Cognition is often affected early in Parkinson’s disease (PD). Lewy body and amyloid β (Aβ) pathology and cortical atrophy may be involved. The aim of this study was to examine whether medial temporal lobe structural changes may be linked to cerebrospinal fluid (CSF) biomarker levels and cognition in early PD. PD patients had smaller volumes of total hippocampus, presubiculum, subiculum, CA2–3, CA4-DG, and hippocampal tail compared with normal controls (NCs). In the PD group, lower CSF Aβ38 and 42 were significant predictors for thinner perirhinal cortex. Lower Aβ42 and smaller presubiculum and subiculum predicted poorer verbal learning and delayed verbal recall. Smaller total hippocampus, presubiculum and subiculum predicted poorer visuospatial copying. Lower Aβ38 and 40 and thinner perirhinal cortex predicted poorer delayed visual reproduction. In conclusion, smaller volumes of hippocampal subfields and subhippocampal cortex thickness linked to lower CSF Aβ levels may contribute to cognitive impairment in early PD. Thirty-three early PD patients  and NC had 3 T magnetic resonance imaging (MRI) scans. The MRI scans were post processed for volumes of hippocampal subfields and entorhinal and perirhinal cortical thickness. Lumbar puncture for CSF biomarkers Aβ38, 40, 42, total tau, phosphorylated tau (Innogenetics), and total α-synuclein  were performed. Multiple regression analyses were used for between-group comparisons of the MRI measurements in the NC and PD groups and for assessment of CSF biomarkers and neuropsychological tests in relation to morphometry in the PD group. Approximately, 25% of non-demented PD cases have mild CI (MCI), which may precede dementia.2–4 Hippocampal atrophy has been associated with CI, especially poorer memory, in PD with and without dementia.3Imaging studies in PD have shown different patterns of magnetic resonance imaging (MRI) cortical gray matter atrophy. In PD without dementia, little or no regional thinning has been found in hippocampus, temporal, limbic, frontal, parietal, or occipital cortex, but there are more pronounced changes in PD-MCI and widespread atrophy is described in PD with dementia.6 The hippocampal subfields are in varying degree present within the head, body, and tail of the hippocampus.7 The hippocampus proper includes the dentate gyrus (DG) tightly connected to CA4, and the remaining subfields of cornu ammonis (CA3-1)5 filled by densely packed pyramidal cells6 similarly layered as the neocortex. As described in primate models, associative cortical input is transferred mainly through PRC and ERC, and processed through the hippocampal subfields by way of the highly plastic connections in the polysynaptic loop to CA1, subiculum, and presubiculum.6 These output structures project back to ERC, PRC and prefrontal, thalamic, parahippocampal, and retrosplenial regions.5Learning, memory, and visuospatial functions are related to hippocampus and subhippocampal regions , and perirhinal cortex (PRC)) of the medial temporal lobe.8–12 that are related to poorer memory functions.9–11 Selective vulnerability of different hippocampal regions in PD with and without dementia have been reported in a few studies that found atrophy in the hippocampal head,14 CA1-4, DG, subiculum, the whole hippocampus, and ERC in association with poorer memory functions.15–18MCI and Alzheimer’s disease (AD) have been linked to medial temporal lobe affection including CA1-4, DG, subiculum, presubiculum, ERC, and PRC19 Neuropathological changes in the hippocampus have been shown in postmortem PD brains with and without dementia, including Lewy neurites, especially in CA2  plaques.A2 refs. ,21, Lewy23 On a group level, amyloid species  are lower than in controls and associated with CI in PD.24–29 In most studies, tau levels are reduced or unchanged in PD without dementia.29 CSF total α-synuclein (t-α-syn) is reduced or unchanged in PD,29 but its association with CI is not yet clarified.27–31Aβ, tau, and α-synuclein neuropathologies are reflected as neurochemical changes in the cerebrospinal fluid (CSF), and CSF Aβ42, total tau, and phosphorylated tau have been established as biomarkers for AD diagnosis.8 Studies in AD have found associations between lower CSF Aβ42 and CA1 deformation, and hippocampal atrophy and higher T- and phosphorylated tau, and hippocampal, parahippocampal, and ERC atrophy.32 However, very few studies have assessed the relationship between CSF biomarkers and atrophy in PD. Aβ species and total tau have been associated with ventricular enlargement, but not with hippocampus volume.33 Associations between lower Aβ42 and higher tau and frontal, parietal and temporal cortical atrophy were found in PD with dementia,34 and lower Aβ42 in non-demented PD patients was prospectively associated with parietal and occipital thinning.26 An association between low t-α-syn and frontal cortical thinning in PD without dementia has also been reported.31 To our knowledge, no other study has evaluated the hippocampal subfields in relation to CSF biomarkers in PD.In AD and normal controls (NCs), links between brain atrophy and CSF biomarkers have been reported, supporting the hypothesis that the biomarkers reflect regional or global neurotoxicity related to protein dysmetabolism.The aims of this study were to measure structural changes of hippocampal subfields and subhippocampal regions and explore possible associations with CSF biomarkers and neuropsychological functions in early, non-demented PD patients with and without MCI. We hypothesized that PD patients have smaller hippocampal subfield volumes compared with NC, and that these changes are associated to CSF biomarkers of neurodegeneration as well as to learning, memory, and visuospatial functions.Descriptive data and CSF measurements of the study participants are presented in P=0.558).Associations between CSF biomarkers and MRI variables in the PD group are shown in Finally, we assessed the MRI variables and CSF biomarkers as predictors of the neuropsychological tests . Lower A11The present study is the first study to assess hippocampal subfields and subhippocampal regions in relation to CSF biomarkers in PD patients, proposing Aβ species as early predictors of PRC thickness and as predictors of CI in conjunction with hippocampal subfields. It confirms selective vulnerability of the hippocampal area in early PD patients without dementia. Specifically, we found that subiculum and the hippocampal tail were significantly smaller in PD compared with NC. When exploring the association with cognitive domains in the PD group, we found that smaller subiculum and presubiculum predicted poorer verbal learning and delayed verbal recall, and together with Aβ42, they predicted cognition even better. Lower total hippocampus, presubiculum, and subiculum volumes were associated with poorer visuospatial copying. None of the hippocampal subfields showed associations with the CSF biomarkers. Our findings are in line with studies in primate models and patients with amnestic MCI that have shown associations between hippocampus, subiculum, and presubiculum, and memory and visuospatial functions.15 This is partly in line with our study, and the discrepancies may be due to different PD sample types. For example, our sample had shorter disease duration, fewer with MCI, no one had hallucinations, and there were partly different neuropsychological tests.One earlier study in PD using the same subfield segmentation technique found volume loss in CA2–3, CA4-DG, subiculum, and the whole hippocampus, and this correlated with verbal learning.16 They did not find any significant associations between CSF amyloid or tau species and hippocampal radial distance thickness.33 The discrepancies with our study may be due to the use of different segmentation techniques, radial distance mapping, and neuropsychological tests.Another study in early PD with and without CI found associations between radial distances as a measure of atrophy in CA1, CA3, and subiculum, and poorer verbal recall and recognition, suggesting that these functions are impaired at least partly due to structural hippocampal changes.Even though PRC thickness was not different in PD compared with NC, lower Aβ38 and 42 were significant predictors for thinner PRC in the PD group only. Both lower Aβ38, 40, and thinner PRC predicted poorer delayed visual reproduction, but these associations disappeared when analyzed together. This may indicate that there is a relationship between Aβ species and PRC and delayed visual reproduction, but the findings are not consistent and more research are needed with larger sample sizes to confirm the result. To our knowledge, this is the first study to assess PRC thickness in PD.35 and is also affected in PD.22 Aβ may contribute to hyperphosphorylation of tau, which is the main component of neurofibrillary tangles.36 PRC have been linked to visual recognition memory,5 and CSF Aβ42, visual recognition memory, and PRC are all affected early in patients with amnestic MCI and AD.37 A study in early PD found correlation between low CSF Aβ42 and poorer delayed visual pattern recognition memory in early PD.28 On the basis of this and our findings that lower Aβ42 and 38 are predictors for thinner PRC in early PD, we hypothesize that Aβ pathology leads to thinning of PRC, possibly through development of neurofibrillary tangles, and subsequently impaired visual recognition memory. As the present study has a low number of cases with potential bias possibilities, it is important to explore this theory in a larger study population and also include tests of visual recognition memory.PRC is one of the first areas affected by neurofibrillary tangles in pre-dementia AD,38 However, longitudinal follow-up is needed to test this hypothesis in more detail.A recent amyloid PET study in cognitively normal elderly showed that cortical Aβ load was related to functional MRI disruption of functional connectivity of the PRC, supporting the hypothesis that dysfunction in PRC may be a very early sign of cortical amyloid deposition and predict memory impairment and dementia.32 our findings are not unique to early PD, but may reflect concomitant AD or at least shared Aβ and tau pathology.As hippocampal subfields and subhippocampal regions are affected and related to poorer memory functions and CSF Aβ42 and T- and phosphorylated tau in MCI and AD,18 We found no such association and the discrepancy may be due to longer disease duration in other studies that most of the patients included already had MCI or dementia or also different neuropsychological tests used in these studies.ERC has been reported to be smaller in PD and is associated with poorer learning and memory.27–31 but a possible enhancing interaction with Aβ has been proposed.40 We found no association between t-α-syn CSF levels and cognition or hippocampal subfield atrophy. More studies are needed to explore the role of t-α-syn and cognition, and CSF studies