leukoreduction, may interfere in immunocompromised recipients.Given the efficiency of pre-storage leukoreduction systems, we did not anticipate that an LR transfusion would lead to significant DNA contamination from the donor, which was supported by the negative results observed in all of the simulations of LR product to both immunocompetent and leukopenic recipient combinations. Previously published experiments have reported similar findings using LR products; a number of these, however, were limited by patient population  and access to only the recipient’s DNA ,9,18,19.Despite the fact that no donor DNA was detected in the recipients of LR units, the analysis of the simulated PLR transfusions did reveal low but appreciable levels of donor DNA in the immunocompetent recipient. For the immunocompetent recipient, these results can realistically only be extrapolated to suggest the potential to interfere with germline genetic testing in situations where (1) the recipient is a neonate; (2) the volume transfused is close to a 1:1 ratio with their TBV; and (3) the subsequent genetic test has a limit of detection below 2–3%. While this would not apply to NGS-based hereditary assays, which are commonly indicated in these situations, it may impact the targeted genotyping platforms. These considerations are true as well for the leukopenic recipient of a PLR unit, but from a laboratory perspective, of greater significance was the observation that sequentially increasing levels of donor DNA were detected as the number of units transfused to the leukopenic recipient also increased, starting with just the two-unit aliquot.RPS19 variant, a large quantity of donor DNA that is negative for the variant (wild type) could decrease the signal for the variant nucleotide. However, a significant quantity of donor DNA would be required, given that the recipient’s DNA would contain 50% variant and 50% wild-type sequence at that locus; however, if the patient presented with bone marrow failure due to the presence of a heterozygous FANCA variant and is leukopenic, a much lower quantity of donor DNA would be required to decrease the variant from the expected 50%. In contrast, if the recipient is a neonate undergoing diagnostic exome sequencing for failure to thrive, and the donor is heterozygous for a KCNQ1 variant that would predispose to long QT syndrome, the presence of donor DNA may result in a signal; however, again, significant donor DNA would need to be present to reach a level near 50% variant frequency to result in a false positive clinical call. More likely, the laboratory would note the unusual variant frequency and contact the ordering provider for more clinical and family history to understand the unexpected findings and may recommend confirmatory testing with a new blood draw. An adult recipient would only ever receive up to two units from a single donor (a double red blood cell unit), which are often processed using a single filter, due to the regulations surrounding RBC donation. Therefore, for an adult recipient, false positives due to donor variants would be unlikely; however, if the recipient is leukopenic, the 6.3% donor DNA observed could potentially interfere with a sensitive assay. For both immunocompetent and leukopenic recipients, false negatives would be unlikely, unless the patient had low level mosaicism and a massive transfusion. Additionally, as mentioned above, in germline testing, most variants are expected to be present at 50% for heterozygotes or 100% for homozygotes, so the identification of 6.3% donor DNA may not impact results, unless incorrectly attributed to naturally occurring mosaicism.The presence of donor DNA could result in either the detection of a variant in the donor, but not present in the recipient , or could obscure the presence of a variant in the recipient and not present in the donor(s). For example, if the recipient was being tested for Diamond-Blackfan anemia due to pure red cell aplasia, and has a heterozygous The focus of this manuscript is germline testing, but similar considerations would apply to somatic testing. Somatic testing is typically directed toward specific variants, which are expected to be present in a healthy blood donor; therefore, false positives would be highly unlikely. If the recipient had a somatic variant, the presence of donor DNA  may lead to a lower variant allele frequency; however, unless the variant was present at a frequency close to the limit of detection, this would require the presence of a significant amount of donor DNA to have a clinically significant impact.6 for RBC and apheresis or pooled platelets and to 8.3 × 105 in whole-blood-derived platelets, demonstrated in >95% of units sampled. The Council of Europe (EC) has an even lower threshold of <1 × 106 leukocytes demonstrated in >90% of WBC-reduced RBC and single-donor platelets. Filtration failures (defined by the inability to meet thresholds of <5 × 106 or <1 × 106 leukocytes) can indeed occur and are attributed to the age of unit, temperature, height of the bag at the time of filtration, filter malfunctions and flow rates [6 WBCs per unit while approximately 8% exceeded the EC threshold of 1 × 106 [7 range, indicative of filtration malfunctions; our PLR had a WBC count of 1.7 × 107. Importantly, these numbers fulfilled all quality expectations and were still well within the required passing thresholds of 95 and 90%. Additionally, based on the exceedingly low likelihood of receiving a PLR unit, in practice this possibility should only be considered if contamination is observed in the raw data results and all other causes have been sufficiently ruled out.When applying these results to realistic clinical scenarios, it is important to understand the probability of receiving a PLR unit in practice. As mentioned, AABB standards, per the FDA, mandate that LR blood and blood components shall be prepared by a method known to reduce the leukocyte number to <5 × 10ow rates ,21,22,23 1 × 106 . ImportaAs expected, all of the NLR combinations displayed increasing amounts of donor DNA in both recipients. The two-unit transfusion in the immunocompetent recipient had the least amount of donor DNA at 13.3%, a level certainly capable of confounding genetic testing and a clinically realistic scenario since leukoreduction is not a universal practice in the United States and NLR whole blood products are often used in the setting of trauma. The other scenarios are only clinically realistic for a neonate, since, again, an adult patient would not receive greater than two units from the same donor.5, which may be different from that of other institutions. That being said, Terumo publishes an average residual leukocyte count of 2 × 105 for its BCT Immugard III-RC filter for whole blood and red cells, so our institutional average seems to be on par with the industry standard. Despite these limitations, similar results have been seen when analyzing the impacts of transfusion on the HLA typing of deceased donors, further confirming our results. [This study was limited by the fact that it was an in vitro model and cannot account for the size difference of recipients, immunologic variability, the sequestration of WBCs, laminar flow and turbulence, or the natural physiologic changes that occur with in vivo transfusions. In vivo, an immunocompetent recipient’s immune system would presumably recognize any donor WBCs present in the transfused product and eliminate it. One study using allele-specific PCR assays suggested that 99.9% of donor leukocytes were cleared within the first two days after transfusion. Therefore, most of these factors would not lead to an increase in the quantifiable donor DNA, but rather, a decrease such that the in vitro model presented here represents the highest concentration possible from a transfusion. However, it is important to note that a transient 1-log expansion has been observed on days 3–5 in a previously published study.  