The contrast sensitivity function (CSF) relates the visibility of a spatial pattern to both its size and contrast, and is therefore a more comprehensive assessment of visual function than acuity, which only determines the smallest resolvable pattern size. Because of the additional dimension of contrast, estimating the CSF can be more time-consuming. Here, we compare two methods for rapid assessment of the CSF that were implemented on a tablet device. For a single-trial assessment, we asked 63 myopes and 38 emmetropes to tap the peak of a “sweep grating” on the tablet’s touch screen. For a more precise assessment, subjects performed 50 trials of the quick CSF method in a 10-AFC letter recognition task. Tests were performed with and without optical correction, and in monocular and binocular conditions; one condition was measured twice to assess repeatability. Results show that both methods are highly correlated; using both common and novel measures for test-retest repeatability, however, the quick CSF delivers more precision with testing times of under three minutes. Further analyses show how a population prior can improve convergence rate of the quick CSF, and how the multi-dimensional output of the quick CSF can provide greater precision than scalar outcome measures. While acuity is well established and can be very useful e.g. for adjusting optical correction, it has at least two issues in domains where precision is paramount, such as in clinical trials. First, the variability of repeated measurements makes the method insensitive to subtle changes in vision, e.g. due to disease progression or treatment effects456A more comprehensive assay of visual performance is provided by the contrast sensitivity function (CSF). The CSF relates an observer’s ability to recognize a spatial pattern not only to its size, but also to its contrast; the behavioural CSF is also reflected in receptive field analysis of individual neurons9In psychophysical laboratories, the CSF is typically assessed by estimating individual thresholds for a range of spatial frequencies, using one-dimensional Bayesian adaptive methods1517M a four-dimensional tuple describing a CSF and D the set of trials. The range of different CSFs, each described by a different M, spans the entire gamut from very low to excellent vision, and initially , the algorithm assumes the same probability for each of these CSFs; alternatively, other sources of information, such as knowledge about the distribution of CSFs in a particular subject population, can be used to model a more informative initial probability distribution, or prior. For each trial, the algorithm then chooses that combination of stimulus frequency and contrast that maximizes expected information gain about this CSF distribution. For example, the very first trial should not be trivially easy (such as in acuity charts that start with the largest letter) because the outcome (a correct response) is so likely for almost every subject  that it is probably uninformative for this particular subject, and the likelihood of different CSFs. Instead, the algorithm typically chooses a medium-sized, medium-contrast stimulus for the first trial , and the probability distribution over all CSFs is updated according to the subject’s response. For example, an incorrect response to such a medium-difficulty stimulus would make CSFs that represent excellent vision less likely, and thus the stimulus for the subsequent trial should not be difficult. Over the time course of the test, the quick CSF repeats these steps to home in on stimuli that are near the subject’s threshold and that provide the most information about the probability space of different CSFs. This means that a high resolution in the stimulus space  is useful because it allows the algorithm to adapt more finely to the current subject; however, not all possible stimuli need to be presented during the experiment. Finally, a sample is taken from the probability distribution of CSF models M and the median sensitivity over this sample can be computed for any spatial frequency based on the model parameters.However, advances in computational power and display technology have opened avenues for novel methods of visual assessment to overcome these issues. For example, the quick CSF method2022We further extended our tablet-based implementationIn order to establish a baseline for how much information can be captured ultra-rapidly, in a single trial, we also collected a single-trial assessment of the peak of the CSF: using the iPad’s touch screen, observers were asked to indicate the point of highest sensitivity on a “sweep grating” picture that varies spatial frequency and contrast along the two image dimensions. We analyze the relationship between quick CSF results on the one hand and single-trial assessment of contrast sensitivity on the other hand, and also compare the test-retest variability of both assessments, using a method from information retrieval that is less prone to artefact than commonly used test-retest measures.A necessary requirement for a precise test is that two measurements of the same true value return similar results; in other words, a precise test should be highly repeatable. However, this requirement is not sufficient, as reliable tests are not necessarily preciseN of subjects, 1/N). Generally, each subject is assigned the fractional precision (1 − (rank − 1)/N) of their individual retest value when all subjects’ retest values are sorted by their similarity (e.g. Euclidean distance) to the individual test. We repeat this for each subject and FRP then is the average of all subjects’ precisions. If test and retest scores are distributed randomly, the expected FRP will be 0.5. Notably, FRP also takes into account that identical test outcomes for different subjects, e.g. due to large step sizes, reduce the ability to identify the test-retest pair. For example, in typical healthy, best-corrected cohorts, almost all subjects have one of very few different logMAR scores at or near 20/20. In the extreme, a very coarsely resolved test, such as light perception in a sighted cohort , would yield an FRP of 0.5, i.e. chance performance.We instead propose to assess test-retest variability based on concepts from information retrieval, namely by a measure we call Fractional Rank Precision (FRP)As an FRP example, imagine three subjects who have test scores of 1.0, 1.5, and 1.2, and retest scores of 1.0, 1.3, and 1.4, respectively. The absolute differences of the three retest scores to the test score (1.0) of the first subject are  and thus the first subject’s retest is closest to its test score, i.e. has rank 1. For the test score of the second subject (1.5), the absolute differences are  and thus the second subject’s retest has rank 2. For the third subject (test score 1.2), the absolute differences are , and its retest has rank 2.5 (because first and third subjects’ tests tie for rank 2). The Fractional Rank Precision therefore is 1/3 × (1 + 2/3 + 1/2) = 0.72. This formulation has the benefit that it expresses test-retest variability in terms of inter-subject variability, without resorting to absolute values that may make it difficult to compare the repeatability of different tests with different absolute score ranges. A further benefit is the penalty for test scores that are quantized at the cost of precisionp ≪ 0.01, R2 = 0.34 for peak sensitivity and R2 = 0.40 for peak frequency); multivariate regression explains more of the variance  with no statistically significant interaction between peak sensitivity and peak frequency (p = 0.59).The two panels in Bland-Altman Coefficients of Repeatability were 0.497 and 0.476 log10 units for peak sensitivity and frequency, respectively. Fractional Rank Precision for both features was 0.715 and 0.681, respectively; combining the two features and performing a nearest-neighbour search improved FRP to 0.769.Test times for the quick CSF are shown in p = 0.06, R2 = 0.15). A particularly strong difference between the two metrics can be seen in the bottom right of p = 0.46, R2 = 0.01); in other words, subjects with poor vision exhibit similar test-retest variability as subjects with excellent vision.Fractional Rank Precision values for different features of the quick CSF are shown in Contrast sensitivity has been recognized as a more informative outcome measure than visual acuity, the currently prevalent measure of visual function45293031173334A multitude of studies have investigated the repeatability of established contrast sensitivity charts as a proxy to precision. Absolute numbers are not necessarily comparable across studies because of different scoring rules363740In our present study, we used two different measures of contrast sensitivity that have very short testing times, and evaluated their validity in a cohort of healthy observers with varying refractive error. The first measure required subjects to tap a tablet device only once, and therefore probably constitutes the lower limit of testing time that is practically achievable. Recently, other groups have employed a similar technique to assess contrast sensitivity434445R2 > 0.5 and a test-retest precision that corresponds to about 10–15 trials of quick CSF. However, the Bland-Altman CoR was more than twice as high for peak-tapping as for the quick CSF after 50 trials, and the quick CSF in principle could be run for more trials to achieve even greater precision. Furthermore, it should be noted that the position of the sweep grating was not randomly shifted before each test session, so that subjects might have remembered (and reproduced) the location they had tapped on the screen in previous sessionsDespite these fundamental problems, single-trial assessment correlated reasonably with quick CSF assessment, with an One potential use of such ultra-rapid screening tool might be the initialization of the quick CSF method with a subject-specific prior. We demonstrated in  that theet al.Kim The quick CSF method estimates sensitivities for a wide range of spatial frequencies. The AULCSF already yields a useful summary statistic4950515253224453We performed FRP analysis on higher-dimensional combinations of CSF features that might capture CSF variation more comprehensively. To this end, we used a simple nearest-neighbour approach with unit-normalized feature scales, and indeed FRP scores improved over those for scalar features such as AULCSF or CSF acuity; we note that more sophisticated Machine Learning techniques that differentially weight the individual CSF features might improve FRP scores even further. In our present analysis, best results were achieved with a combination of low-, medium-, and high-SF sensitivities (1.5 and 6 cpd and CSF acuity) and the summary statistic AULCSF; notably, this four-dimensional combination also performed better than the combination of (near-peak) sensitivity at 1.5 cpd and CSF acuity that would correspond to a two-point measurement.In summary, we here showed that rapid assessment of the whole contrast sensitivity function on a portable device is feasible, and that the quick CSF method delivers highly precise results in testing times of less than three minutes. This efficiency can be further improved by an informed population