The process of neurovascular coupling ensures that increases in neuronal activity are fed by increases in cerebral blood flow. Evidence suggests that neurovascular coupling may be impaired in Multiple Sclerosis (MS) due to a combination of brain hypoperfusion, altered cerebrovascular reactivity and oxygen metabolism, and altered levels of vasoactive compounds.Here, we tested the hypothesis that neurovascular coupling is impaired in MS. We characterized neurovascular coupling as the relationship between changes in neuronal oscillatory power within the gamma frequency band (30–80 Hz), as measured by magnetoencephalography (MEG), and associated hemodynamic changes  as measured by functional MRI. We characterized these responses in the visual cortex in 13 MS patients and in 10 matched healthy controls using a reversing checkerboard stimulus at five visual contrasts.There were no significant group differences in visual acuity, P100 latencies, occipital gray matter (GM) volumes and baseline CBF. However, in the MS patients we found a significant reduction in peak gamma power, BOLD and CBF responses. There were no significant differences in neurovascular coupling between groups, in the visual cortex.Our results suggest that neuronal and vascular responses are altered in MS. Gamma power reduction could be an indicator of GM dysfunction, possibly mediated by GABAergic changes. Altered hemodynamic responses confirm previous reports of a vascular dysfunction in MS. Despite altered neuronal and vascular responses, neurovascular coupling appears to be preserved in MS, at least within the range of damage and disability studied here. In the healthy brain, neuronal activity and cerebral blood flow (CBF) have a close spatial and temporal relationship: increases in neuronal activity are associated with local increases in CBF via changes in blood vessel tone, a process known as neurovascular coupling. Neurovascular coupling is mediated by the interaction of neuronal, glial and vascular cells  and is tIn Multiple Sclerosis, hypoperfusion is seen in both GM and normal appearing WM , as wellHere, we investigated neurovascular coupling in MS using two complementary non-invasive imaging modalities: magnetoencephalography (MEG) and functional Magnetic Resonance Imaging (fMRI). fMRI signals are based on the local vascular response, a process known as functional hyperaemia. MEG directly measures magnetic fields generated by the electrical currents produced by synchronous activity of thousands of neurons, the local field potential . Both MEBy displaying the same reversing checkerboard stimulus at five levels of contrast, we probed neuronal and hemodynamic responses in the visual cortex, expecting these responses in the early visual areas to increase monotonically with increasing contrast . We usedMEG studies investigating MS have mostly used resting-state paradigms, showing clear network disruption across theta, alpha and beta frequency bands e.g., . At the We report no significant group differences in visual acuity scores, P100 latencies, occipital GM volumes and baseline CBF. However, in the MS patients we found a significant reduction in peak gamma power, the blood oxygen-level-dependent (BOLD) response and CBF response to visual stimulation. The patient group presented with more varied neurovascular coupling relationships than the controls, but there was no significant group difference in neurovascular coupling, in the early visual cortex.Patients with a diagnosis of MS  were recAll participants had a behavioral session, a MEG and an MRI scan performed on the same day, except for one control who returned on a different day for the MRI scan.Expanded Disability Status Scale (EDSS) (Nine-Hole Peg Test (9-HPT) for upper limb motor function, the Timed 25 Foot Walk (T25-FW) for mobility and walking, and the Paced Auditory Serial Addition Test (PASAT) 2 and 3 s as a measure of sustained attention. Visual acuity was assessed, in each eye separately, with a SLOAN letter chart (Precision Vision) at 100%, 25%, 10%, 2.5%, 1.25% and 0.6% contrast, expressed as a decimal that represented viewing distance divided by the letter size (in M-units). All participants except five required corrective lenses for daily use and wore them throughout the testing sessions.Patients’ disability was assessed using the e (EDSS) . Tests fe (EDSS)  were carIdentical stimulation parameters were used for fMRI and MEG. The visual stimulus consisted of a black and white checkerboard, polarity reversing every 250 ms. Checks were squares, with a spatial frequency of 1 cycle per degree. The checkerboard was displayed on a mean luminance background, with a small red fixation circle in the center. The rest conditions consisted only of this background and fixation. For both scanning modalities, the entire stimulus field was 16 × 16° of visual angle and the stimulus was projected on screens with a 1024 × 768 resolution and 60-Hz refresh rate. The checkerboard was displayed at 5 Michelson contrast levels: 6.25%, 12.5%, 25%, 50% and 100%. Stimuli were displayed