Thereforresults. ,10Leukoreduced blood products are exceedingly unlikely to affect genetic testing in patients of any age. Information regarding this can be easily obtained from the institution’s blood bank or laboratory director;Transfusion of non-leukoreduced products can lead to appreciable amounts of donor DNA and these situations need further investigation including information such as the number of products transfused, the length of time since the last transfusion, and the clinical scenario.With some exceptions, our in vitro results suggest that STR analysis with a limit of quantification of 1% is sufficient to be reliably applied as a surrogate marker of contamination risk for the majority of commercially available molecular assays and the following recommendations can be applied when triaging these situations both pre and post analytically:In conclusion, the standard transfusion of two leukoreduced RBC units to an adult immunocompetent or immunocompromised recipient is unlikely to impact genetic testing results; however, in other scenarios  there is a potential for donor DNA to impact the genetic testing results. Consultation with the genetic testing laboratory and the blood bank, along with clinical correlation if testing is performed, is recommended.


The incidence of angiostrongyliasis is increasing in recent decades due to the expanding endemic areas all over the world. Clinicians face tremendous challenge of diagnosing angiostrongyliasis because of the lack of awareness of the disease and less effective definitive laboratory tests.Angiostrongylus cantonensis (A. cantonensis) with both the CSF and the serum were all negative. In contrast, the metagenomic next-generation sequencing (mNGS) was applied with the serum sample and the CSF sample in the middle phase. The central nervous system (CNS) angiostrongyliasis was diagnosed by mNGS with the mid-phase CSF, but not the mid-phase serum. At the same time, the CSF analysis revealed eosinophils ratio up to 67%. The discovery of A. cantonensis was confirmed by PCR with CSF later. Unfortunately, the patient died of severe angiostrongyliasis. During his hospitalization, mNGS was carried out repeatedly after definitive diagnosis and targeted treatment. The DNA strictly map reads number of A. cantonensis detected by mNGS was positively correlated with the CSF opening pressure and clinical manifestations.A 27-year-old man initially manifested skin itching, emesis, myalgia and quadriparesis. With progressive weakness of four limbs and elevated protein in the cerebrospinal fluid (CSF), he was diagnosed as Guillain-Barré syndrome and treated with intravenous methylprednisolone and immunoglobulin. However, the patient deteriorated with hyperpyrexia, headache and then persistent coma. The routine tests for A. cantonensis infection highlights the benefit of mNGS as a target-free identification in disclosing the rare CNS angiostrongyliasis in the unusual season, while solid evidence from routine clinical testing was absent. The appropriate sample of mNGS should be chosen according to the life cycle of A. cantonensis. Besides, given the fact that the DNA reads number of A. cantonensis fluctuated with CSF opening pressure and clinical manifestations, whether mNGS could be applied as a marker of effectiveness of treatment is worth further exploration.The case of  Angiostrongylus cantonensis (A. cantonensis) is a zoonotic pathogen, which occasionally causes human angiostrongyliasis with the main clinical manifestation as eosinophilic meningitis [A. cantonensis is expanding, and the incidence of angiostrongyliasis among humans is increasing, too [A. cantonensis would appear in the early phase of angiostrongyliasis [ningitis . Althouging, too . Angiosting, too . The pating, too . Howevering, too –6. Moreoing, too . Herein,gyliasis , not mengyliasis . In thisA 27-year-old male officer, who was previously healthy and lived in South China, was presented to a local clinic in early November after skin itching, emesis, generalized myalgia and slight weakness in all limbs , the patient manifested progressive pain and weakness of all limbs but alleviated itching. Upon admission to a local hospital, he presented muscle strength of Grade 3 for all limbs in physical examination. Transient rashes on his trunk were observed on DAI 8 Fig. a. The CSOn DAI 15, the patient’s condition suddenly deteriorated with fever, generalized flaccid paralysis, gatism, and mental aberrations, including delusion and raves. The second LP was performed immediately to find CSF opening pressure remaining at 230mmH2O. CSF analysis revealed WBC 278 × 10^6/L (85% monocytes and 15% multinucleated cells). The CSF protein was 620 mg/L and glucose was 3.2 mg/L with CSF/blood glucose ratio of 58.6%. Cytology of CSF was not recorded. The mNGS for pathogen detection from the serum was carried out on DAI 16 and reported negative on DAI 18  for further treatment. He was sleepy but alert, complained of severe weakness, mild headache and stomachache. Neurological examination was remarkable for severe quadriparesis (muscle strength of Grade 1) and positive meningeal irritation signs. Extensive microbiological tests of the serum were negative on admission. He was intubated on DAI 20. The LP was performed again on DAI 20 and revealed raised CSF opening pressure of 520mmH2O and mNGS with the CSF sample was carried out for the first time. Of note, the CSF showed the coconut-like appearance  of 13,492. The result was verified by quantitative polymerase chain reaction (qPCR) later with the same CSF sample for mNGS  until DAI 31.On DAI 22, Unfortunately, methylprednisolone was suspended on DAI 26 due to severe bleeding of pre-existing hemorrhoids and then the intrathecal dexamethasone was tried on DAI 31 because the miserable fact that the CSF opening pressure increased to more than 1100 mm H2O Fig. . On the A. cantonensis [Although most human angiostrongyliasis manifests as eosinophilic meningitis and is of good prognosis