should measure other α-synuclein species as well.Although α-synuclein is a synapse protein and a major component of Lewy bodies, the role in CI is unclear,4There was no cortical gray matter thinning in other brain regions, suggesting that differential hippocampal atrophy precedes gray matter changes in early PD. This is in line with another study describing hippocampal atrophy as a biomarker of initial CI in PD.This is the first study to assess hippocampal subfields and subhippocampal regions with 3 T MRI in relation to CSF biomarkers and cognition in early PD patients. Another strength of this study is CSF t-α-syn measurement in addition to the traditional biomarkers.42 but we only included the larger hippocampal structures with the best Dice overlap coefficients with manual segmentation.41 Anatomical subfields differences may arise due to different segmentation techniques, and a unified segmentation protocol is needed.3 -T MRI high-field strength employed here allows more detailed imaging of hippocampal substructure. All segmentations were manually inspected, and the image and segmentation quality and consistency were considered good. The segmentation technique was automated, and whether manual segmentation is superior to automated is debated,15 required 19–41 subjects in each group. Power calculations for subiculum and presubiculum only demanded more participants than in our study. This suggests that the number of participants is adequate to see the most relevant differences. The cross-sectional design is another limitation, and subsequent longitudinal data from this study might inform about the relation between progression of CSF biomarkers, atrophy, and cognition.Another limitation of the study is the low number of participants. Power calculations for the hippocampal subfields to detect a difference of 7.5% between PD and NC, based on a power of 80%, a significant alpha of 0.05 and s.d.’s for the different hippocampal subfields available from another study,In conclusion, regional hippocampal atrophy is present early in PD patients and related to impairments in verbal learning and memory and visuospatial functioning, and probably connected to Aβ pathology. The perirhinal cortex might be the first affected area in PD contributing to impairment of visuospatial memory.The patients in this cross-sectional study were consecutively recruited from a university hospital based neurological outpatient clinic from 2011 to 2014 ref. .43 Hoehn and Yahr stage <3 and disease duration ⩽6 years were included. Neurological examination and diagnosis, the unified PD rating scale part III motor examination and Hoehn and Yahr staging were performed by trained research physicians and movement disorder specialists. All PD patients had pathological ioflupane (123I) single-photon emission computed tomography (DaTSCAN) to support the clinical diagnosis of PD. The 15-item Geriatric Depression Scale was used to assess depression. Total daily levodopa equivalent dose was calculated.44 Exclusion criteria were dementia, and other severe somatic or psychiatric comorbidity that could contribute to CI, including cancer under treatment, ischemic stroke, drug abuse, solvent exposure, and moderate to severe depression.Thirty-three patients diagnosed with PD according to the UK Parkinson’s Disease Society Brain Bank clinical diagnostic criteria,45 Associations between hippocampal subfields and neuropsychological tests were explored in 30 patients using raw scores as previously described;29 verbal learning and delayed recall  were measured by Rey Auditory Verbal Learning Test. Visuospatial ability and delayed visual reproduction after 30 min were measured by Rey Complex Figure Test. Aspects of executive functions were assessed by tests measuring divided attention (Trail-Making Test-B), response inhibition (Stroop Color Word), and verbal fluency . High scores on these tests represent good achievement, except for Trail Making Test-B and Stroop Color Word where high scores represent poor performance.Cognitive screening tests were performed in all PD patients and used to categorize the patients into groups of no CI, SCI, MCI, or dementia (exclusion criterion) as described earlier.n=16) and from subjects undergoing elective orthopedic surgery in spinal anesthesia (n=16). Inclusion criteria were as previously described29 normal cognition based on Mini-Mental State Exam ⩾28, Rey Auditory Verbal Learning Test delayed recall, Trail Making Test-B, and Controlled Oral Word Association Test. Exclusion criteria were as for PD, in addition to relevant subjective cognitive memory symptoms, PD and AD.Thirty-two neurologically healthy NCs were recruited from volunteers, spouses, or relatives of participating patients . In PD patients, Aβ38, 40, and t-α-syn levels were determined by electrochemiluminescence using anti-Aß 6E10 detection antibody . As red blood cells contain α-synuclein antigenicity, a cutoff value of 350 ng/ml for hemoglobin was determined, and samples with higher hemoglobin concentrations were excluded from the further analysis as previously recommended,http://surfer.nmr.mgh.harvard.edu/) as previously described.12 This includes segmentation of the subcortical white matter and deep gray matter volumetric structures,47 and parcellation of the cortical surface according to a previously published parcellation scheme.48 This labels cortical sulci and gyri, and thickness values are calculated in the regions of interest (ROI). The thickness values of the ERC and PRC were calculated using methods based on ultrahigh resolution ex vivo applied to in vivo MRI, as implemented in FreeSurfer.50 Further, an automated procedure for segmentation of the subfields of the hippocampus, as implemented in FreeSurfer, was employed.41 From the hippocampal segmentation, we chose to analyze volume of the following subregions; subiculum, presubiculum, cornu ammonis sector 1 (CA1) separately, sector 2 and 3 combined (CA2–3), sector 4 combined with the dentate gyrus (CA4-DG), and the hippocampal tail, the posterior part of hippocampus that cannot be segmented into the different subfields.41 In addition, we analyzed the 34 cortical thickness regions of interests48 to differentiate this to hippocampal changes.All participants had Philips Achieva 3-T MRI scanning . A single three-dimensional turbo field echo sequence was acquired for morphometric analysis with the following sequence parameters: repetition time/echo time/inversion time/flip angle=4.5 ms/2.2 ms/853 ms/8°, matric=256×213, 170 slices, thickness 1.2 mm, in-plane resolution of 1×1.2 mm. Cortical reconstruction and volumetric segmentation were performed with the FreeSurfer image analysis suite version 5.3.0  χ2-test was used. The sum of the left and right side MRI subfield volume variables were calculated and were normalized to ICV by dividing the volume variables by ICV. The average of the left and right side PRC, ERC, and 34 cortical thickness variables, and the normalized subfield volume variables were used as dependent variables in multiple linear regression analyses with group (PD or NC), gender, and age as independent variables. The multiple comparisons were corrected for by Bonferroni. They were further assessed as dependent variables in multiple regression analyses with CSF biomarkers as predictors adjusting for age and gender (independent variables) in the PD group. Finally, we performed multiple regression analyses with neuropsychological tests as dependent variables and MRI variables as predictors adjusting for age, gender, and education. CSF biomarkers, unified PD rating scale part III, and levodopa equivalent dose that proved to be significant predictors of a neuropsychological test were included in the multiple regression analysis together with the MRI variables. P-values⩽0.05 were considered significant.Statistical analyses were performed by using IBM SPSS version 20 . Continuous variables of demographical and clinical data were compared by Mann–Whitney


The goal of this study was to formulate and evaluate side effects of transdermal delivery of proniosomal gel compared to oral tolterodine tartrate (TT) for the treatment of overactive bladder (OAB). Proniosomal gels are surfactants, lipids and soy lecithin, prepared by coacervation phase separation. Formulations were analyzed for drug entrapment efficiency (EE), vesicle size, surface morphology, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, in vitro skin permeation, and in vivo effects. The EE was 44.87%–91.68% and vesicle size was 253–845 nm for Span formulations and morphology showed a loose structure. The stability and skin irritancy test were also carried out for the optimized formulations. Span formulations with cholesterol-containing formulation S1 and glyceryl distearate as well as lecithin containing S3 formulation showed higher cumulative percent of permeation such as 42% and 35%, respectively. In the in vivo salivary secretion model, S1 proniosomal gel had faster recovery, less cholinergic side effect on the salivary gland compared with that of oral TT. Histologically, bladder of rats treated with the proniosomal gel formulation S1 showed morphological improvements greater than those treated with S3. This study demonstrates the potential of proniosomal vesicles for transdermal delivery of TT to treat OAB. Overactive bladder (OAB) syndrome was defined as “urgency, with or without urge incontinence, usually with frequency and nocturia,” by the International Continence Society . This leAntimuscarinics most currently used to treat OAB are oral oxybutynin, tolterodine, and trospium, which have associated side effects such as dry mouth, constipation, headache, and blurred vision, often leading to discontinuation of therapy ,5. TolteVesicular systems, especially proniosomes, self-assembled uni- or multilamellar bilayered spheroidal structures covered with non-ionic surfactants, are becoming increasingly popular ,12,13. T® surfactants, cholesterol, soy lecithin, glycerine, acetic acid, pilocarpine and neutral buffered 10% formalin solution were from Sigma–Aldrich . Glyceryl distearate , Wecobee® M, a triglyceride derived from fully hardened palm kernel oil (conforms USP/NF monograph) from Stepan  and isopropyl alcohol (IPA) from Merck .TT was provided by Aurobindo Pharmaceuticals . SpanProniosomal gels were prepared by coacervation phase separation method . Surfactg at 5 °C for 30 min . The supernatant layer was collected to measure free drug by high-performance liquid chromatography (HPLC) [Proniosomal gel (100 mg) was dispersed in 10 mL phosphate buffer saline, pH 7.4 (PBS). The vesicles were pelleted by centrifugation at 12,000× y (HPLC) . The perThe particle size, polydispersity index (PDI), of pronisomes were measured using a Malvern Zetasizer  at 25 °C and a 90° scattering angle. A 1:100 dilution of the sample was made using double distilled water before the measurements to get 50–200 optimum kilo counts per second (Kcps) for measurements. Results are shown in A thin layer of proniosomal gel formulation was spread on a glass slide, a drop of distilled water was added, and the slide was examined under an optical microscope to assess whether the sample formed vesicles or had precipitated drug. With confirmation of vesicle formation, the same procedure was followed to place the gel sample on a holding disk for analysis inside an Environmental Scanning Electron Microscope  chamber. Water within the sample was allowed to evaporate before examination and photographs were taken at a 10 kV accelerating voltage .The pH of the proniosomal gel were determined by a digital pH meter . 