prior or even further by the use of an ultra-rapid, single-trial prior assessment. Ultimately, the real value of a vision test is shown by its ability to detect small changes in visual function due to disease progression or treatment, and further studies are needed to evaluate the sensitivity of the quick CSF.Experimental data were collected from 101 subjects (aged 14–75 years with a mean of 22) who were recruited among students and faculty of the Institute for Psychology and Behavior, Jilin University of Finance and Economics, Changchun, Jilin Province, China. Subjects gave informed consent and the experimental design followed the principles of the Declaration of Helsinki and was approved by the local Ethics Committee (protocol IPBWH1101). Based on self-reported optical refraction, 63 subjects were myopes  and 38 subjects were emmetropes .Subjects were tested in all applicable combinations of {right eye, left eye, both eyes} and {without correction, with correction}. To assess repeatability of our device, one randomly chosen condition was then repeated at the end of the session. While repeat measures taken over a longer time interval may be closer to clinical practice, intra-session repeatability measurement excludes subject variability over longer time scales and is thus a common method to assess the measurement itself 3438404361http://www.michaeldorr.de/quickcsf.Overall, 593 data sets were collected; these are publicly available at m2. Prior to each tested condition, the single-trial assessment was recorded; to this end, subjects were shown a sweep grating as in During the experiment, subjects were seated and held an iPad 4 in portrait orientation at a viewing distance of 60 cm. Mean screen luminance was set to 185 cd/For each of 50 trials, one of 11520 possible stimuli  was selected by the quick CSF method. The stimulus was then briefly presented (500 ms), followed by an array of letters from which subjects had to choose their response on the touch screen.prior probability distribution over the parameter space. Such a prior could be derived from previous knowledge, e.g. the statistics of visual function in the general population or an individual’s earlier test results. In our experiment, however, we approached data collection in the most principled way and chose a uniform distribution over the parameter space. While this makes the least assumptions, a drawback of this approach is that physiologically implausible or even impossible parameter combinations such as a very high peak frequency together with a very low bandwidth initially are equally likely as more plausible combinations, and thus convergence may be slower than theoretically possible. In order to investigate this effect after data collection had concluded, we re-scored all subjects’ responses with an additional run of the algorithm per test session. For this analysis, the posterior over the whole test set (593 data sets) was used as the prior, and we report results both for this ‘population’ prior and the original ‘uniform’ prior.The quick CSF uses Bayesian inference to update its belief (i.e. the best estimate of the true CSF) after each trial. Before the first trial, however, no data have been collected and the initial belief has to be described by a fsubject, we sorted all the N observers’ retest values fn by their distance to fsubject, and assigned a rank r(observern) accordingly. The fractional rank for one subject then was From the final estimates of the CSFs, we computed threshold sensitivity for each of the 24 spatial frequencies that corresponded to our stimulus set . For a broad summary statistic, we also computed the area under the log CSF (AULCSF) in the spatial frequency range from 1.5 to 18 cpd. For these 25 features, we computed Coefficients of Repeatability (1.96 times the standard deviation of test–retest differences) and the Fractional Rank Precision metric (FRP): for each subject’s test feature For increased robustness, FRP analysis was run in both directions and results were averaged, i.e. all retests were ranked by their distance to each test once, and all tests were ranked by their distance to each retest once.By definition, the CSF is a multi-dimensional function. While the AULCSF can already provide a very useful summary statistic in many cases, higher-dimensional descriptions of the whole CSF may more precisely capture changes in the CSF and thus track an individual’s performance. We therefore performed FRP analysis on multi-dimensional feature vectors and performed a nearest-neighbour search to identify possible test-retest pairs.In order to keep the number of possible dimensions small, we did not use all 25 features from above. Instead, we computed the following features on the CSF: i) AULCSF; ii) CSF acuity, the spatial frequency for which contrast threshold reaches 100%; iii) sensitivities for six spatial frequencies as mandated by the FDAHow to cite this article: Dorr, M. et al. Evaluation of the precision of contrast sensitivity function assessment on a tablet device. Sci. Rep.7, 46706; doi: 10.1038/srep46706 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort.We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure . CSF, clinical, and biomarker data from the earliest available time point were analyzed. Cell-free mtDNA associations with CSF inflammation and iron-related biomarkers , VL, and GDS were evaluated by multivariable regression.p < 0.001) or CSF (p < 0.001) and in those on antiretroviral therapy . Participants on ART with undetectable VL in both CSF and plasma had lower mtDNA levels than those with detectable VL in both compartments (p = 0.001). Higher mtDNA levels were observed in participants in the highest vs. lowest tertile (T3 vs. T1) of CSF CXCL10  and TNF-a  in unadjusted analyses. MtDNA levels also correlated with CSF leukocyte count. After adjusting for CSF leukocyte count and VL, mtDNA levels were also associated with other inflammation- and iron-related biomarkers in CSF, including TF  and CP . With additional correction for ART use, mtDNA was also negatively associated with CSF VEGF (p < 0.05) and IL-6 (p = 0.05). We observed no associations of CSF mtDNA levels with age or GDS-defined NC impairment.CSF cell-free mtDNA levels were significantly lower in participants with undetectable (vs. detectable) VL in either plasma  contains supplementary material, which is available to authorized users. The pathogenesis of HIV-associated neurocognitive impairment (NCI) remains incompletely understood. Neuroinflammation due to chronic immune activation and viral replication in the central nervous system (CNS) is important in promoting HIV-associated NCI (reviewed in  and 2]))2]). In Mitochondria are intracellular organelles, critical for energy production and a host of metabolic processes, which contain their own distinct DNA genome (mtDNA). Most mammalian cells contain multiple copies of mtDNA  , 6. BraiMitochondrial damage and mtDNA copy number within brain tissue have been associated with several neurodegenerative diseases, as well as with aging –10. FeweDisruption of iron homeostasis is also a characteristic feature of neurodegenerative disorders, including AD and PD –17, and n = 107), and a protease inhibitor-based regimen (n = 152). The study was approved by institutional review boards at each site, and all participants provided written informed consent.CHARTER is a prospective, observational study that enrolled ambulatory, HIV-infected adults from 2003 to 2007 at six USA locations: Johns Hopkins University, Baltimore, MD; the Icahn School of Medicine at Mount Sinai, New York, NY; University of California, San Diego, CA; University of Texas Medical Branch, Galveston, TX; University of Washington, Seattle, WA; and Washington University, St. Louis, MO. Standardized, comprehensive neuromedical, neurobehavioral, and laboratory assessments were performed, as previously described , 24. ThiHIV-associated neurocognitive disorder (HAND) was diagnosed when NCI and functional impairment were determined to likely be due to HIV-related effects on the brain rather than to comorbid conditions . DetaileHIV infection was diagnosed by ELISA with western blot confirmation. HIV viral load  were measured in plasma and CSF by reverse transcriptase PCR  performed at a central lab .g for 15 min to remove cells and cellular debris, and the supernatant was stored at −80 °C. Total genomic DNA was extracted from CSF supernatant samples using QIAamp DNA Mini Kit  according to the manufacturer’s protocol, and was digested using the BamHI HF  restriction enzyme. MtDNA was then quantified by droplet digital PCR  using a primer-probe combination targeting the NADH dehydrogenase 2 gene  found within the mitochondrial genome. A genomic DNA (gDNA) control was also measured by targeting the ribonuclease P protein subunit p30 gene . Quantification was performed according to manufacturer’s instructions and was carried out in triplicate in 20 μL reactions, consisting of 10 μL of 2× Bio-Rad supermix for probes, 1 μL of either 20× Primer/FAM ND2 mix or 20× Primer/VIC RPP30 mix, 4 μL of molecular grade water, and 5 μL of DNA. Droplet generation and amplification were performed per protocol, and after analysis of the droplets, mtDNA copy number per mL of CSF was calculated using QuantaSoft .CSF samples, obtained at the baseline visit, were analyzed for cell-free mtDNA content. Briefly, CSF was centrifuged at 250×Concentrations of inflammation and angiogenesis biomarkers were measured in CSF according to manufacturer’s protocol by commercial high-sensitivity multiplex (interleukin (IL)-6, IL-8, tumor necrosis factor-alpha (TNF-α) or single-plex (CXC motif chemokine 10 (CXCL10), vascular endothelial growth factor (VEGF) bead-based immunoassay arrays . Ten percent of all assays were repeated to assess operator and batch consistency.Quantification of CSF iron content was performed using the Quantichrom Iron Assay , following the manufacturer’s protocol. CSF transferrin content was determined by enzyme-linked immunoabsorbent assay , Abcam, Cambridge, MA), performed according to the manufacturer’s protocol. CSF samples were centrifuged, and duplicate samples were diluted 1:1000 with mix diluent. The human heavy chain ferritin (H-ferritin) content of CSF samples was determined by ELISA assay  performed according to the manufacturer’s protocol. Additional iron-transport-related biomarkers quantified in CSF included ceruloplasmin (CP) and haptoglobin (HP). Levels of these proteins were measured using commercially available bead-based immunoassays (Millipore) validated for CSF.t test was used to determine the difference in log mtDNA levels between participants who were on ART and those who were not on ART. Within the ART-treated subset, analysis of variance (ANOVA) was used to determine differences in mtDNA levels between the following four groups: (1) detectable plasma and CSF viral load (n = 27), (2) detectable CSF viral load but undetectable plasma viral load (n = 5), (3) detectable plasma viral load but undetectable CSF viral load (n = 56), and (4) undetectable plasma and CSF viral load (n = 147). The small number of subjects with detectable HIV in CSF but not plasma limited the power to compare this category with