with in-time treatment with corticosteroids and/or anthelmintics, CNS angiostrongyliasis with delayed treatment or untreated may developed into fatal meningoencephalitis. Given the fact that there is no valid treatment of severe CNS angiostrongyliasis, the early diagnosis and intervention accordingly becomes extremely essential for patients with angiostrongyliasis to survive. Although the discovery of larvae from the CSF or eye chamber is the gold standard for the diagnosis of CNS angiostrongyliasis, these findings are quite rare even in the severe cases . Currenttonensis –13. Howetonensis , 15. Revtonensis . Moreovetonensis , 16. Lastonensis . In recetonensis , 17, 18.tonensis . Comprehtonensis . In one A. cantonensis was first detected by mNGS from the cerebrospinal fluid on DAI 20. After the identification of A. cantonensis by mNGS in the CSF, other definitive testing, including specific antibody, Charcot-Leyden crystals, reported positive. It is suggested that the mNGS showed high sensitivity in detecting A. cantonensis compared with other concurrent laboratory testing, and the target-independent identification of pathogens could aid clinicians to achieve definitive diagnosis even the clinician was unaware of the etiology. Therefore, mNGS did facilitate the identification of pathogens in the case. On the other hand, although A. cantonensis was reported to be detected with mNGS in a previous case, its existence was not confirmed with PCR [A. cantonensis by mNGS was verified by PCR, which provided solid evidence to support the workability of mNGS in identifying A. cantonensis.In the current case, with PCR . In our A. cantonensis by mNGS can be explained by the absence of nucleic acid of the causative pathogen in the mid-phase serum, where all larvae had left and migrated into the CNS. It is suggested that the effectiveness of samples may have a time window which is based on the life cycle of parasites. For detection of A. cantonensis, we suggest the CSF samples must be included for mNGS and the serum samples should not be tested alone once beyond the time window of two weeks.Interestingly, the mNGS with serum sample on DAI 16 was negative in this case. A previous study has proved that mNGS with serum sample along could be false negative in meningitis and encephalitis . As knowA. cantonensis does not multiply but migrates, grows and dies in the CNS of humans [A. cantonensis by mNGS since the definitive diagnosis in order to deepen the understanding of angiostrongyliasis and its treatment (Fig. A. cantonensis varied in general accordance with the CSF opening pressure. The relationship between fluctuation of DNA SMRN and clinical outcome is worth further exploring.f humans . The natf humans . In our ent Fig. . The incA. cantonensis infection highlights the benefit of mNGS as a target-free identification in disclosing the rare CNS angiostrongyliasis in the unusual season, while solid evidence from routine clinical testing was absent. Given the expanding endemic area of A. cantonensis due to the increasing interaction between districts and global warming, clinicians should be aware of the possibility of angiostrongyliasis and choose appropriate samples for mNGS according to the life cycle of A. cantonensis. Whether the DNA SMRN, which varied with CSF opening pressure and clinical manifestations, could be applied to monitor disease progression and treatment effectiveness is worth further observation.The case of Additional file 1 The cover chart of serum mNGS. Additional file 1 provided the cover chart of serum mNGS on DAI 16 which was negative in revealing A. cantonensis.Additional file 2. Procedure of mNGS and PCR. Additional file 2 provided detailed procedure of mNGS and PCR.


Recording the neural activity of cells in the brain of patients with Parkinson's disease challenges long-standing assumptions about how this disease manifests at the cellular level. Related research article Valsky D, Heiman Grosberg S, Israel Z, Boraud T, Bergman H, Deffains M. 2020. What is the true discharge rate and pattern of the striatal projection neurons in Parkinson's disease and Dystonia?. eLife9:e57445. doi: 10.7554/eLife.57445A group of structures deep inside the brain are thought to be responsible for the progression of Parkinson’s disease. These structures, known as the basal ganglia, play an important role in coordinating movement via two opposing motor pathways: the ‘indirect pathway’ which suppresses movement, and the ‘direct pathway’ which promotes movement. It has been reported that overactivation of the indirect pathway and underactivation of the direct pathway lead to the motor impairments associated with Parkinson’s disease .Previous studies investigating the cellular mechanisms that cause these abnormalities have largely focused on spiny projection neurons (SPNs for short), a group of cells found in a basal ganglia structure known as the striatum. These cells express one of two types of dopamine receptors called D1 and D2. SPNs expressing D1 are frequently referred to as the origin of the direct pathway, whereas SPNs expressing D2 are referred to as the origin of the indirect pathway. These pathways then pass this signal between multiple structures of the basal ganglia until they reach a group of cells known as the output nuclei.The striatum receives most of its dopamine from an area in the brain that degenerates in Parkinson’s disease. This loss in dopamine is thought to reduce the activity of D1-SPNs and increase the activity of D2-SPNs, causing neurons in the striatum to fire at different rates: this is predicted to drive the excessive activity of the indirect pathway and reduced activity of the direct pathway, which leads to pathological activity throughout the basal ganglia . This hyNow, in eLife, Marc Deffains (University of Bordeaux) and colleagues – including Dan Valsky  as first author – report experiments investigating the firing rates of SPNs in patients with Parkinson’s disease . The teaThese results were in contrast to the only other human study which supports the predictions made by the rate model . HoweverThe fact that Valsky et al. were not able to find evidence for the rate model within the striatum was not entirely unexpected, as the exceptions and limitations of this model have become increasingly documented . Other mThese findings raise a lot of questions about the role the striatum plays in the motor impairments associated with Parkinson’s disease. However, two critical variables that were not addressed in this study are synchrony (non-oscillatory), and total neuron recruitment within the striatum. When multiple SPNs fire simultaneously, this relays a more powerful signal to downstream areas of the brain than if SPNs are activated independently. Therefore, if dopamine depletion enabled more D2-SPNs to activate simultaneously, or in greater numbers, this could lead to increase indirect pathway output from the striatum. However, the techniques used to record neuronal activity in this study means it is not possible to determine whether this change occurred.These findings highlight the need for a critical reassessment of long-standing assumptions about the cellular mechanisms involved in the onset of Parkinson’s disease. Even if this study does not completely rule out the striatum as a source of basal ganglia abnormalities in this condition, it narrows down the types of changes that might be responsible. It also emphasizes the roles of brain structures that are traditionally considered to be ‘downstream’ of the striatum in generating the neuronal defects associated with Parkinson’s disease . Further