1 g of gel was dissolved in 20 mL of distilled water and the electrode was then dipped into gel formulation and constant reading was noted. The measurements of pH of each formulation were replicated three times.Optimized formulations S1 and S3 were stored at three different temperatures: refrigeration (2–8 °C), room (25 ± 2 °C) and oven (45 ± 2 °C) . After t−1 and in the frequency range of 4000–650 cm−1. Spectra are presented in The spectra of gel formulations and individual components  were obtained on a Magma-IR™ Spectrometer 750  equipped with a Golden Gate™ Single Reflection Diamond Attenuated Total Reflectance (ATR) system. The spectra were determined from an average of 20 scans with a resolution of 4 cm2 donor compartment, were used for in vitro skin permeation studies. An aliquot (0.1 gm) of proniosomal gel was applied to a Strat-M™ membrane , an alternative to animal skin. The receptor cell (12 mL capacity) was jacketed to maintain 32 °C and had a magnetic bead for uniform stirring. The sample (1 mL) from the receptor compartment was collected every hour for 8 h and an equal volume of fresh phosphate buffer saline, pH 7.4, was added immediately [v/v), measured at 281 nm with a ultraviolet (UV) detector [Vertical Franz Diffusion Cells , with an area of 1.7671 cmediately ,17. The detector . Likewisv/v) aqueous formalin solution, the second group was the control (untreated), and the third group was administered the S1 gel, while the fourth group received S3 gel, to evaluate skin irritation. The optimized gel (1 g) or formalin solution (1 mL) was applied to the posterior side of the shaved dorsal region at the back of rats (5 cm2 area) once a day for three consecutive days. The observed localized reactions, edema and erythema, daily for three days are as shown in Table 4.The skin irritancy test was performed with slight modifications to the mentioned method on male albino rats 150–180 g) to determine localized skin reaction [0 g to deAcetic Acid (ACA) solution (0.75%) was administered to unconscious rats by a urinary catheter. In this model, chemical irritation caused by ACA produces acute inflammation of the urinary bladder and, consequently, facilitates micturition in rats . BladderMeasurement of salivary secretion study was performed after the rats were anesthetized with diethyl ether . We usedThe urinary bladder was removed from each rat and immediately immersed in 10% neutral buffered formalin solution. After processing, embedding, and sectioning, 5-mm-thick tissue sections were stained with hematoxylin followed by eosin and observed under a light microscope.t-test were applied. p < 0.05 was considered significant. Values are expressed as means ± standard deviation.Statistical analysis of data was performed with SPSS v8.0 . Analysis of variance and the paired ® M, in S4, led to a marked decrease in EE (p < 0.05), compared with that of S3. S1 had a significantly higher EE than S2 (p < 0.05), the latter containing twice the amount of cholesterol. Thus, the optimum Span: cholesterol ratio was 20:1. S2 and S3 had the same composition of surfactants but different lipids, cholesterol, and glyceryl distearate, respectively. However, their EE values were not significantly different (p > 0.05). The pH of the formulations is suitable for skin . Insoluble drug crystals were observed with higher drug (40 mg) as seen in ® M. From the eSEM analysis of S1, the micelle surfaces appeared to be coated with drug [Increasing the drug load from 20 to 40 mg in the S1 formulation resulted in a non-significant decreasing trend of EE from 91.68% to 89.45% . The results are shown in p < 0.05). With oral TT the total saliva collected (p < 0.05) was less at all time points  (n = 6) or S3  was significantly more than in the rats receiving the oral formulation . The amounts of total saliva secreted in the first 10 min in control, oral TT treated, S1 treated, and S3 treated groups were similar after pilocarpine stimulation  A, comparmulation A.p < 0.05). However, the oral TT resulted in a more marked decrease in total saliva collected (p < 0.05) at all time points (p < 0.05) than those after oral administration of TT. However at 24 h the weights of saliva collected were markedly higher for S3 compared with S1, indicating that gel administration for the latter is required after 12 h. Compared with the S1 formulation, S3 showed less drug permeation in vitro  ,22. TT i® M, to Span formulations led to a marked decrease in EE probably because of an increase in bilayer hydrophobicity and larger vesicle size, both contributing to a lower EE [Cholesterol interacts with surfactants, influencing their physical properties and vesicle structures by modulating cohesion and mechanical strength of bilayers, causing an increased EE . Additiolower EE . Additiolower EE ,29. Thislower EE .Glyceryl distearate plays the same role as cholesterol in influencing this parameter. Formulation with Span 40 have a lower EE than Span 60, perhaps because the shorter alkyl chains and lower phase transition temperature (Tc) of the former would form less ordered bilayers, decreasing stability and, therefore, EE ,21. EE wAt higher temperature, there is a higher leakage from proniosome formulation vesicles, whereas good stability was observed at lower temperatures (2–8 °C). This may have been the result of vesicle loosening and higher fluidity of lipid bilayers at higher temperature . Cholest® M and glyceryl distearate; While the absence of Wecobee® M alone, produced less loose structures. Also because of the higher hydrophobicity of Span, spherical vesicles are not formed with the addition of water. Moreover, higher hydrophobic surfactants become too bulky and do not fit into a spherical shape when contacted with aqueous media. In addition, the packing of vesicles appeared like a hexagonal system with more hydration. The hydrophilic TT is attracted to the hydrophilic region on the outer surface of micelles, which can contribute to an increased EE, leading to larger surface areas enabling higher drug loading.Niosomes are formed by increasing hydration of proniosomes in aqueous media. Span formulations containing cholesterol and lecithin had loose structures probably because of the combined absence of Wecobee−1; there were bands recorded for –C–H stretching (aromatic) at 3040.10 and 3012.07 cm−1; while –CH3 stretching  resulted in 2685.15 and 2934.90 cm−1; furthermore C=C stretching (aromatic) resulted 1604 and 1454 cm−1 bands. The respective shifts from surfactants span 20 and 60 at 1750, 2950 and 1450 cm−1, 2900 cm−1 were also found in the gels.Interactions between cholesterol and Span 60 in the bilayer of niosomes involve hydrogen bonding , leadingIn vitro permeation studies should be performed in a non-occlusive environment to better simulate the topical application of proniosomal gels . Span baConcerning surfactant and cholesterol desorption of the drug from the surface of the vesicles. The following slower, permeation phase might have indicated drug diffusion from swollen bilayers in contact with water on the buffer saline solution . In all p > 0.05), suggesting that these lipids play similar roles in promoting drug permeation. The formulation with the lower amount of cholesterol had better drug permeation, unlike the formulation with the higher amount. Perhaps, this might be attributed to its higher amount of cholesterol which may decrease the leakage and permeability of TT from the proniosomal vesicles leading to lesser permeation. Glyceryl distearate may disrupt the lipids in the stratum corneum allowing vesicle penetration [In the Span series, glyceryl distearate or cholesterol addition led to similar permeation percentages (etration . GlyceryIt is essential to have good skin compatibility without irritation for transdermal delivery formulation as it stays in the skin longer. Thus the obtained results clearly signify that the proniosomal gel achieved the objectives of controlled drug release. The Span surfactants of the proniosomal gels had longer alkyl chain length leading to gradual release of the drug into the skin layer over longer period of time. This was helpful to reduce the irritation and toxicity of drug .In the salivary secretion model, orally administered TT was reported to bind more strongly to the salivary gland, reach the systemic circulation and act at the cholinergic receptor more than transdermally administered TT . Total sFrom the histopathological analysis, there was a clear difference between rats in the control group, with a normal intact urinary bladder, and those in the acetic acid treated group, which showed transitional cell necrosis as well as mucosal ulceration and inflammatory infiltration indicating acute cystitis. The inflammatory infiltrates consisting of neutrophils and scattered lymphocytes, in submucosal and muscle layers, suggest an acute form of inflammation in the bladder tissue that extended throughout its thickness. The bladders in rats receiving oral TT showed nearly normal bladder structures, indicating complete bladder healing. Regenerative surface is one of the indications of the treatment modality efficacy.However, the mucosal surfaces in bladders of transdermal TT treated rats had numerous papillary folds clustered with transitional cells. Furthermore, the submucosa in these bladders showed evidence of fibrosis and blood vessels, suggesting healing scar tissue. S3 formulation appeared to induce rapid transitional epithelium regeneration with mild mucosal ulceration; however, complete regeneration may be possible with long term treatment.Our findings suggest that, among the formulations studied, the S1 gel had the most effective combination of surfactant, cholesterol, and aqueous phase. This formulation had the highest EE and drug permeation properties. In the in vivo studies, rats administered with S1 had higher salivary secretion, compared with oral and S3 formulations; moreover, we observed faster regeneration of epithelium in the bladder for S1 compared with the S3 formulation. Thus, our study has established that transdermal TT formulations can have efficacies comparable with those of oral formulations for OAB and, also, cause less dry mouth effects. These results can serve as a foundation for future studies on the potential of proniosomal gels for transdermal TT delivery.