other groups. Pearson’s correlations were calculated to evaluate the relationship between mtDNA level and CSF leukocyte count. To evaluate the relationship between mtDNA and neurocognitive performance, Wilcoxon tests were performed to compare mtDNA levels between the individuals with no NCI, and those with asymptomatic NCI (ANI) and mild NCI or neurocognitive disorder (MND). Too vfew participants had HIV-associated dementia  to power comparisons with the other groups. In order to determine if associations with mtDNA were independent of other factors that may relate to neuroinflammation and NCI, associations between cell-free mtDNA levels and neurocognitive performance were assessed in multivariable linear regression models, stratified by ART status (on vs. off) and adjusted for nadir CD4+ T-cell count, CSF leukocyte count, and CSF virus detectability, or adjusted for nadir CD4+ T-cell count, CSF leukocyte count, and plasma virus detectability [p values <0.05 were considered statistically significant.Univariate linear regressions were performed to assess associations between mtDNA levels and clinical variables, including demographic characteristics, ART use, CSF HIV viral load and biomarkers of inflammation, angiogenesis, and iron transport in CSF. To fulfill the normality assumption of linear regression, mtDNA copy number was log-transformed. CSF biomarkers other than mtDNA, which were also continuous but non-normally distributed variables, were categorized as tertiles to improve sensitivity of statistical association tests. Student’s load n = , (2) dettability . Similartability  and SAS 10 copies/ml and 1.7 in CSF (IQR 1.7–2.3) log10 copies/ml. Thirty-four percent (plasma) and 60% (CSF) had undetectable HIV RNA. The median (IQR) current CD4+ T-cell count was 455 (270–636) cells/mm3. Thirty percent of participants were classified as having NCI based on the GDS [p = 0.02).Unimpaired participants were also less likely to have a self-reported diagnosis of diabetes (Type I or II) . Demographic and clinical characteristics of study participants are presented, overall and stratified by NCI status, in Table The mean age of the study population was 43 years (standard deviation 8.2); 77% were men, and 54% reported non-white race/ethnicity. Sixty-nine percent of participants were on ART at the time of CSF sampling. The median HIV RNA concentrations were 1.9 in plasma (Interquartile range (IQR) 1.7–4.0) log the GDS , and the10 copies/ml. Individuals on ART at the time of CSF sampling had significantly lower cell-free CSF mtDNA levels than those who were ART-naïve (p = 0.03) or who had previously received ART (p = 0.02). Additionally, participants with detectable HIV RNA in either plasma or CSF (grouped together) had significantly higher cell-free CSF mtDNA levels than those with undetectable HIV RNA in either plasma (p < 0.001) or CSF (p < 0.001) (data not shown). Higher cell-free CSF mtDNA was seen with lower nadir CD4+ T-cell counts (p < 0.01). Higher CSF WBC and CSF HIV RNA were also associated with higher mtDNA levels (both p < 0.001), with a modest positive correlation between CSF WBC and cell-free CSF mtDNA . No associations between CSF cell-free mtDNA levels and age, self-reported race/ethnicity, body-mass index, lifetime diagnosis (self-report) of diabetes mellitus, or depression (Beck’s Depression Inventory Score) were identified.Overall median (IQR) CSF cell-free mtDNA was 3.92 (3.67–4.27) logn = 193, 4.17 vs. 3.97 log10 copies/ml; p = 0.04) or to those with ANI . No individuals with MND had a mtDNA level less than 3.69 log10 copies/ml .We did not observe any association between the CSF mtDNA level and the trajectory of neurocognitive status, as n = 332) comparing CSF mtDNA levels in the third (T3) and first (T1) tertiles of each biomarker, mtDNA levels were positively associated with CXCL-10  and TNF-α  , lower cell-free CSF mtDNA levels were associated with higher levels of CSF transferrin , and CP . Age did not contribute to any of these models. Associations with CXCL-10 and TNF-α did not persist after adjustment for CSF WBC and HIV RNA.In univariate analyses (d first T tertilesn = 294), CSF mtDNA was again negatively associated with CSF transferrin (p < 0.01), CP (p = 0.06), VEGF (p = 0.03), and IL-6 (p = 0.05). Since comorbid conditions can influence neurocognitive performance, we also stratified by the severity of comorbidities present. Among participants with comorbidities that were assessed to adversely affect neurocognitive performance, cell-free CSF mtDNA levels continued to be negatively associated with CSF transferrin (p = 0.02), CP (p < 0.01), VEGF (p < 0.05), and IL-6 (p < 0.05) (n = 134 except for iron biomarker levels (n = 80)), even after adjusting for CSF WBC. Among individuals who only had comorbidities deemed to be incidental, no associations with CSF mtDNA were identified.We also addressed potential confounding due to non-suppressed HIV infection by analysis restricted to individuals on ART. In this subgroup , supporting novel mechanisms of CNS injury in HAND. CP plays a critical role through its ferroxidase activity which facilitates binding of iron to transferrin. In addition, both CP and VEGF have neuroprotective as well as strong angiogenic properties , 40. CP In persons with HIV RNA in the CSF, pleocytosis was more common than in those with suppressed HIV replication on ART –48, and Limitations of this study include the cross-sectional design, some degree of heterogeneity of treatment regimens in cohort participants on ART, and inability to extrapolate from cell-free mtDNA levels in CSF to intracellular mtDNA content in the brain. Nevertheless, this study provides new insights into the complex pathophysiology of neuroinflammation and mitochondrial damage during HIV infection.In conclusion, cell-free CSF mtDNA was inversely associated with biomarkers of iron transport in HIV-infected persons when controlling for CSF HIV RNA and WBCs. Given the importance of iron homeostasis in neuronal function  and also


Atrial fibrillation (AF) is a common diagnosis of patients presenting to the emergency department (ED). Intravenous (IV) diltiazem bolus is often the initial drug of choice for acute management of AF with rapid ventricular response (RVR). The route of diltiazem after the initial IV loading dose may influence the disposition of the patient from the ED. However, no studies exist comparing oral (PO) immediate release and IV continuous infusion diltiazem in the emergency setting. The objective of this study was to compare the incidence of treatment failure, defined as a heart rate (HR) of >110 beats/min at four hours or conversion to another agent, between PO immediate release and IV continuous infusion diltiazem after an initial IV diltiazem loading dose in patients in AF with RVR.This was a single-center, observational, retrospective study conducted at a tertiary academic medical center. The study population included patients ≥18 years old who presented to the ED in AF with a HR > 110 beats/min and received an initial IV diltiazem loading dose. We used multivariate logistic regression to assess the association between routes of administration and treatment failure.A total of 111 patients were included in this study. Twenty-seven percent (11/41) of the patients in the PO immediate-release group had treatment failure compared to 46% (32/70) in the IV continuous-infusion group. The unadjusted odds ratio (OR) of treatment failure with PO was less than IV at 0.4 . When we performed a multivariate analysis adjusted for race and initial HR, PO was still less likely to be associated with treatment failure than IV with an OR of 0.4 . The median dose of PO diltiazem and IV continuous infusion diltiazem at four hours was 30 mg and 10 mg/h, respectively.After a loading dose of IV diltiazem, PO immediate-release diltiazem was associated with a lower rate of treatment failure at four hours than IV continuous infusion in patients with AF with RVR. Atrial fibrillation (AF), a supraventricular tachyarrhythmia, is the primary diagnosis for over 467,000 hospitalizations each year.The AFFIRM trial compared rate and rhythm control in 4,060 chronic AF patients. It found no difference in overall mortality, but there were fewer hospitalizations with rate control compared to rhythm.1Diltiazem, a non-dihydropyridine calcium channel blocker, is a common initial choice in the management of AF with rapid ventricular response (RVR) due to its ability to be given as an intravenous (IV) push, continuous infusion, and oral (PO) immediate-release or extended-release tablet. In the ED a loading dose (LD) of IV diltiazem is usually administered followed by PO immediate-release tablet or IV continuous infusion. Both options allow for dose titration in the short term before converting to a longer-acting PO formulation for discharge. The PO immediate-release diltiazem tablet has an onset of action of 30–60 minutes and is dosed every six hours.The route of diltiazem after the initial IV LD can influence the disposition of the patient from the ED, the level of care needed, and hospital length of stay (LOS). Patients who receive only the PO immediate-release diltiazem absorb a therapeutic dose quickly and can generally be discharged or admitted to a general medicine floor, but cannot be titrated more frequently than every six hours. Patients who received the IV continuous infusion must have their dose frequently titrated by nursing and often require stepdown care. No studies exist comparing the efficacy of PO immediate-release and IV continuous-infusion diltiazem in the emergent management of AF with RVR. The objective of this study was to compare the incidence of treatment failure at four hours between PO immediate-release and IV continuous-infusion diltiazem after an IV LD.This was a retrospective, observational, medical record review conducted with data from Virginia Commonwealth University Health, a tertiary medical center ED that treats over 95,000 patients annually. We retrospectively identified cases of ED diltiazem use from July 1, 2014, to July 1, 2015, from electronic medical records. Inclusion criteria included the following: patients ≥ 18 years old who presented to the ED in AF, with a HR > than 110 beats/min, who received an initial diltiazem IV LD and then subsequently PO immediate-release or IV continuous-infusion diltiazem. Of note, no ED AF protocol existed at the time of the study. Diltiazem dose and route selection were at the discretion of the ED provider. We excluded patients if they were pregnant or a prisoner. We also excluded patients if they had received electrical cardioversion or other rate control or antiarrhythmic medication in the prehospital or ED setting before being administered an IV LD of diltiazem. This study was approved by the institutional review board.What do we already know about this issue?Diltiazem is often used in the acute, emergent management of atrial fibrillation. No studies exist comparing oral (PO) immediate release and intravenous (IV) continuous infusion diltiazem.What was the research question?Compare the incidence of treatment failure at four hours between PO immediate release and IV continuous infusion diltiazem.What was the major finding of the study?PO immediate-release diltiazem was associated with a lower rate of treatment failure at four hours than IV continuous infusion.How does this improve population health?If PO immediate-release diltiazem is associated with less treatment failure, it may permit the disposition of emergency department patients to a less resource-intensive setting.