Background: The COVID-19 pandemic has resulted in worldwide morbidity at unprecedented scale. Troponin elevation is a frequent laboratory finding in hospitalized patients with the disease, and may reflect direct vascular injury or non-specific supply-demand imbalance. In this work, we assessed the correlation between different ranges of Troponin elevation, Electrocardiographic (ECG) abnormalities, and mortality.Methods: We retrospectively studied 204 consecutive patients hospitalized at NYU Langone Health with COVID-19. Serial ECG tracings were evaluated in conjunction with laboratory data including Troponin. Mortality was analyzed in respect to the degree of Troponin elevation and the presence of ECG changes including ST elevation, ST depression or T wave inversion.Results: Mortality increased in parallel with increase in Troponin elevation groups and reached 60% when Troponin was >1 ng/ml. In patients with mild Troponin rise (0.05–1.00 ng/ml) the presence of ECG abnormality and particularly T wave inversions resulted in significantly greater mortality.Conclusion: ECG repolarization abnormalities may represent a marker of clinical severity in patients with mild elevation in Troponin values. This finding can be used to enhance risk stratification in patients hospitalized with COVID-19. Coronavirus Disease COVID19) pandemic, induced by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is reaching now historical magnitude as one of the deadliest outbreaks in modern history 9 pandemi, 4 reveaThis is a retrospective study performed at NYU Langone Medical Center, New York, USA. We included 204 consecutive adult patients hospitalized at NYU Langone Medical Center with COVID19 disease. Medical records were reviewed to obtain baseline characteristics, laboratory data, and ECGs. Troponin I concentrations were assessed via the Abbott Architect method  wherein the 99th percentile for a normal population is 0.05 ng/mL and the maximal Troponin level was recorded. Descriptive analyses were performed by troponin levels stratified into normal (0.00–0.05 ng/mL), mildly elevated (0.05–1 ng/mL), and significantly elevated (>1 ng/mL). The first, presenting ECGs were reviewed and interpreted by five senior cardiologists who were blinded to the clinical status of the patients. Data reviewed from each ECG included heart rate, rhythm categorized as normal sinus rhythm or atrial fibrillation/flutter (AF), atrioventricular block (AVB), right bundle branch block (RBBB), left bundle branch block (LBBB), a non-specific intraventricular conduction block (QRS duration >120 ms), the presence of ST segment or T-wave changes . The closing date of follow-up was April 15th 2020. Collected data on the closing date included arrhythmic events and mortality. The study was reviewed and approved by the NYU Institutional Review Board and Quality Improvement initiative in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments, with a waiver of informed consent.t-test if samples had normal distributions and Mann-Whitney U test if samples did not have normal distributions. Hypothesis testing for categorical variables was performed using Fisher's exact test. Significance testing for Kaplan-Meier curves was performed using log-rank test. For predictors of mortality, univariate analysis was performed using Cox proportional hazards regression, and significant univariate predictors were included in the multivariate analysis.Statistical analysis was performed using IBM SPSS Statistics 26, and figures were constructed using GraphPad Prism 8. Continuous variables are expressed as mean ± standard deviation or median (25th−75th percentile), and categorical variables are expressed as count (percentages). Normality of data samples was assessed using Shapiro-Wilk test. Two sample hypothesis testing for continuous variables was performed using Student's p < 0.01; p = 0.04) in the intermediate Troponin group.We included 204 patients in our cohort with a mean follow up time of 24.2 ± 7.4 days. The clinical and epidemiological characteristics stratified by ECG abnormalities are presented in Multiple mechanisms have been shown to explain the frequent COVID-19 induced cardiovascular injury. These include direct injury to the myocardium induced by a cytokine storm resulting from a hyperinflammatory state, microvascular damage resulting from abnormal activation of the coagulation cascade including disseminated intravascular coagulation and thrombosis, supply-demand mismatch resulting from respiratory induced tissue hypoxia in conjunction with increase in metabolic demand of infection and inflammation, and myocardial injury by direct entry of SARS-CoV-2 into cardiomyocytes expressing the ACE2 receptor –9. In thThough troponin elevation above the 99th percentile of the upper reference limit is considered the central marker of “myocardial injury,” mild elevation between 0.05 and 1 is often non-specific and associated with non-vascular etiologies such as strain, myocyte necrosis and increased cell membrane permeability –13. IndeOur study has several limitations. This is an observational, retrospective study. Because of its retrospective nature, the study is subject to selection bias, and its results imply association, not cause and effect. Relatively short follow-up time after was available. The study was not aimed at providing mechanistic insight for the cause of repolarization changes and Troponin elevation. We did not asses structural information from echo due to limited number of tests performed. However, this study was directed at assessing the presenting ECG as a readily available tool for risk stratification in combination with Troponin, a simple blood test.lior.jankelson@nyumc.org.The data analyzed in this study is subject to the following licenses/restrictions: Data sharing will be considered pending request. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by Quality Control Act With Accordance to the NYU Langone Health IRB. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LJ and EC: concept, data collection, and manuscript. MD and EK: statistics. LW, ES, CN-R, RK, and RB-C: data collection. CB, AA, DH, SB, MS, DP, and LC: manuscript writing and review. All authors contributed to the article and approved the submitted version.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.