This study evaluates the association between high sensitivity troponin I (hsTnI) and T (hsTnT) and the morphology of coronary artery plaques detected by coronary computed tomography angiography (CCTA) in patients with suspected coronary artery disease (CAD).  Patients undergoing CCTA were prospectively enrolled. CCTA was indicated by a low to intermediate pretest probability for CAD during routine clinical care. Within 24 hours of CCTA examination, peripheral blood samples were taken to measure hsTnI, hsTnT, and N-terminal probrain natriuretic peptide (NT-proBNP). p = 0.0001). In multivariate logistic regression models, hsTnT, but not hsTnI, was still significantly associated with mixed coronary plaque morphology .  A total of 99 patients were enrolled with 43% without CAD, 9% with noncalcified plaques, 28% with calcified plaques, and 19% with mixed type plaque lesions. Both hsTnI and hsTnT levels were able to discriminate significantly between the groups, especially in the presence of mixed coronary plaques (AUC range: 0.741–0.752; NCT03074253. Both hsTnI and hsTnT are able to discriminate between different coronary artery plaques morphologies, whereas hsTnT was significantly associated with mixed coronary plaques in patients with suspected CAD. This trial is registered with  From a clinical perspective, cardiac troponins represent the main diagnostic parameter to diagnose patients suffering from an acute coronary syndrome (ACS). Contemporary troponin assays aim to detect the release of troponins from cardiomyocytes undergoing ischemia or necrosis. In the past decades, high sensitivity (hs) troponin assays were developed, which might be able to detect even the presence of subclinical coronary artery disease (CAD). Several studies demonstrated the potential value of hs troponins for screening of CAD or risk prediction for adverse cardiovascular events in patients with stable CAD –5.An acute ST-segment elevation myocardial infarction (STEMI) is a consequence of acute coronary arterial thrombosis at the site of a vulnerable ruptured coronary plaque. Therefore, the composition of coronary arterial plaque might represent to be even more important compared to its stenosing character. Histologically, a coronary plaque is prone to rupture when possessing a thin fibrous cap, a large necrotic core, and a high content of macrophages , 7. DeteCoronary computed tomography angiography (CCTA) has become the main noninvasive imaging technique being able to detect the presence of CAD or to predict adverse coronary events –12. HoweMoreover, CCTA might also be able to characterize the morphology of coronary plaques , 16. PreHowever, whether hs troponin levels might reflect the presence of different plaque morphologies has not been well investigated. Therefore, this study aims to evaluate associations between hs troponin I (hsTnI) and hs troponin T (hsTnT) and CCTA-based coronary plaque morphology in patients at low to intermediate CAD risk. Additionally, the study evaluates whether combining both hs troponins with NT-proBNP reveals an additional value.NCT03074253) represents a clinically prospective, controlled, and monocentric study conducted at the University Medical Center Mannheim, Germany. The research adhered to the principals outlined in the Declaration of Helsinki and was approved by a regional ethics committee. Written informed consent was obtained from all patients. For the present study, patients undergoing CCTA during routine clinical care were included prospectively at the University Medical Centre Mannheim (UMM), Germany. The study was carried out according to the principles of the Declaration of Helsinki and was approved by the local ethics commission II of the Faculty of Medicine Mannheim, University Heidelberg, Germany. All patients were indicated for CCTA due to a low to intermediate PTP of 15–50% presenting with typical or atypical angina pectoris. Patients suffering from an acute myocardial infarction were excluded. Patients with severe chronic kidney disease being defined as an estimated glomerular filtration rate (eGFR) below 40 ml/min were excluded. To determine the eGFR, the Modification of Diet in Renal Disease (MDRD) formula was used.The “Cardiovascular Imaging and Biomarker Analyses” (CIBER) study . Prior to CCTA, all patients underwent a noncontrast-enhanced cardiac CT for the evaluation of coronary calcifications using the Agatston method. The CCTA technique was chosen individually for each patient depending on heart rate and/or rhythm and body mass index, with the goal of minimizing radiation exposure while maximizing diagnostic image quality. Image acquisition techniques included traditional retrospective electrocardiographic (ECG) gating with default use of ECG-dependent tube current modulation, prospective ECG triggering, and prospectively ECG-triggered high-pitch spiral acquisitions. Tube voltage was selected by using anatomic based automated tube voltage selection  in combination with automated tube current modulation. In the absence of contraindications, patients received 0.4 mg of sublingual nitroglycerin before image acquisition. B-blockers (5–20 mg intravenous metoprolol tartrate) were used to lower heart rates to less than 65 beats per minute in patients undergoing prospectively ECG-triggered high-pitch spiral acquisitions.Contrast enhancement was achieved by using a bolus-tracking protocol where image acquisition began once a threshold of 100 HU had been exceeded within a region of interest in the descending thoracic aorta. A volume of 80 cc of iomeprol  was used with a fixed flow rate of 5 ml/s through an 18-gauge intravenous antecubital catheter, followed by 50 ml of saline at the same flow rate.Noncontrast enhanced calcium scoring studies were reconstructed at a section thickness of 3 mm by using a dedicated algorithm . The calcium score was calculated by using dedicated software according to the Agatston method. All contrast-enhanced CCTA data were reconstructed with a section thickness of 0.5 mm in the axial plane with a third-generation iterative reconstruction technique  and a corresponding vascular algorithm (Bv40).All CCTA studies were evaluated on a 3D workstation  using standard MPR as well as centerline curved MPR. Observers were blinded for biomarker levels. In the first step, the number of plaques that could be identified on CT images in the correct location was recorded to assess the sensitivity of different atherosclerotic plaque types. Those plaques, which were found on CT, were further analyzed. Finally, the attenuation within the coronary artery lumen was measured in three ROIs, as described previously .Plaque analysis was performed offline using dedicated software . Window level and width were determined using a standard window level setting. The study population was divided into four different groups—patients who did not show any sign of plaque or stenosis (no CAD), patients with noncalcified plaques defined as lesions without any detectable calcification, patients with calcified plaques defined as lesions with 50% or greater calcium, and patients with mixed plaques classified as lesions with calcification less than 50% .All expressed biomarkers were measured in the serum of patients' blood. Within 24 hours before or after the CCTA, peripheral venous blood samples were taken from each patient and collected in serum monovettes® tubes and centrifuged at 2500 ×g for 10 minutes at 20°C. The aliquoted samples were cooled down with liquid nitrogen before being stored at −80°C until analysis. The whole processing took part within two hours after blood extraction. After thawing, the samples were mixed gently by inverting and centrifuged at 2500 ×g for 10 minutes at 20°C for troponin T and N-terminal probrain natriuretic peptide (NT-proBNP) analysis. Troponin T was measured with the troponin T hs STAT assay on a cobas e 602 analyzer . The limit of blank (LoB) for this assay was 0.003 ng/ml and the limit of detection (LoD) 0.005 ng/ml as described in the instructions for use . For TnIp was <0.05.Data were analyzed using the software IBM-SPSS version 22.0. Categorical variables are expressed as absolute numbers and percentage, whereas continuous variables are shown as mean and range. All biomarkers are presented as the median and interquartile range. For univariate correlations, we used the Spearman-Rho test. To analyze the relation between CAD groups and biomarkers, the nonparametric Kruskal-Wallis Test was used because the values did not show a normal distribution. To test the presence of a Gaussian distribution, the Kolmogorov-Smirnov test was applied. Receiver-operating characteristic curves (ROC) with the area under curves (AUC) were generated to associate hsTn and NT-proBNP with groups of patients with different plaque types. Multivariate regression models were calculated with backward elimination (Forrest plot). The odds ratios for hsTns and cardiovascular risk factors were calculated by binomial logistic regression. All analyses were considered significant when A total of 99 patients was included prospectively with their baseline characteristics being outlined in Median left ventricular ejection fraction (LVEF) being measured by functional computed tomography was normal with 62%. 57% of the patients were found to have a coronary artery disease (CAD) that was morphologically relevant as being assessed by CCTA, whereas 43% had no signs of CAD and no evidence of CT-based coronary arterial plaques. 9% revealed noncalcified plaques, 28% showed calcified plaques, and 19% had mixed type plaque lesions with calcified and noncalcified amounts. In the present cohort, measurable concentrations of hsTnI were present in all 99 patients (100%) and of hsTnT in 78% (77/99) of the study patients.p = 0.0001) between the different groups of patients without coronary plaque and with noncalcified plaques, calcified plaques, and mixed plaques.As presented in Figures n = 43). Highest hsTn levels were found in patients with mixed coronary plaques , followed by decreasing levels in patients with calcified plaques , and with noncalcified plaques .Slight increases of hsTnI and hsTnT were already detectable in patients without coronary plaque . No significant correlations were found between hsTns and calcium and triglycerides.As presented in p = 0.001) and were significantly higher than the AUC of NT-proBNP . Combining hsTnT plus NT-proBNP revealed the highest AUC compared to each biomarker alone , whereas the AUC of hsTnI plus NT-proBNP did not reveal improved discrimination over hsTnI .As presented in Figures Both hsTns were adjusted within multivariable logistic regression models to predict the presence of patients with mixed coronary plaques. Besides the inclusion of hsTns, multivariable models comprised nine clinical known risk factors for CAD, such as age, gender, creatinine, LDLC, arterial hypertension, cardiac family history, smoking, diabetes, and log NT-proBNP.p = 0.004) even after adjusting for all of these cardiovascular risk factors. Noteworthy, log-transformed hsTnI levels were no longer associated with the presence of coronary mixed plaques in this multivariable regression model  (= 0.165) .The present study evaluates whether the concentration of hsTnI and hsTnT correlates with the presence of noncalcified, calcified, and mixed coronary artery plaque composition in patients with suspected CAD being detected by CCTA. It was shown that both hsTnI and hsTnT differentiated significantly between patients with no coronary plaques, noncalcified, calcified, and mixed coronary plaques. Noteworthy, the combination of hsTnT and NT-proBNP best discriminated the presence of mixed coronary artery plaques. In multivariable logistic regression models, only hsTnT was still significantly associated with the presence of mixed coronary plaques, whereas hsTnI was not.These findings are in accordance with the previous studies –31 showiNot only mixed calcified plaques but also noncalcified plaques are known as high-risk plaques being prone to rupture , 37–39. Furthermore, calcification is known to occur more likely in advanced stages of atherosclerosis , 19, 40.Being specifically expressed in myocardial muscle, cells troponin I and T are released from the myocardium due to cardiac injury (from ischemia or various other causes). The levels of concentrations in the blood are related proportionally to the degree of damage in the