® electronic data capture tools.We collected and managed study data using REDCap® that included study definitions to ensure consistency between the investigators. Investigators were not blinded to the study outcome. Any discrepancies between abstractors resulted in a collaborative review of the chart by both investigators until discrepancies were resolved. As a result, interrater reliability was not determined.Two of the study’s investigators abstracted all available data independently. Both were involved in the study design and used a standardized data collection form in REDCapThe primary endpoint of the study was the percentage of patients with treatment failure at 4 ± 1 hour after initiation of PO immediate-release diltiazem or continuous IV diltiazem infusion. Treatment failure was defined as HR of > 110 beats/min at 4 ± 1 hour, a switch in therapy from PO immediate-release diltiazem to IV continuous infusion diltiazem, the requirement of an additional IV diltiazem bolus within four hours from the start of PO or IV continuous infusion, or addition/switch of therapy to another rate control or antiarrhythmic agent within four hours. A clinical endpoint of 4 ± 1 hour was selected to give time for both the PO and the IV diltiazem to have therapeutic effect. It was also concluded that this was a reasonable amount of time for the ED provider to determine disposition. We made the decision not to include time points extending beyond four hours due to the increased number of confounding factors, including the conversion to PO β-blockers or extended-release PO diltiazem.Patient characteristics collected included age, weight, race, sex, initial HR and BP, and initial diltiazem LD. We assessed the safety endpoint of clinically significant hypotension by recording the indication for diltiazem discontinuation and the need for vasopressors administration for hemodynamic support.No power calculation was done due to the study’s exploratory nature. We based dates for study inclusion on when diltiazem PO immediate-release tablets became readily available in the ED medication-dispensing unit. If included patients presented to the ED multiple times during the study period, only the most recent encounter was considered.2 or Fisher’s exact test, and we compared continuous variables using Student’s t-test. We used univariate logistic regression to identify those characteristics associated with treatment failure and therefore eligible for inclusion in a final, multivariable model. Per the modeling strategy presented by Hosmer et al., a liberal p-value of 0.15 was used to identify these potential confounders.We analyzed data using Excel, R 3.2.2, and JMP 11.0.0 . Nominal variables were evaluated with ΧWe reviewed 324 patients for study inclusion and excluded 213 . The mosFor the primary endpoint of treatment failure at four hours, 27% of patients (11/41) in the PO immediate-release diltiazem group met criteria compared to 46% of patients (32/70) in the IV group, a difference of 19% (p=0.049). The unadjusted odds ratio (OR) of treatment failure with PO when compared to IV was 0.4  . We perfFifty-three percent of patients in the PO immediate-release diltiazem group received an initial dose of 30 mg and 41% received 60 mg. The median dose of IV continuous-infusion diltiazem at four hours was 10 mg/h (range 2.5 mg/h to 20 mg/h). Patient disposition from the ED can be seen in From a safety standpoint, no patients required vasopressors for BP support or had their diltiazem therapy discontinued for hypotension. Diltiazem was stopped for only two indications in both the PO and IV group-change in agent and lack of indication . In one case, the discontinuation reason was unknown.In the emergent setting, diltiazem has been shown to be superior to digoxin, metoprolol, and amiodarone in the initial management of AF and flutter.In our study, we found that PO immediate-release diltiazem resulted in a 0.4 (95% CI 0.15–0.94) OR of treatment failure when compared to IV continuous infusion. In other words, PO immediate-release diltiazem resulted in an odds of heart rate control 2.6 times greater than IV continuous infusion at four hours. This is a surprising result given the higher bioavailability of the IV route compared to the oral formulation. A possible reason for this difference in treatment failure may be that IV continuous infusion was sub-optimally titrated. In our sample, the median hourly dose of the IV continuous infusion at four hours was only 10 mg/h, well below the maximum dose of 15 mg/h. Slow titration to sub-maximal doses may have resulted in suboptimal diltiazem plasma concentrations in comparison with patients who were given immediate-release PO diltiazem. In theory, PO dosing may have achieved a higher plasma concentration as a result of the entire diltiazem dose being given at once. Therefore, our results may not reflect the comparison of two treatment regimens at optimal dosing capacity, but rather the real-world practice in which medication titration is not always optimized.PO diltiazem was associated with statistically significant higher odds of being admitted to the general floor and lower odds of being admitted to stepdown or the ICU. Patients who received PO also had a two-day shorter median LOS compared to IV. While the differences in these two parameters cannot be ascertained in a definitive manner due to the retrospective nature of the study, it is possible that the extended time needed to transition patients from IV to PO diltiazem before discharge may have played a contributing factor. Patient disposition and decreased LOS represent a possible area of healthcare cost savings that should be investigated in future prospective studies.Providers may choose IV continuous-infusion diltiazem if they want to titrate to lower doses in patients with borderline hemodynamic stability. In our study, however, clinically significant hypotension (defined as hypotension requiring discontinuation of the therapy and/or vasopressors) did not occur in the PO or IV group. Overall, our findings call in to question the primacy of IV continuous-infusion diltiazem for AF. PO diltiazem was associated with a lower rate of treatment failure and higher rate of heart control than IV continuous infusion and with similar safety. Importantly, these findings are the result of a retrospective study with limited sample size and therefore must be confirmed in a larger, prospective, randomized controlled trial.This study has several limitations. Its retrospective nature limited sample size and abstraction. Incomplete documentation prevented characterization of the severity of the patient’s symptoms, past medical history of AF, and home medications. In addition, identifying the total amount of diltiazem received via the continuous infusion route to allow for summative dose comparisons against oral was not possible due to inconsistent documentation of IV titrations. The statistically significant difference between groups in baseline HR suggests a potential selection bias against IV continuous-infusion diltiazem as providers may have selected this route of administration for more acutely ill patients and reserved PO diltiazem for milder cases. While we accounted for a select number of patient-specific factors in our logistic regression model, the potential for additional, unmeasured confounders still exist, which could mean the study showed only association, not causation. Our primary endpoint measured treatment failure at 4 ± 1 hour to give time for both the PO and the IV diltiazem to have therapeutic effect. A majority of patients failed due to HR > 110; our study may have excluded other time points where HR control was achieved. Lastly, the small sample size and low power resulted in large CI for the odds ratios.After a loading dose of IV diltiazem, PO immediate-release diltiazem was associated with a lower rate of treatment failure at four hours when compared to IV continuous infusion in patients with atrial fibrillation with rapid ventricular response.


Normal pressure hydrocephalus (NPH) is a syndrome characterized by the triad ofgait disturbance, mental deterioration and urinary incontinence, associated withventriculomegaly and normal cerebrospinal fluid (CSF) pressure. The clinicalpresentation (triad) may be atypical or incomplete, or mimicked by otherdiseases, hence the need for supplementary tests, particularly to predictpostsurgical outcome, such as CSF tap-tests and computed tomography (CT) ormagnetic resonance imaging (MRI). The CSF tap-test, especially the 3 to 5 dayscontinuous external lumbar drainage of at least 150 ml/day, is the onlyprocedure that simulates the effect of definitive shunt surgery, with highsensitivity (50-100%) and high positive predictive value (80-100%). According tointernational guidelines, the following are CT or MRI signs decisive for NPHdiagnosis and selection of shunt-responsive patients: ventricular enlargementdisproportionate to cerebral atrophy (Evans index >0.3), and associatedballooning of frontal horns; periventricular hyperintensities; corpus callosumthinning and elevation, with callosal angle between 40º and 90º; widening oftemporal horns not fully explained by hippocampal atrophy; and aqueductal orfourth ventricular flow void; enlarged Sylvian fissures and basal cistern, andnarrowing of sulci and subarachnoid spaces over the high convexity and midlinesurface of the brain. On the other hand, other imaging methods such asradionuclide cisternography, SPECT, PET, and also DTI or resting-statefunctional MRI, although suitable for NPH diagnosis, do not yet provide improvedaccuracy for identifying shunt-responsive cases. This review was based on aPubMed literature search from 1996 to date.Normal pressure hydrocephalus (NPH) is a syndrome characterized by the triad of gaitdisturbance, mental deterioration and urinary incontinence, which are associatedwith enlargement of the ventricular system and normal cerebrospinal fluid (CSF)pressure. In NPH, CSF pressure may be normal at one spinal tap, but episodes ofincreased CSF pressure can occur, and hence NPH is also termed "intermittentpressure hydrocephalus". It is caused by excessive accumulation of CSF in theventricular system due to an impairment of its flow distally to the fourth ventricle. About 50% of cases with communicating NPH have aknown cause , such as meningitis,subarachnoid hemorrhage, or cranial trauma, while the remaining 50% of cases areidiopathic (INPH), usually presenting in the 7th decade of life. Epidemiologicaldata on NPH incidence and prevalence are scarce, but surveys in Germany, Norway,Sweden and Japan have estimated the annual incidence of INPH to be between1.8/100,000 and 5.5/100,000 inhabitants, with a prevalence ranging from 0.2% to 2.9%among individuals aged 65 years or older,The diagnosis of NPH is based on the following criteria:[1] a history of gait disturbance, progressive mental