However, the mechanisms underlying this process remain unclear. This study aimed to determine whether the improvement of hepatic steatosis by G-CSF was associated with autophagy in a rat model of diabetes.  We previously reported that the granulocyte colony stimulating factor (G-CSF) ameliorated hepatic steatosis with the enhancement of μg/kg/day; diabetes mellitus (DM)/G-CSF) or saline  for 5 consecutive days. Four weeks later, serum biochemical and histology analyses were conducted. The expression of autophagy-associated proteins was determined by Western blotting. The mRNA expression of β-oxidation-related genes was determined by quantitative real-time polymerase chain reaction. HepG2 cells were cultured under high glucose (HG) conditions with G-CSF treatment, followed by Oil Red O staining for quantification of lipids.  Eight rats were fed a standard diet, and 16 rats were fed high-fat diet (HFD) for 5 weeks. All HFD-fed rats were then injected with streptozotocin (STZ). One week later, HFD rats injected with STZ were randomly treated with either G-CSF (200 β-oxidation-related genes was higher in DM/G-CSF rats than in the DM/saline-treated rats. Quantification of lipid levels in HepG2 cells cultured with HG and G-CSF treatment revealed no significant differences.  Histological analysis showed lower lipid accumulation in the DM/G-CSF group than in the DM/saline-treated rats. Protein levels of LC3 and beclin-1 were higher, and those of p62 were lower in the DM/G-CSF rats than in the DM/saline-treated rats. The mRNA expression of  Our data suggested that G-CSF potentially improves hepatic steatosis and autophagy in the liver of diabetic rats. Nonalcoholic hepatic steatosis is an initial stage of nonalcoholic fatty liver disease (NAFLD) and is defined as the accumulation of triglycerides (TG) without significant alcohol consumption in the liver . Severe Autophagy is a process of lysosomal degradation that can degrade intracellular organelles. It can remove damaged cellular components to maintain the homeostasis of cellular energy . Previouβ-oxidation-related genes, the detailed mechanisms underlying the therapeutic effects of G-CSF on hepatic steatosis remain unclear.The granulocyte colony stimulating factor (G-CSF) is a regulator of neutrophil development and formation that is widely used to mobilize bone marrow-derived stem cells in the peripheral blood . G-CSF aIn this study, we explored whether the mechanisms of the therapeutic effect of G-CSF were associated with autophagy in a rat model of diabetes. In addition, to identify whether the effect of G-CSF was direct, we investigated the lipid accumulation in HepG2 cells treated with G-CSF.This study was performed in accordance with the ARRIVE guidelines on animal research . The Hann = 8) with 5.4% fat content or a high-fat diet  with 60% fat content for 6 weeks. After 5 weeks of feeding, all the rats fed with the HFD were injected intraperitoneally with a single low-dose of streptozotocin  diluted in 0.1 mol/L citrate buffer. Normal control rats were injected with equivalent volumes of citrate buffer. One week later, the HFD-fed rats injected with STZ were randomly selected to receive human recombinant G-CSF (diabetes mellitus (DM)/G-CSF; 200 μg/kg/day; Leucostim, Dong-A Pharmaceutical, Seoul, South Korea; n = 8) or an equal volumes of saline  intraperitoneally for 5 consecutive days. Body weight was determined every week, and blood glucose level was measured at baseline, pretreatment, and posttreatment. The rats were sacrificed under CO2 anesthesia for histology and biochemical studies 4 weeks after the beginning of the treatment.The experimental design is shown schematically in μU/mL) × fasting glucose (mmol/L)/22.5 [Blood samples were collected from the tail vein after 8 hours of fasting, and serum was obtained by centrifugation and stored at −70°C. The levels of serum glucose, total cholesterol (TC), TG, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and free-fatty acids (FFAs) were measured by using a model AU400 auto analyzer  . Insulin/L)/22.5 .μm-thick sections for hematoxylin and eosin (H&E) staining. To evaluate the lipid accumulation, frozen 4 μm-thick sections of liver tissues were stained with Oil Red O as previously described [To examine the liver morphology, liver tissues were fixed in 10% formalin solution, embedded in paraffin, and then cut into 3 escribed . Five fiμg) was reverse-transcribed with reverse transcriptase . The mRNA expression levels were quantified using a Light Cycler® 480 System  with a FastStart DNA Master SYBR Green I kit (Roche). The genes of interest included those for AMP-activated protein kinase- (AMPK-) 1α, AMPK-2α, carnitine palmitoyltransferase (CPT)1, and peroxisome proliferator-activated receptor gamma coactivator- (PGC-) 1α. The primers used are shown in Total RNA was isolated from the liver tissue using QIAzol reagent  following the manufacturer's instructions. The RNA  with a protease inhibitor . Forty micrograms of proteins were resolved by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane . After blocking with 5% bovine serum albumin, the membrane was incubated overnight at 4°C with primary antibodies against LC3, beclin-1, p62, and GAPDH . GAPDH was used as a protein loading control. Protein bands were visualized and quantified using an Image lab 5.0 image analyzer (Bio-Rad).2 condition. A cell model for accumulation of intracellular lipids was created by exposure to a high glucose  for 36 hours. For pharmacological studies, HepG2 cells were incubated with either normal glucose (5.5 mM) or HG (45 mM) medium supplemented with indicated concentrations of G-CSF .HepG2 cells were obtained from the American Type Culture Collection  and cultured in DMEM  with normal glucose (5.5 mM) containing 10% FBS (Life Technologies) and 1% streptomycin/penicillin (Life Technologies). Cells were incubated at 37°C in a humidified atmosphere under 5% COHepG2 cells were cultured overnight, and then incubated with increasing