myocardium, as demonstrated in a prospective single-center study in patients presenting with an ST elevation myocardial infarction . But witThe present study delivers additional knowledge about the release of both hsTnT- and hsTnI-suspected CAD patients to differentiate, as well as CCTA, between different coronary plaque morphologies. To our knowledge, it is the first study demonstrating a correlation between hsTnI and CCTA-derived plaque morphology. Of note, hsTnI was, unlike hsTnT, no longer significantly associated with the presence of mixed plaques in multivariable regression models. This might be explained due to the fact that hsTnT was detectable in only 78% of the patients, whereas hsTnI was present in all patients. Therefore, the analysis within the multivariate regression might be influenced.In conclusion, the findings of the present study are in line with previous studies, demonstrating that levels of hsTn can differentiate between the different characteristics of coronary plaques and might become potentially useful biomarkers in detecting vulnerable coronary plaques. These results accomplish the arguments concerning the discriminative capacity between hsTn and the presence of a subclinical CAD. hsTn is able to detect mild CAD and might therefore bear the potential to classify the morphology of coronary lesions with regard to their amount of calcium as a noninvasive method without radiation unlike CCTA.The present study is limited by its small sample size, thereby restricting propositions for the general population. However, the present study applies a step-wise statistical approach including all measured numerical hsTn results and avoids a too early simplification of statistics by using single and combined C statistics as well as univariate and multivariable regression models. Regarding the probability of hsTn to predict the plaque morphology, the present study shows further limitation since CCTA is not as precise as conventional angiography combined with imaging techniques such as IVUS and OCT. However, experienced observers and a high quality of CCTA images outweigh potential bias assuming that the actual plaque composition matches to the presented results. Classifying plaques only with regard to their amount of calcium is common in research with CCTA –45, but


The aim of this report is to present a case of a patient, metal foundry worker, who had been exposed to industrial silver salts for over 20 years. It is well established that chronic exposure to silver compounds can cause accumulation of silver deposits in various tissues. This condition is referred to as argyrosis or argyria, whereas changes related to eye tissues are defined as ocular argyrosis.A complete eye examination, corneal confocal microscopy, kinetic and static visual field test, posterior segment optical coherent tomography, pattern visual evoked potentials (PVEP), flash visual evoked potentials, multifocal electroretinogram, pattern electroretinogram (PERG), full-field electroretinography (FERG) and electrooculogram were all performed.Eye examination revealed decreased visual acuity, corneal deposits and drusenoid changes within the macula. Although electrophysiology tests did not show changes in the function of retinal pigment epithelium, they revealed abnormal function of photoreceptors in the central and peripheral retina. PERG abnormalities and delayed latency of P100 wave in PVEP confirmed impaired function of the inner layers of the retina in the macular region.Corneal confocal microscopy and electrophysiological tests may help confirm the diagnosis of ocular argyrosis. Silver is a naturally occurring element with high thermal and electrical conductivity. It is harder than gold, very ductile and malleable, and it has antimicrobial effects.The properties of silver have been known since antiquity. Currently, the metal is widely used in metallurgy, photography, medicine, and for the production of jewelry, coins and mirrors; it is also utilized as a water disinfectant .Argyrosis involves the accumulation of silver in various tissues, leading to their dysfunction. Argyrosis is a rare dermatological and systemic disease caused by the accumulation of silver deposits mainly in the skin, mucous membranes and internal organs following chronic exposure to silver or silver compounds.The disease can be diagnosed based on blood and urine tests. Correct diagnosis is facilitated by skin biopsy, which reveals small gray-brown or brown-black granules, approximately 1 mm in size, scattered mainly in the dermis and eccrine sweat glands .Chronic exposure to silver compounds through ingestion, inhalation or skin contact results in the accumulation of silver in the eyes and in the adjacent tissues . This coThe deposition of silver salts results in skin discoloration around the eyes; the affected area becomes gray or bluish-gray. Silver precipitates can be distributed in the elastic fibers of the connective tissue and basement membranes  includinOcular argyrosis diagnosed during a thorough ophthalmic examination may be the first sign of generalized argyria.Adverse health outcomes of silver depend on the dose, duration and form of exposure  as well as on individual characteristics .Under normal circumstances, people are exposed to small amounts of silver present in food, drinking water, soil and sometimes also in the air.Consumption of large doses of colloidal silver can cause coma, pleural edema and hemolysis. Silver is also toxic to bone marrow and may cause agranulocytosis. Ingestion of high doses of inorganic silver yields effects similar to those of corrosive solutions, i.e., burning of the throat and epigastric area leading to abdominal pain, vomiting and diarrhea. A single lethal dose of silver nitrate estimated by the World Health Organization is about 10 grams. Silver salts are more toxic than silver proteins or colloidal silver, very popular in alternative medicine .A 71-year-old patient was referred to the Corneal Unit of the University Eye Hospital in Katowice, Poland, due to gradual deterioration of distance and near visual acuity, night blindness  being the most remarkable complaint in the last few years. He also had corneal changes in both eyes. The patient admitted having been exposed to industrial silver salts in a metal foundry where he had worked for over 20 years. He was retired for 5 years, and during that time, he was no longer exposed to silver.Uncorrected distance visual acuity was 0.3 in the right eye (RE) and 0.4 in the left eye (LE) on the Snellen chart. Correction did not improve distance visual acuity. Best corrected near visual acuity was 1.0 for the RE with correction 2.5 Dsph and 0.75 for the LE with correction 2.5 Dsph. Intraocular pressure was 19 mm Hg and 17 mmHg in the RE and LE, respectively.Slit lamp examination revealed discoloration of the eyelid skin and conjunctiva in the anterior segment, more prominent in the medial canthal region. Corneal stromal haze was present in the center and periphery, with confluent whitish gold-like deposits extending from the anterior and deep stroma to Descemet’s membrane. The patient also had nuclear cataract in the RE; his LE was pseudophakic with pseudoexfoliative syndrome. Dilated-pupil fundus examination showed drusenoid changes in the macular region and rearrangement in retinal pigment epithelium (RPE). Optic nerves were normal Fig. . The skiConfocal microscopy, CS4 , showed characteristic changes. Confluent small silver deposits were seen in the center and periphery of the cornea. Descemet’s membrane thickening was also found. Due to disease progression, numerous punctate hyper-reflective silver deposits hindered accurate endothelial cell assessment. Changes in the cornea resembled accumulated gold deposits Fig. .Fig. 2CoGoldmann kinetic perimetry  revealed visual field defects in the upper, nasal and temporal quadrants of the RE, while in the LE the loss was limited to upper and temporal quadrants. Octopus 1-2-3 static perimetry  showed reduced retinal sensitivity and relative scotomata—predominantly in the RE  revealed drusenoid changes in RPE. The ganglion cells complex thickness map revealed a slight increase from the mean values Fig. .Fig. 4OCIn pattern visual evoked potentials (PVEP), P100 latencies were delayed to 110 % after stimulation with 1°, and to 130 % after stimulation with 15′. P100 amplitudes were within normal limits. Flash visual evoked potentials (FVEP) revealed normal amplitudes and latencies Fig. . PatternFull-field electroretinography (FERG) under both scotopic and photopic conditions revealed a decrease in a- and b-wave amplitudes to about 30 % of the normal values (a-waves were more distorted) as well as a- and b-wave implicit time delay. Amplitudes of oscillatory potentials were very low Fig. . ElectroSilver deposits cause gray-blue discoloration of skin areas exposed to direct sunlight. A relationship was shown between skin pigmentation levels and the duration of exposure to silver compounds .Argyrosis mainly affects people who ingest silver-based products and those who work in metal foundries, silver mines and factories with silverware .Silver may accumulate in various tissues with the highest concentrations found in the skin, liver, spleen and adrenal glands .At present, ocular argyrosis is less frequently diagnosed, which is associated with the withdrawal of silver-containing substances  by drug control agencies. Nevertheless, silver-containing products are still sold as dietary supplements and ingredients in alternative medicine. This leads to the emergence of new cases of silver poisoning .Confocal microscopy is a noninvasive examination facilitating the diagnosis of ocular argyrosis, especially when corneal biopsy cannot be taken. A relationship has been reported between the severity of argyrosis demonstrated on corneal confocal microscopy and advancement of generalized argyrosis . Dense dSpencer et al. concluded that silver deposits were inert and did not cause visual deficit , 15. SomIn our patient, night blindness was related to photoreceptor damage. Fundus examination showed drusenoid changes in the macular region and RPE rearrangement, similar to the findings of Stafeeva et al.  and MossTo our best knowledge, there are not many reports in the literature on electrophysiology testing in ocular argyrosis, and none of those we found presented detailed results of electrophysiological tests.Stafeeva et al. described a patient with ocular argyrosis manifested with a decrease in visual acuity and nyctalopia. Electroretinography did not reveal functional changes in scotopic and photopic amplitudes, latency or in oscillatory potentials and 30-Hz flicker testing . Moss etOn the contrary, Flögel et al.  describeDetailed electrophysiological examinations performed in our patient did not reveal changes in RPE function; however, they revealed abnormal function of photoreceptors in the central and peripheral retina. PERG abnormalities confirmed impaired function of the inner layers of the retina in the macular region. Delayed latency of P100 wave in PVEP might have resulted from problems with visual transmission in central retina. Retinal dysfunction with abnormal ERG results might be associated with long-lasting argyrosis.Pala mentioned numerous corneal conditions that should be taken into account in the differential diagnosis of ocular argyrosis, including pre-Descemet dystrophy, X-linked ichthyosis as well as deposits of heavy metals  or drugs  and conditions with abnormal eye pigmentation, such as malignant melanoma . If retiIn the course of argyria, silver deposits may build up in various eye tissues. Confocal microscopy, OCT of the macula and electrophysiological tests are noninvasive tools, which help confirm the diagnosis of ocular argyrosis. Confocal microscopy may enable ophthalmologists to obtain in vivo cross-sectional images of the corneal layers, while OCT and electrophysiological tests may reveal retinal changes. Detailed patient history is also very important.Until now, there have been few reports on retinal abnormalities in patients with ocular argyrosis. Changes in retinal activity occurred in photoreceptors and across to the inner layers of the retina; the most advanced changes were found in the macula. Corneal deposits and RPE changes that can cause dysfunction of the retinal adaptation to darkness and light result in considerable problems with functional vision.