deterioration, andurinary urgency or incontinence;[2] hydrocephalus, defined as Evans' ratio >0.30on computed tomography(CT) or magnetic resonance (MR) imaging; and[3] a CSF opening pressure (appropriately measured) of <24 cm ofwater.5Differential diagnostic difficulties may arise when the clinical manifestations ofthe triad are atypical or incomplete, or when they are mimicked by other diseases.Indeed, many other conditions may cause the complete triad, such as vasculardementia, parkinsonism, Lewy body disease, corticobasal degeneration, progressivesupranuclear palsy, multiple system atrophy, neurosyphilis, medication side effects;or in combination with other diseases, particularly cerebrovascular and Alzheimer'sdisease, which are present in up to 75% of patients with INPH.Shunt surgery can improve all NPH symptoms and quality of life in up to 80% ofproperly selected cases, but has complications rates (35-52%) that dissuade us fromshunting every suspected case. Surgical decision may be difficult and yield varyingresults, particularly in the elderly with comorbidities or vascular or degenerativebrain diseases, which can mimic or worsen NPH symptoms. Therefore, in order toimprove diagnosis and management of NPH cases, complementary tests have to be used,especially neuroimaging and CSF tap tests.7 A 'positive' RC(with ventricular reflux and convexity block) can be seen in other dementiadisorders and even in healthy subjects, thus having questionable predictive value.Most clinicians suggest it should no longer be performed,8 and the International INPH Guidelines9 have not included it as an option,since it does not improve the diagnostic accuracy of identifying shunt-responsivecases.Radionuclide cisternography (RC), intracranial pressure monitoring (ICP) and lumbarinfusion tests can show CSF dynamics malfunction, but none are able to confirmwhether the patient will benefit from surgery.11 Since the one tap CSF-TT has lowsensitivity (26-61%), a "negative" result cannot be used to exclude patients fromsurgery. In such cases, the alternative is a repeated CSF-TT (RTT) performed onthree consecutive days with a minimum of 30 to 40 ml CSF removed; or continuousexternal lumbar drainage (ELD) for 3 to 5 days, with a minimum of 150 ml CSF draineddaily. Due to ELD's high sensitivity (50-100%) and high positive predictive value(80-100%), it is considered by the 2005 International INPH Guidelines the mosteffective test for identifying shunt-responsive cases, even though it requireshospital admission and is associated with higher complication rates .The CSF tap test (CSF-TT) consists of quantitative testing of gait and cognitivefunctions before and after the drainage of 40-50 ml lumbar CSF. It is the onlyprocedure that can temporarily simulate the effect of a definitive shunt, and canpredict not only the outcome of surgery but also the degree ofimprovement.In NPH, computed tomography (CT) and magnetic resonance imaging (MRI) showventricular enlargement disproportionate to cerebral atrophy, with associatedballooning of frontal horns, periventricular hyperintensities, thinning andelevation of the corpus callosum, and widening of temporal horns without evidence ofhippocampal atrophy. Although diagnosis can be made on the basis of CT findingsalone, MRI is more accurate for disclosing associated pathologies (such ascerebrovascular disease) and also for detecting NPH typical signs of prognosticvalue, besides avoiding exposure to ionizing radiation.The International Guidelines have recommended the following key imaging features fordiagnosis of INPH and selection of shunt-responsive patients:Ventricular enlargement not entirely attributable to cerebral atrophy orcongenital enlargement (Evans index >0.3).No macroscopic obstruction to CSF flow.At least one of the following supportive features:Enlargement of the temporal horns of the lateral ventricles notentirely attributable to hippocampus atrophy;Callosal angle of 40º or greater;Evidence of altered brain water content, includingperiventricular signal changes on CT and MRI not attributable tomicrovascular ischemic changes or demyelination;An aqueductal or fourth ventricular flow void on MRI.12 alsoinclude the following key imaging features:The Japanese INPH Guidelines(1) enlarged Sylvian fissures and basal cistern; and(2) narrowing of the sulci and subarachnoid spaces over the highconvexity and midline surface of the brain. Unlike the InternationalGuidelines, periventricular changes are not considered essential.13 Newer MRI techniques such as DTI14 and resting-state functional MRI15 have also been studied in NPHallowing the possibility of detecting biomarkers and improving NPH diagnosis.However, the diagnostic and prognostic value of all these neuroimaging techniques isnot well established and they are not part of the routine selection procedures forshunt surgery.Other neuroimaging methods have also been used in NPH. Single photon emissioncomputed tomography (SPECT) and positron emission tomography (PET) can showreduction of cerebral blood flow and metabolism, mainly in frontobasal and anteriorperiventricular regions, even with improvement of regional cerebral metabolic rateof glucose in shunt responsive patients.16 which is the ratio between themaximal width of the frontal horns and the maximal width of the inner table of thecranium at the level of the frontal horns; or by an equivalent measure, such as bydividing the diameter of the frontal horns by the widest brain diameter  reliably determine ventricular enlargement,without the need for expensive, time-intensive and technically challenging computersoftware, unavailable in many healthcare services.The reliability of the Evans index has been questioned by some studies using modernbrain imaging techniques (FreeSurfer) for ventricular volumetric analysis.19Periventricular signal changes may be associated with subcortical vascularencephalopathy  in NPH, but this does not predictpoor surgical outcome and should not exclude patients from shunting.21 In normal aging and degenerative diseases , the thinning of the gyri and the corresponding dilation of the sulci aremore generalized, occurring to a similar degree in the affected brainregions.22In typical NPH cases, the ventricles are disproportionately more dilated than thecortical sulci, which are narrow or obliterated at the high convexity and midline,with local narrowing of the subarachnoid space surrounding the outer brain surface,as can be seen in a MRI coronal section at the level of the posterior commissure and 3. I5 as occurs in degenerative diseases such as Alzheimer's and Lewybody dementia.23 In a recentstudy, shunt-responders had a significantly smaller mean preoperative CA comparedwith non-responders (59% versus 68%), with a cut-off value of 63% providing the bestprognostic accuracy.24 Anotherconsistent finding in NPH, best recognized with mid-sagittal MRI, is callosal upwardelevation, stretching and thinning, usually showing total or partial recovery aftershunting.25Callosal angle (CA) as well as temporal horns and hippocampus are best evaluated withcoronal MRI. CA is the angle between the lateral ventricles , and is 26 althoughnarrow temporal horns are also compatible with NPH diagnosis.27 Evaluation of temporal horns andhippocampal/perihippocampal structures may distinguish hydrocephalic enlargement ofthe ventricle, as occurs in NPH  from ventriculomegaly secondary to cerebral atrophy .28Measurement of the hippocampus volume may be of diagnostic and prognostic value incases of suspected NPH versus Alzheimer's disease (AD), in which the hippocampus issignificantly smaller.29 Diffusiontensor imaging (DTI) of the fornix may also differentiate NPH from AD. Fornixvolume, cross-sectional area, and fractional anisotropy are smaller both in NPH andin AD relative to controls. Fornix length, however, is significantly greater in NPHthan in controls yet not altered in AD, probably explained by mechanical stretchingdue to lateral ventricular dilation and corpus callosum deformation in NPH and bydegeneration secondary to hippocampal atrophy in AD.30Wide temporal horns are typical of NPH and represent significant predictors of apositive shunt outcome,32 showed correlation between voidsign in the aqueduct and shunt results, while others33 did not, with the same frequency of void signoccurring in NPH patients and healthy controls.Aqueductal flow void is a loss (increased hypointensity) of signal seen within theaqueduct and neighbouring third and fourth ventricles, particularly on T2-weightedMRI images. The signal loss is usually increased in states of hyperdynamic CSFmotion, with higher velocities, turbulent and accelerated flow, which occur mainlyin passages with smaller cross-sectional areas such as the aqueduct. In NPH there isgreater outflow of CSF through the aqueduct with subsequent increase in signal loss(void sign). The first studies35 Theaqueductal CSF stroke volume  is then calculated. Initialstudies36 found that NPHpatients who responded to shunting had aqueductal stroke volume> 42 µL or at least twice the ACSV of healthyelderly subjects, while others did not.38 For this reason,a single PC-MRI measurement of stroke volume cannot reliably predict which patientswill improve after shunting.35 Inone study, 14% of patients who did not improve after a high-volume lumbar tap testhad significantly higher aqueductal CSF flow rates than patients whoimproved.38Further advances in CSF flow imaging have enabled quantification by means of cinephase-contrast MRI (PC-MRI) throughout the cardiac cycle. With this technique, theslice is positioned on an angled axial plane perpendicular to the aqueduct usinghigher spatial resolution MRI because of the small size of this structure, as wellas short TR to achieve adequate temporal resolution.39On the other hand, the combination of PC-MRI with the tap test by measuring the peakCSF flow velocity at the level of the aqueduct, before and after lumbar CSFdrainage, has been shown to be a sensitive method to support the diagnosis of NPHand select patients who are likely to benefit (or not) from shunt surgery.Thus, the findings of aqueductal CSF flow void alone, or of a single PC-MRImeasurement, have long been observed even in healthy persons, and cannot safelysupport NPH diagnosis or postsurgical prognosis.40have been introduced, but their predictive value for selecting shunt-responsive NPHpatients needs to be further investigated with more extensive studies.35More recent MRI techniques for CSF flow study, such as time-spatial labelinginversion pulse (Time-SLIP),In conclusion, NPH is a treatable cause of dementia, with the best shunting resultsoccurring when the CSF tap test is positive and CT or MRI show signs of highdiagnostic and predictive value, such as: ventricular enlargement disproportionateto cerebral atrophy (Evans index > 0.3) with ballooning of frontal horns;periventricular hyperintensities; corpus callosum thinning and elevation, callosalangle between 40º and 90º; widening of temporal horns not entirely explained byhippocampal atrophy; aqueductal or fourth ventricular flow void; enlarged Sylvianfissures and basal cistern, and narrowing of sulci and subarachnoid spaces over thehigh convexity and midline surface of the brain.