concentrations  of G-CSF for 36 hours. Subsequently, cells were treated with methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (0.5 mg/mL) diluted in the medium and incubated for an additional another 2 hours. The culture medium was removed and the intracellular formazan product was dissolved in dimethylsulfoxide (DMSO). The absorbance was measured at a wavelength of 595 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad). MTT assays were performed in triplicate.To prepare Oil Red O solution, 0.35 g Oil Red O  per 100 mL of 100% isopropanol was mixed to yield an isopropanol concentration of 60%; this solution was then filtered. Briefly, the HepG2 cells were washed with PBS, followed by fixation with 10% formalin for 30 minutes. After washing with distilled water, fixed cells were washed with 60% isopropanol shortly and then stained with Oil Red O solution for 30 minutes at room temperature. Subsequently, after washing with distilled water, the Oil Red O was eluted with 100% isopropanol for 15 minutes at room temperature, and it is quantified by measuring the absorbance at a wavelength of 490 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad).P values <0.05 were considered statistically significant.All statistical analyses were conducted using SPSS 21.0 for Windows . The results are presented as the means ± standard deviations, except for the results of the histological analyses, which are presented as the means ± standard errors of the means. Comparisons of parameters among groups were performed using one-way analysis of variance (ANOVA) followed by a post hoc Tukey's test. F = 15.682, P < 0.05). HOMA-IR was significantly lower in the DM/G-CSF group than in the DM/saline group and did not differ between the normal control group and the DM/G-CSF group  = 13.494, P < 0.05). Finally, levels of circulating TG, AST, ALT, and FFAs were similar for all groups. TC levels were significantly higher in the DM/saline group than in the normal control group  = 79.387, P < 0.05).The progression of hepatic steatosis was verified by H&E and Oil Red O staining. At the end of the experiment, the liver tissues from the DM/saline group showed severe micro- and macrovesicular steatosis and numerous ballooning hepatocytes. In contrast, the liver tissues from the DM/G-CSF group exhibited no fat accumulation; this was not different from the case for the liver tissues of rats from the normal control group . The accF = 15.552, P < 0.05). The protein level of beclin-1 was significantly higher in the DM/G-CSF group than in the DM/saline group and the normal control group  = 16.446, P < 0.05). The protein level of p62 was significantly lower in the DM/G-CSF group than in the DM/saline group, and it was not significantly different from that in the normal control group  = 16.457, P < 0.05).To confirm whether the ameliorative effects of G-CSF on hepatic steatosis were associated with autophagy, we examined the protein expression of autophagy markers, including LC3, beclin-1, and p62 by Western blotting. The protein level of the LC3, as determined by calculating the LC3 conversion (LC3-II/LC3-I), was significantly higher in the DM/G-CSF group than in the DM/saline group and the normal control group   = 8.615, P < 0.05). The mRNA level of AMPK-2α was significantly higher in the DM/G-CSF group than in the DM/saline group and the normal control group; there was a significant difference between the mRNA level of AMPK-2α in the DM/saline group and the normal control group  = 20.619, P < 0.05). The mRNA level of CPT1 was significantly higher in the DM/G-CSF group than in the DM/saline group and the normal control group  = 17.477, P < 0.05). The mRNA level of PGC-1α was significantly higher in the DM/G-CSF group than in the DM/saline group and the normal control group  = 11.673, P < 0.05).To investigate the alteration of the expression of ol group   were added to the culture medium of HepG2 cells. After 36 hours, cell viability was measured using the MTT assay. Treatment with G-CSF at concentrations up to 100 ng/mL had no cytotoxic effects on HepG2 cells. However, treatment with G-CSF at a concentration of 1000 ng/mL reduced cell viability, as observed by comparisons with the untreated group . To deteβ-oxidation as well as hepatic steatosis in the liver tissue. Moreover, G-CSF induced autophagy activation, which provides a molecular explanation for the therapeutic effects of G-CSF on hepatic steatosis. We also confirmed that G-CSF did not affect the lipid accumulation in the HepG2 liver cancer cells, which indicates that G-CSF may not have a direct beneficial effect on hepatic steatosis by signaling through the G-CSF receptor.In the present study, we examined the effects of G-CSF treatment on hepatic steatosis in a rat model. We demonstrated that G-CSF treatment emeliorated Growing evidence has demonstrated the contribution of autophagy in the regulation of lipid metabolism. There have been studies that activation of autophagy could reduce the number of lipid droplets resulting in therapeutic effects against NAFLD , while dβ-oxidation, a process that is critical for the disposal of fatty acids [β-oxidation has an important role on the development of fatty liver disease and nonalcoholic steatohepatitis [β-oxidation can lead to the accumulation of fatty acids, which causes NAFLD [β-oxidation and the autophagy activity would be ideal targets for NAFLD therapy. Presently, G-CSF increased the expression of β-oxidation genes, such as AMPK-1α, AMPK-2α, CPT1, and PGC-1α, accompanied with the enhancement of autophagy in the rat liver. Further studies are needed to determine whether increased β-oxidation is dependent on G-CSF-induced autophagy, and whether protein expression levels of these genes are similar to the corresponding mRNA expression.Autophagy contributes to the generation of fatty acids by breaking down lipid droplets and formation of the autolysosome, which is an autophagosome combined with a lysosome , 26. Thety acids , 28. β-oepatitis . In condes