ICH).To explore the association, if any, between the relationship between cardiac troponin and cerebral herniation after intracerebral hemorrhage  scans at admission and follow‐up, and clinical outcomes were obtained. Using multiple logistic regression to identify the relationship of troponin and herniation. The association between troponin and hematoma volume was assessed using bivariate correlation and linear regression.Six hundred and eighty‐seven consecutive CT at admission and follow‐up was 4 and 30.25 hr, respectively. In multivariate analysis, elevated troponin was independently associated with cerebral herniation . And those with elevated troponin had larger hematoma volume at follow‐up in bivariate correlation  and linear regression , higher National Institutes of Health Stroke Scale score  and lower Glasgow Coma Scale score  than those without.Among 188 (27.4%) patients who underwent the test of serum cardiac troponin at admission, 16 (8.5%) demonstrated cerebral herniation. The median time from symptom onset to ICH patients with large hematoma volume and elevated troponin.Elevated cardiac troponin was associated with an almost five‐fold increased risk of cerebral herniation, but not in‐hospital mortality. The possibility of cerebral herniation should be considered when  Traumatic ICH, primary SAH and Hemorrhagic transformation of cerebral infarct were excluded. Among the 687 ICH patients, 188 (27.4%) had the test of serum cardiac troponin at admission. The methodology of our study was in accordance with local ethics criteria for human research. All patients or their guardians provided informed consent for participation.2.2We derived data on demographics, doubtful risk factors for ICH, data of electrocardiogram, clinical characteristics and time to CT from the prospective database of the National Key Technology R&D Programme of the 12th Five‐Year Plan which had been reported previously , and serum ions. Blood samples were taken and processed as soon as possible while at admission. Imaging information  is limited to CT scans or CTA. Etiology of ICH was categorized as hypertension, aneurysm, arteriovenous malformation, moyamoya disease, severe systemic disease, or unknown. Data on in‐hospital mortality was also collected. Available CT scans were independently reviewed by M.X. and J.L.2.3ABC/2 where A is the maximum diameter of the greatest cross section of the hematoma, B is perpendicular to A and C represents the slice thickness multiplied by the number of CT slices −0.203 ×  was defined as eGFR < 15 ml/min per 1.73 m2.On CT scans, cerebral herniation was characterized by uncontrollable diffuse brain swelling or mass effect resulted from predominantly unilateral brain swelling, often combined with obliteration of perimesencephalic cisterns  patients were enrolled. Among all these 188 subjects with complete data, mean age was 56.34 years. 60.6% were men, and 66 (35.1%) had elevated serum cardiac troponin. The median time from symptom onset to CT at admission and follow‐up was 4 and 30.25 hr, respectively. The mean hematoma volume was 30.04 ml  and 39.9% were originated in deep.Among these 66 patients with elevated troponin, one had cardiac ischemia characterized by atrial premature beats, the change of T‐wave and left anterior fascicular block on electrocardiogram; other electrocardiogram abnormalities included atrial fibrillation in one patient and supraventricular tachycardia in one patient. In addition, 5 of the 66 patients had pre‐existing heart disease which included coronary heart disease, myocardial infarction and congenital heart disease.p = .003), higher NIHSS score (p = .003), higher proportion of elevated serum troponin (p = .016) and larger hematoma volume (p = .002) compared with patients without cerebral herniation.Table p = .037). After adjusting for additional confounders, elevated serum troponin was still associated with the presence of cerebral herniation .After adjusting for age, sex, GCS and NIHSS, those with cerebral herniation were more likely to have elevated troponin , more likely to have diabetes mellitus (p = .016), had higher systolic blood pressure and diastolic blood pressure . As for the hematoma site, patients with elevated troponin had a higher proportion of hematoma in deep compared with those with normal troponin , and were more likely to be accompanied by hypertensive ICH (p < .001), whereas normal troponin was more likely to be accompanied by secondary ICH such as arteriovenous malformation (p = .034). In addition, patients with elevated troponin had lower GCS score and higher NIHSS score . And in‐hospital mortality was also higher among elevated troponin group compared with normal troponin group . After adjusting for only age and sex, elevated troponin was significantly associated with GCS < 8, as well as NIHSS > 10, whereas the relationship of elevated troponin with in‐hospital mortality and deep location of ICH became no longer significant (Table p = .016) and NIHSS > 10 .Next, the association between elevated troponin and the location of hematoma, as well as the etiology and the severity of ICH, was analyzed Table . Patientp = .003) and linear regression  than those with normal troponin. However, elevated troponin was not associated with hematoma volume at admission and the change in hematoma volume from diagnostic to follow up CT. Figures As for hematoma volume, those with elevated troponin had large hematoma volume at follow‐up in bivariate correlation (correlation coefficient, .375, 4Cerebral herniation is a rare and devastating complication in ICH patients. Intracerebral hematoma could lead to the rapid increase of cerebral pressure and result in the downward displacement of brain tissue through the notch (Wu et al., In this retrospective cohort study, we found that elevated troponin was not just associated with the frequency of cerebral herniation, but also large hematoma volume at follow‐up, suggesting that serum troponin levels played a pivotal role in ICH patients and are associated with the severity of stroke. Furthermore, we observed that ICH patients with elevated troponin were more likely to have a higher systolic blood pressure levels, a higher frequency of CKD and ESRD and a more frequent deep location, possibly shared the pathophysiology of cerebral herniation. However, the change in hematoma volume from diagnostic to follow up CT between patients with and without elevated troponin, as well as between those with and without cerebral herniation, was not significant. Previous study reports that approximately one‐third of ICH patients had significant change in hematoma volume (Brott et al., The mechanisms that can explain the association of elevated troponin with high frequency of brain herniation are complex and incompletely understood. First, Cardiac troponin is a highly sensitive and specific marker of myocardial injury (Agewall, Giannitsis, Jernberg, & Katus, Our study indicated that ICH patients with elevated troponin had a higher proportion of in‐hospital mortality. Whereas the relationship of elevated troponin to in‐hospital mortality became no longer significant after adjusting confounding factors.Limitations of this study include the single‐center design and retrospective analysis with exclusion of those subjects without the data of serum troponin. Therefore, there might be selection bias in this study. Other limitations were small sample size for multivariable analysis, single measurement of troponin and the possible existence of the unmeasured confounding factors which may explain some of our findings. Future prospectively large studies are warranted to make this distinction.5Elevated troponin, even after adjusting potential confounders, is associated with an almost five‐fold increased risk of cerebral herniation. The possibility of cerebral herniation should be considered when ICH patients with large hematoma volume and elevated troponin.None declared.


E. coli and bacteriophage Phi6 were used to evaluate handwashing with and without organic load added to simulate bodily fluids. Hands were inoculated with test organisms, washed, and rinsed using a glove juice method to retrieve remaining organisms. Impact was estimated by comparing the log reduction in organisms after handwashing to the log reduction without handwashing. Rinse water was collected to test for persistence of organisms. Handwashing resulted in a 1.94–3.01 log reduction in E. coli concentration without, and 2.18–3.34 with, soil load; and a 2.44–3.06 log reduction in Phi6 without, and 2.71–3.69 with, soil load. HTH performed most consistently well, with significantly greater log reductions than other handwashing protocols in three models. However, the magnitude of handwashing efficacy differences was small, suggesting protocols are similarly efficacious. Rinse water demonstrated a 0.28–4.77 log reduction in remaining E. coli without, and 0.21–4.49 with, soil load and a 1.26–2.02 log reduction in Phi6 without, and 1.30–2.20 with, soil load. Chlorine resulted in significantly less persistence of E. coli in both conditions and Phi6 without soil load in rinse water (p<0.001). Thus, chlorine-based methods may offer a benefit of reducing persistence in rinse water. We recommend responders use the most practical handwashing method to ensure hand hygiene in Ebola contexts, considering the potential benefit of chlorine-based methods in rinse water persistence.To prevent Ebola transmission, frequent handwashing is recommended in Ebola Treatment Units and communities. However, little is known about which handwashing protocol is most efficacious. We evaluated six handwashing protocols , and 0.05% sodium dichloroisocyanurate, high-test hypochlorite, and stabilized and non-stabilized sodium hypochlorite solutions) for 1) efficacy of handwashing on the removal and inactivation of non-pathogenic model organisms and, 2) persistence of organisms in rinse water. Model organisms  The Ebola Virus Disease (EVD) outbreak in West Africa from 2013–2016 was the largest to date, resulting in 28,638 cases, 11,316 deaths, and establishing the disease as endemic in the region –6.Ebola is an enveloped, non-segmented, negative sense, single-stranded RNA filovirus with seven genes. The filamentous particles are approximately 80nm in diameter and range from 300–1,100 nm long ,8. The v8/mL [EVD is spread through contact with infected individuals, their bodily fluids, or contaminated surfaces such as hands . Contact8/mL ,13. With8/mL . For Ebo8/mL –18, alth8/mL . The Wor8/mL –19. In tThe purpose of handwashing is to remove potentially harmful organisms from hands, preventing their transmission from person to person. Handwashing may inactivate organisms, but this is not always necessary to prevent disease transmission. Inactivation is not expected for handwashing protocols using plain soap, but is expected for protocols using chlorine. Although handwashing is widely considered to be an important part of disease prevention and studies show that it is effective for preventing transmission of infection –23, studA recent systematic review on evidence for handwashing with chlorine in Ebola outbreaks found only four studies investigating the efficacy of chlorine for handwashing , and we Along with safety and efficacy considerations, there are benefits and drawbacks to each handwashing protocol in outbreak settings, including ease of transport, local availability, cost, and acceptability . Soap isThe selection of a safe, efficacious, and practical handwashing protocol is essential during an EVD outbreak, and is currently hampered by limited evidence on the efficacy of commonly used handwashing protocols and lack of evidence about persistence in rinse water. To address these research gaps, we evaluated six commonly recommended handwashing protocols for: 1) efficacy at the removal of non-pathogenic model organisms from hands and, 2) ability to reduce persistence of these organisms in rinse water.We conducted testing with human subjects to investigate the reduction of non-pathogenic model organisms on hands after washing and the persistence of organisms in rinse water using a matrix including six handwashing protocols and two controls, two surrogate organisms, and two soil load conditions. This was a crossover randomized trial in which surrogate organisms and soil load status were tested on the same subjects at different times, and in which the order of application of the handwashing protocols was randomized for each subject at each time. The study enrollment and baseline, materials used, and experimental procedure are described below.The study took place on the Tufts University campus in Medford, MA, USA beginning with recruitment on April 2, 2016 and concluding with the final experiments on May 5, 2016. The study was approved by the Institutional Review Board at Tufts Medical Center and Tufts University Health Sciences Campus (#12018); Harvard University ceded review to the Tufts Institutional Review Board.