Treponema pallidum particle agglutination (TPPA) titer, CSF leukocyte count, and CSF protein concentration were correlated with both serum RPR and TPPA titers. At 6 months after treatment, 28 and nine patients achieved serological responses of RPR and TPPA tests, respectively. The sensitivities of the serological response of RPR and TPPA tests for identifying the normalization of CSF abnormalities were 60.0∼83.3% and 17.1~22.2%, respectively; and 75.0∼91.3% of patients showing serological response of RPR test also achieved CSF normalization, suggesting that the serological response could predict CSF normalization to some degree. Particularly, in patients with ≥8-fold decreases in the serum RPR titer, the CSF RPR, CSF leukocyte count, and CSF protein concentration had normalized, and follow-up lumbar puncture could be reduced considering the resolution of neurological symptoms.This study aimed to determine whether a serological response could predict the normalization of cerebrospinal fluid (CSF) abnormalities at 6 months after treatment in human immunodeficiency virus (HIV)-negative neurosyphilis patients. A total of 123 neurosyphilis patients were recruited at baseline, 58 of these patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study. Before treatment, the CSF rapid plasma reagin (RPR) titer, CSF  Treponema pallidum, which may occur at any stage of infection. Conventionally, the diagnosis of neurosyphilis is based on a reactive non-treponemal test , pleocytosis and/or elevated protein in cerebrospinal fluid (CSF) collected by lumbar puncture. In recent years, the serological activity of the RPR test was found to be associated with the development of neurosyphilis. In both human immunodeficiency virus (HIV)-infected and HIV-uninfected syphilis patients, those with higher serum RPR titer were reported to have a higher likelihood of neurosyphilis1. Therefore, the results of serum RPR tests have been recommended as predictors of neurosyphilis to avoid unnecessary lumbar puncture2.Neurosyphilis refers to infection of the central nervous system by 3. However, performing a lumbar puncture in a clinical setting is logistically difficult, and patients often refuse the procedure. Thus, there is an urgent need to find a surrogate for the normalization of CSF abnormalities after neurosyphilis treatment. Recently, researchers found that a 4-fold decrease in the serum RPR titer or reversion of the test to a nonreactive status could be a strong indicator of a successful neurosyphilis treatment4. In that study, >90% of patients demonstrating normalization of the serum RPR titer also showed normalization of clinical and CSF abnormalities, with the exception of the CSF protein concentration, at 13 months after treatment. However, 78% of the study participants had HIV infection and therefore presented characteristics different from HIV-negative syphilis patients5. Whether the HIV status influences the relationship between the serum RPR titer and treatment success remains unclear. Additionally, although treponemal tests should not be used to assess disease activity and treatment outcomes6, our preliminary study found that the likelihood of neurosyphilis was positively associated with the reactivity of serum Treponema pallidum particle agglutination (TPPA) tests7. In the present study, we assessed whether the serological response of the RPR and TPPA tests could help predict the normalization of CSF abnormalities after treatment in HIV-negative neurosyphilis patients.In addition to the diagnosis of neurosyphilis, the efficacy of neurosyphilis treatment is mainly assessed by CSF examination. A CSF examination is recommended to be repeated every six months to evaluate changes in CSF parameters and determine the need for retreatmentThe baseline characteristics of 123 neurosyphilis patients are shown in Table rs = 0.604, P < 0.001), CSF TPPA titer , CSF leukocyte count  and CSF protein concentration . Similarly, the serum TPPA titer was correlated with the CSF RPR titer , CSF TPPA titer , CSF leukocyte count  and CSF protein concentration . Neither the serum RPR titer nor the serum TPPA titer was correlated with CSF albumin, CSF glucose, CSF chloride, CSF lactate or CSF lactate dehydrogenase.The correlations between each CSF parameter and the serum RPR or TPPA titer were evaluated before neurosyphilis treatment Table . Serum RFifty-eight neurosyphilis patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study. Fifty-three of these patients received the recommended penicillin therapy; the other five were allergic to penicillin and were treated with ceftriaxone or doxycycline. These patients were divided into early syphilis and late syphilis subgroups. There were only three patients in the early syphilis subgroup; of these, none achieved a serological response of RPR test, and one achieved a serological response of TPPA test. No patients had reactive baseline CSF RPR. Two patients had an abnormal baseline CSF leukocyte count, and one of them exhibited normalization at 6 months after treatment. One patient had an abnormal baseline CSF protein concentration and exhibited normalization. The early syphilis subgroup was not included in further statistical analyses due to the small sample size.In the late syphilis subgroup, which consisted of 18 late syphilis patients and 37 patients with syphilis of unknown duration, 28 (50.9%) and eight (14.5%) patients achieved serological responses of RPR and TPPA tests, respectively. A ≥4-fold increase in the serum RPR or TPPA titer was not observed during the study period. Of 45 patients with reactive baseline CSF RPR, 26 (57.8%) achieved a response of CSF RPR test. Of 41 patients with an abnormal baseline CSF leukocyte count, 35 (85.4%) exhibited a normalized CSF leukocyte count. Of 33 patients with an abnormal baseline CSF protein concentration, 18 (54.5%) exhibited a normalized CSF protein concentration.P = 0.001). Similarly, the sensitivities of the serological response of the RPR test for identifying normalization of the CSF leukocyte count and CSF protein concentration were 60.0% and 83.3%, respectively. These values were higher than the respective sensitivities of the serological response of TPPA test  achieved CSF RPR responses. Furthermore, 77.8% had a normalized CSF leukocyte count, and 25.0% had a normalized CSF protein concentration. Fourteen patients had a 2-fold decrease, 19 patients had a 4-fold decrease, four patients had an 8-fold decrease, and four patients had a 16-fold decrease in the serum RPR titer. As the serum RPR titer decreased, the normalization of CSF abnormalities increased. In patients whose serum RPR titer decreased ≥8-fold, the CSF RPR, CSF leukocyte count, and CSF protein concentration had normalized  in neurosyphilis patients before treatment. In contrast, treponemal tests are considered irrelevant to disease activity due to persistent positive results for life. As a quantitative treponemal test, the TPPA test is based on agglutination. The quantitation of treponemal antibody is possible but has not been demonstrated to be worthwhile10. However, we found that the serum TPPA titer was also correlated with those CSF profiles before treatment. Our results suggest that serological activity of both the RPR and TPPA tests could reflect CSF abnormalities before neurosyphilis treatment.A high serum RPR titer has been reported to be associated with an increased likelihood of neurosyphilis4 with a cohort that included 78% HIV-infected individuals and showed sensitivities of >90%  for identifying CSF normalization. This inter-study discrepancy might due to the impact of HIV infection. Before neurosyphilis treatment, HIV-infected patients had a higher baseline serum RPR titer and were 2.5-fold less likely to show normalization of CSF-VDRL reactivity than HIV-uninfected subjects after treatment11.Furthermore, we determined whether seroreactivity continues to reflect the CSF profiles after neurosyphilis treatment. Among the 55 patients involved in this analysis, 28 achieved a serological response of RPR test, whereas only eight achieved a serological response of TPPA test at 6 months after treatment. A serological response of RPR test was more sensitive than a serological response of TPPA test for identifying CSF normalization, with sensitivities of 69.2%, 60%, and 83.3% for identifying normalization of the CSF RPR, CSF leukocyte count, and CSF protein concentration, respectively. These findings differ from those of a previous study3. In our study, despite moderate sensitivity, 91.3% (21/23) of patients with a serological response of RPR test also achieved normalization of the CSF leukocyte count. Further analysis showed that as the serum RPR titer decreased from null to 16-fold, a higher proportion of patients achieved normalization of CSF abnormalities. Notably, among patients whose serum RPR titer had decreased ≥8-fold, the CSF RPR, CSF leukocyte count, and CSF protein concentration all normalized. Our results suggest that a serological response of RPR test could serve as a surrogate for the normalization of CSF abnormalities after neurosyphilis treatment and that repeated lumbar puncture could be reduced in patients with ≥8-fold reductions in the serum RPR titer considering the resolution of neurological symptoms. Conventionally, treponemal tests are not used for assessing treatment outcome6. In the present study, 60.3% (35/58) of neurosyphilis patients did not exhibit any change in the serum TPPA titer at 6 months after treatment, suggesting that the development of a serum TPPA response was a long process. Of interest, among those who exhibited a serological response of TPPA test, more than 80.0% also experienced normalization of CSF abnormalities. This finding warrants further consideration and research.The leukocyte count is a sensitive measure of the effectiveness of therapy. The leukocyte count declines earlier than the CSF-VDRL or CSF protein concentration after therapy and determines the need for retreatmentOur study is the first to examine the utility of the serological response in predicting the normalization of CSF abnormalities after neurosyphilis treatment in HIV-negative individuals. However, our findings might be limited due to the inadequate sample size. Only 47.15% (58/123) of neurosyphilis patients underwent treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment, and only eight patients with ≥8-fold decreases in the serum RPR titer were analyzed. Fewer patients made return visits for follow-up at 12 months; accordingly, we failed to conduct a longer follow-up investigation. The baseline serum RPR, serum TPPA, and CSF RPR titer, CSF leukocyte count, and CSF protein concentration of the patients lost to follow-up were compared with those of the 58 study participants, and no significant differences were found. Although the loss to follow-up may have had little impact on the results, analysis of an expanded sample is needed.Our study has practical implications for clinicians caring for HIV-negative neurosyphilis patients. Before neurosyphilis treatment, the serological activity of RPR and TPPA might reflect CSF abnormalities. After neurosyphilis treatment, a serological response of RPR test was more sensitive for predicting normalization of CSF abnormalities than a serological response of TPPA test. In HIV-negative neurosyphilis patients with ≥8-fold decreases in the serum RPR titer, the CSF RPR, CSF leukocyte count, and CSF protein concentration had all normalized, and follow-up lumbar puncture could be reduced considering the resolution of neurological symptoms.Between June 2008 and December 2014, 195 hospitalized patients were diagnosed with neurosyphilis based on clinical and laboratory findings at Zhongshan Hospital, Medical College of Xiamen University. After excluding returning patients, HIV-infected individuals, and patients with unavailable serum RPR test results on admission, 123 neurosyphilis patients were included in the baseline study. Eventually, 58 neurosyphilis patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study  a reactive RPR in CSF, (2) positive TPPA assay and elevated leukocyte count (>10 cells/μL) in CSF, and (3) elevated CSF protein concentration (>500 mg/L) and/or leukocyte count (>10 cells/μL) in the absence of other known causes of these abnormalities, and clinical symptoms or signs consistent with neurosyphilis without other known causes for these clinical abnormalities. Asymptomatic neurosyphilis was defined as syphilis at any stage that met the laboratory criteria for neurosyphilis but without clinical symptoms or signs. Neurosyphilis patients who acquired syphilis infection during the preceding year were staged as early syphilis; patients who had positive serological tests for syphilis for >1 year, or whose duration of syphilis infection was unknown, were staged as late syphilis12.The diagnostic criteria for neurosyphilis complied with the guidelines of the Centers for Disease Control in the USA and EuropeIn this study, at 6 months after neurosyphilis