in 30-s blocks and each contrast level was presented 4 times. The rest blocks were also 30 s long, but were presented 8 times. The block order was pseudorandomized across participants, but for each participant the same block order was used for both MEG and fMRI. The task lasted for 14 min and was repeated twice, once for each eye, with the untested eye covered with a cotton pad. We tested separate eyes because a common initial presentation of MS is optic neuritis, an acute, often unilateral, visual impairment characterized by a reduction in visual acuity and connectivity in visual pathways . The expA 275-channel CTF axial gradiometer system was used to obtain whole-head MEG recordings, sampled at 1200 Hz (0- to 300-Hz band-pass). An additional 29 reference channels were recorded for noise cancelation, and 3 of the 275 channels were turned off due to excessive sensor noise. Fiduciary coils were placed at fixed distances from three anatomical landmarks  and the positions of the coils were monitored continuously. For co-registration, these landmarks were later identified on the subject’s structural MRI and also verified with digital photographs. The MEG data were acquired continuously and epoched offline.MRI data were acquired on a 3T GE HDx MRI system using an eight-channel receiver head coil. A 3D T1-weighted structural scan was obtained for each participant: fast-spoiled gradient recalled echo (FSPGR): acquisition matrix = 256 × 256 × 172, 1 × 1 × 1 mm voxels, TE = 2.9 ms, TR = 7.8 ms.During the visual task, a pulsed arterial spin labeling (ASL) scan sequence was acquired with a dual gradient-echo spiral k-space readout , the first echo being used to estimate CBF changes and the second echo being used for BOLD time series analysis. The proximal inversion and control for off-resonance effects (PICORE) labeling scheme was used, with a label thickness of 20 cm  and 10-mm gap between labeling slab and bottom slice. An adiabatic hyperbolic secant inversion pulse was used with quantitative imaging of perfusion using a single subtraction (QUIPSS II), with a 10-cm saturation band thickness . 191 tagM0) of cerebrospinal fluid for the purposes of perfusion quantification: a single volume with the same acquisition parameters but without the ASL preparation and with an effectively infinite TR (so magnetization fully relaxed). Additionally, a minimum contrast scan was acquired to correct for received image intensity variation with the same previous parameters, except TE = 11 ms, TR = 2 s, and 8 interleaves.Before both pulsed ASL scans, a calibration scan was acquired in order to obtain the equilibrium magnetization  was calculated. Values were then expressed in log(MAR) units, which indicate “visual acuity loss”. A value of 0 indicates no loss so is equivalent to visual acuity at the reference standard (20/20), and an increment increase of 0.1 log(MAR) indicates one line of loss.The 9-HPT, T25-FW, PASAT-2, and PASAT-3 were all scored with the BRB-N manual . Responses from 9-HPT and the T25-FW were measured in seconds to complete, and the PASAT in number of correct trials. For visual acuity, the MAR value , allowing source estimates at brain locations directly comparable across participants. For each grid location inside the brain, the forward model  was calculated for a single-dipole orientation by singular value decomposition, using a single-shell volume conduction model . Source To assess potential alterations in transmission to the visual cortex, latencies of visual-evoked fields (VEFs) were characterized, for the left and right eyes separately, and across all five contrast conditions together. The trials were first re-epoched around the time of reversal (0 s), with the baseline period defined as -0.04 to 0 s, and the stimulus period as 0 to 0.21 s. The data were then low-pass filtered at 15 Hz and baseline corrected. The VEFs were first investigated at the sensor level  by averaging over trials for five posterior occipital sensors. The VEFs were subsequently also characterized in source space  by multiplying the pre-processed sensor-level data with the beamformer weights for the location found to be the peak gamma response to the checkerboard stimuli. For both types of analysis, the latency of the peak amplitude between 0 and 0.21 s was then extracted for each participant.lesion_filling, following the protocol of Battaglini et al., 2012, to improve registration, segmentation and volumetric measures of brain tissue. In brief, a lesion mask was manually created by drawing around any visible lesions on the patient's T1-weighted image. At least one lesion was visible for 11 out of the 14 patients. For these 11 patients, the lesion mask, their T1-weighted image, and a white matter mask (FAST segmentation) was used in order to “fill” the lesion area in the T1-weighted image with intensities that are similar to those in the non-lesioned neighborhood (white matter only).Lesion filling was carried out with the FSL function Brain tissue volume, normalized for subject head size, was estimated with SIENAX