™ Cleansing Bar  to use in place of their usual products. Commercially available heavy-duty vinyl gloves  were also provided for cases in which contact with antimicrobial products was unavoidable (e.g. dishwashing).A total of 18 volunteers were recruited and signed written informed consent forms. This sample size was selected based on the average size of similar efficacy studies, and to allow for standard statistical tests ,36,39,49The six handwashing protocols were HWWS, ABHS, 0.05% HTH solution, 0.05% NaDCC solution, 0.05% NaOCl solution generated with an electrochlorinator, and stabilized 0.05% NaOCl solution produced from laboratory-grade NaOCl stock. These were compared to two controls: no handwashing (control A) and handwashing with water only (control B).For HWWS, commercially-available Dove White Beauty Bar soap  was used. For ABHS, Purell Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol  was used. Chlorine solutions were produced at the Environmental Sustainability Laboratory at Tufts University. Generated NaOCl was produced with laboratory-grade sodium chloride and MilliQ water using an AquaChlor on-site sodium hypochlorite generator . Stabilized NaOCl was produced by diluting a 5.25% laboratory-grade pH stabilized bleach stock solution  with MilliQ water. NaDCC was produced using Klorsept (previously Aquatabs) granules with 50% available chlorine  in MilliQ water. HTH was produced using commercially available granular calcium hypochlorite with 65% available chlorine  in MilliQ water. Concentrations were confirmed to be within 10% of the goal concentration of 0.05% on the day of the experiment using Hach Iodometric Titration Method 8209 .E. coli as a bacterial comparison and potentially relevant indicator for other diseases of public health importance. E. coli has been commonly used as an indicator for hand hygiene studies, and is recommended in the ASTM International Methods [E. coli (ATCC® 25922) onto Luria-Bertani (LB) agar plates and incubated at 35°C for 24 hours to obtain singles colonies before storing at 4°C. The evening prior to an experiment, a single colony from the plate was used to inoculate 10mL of LB broth and incubated overnight at 35°C with shaking. On the morning of the experiment, a fresh culture was launched with 1mL of overnight culture in 20mL fresh broth and incubated for approximately 2.5 hours. A GeneQuant™ 100 spectrophotometer  was used to estimate the concentration of the culture for a target of greater than 108 CFU/mL. This culture was then used to make an inoculate composed of 68% culture and 32% soil load or 0.9% NaCl solution (depending on the condition tested) to spike hands. Concentration of inoculate was confirmed using membrane filtration; samples were passed through a 0.45 μm filter  and incubated on m-ColiBlue24 media  for 24 hours at 35°C. Approximately 20mL of phosphate-buffered saline (PBS) was added to the filtration funnel prior to filtering any samples less than 10mL in volume to ensure uniform filtration of the sample.To extend the relevance of our work beyond Ebola and into other possible organisms of interest, we used non-pathogenic  Methods ,50. In tWe chose Phi6 as a non-pathogenic, biosafety-level 1 bacteriophage surrogate for the biosafety-level 4 Ebola virus  for this experiment. Phi6, like Ebola, is enveloped, and has previously been used as a surrogate for enveloped viruses in environmental studies and has previously been used in studies on human hands –54. As pPseudomonas syringae (HER #1102) host by the double agar overlay method [P. syringae overnight culture were pipetted directly into 6mL of Nutrient Broth Yeast (NBY) soft agar (0.3%) and poured onto plates with NBY hard agar (1.5%). Plates were incubated at 26°C for 24 hours. Phi6 was retrieved by diffusion [7 PFU/mL; 100 μL of diluted sample was pipetted directly into 6mL of NBY soft agar with 100 μL of overnight host culture, poured over NBY hard agar, and incubated at 26°C for 24 hours [The Phi6 bacteriophage (HER #102) was propagated in the y method . Both 10iffusion ; 5mL of 24 hours .E. coli and Phi6 inoculates [Because EVD patients generally shed virus within bodily fluids such as blood, diarrhea, and vomit, and these fluids may alter handwashing efficacy, each condition was also tested with and without added soil load mimicking human bodily fluids. A tripartite soil load was prepared based on ASTM International Standards, containing 7.80 mg/mL bovine serum albumin, 10.92 mg/mL tryptone, and 2.52 mg/mL bovine mucin and added to oculates .E. coli without soil load; 2) E. coli with soil load; 3) Phi6 without soil load; and, 4) Phi6 with soil load. Each condition was administered on a separate day. Before taking samples each day, researchers confirmed eligiblity and fitness. First, volunteers responded to a short survey about their adherence to the antimicrobial-avoidance period and received a visual hand exam to confirm the absence of breaks or abnormalities in the skin. If lack of adherence or skin breakage was noted, volunteers were unable to participate in that day’s experiment. Second, each volunteer was randomly assigned the left or right hand for the day’s experiment and an order in which the six handwashing protocols and two controls  would be tested. Third, a “cleansing wash” step consisted of running through the steps of the experiment described below, but with a blank inoculate. This technique is used to remove any organisms preexisting on hands and strip the hands of excess dirt and oils, which have been shown in previous studies to create systematic differences between the first and subsequent rounds of testing [Volunteers were asked to come to the Environmental Sustainability Laboratory at Tufts University for testing of four conditions: 1)  testing . This stOn each day, testing was performed for all eight handwashing protocols in randomized order according to the same five steps : 1) skinThe pH of skin on the palm and in the webspace between the pointer and middle fingers on each volunteer’s hand was measured before each round of testing using a Hanna Instruments HI 99181 portable pH meter for skin .After pH testing, volunteers’ hands were inoculated with a suspension of the test organism. Volunteers were asked to cup their hands carefully, and 750μL of inoculate was slowly pipetted into each palm for a total of 1.5mL. Volunteers then gently rubbed their hands together until all surfaces of hands were coated with the inoculate. Finally, volunteers held hands still and away from the body for an additional 30 seconds to allow the inoculate to dry.The order in which handwashing protocols were performed was randomly assigned for each volunteer prior to testing. Funnels sized to produce a flow rate of 1.5L/minute (the approximate rate of a tap from a bucket commonly used in outbreak settings) were used to administer water for handwashing. A large, 5.4L capacity Whirl-Pak bag  was placed in a bucket underneath hands while washing to catch all liquid runoff from the washing process . All hanFor control A, no handwashing was performed. For control B, hands were washed using water alone; 500mL of water was run through the funnel in 20 seconds while volunteers rubbed their hands under the water taking care to rub all surfaces of hands, in between fingers, under nails, and both the palm and back of hands. For HWWS, volunteers’ hands were wet with 10mL of water and they were provided with a bar of soap and asked to rub the bar in their hands until lathered (about 5 seconds). Volunteers then rubbed their soapy hands together for an additional 20 seconds, taking care to rub all surfaces, and rinsed their hands thoroughly as 500mL of water was run through the funnel in 20 seconds. For ABHS, a quarter-sized volume of sanitizer (average of 2.75g) was placed on the hands and volunteers were asked to rub their hands together until dry, taking care to rub all surfaces of hands. For all chlorine protocols, subjects were asked to rub all surfaces of their hands thoroughly as 200mL of chlorine solution flowed through the funnel (for approximately 8 seconds). To standardize handwashing as much as possible, the WHO instructions for handwashing were demonstrated to each participant prior to handwashing, and during handwashing participants were prompted to ensure that all surfaces of hands were rubbed.Rinse water was collected during handwashing for the six possible protocols: control, HWWS, and all chlorine solutions. Rinse water collection WhirlPak bags were prepared with 4.5mL of a 12% sodium thiosulfate solution in advance of testing to neutralize any chlorine immediately upon water flowing into the bag. Results from other studies demonstrate that the concentrations of sodium thiosulfate used for sample collection do not have a deleterious effect on bacterial or viral growth ,60.E. coli, a 0.1% sodium thiosulfate solution was used as an eluent; for Phi6, PBS with 0.1% sodium thiosulfate was used. Samples were collected using a modified glove-juice method [After washing was complete and rinse water collected, the hand selected for testing was immediately placed in a smaller WhirlPak bag containing 75mL of eluent up to the wrist. For e method ,61: a reBefore repeating the entire process with the next handwashing protocol, volunteers washed their hands thoroughly with soap and water in a sink and dried with paper towels. Volunteers’ hands were then decontaminated with 70% ethanol. Hands were sprayed front and back until coated with ethanol from a spray bottle, and then rubbed and allowed to dry. This was then repeated. Hands were allowed to dry before starting the next round of testing, leading to a wait time of about 10 minutes. During this time volunteers were instructed not to touch anything. Hand rinse samples taken after decontamination during pre-testing confirmed that no organisms remained on hands following this procedure.E. coli and soft agar plaque assay for Phi6, as described above. Results of zero were replaced with the minimum detection limit of the test as a conservative estimate of the organisms present in the sample.Samples were processed using membrane filtration for Laboratory data were collected on paper forms and entered into Microsoft Excel 15 . Baseline survey data were collected electronically using Qualtrics software  on tablet, mobile phone, and web browser platforms. Difference in the pH of hands prior to testing with each condition was tested used repeated measures ANOVA. The efficacy of the protocols for removal of organisms from hands was defined as the average log reduction in organisms for each handwashing protocol compared to control A (no handwashing), and the log reduction values for each protocol in comparison to no handwashing were then compared to one another. The ability of handwashing protocols to prevent persistence of organisms in rinse water was defined as the average log reduction in organisms compared to control B (handwashing with water only), after which persistence was compared. A one-way repeated measures ANOVA was used to assess the significance of handwashing on log reduction of organisms, and a post-hoc Tukey’s HSD test was used to assess significant differences between each handwashing protocol for models demonstrating significant differences. Bartlett’s test was used to asses each dataset for sphericity, and a Greenhouse-Geisser correction was applied when the test indicated that sphericity was violated. Statistical significance was assessed at the p = 0.05 level. Analysis was performed in STATA 14 .E. coli without soil load, 18 for E. coli with soil load, 12 for Phi6 without soil load, and 17 for Phi6 with soil load. Volunteers were 72% female, ranged in age from 18–22 years, and 