treatment, a serological response of RPR or TPPA test was defined as a ≥4-fold (2 dilutions) decrease in titer  or reversion of the test to nonreactive. A serological nonresponse was defined as a <4-fold decrease or any increase in titer. A response of CSF RPR test was defined as a ≥4-fold decrease in titer or reversion of the test to nonreactive. Normalization of the CSF leukocyte count was defined as a decrease from >10 cells/μL to ≤10 cells/μL. Normalization of the CSF protein concentration was defined as a decrease from >500 mg/L to ≤500 mg/L.16, as follows. Briefly, the samples were subjected to the RPR test with an 18-mm circular test card following a standardized method. The antigen suspension contained carbon particles coated with cardiolipin antigens. Approximately 50 μL of serum was dispensed over the card, followed by a drop of the antigen suspension. The card was placed on a rotating plate at 100 rpm for 8 min to mix the antigen suspension and serum. Readings were obtained immediately with the naked eye through comparisons with negative and positive controls. The serum samples that reacted to RPR were quantified using 2-fold serial dilutions. The TPPA assay is based on the agglutination of colored gelatin particles that have been sensitized (coated) with T. pallidum (Nichols strain) antigen. The initial dilution of the serum samples used in the TPPA reactions was 1:80. Two standard serum samples (including 400 mIU/mL and 80 mIU/mL)  were used as controls for the RPR and TPPA reactions, respectively. Serum samples that produced conflicting or inconclusive results for a particular technique were tested again; the new result was considered the true result.The CSF examination and serological tests for syphilis were performed using RPR  and TPPA  tests according to the manufacturers’ instructions and as previously reportedCSF samples (approximately 2 mL) were collected within a time frame of 2 h. Blood (5 mL) was collected in plain sterile tubes from patients who had fasted for at least 8 h. The blood was then centrifuged within 10 min and analyzed within 4 h. The protein, albumin, lactate, lactate dehydrogenase, chloride, and glucose levels in the CSF samples were measured using a Roche-Hitachi Modular P800 Analyzer . The CSF leukocyte count was measured using an Automatic Blood Cell XE5000 Analyzer .2 transformed for the correlation analysis. McNemar’s test was used to compare the sensitivities of the serological responses of RPR and TPPA tests for identifying the normalization of CSF abnormalities. The statistical analyses were performed using SPSS 19.0 for Windows . A two-sided P-value of <0.05 was considered statistically significant.Spearman’s rank correlation was used to analyze the correlations between each of the CSF parameters and the reactivity of both serum RPR and serum TPPA. Serum RPR and serum TPPA, which are quantitative measures with serial 2-fold dilutions, were log


Meningeosis neoplastica is a diffuse metastatic spread of tumor cells in the subarachnoid space. Although first recognized in 1870, systematic investigations regarding cerebrospinal fluid (CSF) constituents in this condition are scarce.Routine CSF samples analyzed from 2001 to 2012 at the Laboratory of Clinical Neurochemistry, University of Göttingen, were re-evaluated. Patients, whose CSF contained malignant cells were included in this study.-3 (median 17.5 x 10-3). Total CSF protein ranged from 166 to 15,840 mg/l (median 1012 mg/l). CSF lactate was elevated (>2.4 mmol/l) in 65.2% . In 50% of all patients CSF lactate was ≥3.5 mmol/l. The CSF cell counts correlated significantly with the CSF lactate levels and the CSF protein contents. In 56 of 118 CSF samples (47.5%) ferritin was elevated, and in 25 of 65 carcinoma patients (38.5%) an intrathecal production of carcinoembryonic antigen (CEA) was detected. Granulocytes were found in 52.7% of the CSF samples. The percentages of granulocytes and lymphocytes were higher in samples with an elevated cell count.Patients  were identified, whose CSF contained malignant cells. The most frequent primary tumor was breast cancer (32.6%), followed by lung cancer (25.0%) and hematologic malignancies (21.2%). The most frequent clinical symptoms were affections of cranial nerves (41.7%), psychiatric abmormalities (32.6%) and radicular lesions of the lower extremities (20.5%). CSF cell counts ranged from 0 to 4692 cells/μl (median 4 cells/μl) and were elevated in 50%. The CSF-to-serum albumin ratio was abnormal in 69.4%. It ranged from 1.8 to 330 x 10In approximately 50% of CSF samples with meningeosis neoplastica the CSF cell count is not elevated. Diagnosis may be missed when only CSF samples with elevated cell counts are subjected to cytological analysis. CSF lactate and protein and the CSF-to-serum albumin ratio are frequently increased in meningeosis neoplastica. The differential diagnosis between meningeosis neoplastica and central nervous infections, in particular tuberculous or fungal meningitis, can be difficult. Meningeosis neoplastica, the infiltration of the meninges and the subarachnoid space by malignant cells as a consequence of metastatic cancer, was first described by Karl Joseph Eberth as early as 1870 , 3. TumoFrequent clinical symptoms suggesting meningeosis neoplastica are headache, changes in mental status, difficulty in walking, nausea, vomiting, diplopia, lower motor weakness, limb paresthesia, back or neck pain, and radiculopathy . Many anAn early diagnosis of meningeosis neoplastica, before persisting neurologic deficits have developed, permits earlier and potentially more effective treatment, thereby leading to a better quality of life in affected patients . Since tThe medical files including lumbar or ventricular CSF of patients with meningeosis neoplastica, who were treated between January 1, 2001, and December 31, 2012, with different clinical symptoms in the University Hospital Göttingen, in the Protestant Hospital Göttingen-Weende and other regional hospitals, were retrospectively analyzed. The inclusion criterion was the presence of tumor cells as assessed by morphological criteria. Clinical symptoms were assessed by review of the patients’ medical files. We also assessed the results, when cranial computer tomography (CCT) or magnetic resonance imaging (CMRI) was performed. The study was approved by the Ethics Committee of the Medical Faculty of the Georg-August University Göttingen, Germany.After lysis of the erythrocytes, CSF cells were counted manually in a Fuchs-Rosenthal chamber. A cell count of ≤4/μl was considered normal. CSF cell differentiation was performed after cell sedimentation by means of a cytocentrifuge  and May-Grünwald-Giemsa (MGG) staining . Cytology was performed on MGG-stained cytospins. The appearance of tumor cells depended on the origin of the primary tumor. Tumor cells were in general larger than mononuclear hematopoietic cells. Furthermore, they frequently showed a marked increase in the nuclear/cytoplasmic ratio, basophilic cytoplasm, hyperchromasia, irregular nuclear membrane, pseudopodia and increased number of nucleoli. Especially in the case of epithelial cancer, tumor cells frequently lay in clusters and occasionally, in part atypical, mitotic figures were observed. Depending on the primary tumor, tumor cells occasionally showed evidence for mucus production or pigmentation. The CSF protein content was measured by nephelometry after precipitation with trichloroacetic acid in a Dosascat nephelometer  using Gesamteiweiß UC Standard FS  . CSF andIn addition to cytology, protein biomarkers were established especially for carcinomas invading the CNS and the leptomeninges . CarcinoThe CSF concentration of ferritin was measured by nephelometry .S. p values ≤0.05 were considered statistically significant.Statistical calculations were performed with GraphPad Prism software . Data was described by means ± standard deviations (SD), when normally distributed. In the absence of normal distribution data was described by medians and interquartile ranges or as medians and ranges (minima/maxima). For univariate analyses, we used Mann–Whitney U test in the absence of normal distribution. The relation between CSF cell count and CSF lactate concentration and CSF protein was assessed by Spearman’s rank correlation coefficient, rA group of 132 patients with meningeosis neoplastica  was included in this study. In 128 patients lumbar CSF and in four patients ventricular CSF was studied.Clinical symptoms of the patients studied are depicted in Table 97 patients with cytologically proven meningeosis neoplastica received cranial imaging  within a short time period before or after the diagnostic lumbar or ventricular CSF analysis. In 50 of these patients (51.5%), typical signs of meningeosis neoplastica were present on CCT or CMRI.The results of CSF analysis in patients with meningeosis neoplastica are presented in Table Overall, lymphocytes were the most abundant cell population. Granulocytes were found in 52.7% of the CSF samples, in only two patients was the percentage of granulocytes >50%. Both of them had more than 500 cells/µl. In the CSF of patients with >4 cells/μl, lymphocytes and granulocytes were significantly more frequent than in the CSF with ≤4 cells/μl .S = 0.45, p < 0.001)  in 102 of 130 patients (78.5%). The CSF protein was positively correlated with the CSF cell count   in 65.2% . In 66 of 132 patients (50%), CSF lactate was strongly elevated (≥3.5 mmol/l). The CSF lactate concentration was correlated with the CSF cell count  ferritin was elevated, and in 25 of 81 cases (30.9%) an intrathecal production of CEA was detected. No intrathecal CEA production was detected in lymphoma patients. Of the 65 patients with meningeosis carcinomatosa, 25 patients (38.5%) had intrathecal CEA synthesis.Clinical signs and symptoms of the patients included in this study were very similar to those reported by others . CSF anaM. tuberculosis, fungi and other opportunistic pathogens, which also can cause CSF alterations similar to those observed in meningeosis neoplastica.When the CSF cell count is elevated and malignant cells are not present or are overlooked, differentiation between meningeosis neoplastica and infections or autoimmune diseases affecting the CSF space can be difficult. In particular, tuberculous meningitis can be confounded with meningeosis neoplastica, because the clinical symptoms and CSF abnormalities can be similar. Moreover, many patients suffering from malignant diseases are immunosuppressed either by chemotherapy or the underlying disease. They, therefore, are prone to develop CNS infections with Elevated CSF lactate is not specific for certain diseases. Meningeosis neoplastica can cause a CSF lactate elevation , but a cMeningeosis neoplastica frequently is associated with an inflammatory response. The presence of intrathecal IgG, IgA and IgM synthesis as well as the high proportion of lymphocytes in the CSF of patients with meningeosis neoplastica indicate a strong involvement of the adaptive immune system. As in neuroborreliosis, isoelectric focusing was more sensitive for detecting an intrathecal IgG synthesis than the Reiber–Felgenhauer nomogram . The preThe CSF protein concentration correlated positively with the CSF cell count. One explanation for this is an abnormal blood–CSF barrier function with increased permeability for leukocytes and serum-derived proteins. In particular, the leptomeningeal structures and the epithelial cells of the choroid plexus can be infiltrated by neoplastic cells, resulting in an increased permeability for serum compounds . AnotherFerritin CSF concentrations were elevated in 56 of 118 patients (47.5%). CSF ferritin concentrations are elevated after intracranial bleeding and in neoplastic and meningitic CNS diseases, but this parameter is neither specific nor sensitive. An elevated CSF ferritin reliably, but non-specifically indicates severe CNS disease .Carcinoembryonic antigen was measured in 81 patients. In patients with metastatic CEA-producing tumors, diffusion of CEA across the blood–CSF and blood–brain barrier cannot be neglected. The CSF concentrations depend on the respective serum concentration, and absolute cut-off values are not appropriate. Because of the similar molecular mass of CEA and IgA, the Reiber–Felgenhauer nomogram for IgA can be used to detect intrathecal CEA synthesis , 14, 26.The two main findings of this study are: (1) In half of the CSF samples with meningeosis neoplastica the CSF cell count is not elevated. Therfore, diagnosis can be missed when only CSF samples with elevated cell counts are subjected to cytological analysis. (2) CSF lactate and protein and the CSF-to-serum albumin ratio are frequently increased in meningeosis neoplastica. The differential diagnosis between meningeosis neoplastica and central nervous infections, in particular tuberculous or fungal meningitis, can be difficult. For the detection or exclusion of malignant cells in the CSF, cytology performed by an experienced pathologist is necessary.