in FSL . For patRegional GM tissue volumes from the visual cortex were calculated for each subject. These visual ROIs were defined functionally, based on significant group activation to the visual checkerboard stimulus (explained below). The group visual ROI for the left and right eye stimulation was transformed from standard space to T1 subject space and masked with the GM partial volume image to give visual GM ROIs for each subject. Estimates of volume within these ROIs were then normalized with the volumetric scaling factor outputted from the SIENAX analysis.The BOLD signal was isolated by surround averaging the second echo to remove the tag-control signal, as described previously in Z statistic images were thresholded using clusters determined by Z > 2.3 and a (corrected) cluster significance threshold of p = 0.05  to model the effect of group membership. p = 0.05 . A group0 image was registered to this perfusion series and a mask of the lateral ventricles was created, and this was used in the subsequent model to calculate the equilibrium magnetization of blood (M0). A two-compartment kinetic model was fitted to the multi-inversion time data to calculate baseline perfusion, in native space, in ml/100 g/min along with mean arrival time (Tissue Volumes) were transformed to native space in order to estimate the baseline blood flow over GM. The standard-space ROI used in the checkerboard analysis was also transformed to native space, and the baseline perfusion in this region was used to convert fractional estimates of task-induced change in blood flow to changes in absolute blood flow units.As patients with MS are reported to be hypoperfused at rest  a measurval time . IndividWe characterized neurovascular coupling by fitting a linear model that reflected the relationship between the electrophysiological response and the hemodynamic response to the visual checkerboard stimulus. Three coupling models were fitted for each subject: the relationship between gamma oscillations and the relative BOLD signal, the relative CBF signal, and the quantified CBF signal. We used BOLD signal changes, dependent on both metabolism and flow, as this has been the focus of most previous studies relating MEG and fMRI signals. We used CBF signal changes as they reflect direct perfusion to the capillary bed, more localized to the active tissue . We alsoFor each subject, there were 10 data points: one point for each visual contrast and for each eye. We chose not to average across eyes in order to retain useful variance in the responses between eyes, therefore helping us to better model the relationship between MEG and fMRI signals. The gradient of the line, extracted for each participant, was taken to be our coupling measure, indicating the strength of the relationship between these signals.t-tests assessed differences between MS patients and controls on age, behavioral measures, tissue volumes and baseline signals. Mann–Whitney U-tests were used to assess differences between MS patients and controls on the visual acuity scores, for each contrast level tested. Mixed ANOVAs were used to assess the effect of group membership and eye on the latency of the peak (of the VEFs), and the effect of group membership and contrast level on peak gamma power, BOLD and CBF metrics. For the neurovascular coupling measure, gradients and intercepts were extracted from the linear model that was fitted separately for each person, and Mann–Whitney U-tests were used to test the differences in medians between MS patients and controls.Statistical analysis was carried out using IBM SPSS Statistics (Version 20) and R software packages . IndepenU-tests, an exact sampling distribution was used for U  and for each eye . There were no significant group differences between MS patients and controls in visual acuity scores  values were extremely non-normal in their distribution across eyes and groups, and different cells of the design (group vs. eye vs. contrast level) had different variances. Therefore, as the group difference was the focus, separate Mann–Whitney y scores .Table 2MF = 0.001, p = 0.98), and the left eye (156 ± 9) and the right eye (137 ± 8) also did not differ  = 3.04, p = 0.10), for both groups. The results were similar for latencies calculated in source space: there was no main effect of group  = 0.38, p = 0.55) and no main effect of eye  = 1.83, p = 0.19). There was no significant interaction between group and eye for the latencies calculated in sensor space,  = 0.001, p = 0.98), or source space  = 0.16, p = 0.70).We investigated the effect of group  and eye  on the latency of the peak amplitudes of the VEFs, for the sensor and source space analyses. The data were normally distributed, with no outliers. Results reported here are Mean ± Standard Error of the Mean(SEM), expressed in milliseconds. For latencies calculated in sensor space, MS patients (146 ± 9) and controls (147 ± 10) did not have significantly different latencies  = 46.59, p < 0.001 GG; F  = 35.41, p < 0.001; F  = 35.63, p < 0.001, respectively. MS patients