NAFLD . Furtheres NAFLD . Thus, cNAFLD is a common chronic liver disease and is closely associated with type 2 diabetes mellitus (T2DM) . NAFLD aPrevious studies have suggested several potential mechanisms underlying the therapeutic effect of G-CSF on various liver diseases, including the induction of bone marrow-derived cell mobilization and direct effects of the G-CSF receptor-mediated signaling pathway , 36. OurThis study has several limitations. First, we were unable to investigate the therapeutic mechanisms underlying the action of G-CSF on hepatic steatosis, in association with its effects on bone marrow-derived mobilization by G-CSF. This will require further investigations of the bone marrow-dependent effects of G-CSF. Second, treatment regimens including dosage, timing, and duration of G-CSF need to be established. Finally, our study did not analyze the expression levels of other autophagy markers or histological evidence related to autophagy. Additional studies are needed to more precisely evaluate the mechanisms involved in autophagy.β-oxidation and autophagy in a rat model. We speculate that the activation of autophagy by G-CSF could be associated with the reduction in hepatic steatosis through the upregulation of β-oxidation-related genes. In addition, we found that G-CSF did not reduce intracellular lipid accumulation, which implies that the therapeutic effect of G-CSF may be related to bone marrow mobilization.In conclusion, our study showed that G-CSF improved hepatic steatosis and induced the activation of


Genetic data are rapidly becoming part of tumor classification and are integral to prognosis and predicting response to therapy. Current molecular tumor profiling relies heavily on tissue resection or biopsy. Tissue profiling has several disadvantages in tumors of the central nervous system, including the challenge associated with invasive biopsy, the heterogeneous nature of many malignancies where a small biopsy can underrepresent the mutational profile, and the frequent lack of obtaining a repeat biopsy, which limits routine monitoring to assess therapy response and/or tumor evolution. Circulating tumor, cell-free DNA (cfDNA), has been proposed as a liquid biopsy to address some limitations of tissue-based genetics. In cancer patients, a portion of cfDNA is tumor-derived and may contain somatic genetic alterations. In central nervous system (CNS) neoplasia, plasma tumor-derived cfDNA is very low or absent, likely due to the blood brain barrier. Interrogating cfDNA in cerebrospinal fluid (CSF) has several advantages. Compared to blood, CSF is paucicellular and therefore predominantly lacks non-tumor cfDNA; however, patients with CNS-limited tumors have significantly enriched tumor-derived cfDNA in CSF. In patients with metastatic CNS disease, mutations in CSF cfDNA are most concordant with the intracranial process. CSF cfDNA can also occasionally uncover additional genetic alterations absent in concurrent biopsy specimens, reflecting tumor heterogeneity. Although CSF is enriched for tumor-derived cfDNA, absolute quantities are low. Highly sensitive, targeted methods including next-generation sequencing and digital PCR are required to detect mutations in CSF cfDNA. Additional technical and bioinformatic approaches also facilitate enhanced ability to detect tumor mutations in CSF cfDNA. The incidence of primary brain tumors in the United States is approximately 30 per 100,000 persons; one third of these tumors are malignant. The overall 5-year survival for malignant primary brain tumors is 30% and can be as low as ∼5% in the most common aggressive subtype, glioblastoma (GBM) . In chilCurrent techniques for molecular profiling of brain tumors primarily rely on tissue obtained through small biopsies or surgical resection. Brain biopsy is an invasive procedure, which carries a risk of mortality reported to be between 2.8 and 12% depending on technique and patient population . MoreoveLiquid biopsy techniques are fast emerging as non-invasive methods for tumor diagnosis and molecular characterization. Cell-free DNA (cfDNA) refers to DNA that has entered the circulation after cell death. In healthy patients, the majority of this DNA comes from the apoptosis of hematopoietic cells and has a length of ∼167 base pairs. The total amount of circulating cfDNA is increased in patients with solid tumors , and theCirculating tumor-derived cell-free DNA has emerged as a non-invasive modality for genetic characterization of many solid tumors. Advantages of using cell free DNA include that it is present in the plasma of most patients and that it allows serial monitoring of tumor burden and response to therapy. In a study of 640 patients, tumor-derived cfDNA was detected in the blood of >75% of patients with many types of advanced carcinomas (stage III/IV as indicated in the paper) and in approximately 50–75% of patients with localized carcinomas. In contrast, it was detected in only 40% of medulloblastoma patients and in <10% of patients with gliomas. Moreover, the absolute concentration of tumor-derived cfDNA in glioma and medulloblastoma patients was several logs lower than in patients with other tumor types . cfDNA dGenetic analysis of small biopsy specimens may not reflect intratumor heterogeneity or uncover mutations only present at a specific metastatic site . IntratuUsing CSF-derived cell free DNA may overcome the under-sampling bias that may occur with small tissue biopsies. In patients with CNS restricted disease (primary or metastatic), mutations have been found in CSF or plasma cfDNA that are not present in the corresponding biopsy specimen . In the IDH mutation status. Oligodendroglioma is identified by 1p/19q co-deletion, and both astrocytoma and oligodendroglioma are further described as histologic grade II or III, while glioblastoma is by definition grade IV  has been developed that can accurately classify nearly 80% of malignant gliomas based on genetics alone. A retrospective study of 20 glioma patients