Neoplastic invasion into leptomeninges and subarachnoid space, resulting in neoplastic meningitis (NM) is a fatal complication of advanced solid and hematological neoplasms. Identification of malignant involvement of the cerebrospinal fluid (CSF) early in the disease course has crucial prognostic and therapeutic implications, but remains challenging. As indicators of extracellular matrix (ECM) degradation and breakdown of the blood–brain-barrier, Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloproteases (ADAMs) are potential analytes for cerebral pathophysiology and metastatic dissemination of tumor cells into the CSF.We compared protease activities in CSF samples from patients with NM and control individuals using FRET-based metalloprotease substrates with distinct enzyme selectivity profiles in a real-time, multiplex approach termed “proteolytic activity matrix assay” (PrAMA). Protease activity dynamics can be tracked by fluorescence changes over time. By simultaneously monitoring a panel of 5 FRET-substrate cleavages, a proteolytic signature can be identified and analyzed to infer the activities of multiple specific proteases. Distinct patterns of substrate cleavage comparing disease vs. control samples allow rapid, reproducible and sensitive discrimination even in small volumes of CSF.Individual substrate cleavage rates were linked to distinct proteases, and PrAMA computational inference implied increased activities of MMP-9, ADAM8 and ADAM17 (4–5-fold on average) in CSF samples from NM patients that were inhibitable by the metalloprotease inhibitor batimastat (BB-94). The activities of these proteases correlated with blood–brain barrier impairment. Notably, CSF cell counts were not found to directly reflect the protease activities observed in CSF samples from NM patients; this may explain the frequent clinical observation of negative cytology in NM patients.PrAMA analysis of CSF samples is a potential diagnostic method for sensitive detection of NM and may be suitable for the clinical routine.The online version of this article (doi:10.1186/s12987-017-0070-5) contains supplementary material, which is available to authorized users. Leptomeningeal metastasis (LM) resulting in Neoplastic Meningitis (NM) is a spread of malignant cells to the leptomeninges, the subarachnoid space and dissemination of tumor cells within the cerebrospinal fluid (CSF). NM is a complication of patients with progressive cancer (70%) and prognosis is poor once manifest NM with neurological deficits develops . PatientRecently, the combination of neurological examination, radiographic imaging of the neuroaxis and CSF cytology are used to diagnose NM , 4. HoweThe metastatic invasion of tumor cells and dissemination to secondary sites is promoted by deregulated activity of extracellular metalloproteases (MPs), such as matrix metalloproteases (MMPs) and A Disintegrin and Metalloproteases (ADAMs). Both classes of proteases are members the metzincin superfamily, meaning that they utilize a complexed zinc ion in their catalytic site to constitute proteolytic activity. Major roles for MMPs and ADAMs in cancer progression have been reported, in particular, tumor cell invasion, angiogenesis and metastasis , 15. CanIn order to obtain metalloprotease profiles for patients with NM in a pilot study, we utilized a multiplex approach for simultaneous detection of MMP and ADAM activities in the CSF of patients with NM compared to control individuals and tumor patients without NM. The Proteolytic Activity Matrix Analysis (PrAMA) is a combined experimental and mathematical method based on time-lapse fluorescence measurements of a panel of moderately specific FRET-based polypeptides in small volumes of biological samples. The observed cleavage patterns are compared to a standard table of catalytic efficiencies measured from purified mixtures of recombinant metalloproteinases and should reflect changes in specific enzyme activities , 39 in Cg to remove cells. Clarified CSF was aliquoted, snap-frozen in liquid nitrogen and stored at −80 °C for further analysis. Clinicopathological features were documented pseudonymized, patients were grouped in control individuals (crtl), patients with neoplastic meningitis (NM) and patients with brain metastases without neoplastic meningitis (w/o NM) according to neurological examination, contrast-enhanced MRI of the brain and neuroaxis and CSF cytology for clinical diagnosis.This pilot study obtained ethical approval from the local Ethics Committee . Informed consent was provided from patients to use their biological specimens and clinicopathological data for research purposes. No selection criteria were applied, i.e. all available patients with suspected diagnosis of NM and with brain metastases undergoing routine lumbar puncture were recruited into the study. CSF samples in the control group were collected during therapeutic lumbar punctures from patients with normal pressure hydrocephalus (3/12) or idiopathic intracranial hypertension (9/12). Patients with normal CSF cytology and blood–brain-barrier (BBB) physiology without clinical evidence for neuro-infectious, neuro-inflammatory, neuro-degenerative or neoplastic brain pathologies were considered as controls. All CSF samples were collected at the Department of Neurology, Philipps University Marburg, Germany. Lumbar puncture to collect CSF was performed by medical staff according to clinical guidelines. Upon collection, CSF served primarily for diagnostic purposes, remaining CSF was placed on ice and centrifuged at 10002, 20 mM Tris–HCl pH 8.0, 10 mM CaCl2, 150 mM NaCl, 6 × 10−4% Brij-35) was incubated with 50 μl of CSF using 96-well microtiter white opaque plates, each sample was run in technical triplicates. Samples containing sufficient volumes were included for inhibitor studies and repetitive measurements. To some samples, the broad-range MMP/ADAM inhibitor batimastat  was added at a concentration of 1 μM dissolved in DMSO. Fluorescence units versus time were monitored with a Fluostar BMG Optima using excitation and emission wavelengths of 485 and 530 nM at 37 °C, respectively. A non-linear model was used for curve fitting as described previously [Cell-free cerebrospinal fluids were tested for MMP/ADAM activity by using the Proteolytic Activity Matrix Analysis (PrAMA) technique developed by Miller et al. using FRET-based polypeptide substrates PEPDab005, PEPDab010, PEPDab008, PEPDab013 and PEPDab014 , which vary in their specificities towards different ADAM family members and MMPs. PrAMA analysis was performed as described earlier . BrieflyT = 1.4 [t test to compare two sample groups. To compare more than two experimental groups, Analysis of Variance (ANOVA) was used. Values are denoted as not significant , significant * (p ≤ 0.05), highly significant ** (p ≤ 0.01), or very highly significant *** (p ≤ 0.001).The increase in fluorescence resulting from substrate proteolysis was tracked every 5 min for 4 h. For interpretation of time-lapse fluorimetry data, a non-linear curve-fitting model that accounted for substrate depletion and photobleaching decay served to determine cleavage rates. Cleavage rates are all presented in heat maps averaged over technical triplicates, clear outliers were excluded using Dixon’s Q-Test with a 90% threshold. PrAMA inference was performed as described previously with 30% sampling error and threshold σT = 1.4 . Based oTo group the generated datasets, observed average cleavage rates were hierarchically biclustered mean-centered and variance-normalized by row, using Euclidean distance, average linkage and optimal leaf order. Clear patterns emerging from cluster analysis are indicated by dendrograms flanking the array.To investigate the relationship between CSF cell count, blood–brain-barrier impairment and protease activities, Pearson’s correlation coefficients (r) were calculated between these parameters, respectively. Statistical significance (p) was determined as described in the section above.According to the clinical diagnosis of medical professionals at the department of Neurology, patients were assigned to one of three groups: patients with neoplastic meningitis , patients with brain metastases but without leptomeningeal tumor spread  or controls . CSF controls were characterized by normal CSF cytology, physiological protein concentrations, normal albumin and immunoglobulin quotients and absence of oligoclonal IgG bands, thus excluding inflammation, infection and impairment of the blood–brain-barrier. Comparison of baseline demographics for each group did not show significant differences. The mean age was 58.0 years (range 48–60 years) for patients with NM, 53.5 years (range 24–75 years) for patients with brain metastases w/o NM and 41.2 years (range 20–68 years) for controls. In our patient cohort, the NM cases were solely from female patients  since NM is most prevalent in breast cancer patients. Accordingly, we recruited patients w/o NM and control group as far as possible from other female patients so that the proportion of female patients was higher than males .−6 for PEPDab 8; p = 0.00013 for PEPDab 10; p = 0.09 for PEPDab 13; p = 0.0009 for PEPDab 14) whereas there were no significant changes detectable between crtl and patients with brain metastatic cancer w/o NM.Clinically obtained CSF samples from controls, patients with NM and patients with brain metastases but no leptomeningeal metastasis were subjected to time-lapse fluorimetry and analyzed for substrate cleavage rates of FRET peptides PEPDab 5, 8, 10, 13 and 14 in parallel , ADAM8  and ADAM17 . Changes for MMP-2 were not significant, ADAM10 was not identified in the enzyme composition of the CSF samples by PrAMA. Of the proteases identified, PrAMA inference suggests the activities of MMP-9 and ADAM17 were the highest on average  with a high to good correlation coefficient (r) depending on the inclusion of one patient  by ELISA . Molecularly targeted therapies that aim at preventing tumor cells from invasion into the CSF space may offer more efficacy. At present, several pharmacological inhibitors of MPs are already available and ADAM-inhibitors are being developed. After a more detailed analysis of the pathophysiological mechanisms, pharmacological inhibition of MPs and ADAM proteins may be efficient tools to prevent or treat malignant CSF-invasion.In this respect, our results from a multiplexed assay provide a novel perspective into MMP/ADAM contribution to the pathophysiology of NM. More importantly, the simple profiling of protease activities in CSF samples from patients with medical indications seems to reflect individual context-dependent protease function in invasive diseases and should be explored in a larger cohort of NM patients but also in other clinical applications including infectious diseases in the CNS.Based on these pilot data we demonstrated that the simultaneous detection of metalloprotease activities in CSF samples by the PrAMA method could be of diagnostic value to distinguish NM from metastasis without NM and from controls with normal CSF. The observed cleavage profiles are consistently present in patients with a positive follow-up analysis and are modulated by successful treatment of NM. Moreover, we detected ADAM8, MMP-9, and ADAM17 activities in the CSF as major analytes in the CSF of patients with NM.