50% described themselves as “white.”Overall, 18 people registered for the study, completed the baseline assessment, and participated in study activities. One volunteer was unable to complete the Phi6 with soil load session due to scheduling conflicts, and the data for six volunteers was lost from the Phi6 without soil load condition due to problems with growth of the bacterial host culture for titration. The final sample sizes for the four conditions were thus 18 for E. coli without soil load treatment, p<0.001 for all other conditions; HTH and NaDCC in webspaces not significantly different at p<0.05 level). There were no significant differences among hands prior to any handwashing treatment . Overall, pH was higher during Phi6 testing conditions than among E. coli conditions.Across the four testing days, the baseline average pH of hands at the start of the testing day ranged from 4.75–5.06 for palms and 4.79–5.13 for webspaces. The average pH of hands prior to each testing condition ranged from 5.49–6.64 for palms and 5.35–6.50 for webspaces. The pH of hands differed significantly from baseline for 62 of 64 conditions (ANOVA p = 0.009 for webspaces in the E. coli concentration of 1.94–3.01 without soil load and 2.18–3.34 with soil load (Compared to no handwashing (control A), the seven handwashing protocols resulted in an average log reduction in oil load . For botWithin the seven handwashing protocols there were significant differences in efficacy without soil load  = 2.72, p = 0.034). Handwashing with HTH, NaDCC, and stabilized NaOCl all resulted in greater log reduction than handwashing with water only . Bartlett’s test indicated that the assumption of sphericity was violated, so the Greenhouse-Geisser correction was used (p = 0.019). There was also a significant difference among handwashing protocols with soil load  = 3.94, p<0.001), with HTH resulting in a significantly greater log reduction than washing with water only, HWWS, and ABHS .Compared to the condition without any washing (control A), handwashing protocols resulted in an average log reduction in Phi6 concentration of 2.44–3.06 without soil load and 2.71–3.69 with soil load . WithoutWithin the seven handwashing protocols there were no significant differences without soil load  = 2.04, p = 0.073). For handwashing with soil load, there were significant differences found  = 7.01, p<0.001). Handwashing with water alone resulted in greater log reduction than ABHS and stabilized NaOCl , and handwashing with soap resulted in greater log reduction than ABHS, stabilized NaOCl, and generated NaOCl . HTH resulted in greater log reduction than ABHS and stabilized NaOCl  and NaDCC resulted in greater log reduction than ABHS and stabilized NaOCl .E. coli remaining in rinse water of 0.28–4.77 without soil load and 0.21–4.49 with soil load (Compared to washing with water only (control B), handwashing resulted in an average log reduction of oil load . For botE. coli than HWWS .Within the five handwashing protocols there were significant differences with and without soil load  = 331.7, p<0.001 and with soil load F = 162.44, p<0.001). Bartlett’s test indicated that the assumption of sphericity was violated in both models, so the Greenhouse-Geisser correction was used (both p<0.001). In both models all four chlorine solutions resulting in significantly greater log reductions in Compared to washing with water only (control B), other handwashing protocols resulted in an average log reduction in Phi6 remaining in rinse water from 1.26–2.02 without soil load and from 1.30–2.20 with soil load . HWWS reWithin the six handwashing protocols there were significant differences without soil load  = 8.95, P<0.001), and all chlorine solutions resulted in significantly greater log reduction in Phi6 than HWWS . There were no significant differences in the log reduction of Phi6 remaining in wastewater with soil load  = 3.35, p = 0.071). Bartlett’s test indicated that the assumption of sphericity was violated in the model with soil load, so the Greenhouse-Geisser correction was used (p = 0.027).We completed a laboratory-based study in which 18 subjects had their hands contaminated with two surrogate organisms with and without soil load and washed their hands with eight different protocols to: 1) quantify the removal of surrogate organisms from their hands and, 2) quantify the persistence of these organisms in the environment and in rinse water. Across our study, we noted some significant differences between the protocols tested. HTH performed better most consistently across conditions, demonstrating greater log reduction than at least one other method in all three significant handwashing models. HWWS performed well for Phi6 with soil load, outperforming three other conditions. However, these handwashing efficacy differences were small in magnitude. These results demonstrate that concerns that chlorine in particular would not be efficacious due to chlorine demand from skin seem unlikely, though there were small efficacy differences between particular chlorine solutions. Significant differences found for persistence of organisms in rinse water were more pronounced than those for handwashing, and the larger magnitude of differences in log reduction of organisms in rinse water demonstrates that chlorine use may have an added benefit of preventing ongoing disease transmission through rinse water.Despite these statistical differences, we would caution against over interpretation of the data, as confidence intervals for the significant differences in handwashing tended to be large, ranging from < 0.5 log to > 1.5 log reduction. We suspect that the rough similarity in efficacy among protocols is because much of the removal and inactivation from handwashing is due to mechanical action; some studies have shown that the addition of an agent such as soap to handwashing with water alone only marginally improves efficacy ,40. ThusE. coli when chlorine was used for handwashing (mean log reduction > 4 log) but very little reduction with HWWS, and a less dramatic but statistically significant reduction in Phi6 . For Phi6 with soil load, log reduction was similar across all conditions.To our knowledge, this is the first study investigating the persistence of organisms in handwashing rinse water. Rinse water may be less important when water is carried by a drainpipe to a treatment facility or when diseases are less transmissible and virulent. However, it may be an important concern for diseases with outbreak potential such as Ebola and cholera, especially in low-resources or emergency contexts. While there has not been any documented environmental spread of Ebola, the infectious dose is very low and the origin of some cases remains unexplained ,62,63. WE. coli and Phi6 is likely due to differences in mechanisms of disinfection acting on bacteria and viruses. Chlorine inactivates bacteria by lysing the cell [E. coli because the bacteriophage may be more susceptible to the mechanical action of handwashing, interaction with skin, and lipophilic action of soap, but that the reduction was less in chlorine because Phi6 was more resistant to chlorine than E. coli on hands. Because the chlorine was neutralized immediately in collection bags pre-dosed with sodium thiosulfate our results present a very conservative estimate of the ability of chlorine to limit persistence in rinse water, and it is possible this could be increased by longer contact with the chlorine. Therefore, the use of chlorine for handwashing may have an added benefit of inactivating organisms removed from hands and preventing persistence in the environment.The striking difference in significant results and the magnitude of these rinse water results that we observed between the cell , and it the cell ,66. Phi6the cell ,68, and the cell ,70. Howethe cell . We suspE. coli testing. There were no differences among pH of hands prior to any of the handwashing conditions, meaning that the cleansing wash served the purpose of eliminating this potentially confounding difference in pH between handwashing conditions.Our observations on hand pH were consistent with previous research, showing a transient increase in the pH of hands after handwashing. Overall, pH of hands was significantly higher after handwashing than at baseline, as expected due to the stripping of sebum and oils that occurs during the washing process ,73. pH aOur study is limited by differences in the characteristics of the laboratory at Tufts University and hospitals and communities in EVD outbreaks. We conducted the study in rooms maintained at a temperature around 21°C, which is lower than typical conditions in EVD affected areas that experience high humidity and temperatures above 30°C. The temperature of the water used for handwashing, also kept at room temperature in our study, would likely be warmer in a more tropical climate. Previous research has shown that the use of warm or cold water for handwashing has no significant impact on efficacy, given that water hot enough to destroy bacteria would also painfully harm skin ,74. AddiThere also may be differences in handwashing in the laboratory and in the field. While we controlled handwashing conditions to the best of our ability and instructed and observed volunteers carefully, handwashing cannot be replicated exactly from person to person. Furthermore, the efficacy of these protocols in the field would likely be lower as proper handwashing technique might not be adhered to as stringently. We also did not examine different drying methods, although it has been shown that different approaches to drying can reduce contamination further or potentially re-contaminate hands .Finally, the greatest limitation of our study was the need to use surrogates rather than the Ebola virus for the safety of participants; this limitation cannot be ethically overcome, due to the danger the Ebola virus poses to humans. The mechanisms of disinfection and removal of viruses from hands are not clear ,71, so wWhile our study provides valuable insight about handwashing for both viral hemorrhagic diseases such as Ebola and common bacterial organisms, there are many fruitful areas for future research. We assessed six of handwashing protocols most commonly used during the EVD outbreak in West Africa, but there are many other ways of handwashing that could be investigated. These include (but are not limited to) handwashing with differing soap formulas, iodine-based scrubs, alcohol-based foams and wipes, traditional practices such as using ash or sand, and handwashing of gloved hands with any of these approaches. We observed many significant differences in the condition with Phi6 and soil load in particular—as these differences don’t represent one clear pattern and tend to be low magnitude, further research is needed to understand the source and relevance of these differences. For example, the currently recommended handwashing protocols result in different lengths of handwashing among methods, and in the field compliance issues often mean handwashing is not completed for the full recommended time. Further research is also needed to assess critical thresholds for the length of time these handwashing protocols are performed, as handwashing time may have an impact on efficacy. Finally, further research should include a broad range of organisms to add to the small body of literature about handwashing efficacy for different pathogens of concern.Our results suggest all handwashing protocols tested are likely to be similarly efficacious for handwashing in EVD contexts, and that concerns that chlorine in inefficacious for handwashing do not have a strong basis in evidence. HTH may be considered as a particularly good choice for response in which use is acceptable and logistically feasible, as it consistently performed well against both organisms of interest. Overall, chlorine may be preferable in some cases because it may confer a benefit of continued disinfection in rinse water after handwashing. In related work, we investigated the safety of these handwashing methods based on their potential to irritate skin and pose a risk for disease transmission, and found no clinically-relevant differences between these same six methods . Based oS1 Dataset(XLS)Click here for additional data file.S2 Dataset(XLS)Click here for additional data file.