demonstrated that this combined digital PCR and targeted sequencing panel could use CSF cfDNA to correctly classify gliomas in 85% of cases and only failed with low grade tumors or those not in direct contact with the ventricles  is part of the differential diagnosis for many space occupying brain lesions , and in r tumors . A simil20 cases . CSF cytytometry . MYD88 Lh as GBM , making EGFR mutations in the primary tumor, patient-specific EGFR mutations were detected in the CSF cfDNA of all 26 patients. Using CSF cfDNA had greater clinical sensitivity than plasma cfDNA or DNA derived from direct cellular lysis of the CSF . Furthermore, the average mutant allele frequencies were highest in CSF-derived cfDNA , making it more likely that mutations were accurately detected. Many copy number variants were detectable only in the CSF cfDNA and were more abundant in patients who failed tyrosine kinase inhibitor therapy. Common molecular copy number alterations were present in MET, KRAS, ERBB2, BRAF, and ALK. EGFR T790M was identified in the CSF of 30.4% of patients with progression on tyrosine kinase inhibitor treatment vs. 21.7% in plasma  EGFR Mutation Test v2, an approved companion diagnostic for osimertinib, can detect EGFR mutations, including T790M, from the supernatant cfDNA from CSF cytology samples with 87.5% clinical sensitivity and 100% specificity  should be performed as soon as possible after collection, ideally within 6 h, to prevent dilution with leukocyte DNA , alterations were detected in 63% of patients with CNS metastases and 50% of patients with primary brain tumors . The pubof 0.15% . A studyFR exons .a priori knowledge of patient-specific genetic changes. However, in a small study of patients with brain tumors in dangerous anatomic locations, known mutations could not be found in two of four subjects, both of which had variant allele frequencies below 1% (Non-targeted whole genome or exome sequencing has an analytical sensitivity of approximately 5% . At firsbelow 1% . While tbelow 1% . In a stbelow 1% .Cell-free DNA has emerged as a powerful biomarker for the diagnosis and characterization of tumors of the central nervous system. Using CSF as a source has several advantages over plasma, including increased clinical sensitivity and the ability to detect CNS-limited mutations. Moreover, cfDNA from CSF is more likely to represent tumor heterogeneity than tissue biopsy, and if sampled over time, allows for the ability to monitor tumor progression and detect tumor evolution and drug resistance mutations.Most current studies of CSF cfDNA are small and retrospective. A few case reports have demonstrated that CSF cfDNA has been used clinically to guide  and moniIt has recently been shown that the variation of nucleosome positioning between tissue types can be exploited in cell-free DNA to predict tissue of origin. Notably, this observation extends to malignant tissue, which could be leveraged to facilitate determination of tumor origin from cell-free DNA . This maAll authors conceptualized the content and wrote 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.


Bilateral testicular injuries are rare. However, the incidence of these injuries has been increasing in wartime. We describe the case of gunshot wound of the both testicle caused by high velocity bullets. The patient was managed by surgical exploration, debridement and repaired of both testis using tunica vaginalis. During the follow up, the left testis was not viable and there was a need for orchiectomy; in a follow-up of 4 months, ultrasonography showed a viable right testis with minimal atrophic change and the patient reported to have normal erection with borderline hormonal function. We describe this case of bilateral testicular rupture, which was repaired using tunica vaginalis as graft, with attention to the management and outcome of this injury process. Bilateral testicular injury caused by gunshot is rare and represents proximally about 6-26% of all penetrating trauma. Recently, wider incidence of this injury has been noted in wartime ,2. InjurA 20-year-old single male presented to the emergency department with scrotal rupture, left leg laceration and bleeding following a gunshot accident from a distance of 12-16 cm, which penetrated the scrotum and both testes and came out from the medial aspect of the left leg. The patient experienced severe pain in his scrotum and left lower extremity. Nothing significant was mentioned in his previous medical history. On arrival, he was noted to be conscious, and had no vomiting and no abdominal pain. In physical exam, his vital signs seemed normal , his abdomen was soft without tenderness, his scrotum had a big laceration, and both testes were completely ruptured . The exiDebridement of necrotic testicular tissue was subsequently preformed. However, the ruptured testis could not be closed primarily because the extruded seminiferous tubules and the big tissue defect of the tunica albuginea did not allow the apposition of the tunica albugineal edges. Therefore, closure of the tunica albuginea using tunica vaginalis as a graft tissue was done  by using Vicryl suture bilaterally, and the drain was left in situ . Skin waEthical consideration: the patient gave their written informed consent for participation in our study.The incidence of penetrating testicular injuries has been on increase over the years, due to war and violence. Indeed, bilateral testicular injuries are more common in penetrating trauma and the common cause of these injuries is gunshot . Early sIn cases of complete testis amputation, a microsurgical measure should be taken for the best chance of viable reanastomosis . HoweverEmergency assessment, diagnosis, and early scrotal exploration are important for treatment of testicular rupture and using the tunica vaginalis as graft for repaired ruptured testis. Additionally, we did look at whether the patient was able to preserve the function of his testis by assessing erectile function following this event.