had significantly lower signal responses, compare with controls, for BOLD and CBF quantified signals, but this was not significant for CBF percent: F  = 7.97, p = 0.01; F  = 6.62, p = 0.02; F  = 2.76, p = 0.11, respectively. All pairwise comparisons between contrast levels were significant. There was no significant interaction between the effect of contrast and group on BOLD changes  = 0.86, p = 0.42, GG) CBF quantified changes  = 1.25, p = 0.30), or CBF percent changes  = 0.70, p = 0.59).BOLD, CBF quantified and CBF percent signals increased significantly as stimulus contrast increased: F  = 7.87, p = 0.01, GG). Therefore, simple main effects were investigated. There was no significant group difference at the 6.25% or 12.5% contrast levels  = 1.13, p = 0.30; F  = 1.99, p = 0.17, respectively), but MS patients showed significantly lower peak gamma power changes at 25%, 50%, and 100%  = 6.28, p = 0.02; F  = 12.13, p < 0.01; F  = 10.71, p < 0.01, respectively). Peak gamma power signals increased significantly as stimulus contrast increased, for both the control group  = 46.03, p < 0.001, GG) and the patient group  = 22.46, p < 0.001, GG). All pairwise comparisons between contrast levels were significant, except between 6.25% and 12.5%, and 12.5% and 25% in the patient group.For the MEG results, there was a significant interaction between the effect of contrast and group on the peak gamma power changes  to characterize the neuronal response. Broadly, the gamma rhythm is theorized to reflect the balance between excitatory and inhibitory signaling; networks of fast-spiking, parvalbumin-expressing, GABAergic interneurons act on pyramidal cells to bring about synchronous inhibitory post synaptic potentials . A largeDisruptions in gamma oscillations have been reported in many brain disorders . In MS, Although they presented with preserved visual acuity and latency of visual-evoked fields, the MS patients showed significant hemodynamic alterations, in the form of reduced BOLD and CBF responses to the visual checkerboard, in the primary visual cortex. In the context of largely preserved neurovascular coupling, the reduced hemodynamic response is consistent with the reduced electrophysiological response. Changes in the BOLD response to visuomotor tasks have been previously demonstrated, showing that inflammation and white matter structural damage play a role in altering hemodynamic responses in MS . Our altThe relationship between the peak gamma power change and the BOLD/CBF response  was our empirical measure of neurovascular coupling. While this is intuitive, assuming that the blood flow response only reflects a coupling with gamma oscillatory activity is simplistic. The amplitude of gamma oscillations can be modulated by the phase of slower oscillations, termed cross-frequency phase-amplitude coupling , and an milieu. Indeed, the response of blood vessels to neuronal activity is not only mediated by reactivity of the smooth muscle cells, but also by neuronal and glial signaling, involving many chemical mediators. Increased levels of both vasodilators (e.g., nitric oxide, In this study, we could not demonstrate significant differences in neurovascular coupling between the MS patients and controls. While this may be related to the power of the study, it may also reflect the complexity of the biology underlying the relationship between neuronal activity and the hemodynamic response, which in MS is affected by the inflammatory While not significantly different from healthy volunteers, the analysis of the neurovascular coupling showed a trend for the MS group to have lower intercepts and higher gradients, when predicting the BOLD and CBF changes from the peak gamma power changes. An increased blood flow response, for same gamma power change, may seem counterintuitive considering the reports of blood vessels being less reactive in MS . HoweverWe found evidence for reduced neuronal and hemodynamic responses in the early visual cortex in MS in response to visual stimulation, in the absence of substantial functional impairments to visual acuity or delayed visual-evoked fields. We could not demonstrate a significant alteration in neurovascular coupling in MS patients. Further research into neurovascular and metabolic function across the whole brain, and at different disease stages, will help uncover the importance of these processes in MS.


Pulse wave analysis is an emerging approach that analyzes parameters comprising strong predictors of cardiovascular (CV) events and all-cause mortality, especially in patients with high CV risk based on established risk factors. This study used the oscillometric method, provided by the Mobil-o-Graph  device, to compare data regarding the pulse wave analysis parameters in hypertensive nondiabetic and diabetic patients.In this cross-sectional study, 276 individuals were examined in the academic hypertension outpatient care unit of the Federal University of the Triângulo, in Mineiro, Brazil, from January to December 2016. The pulse wave analysis was performed by oscillometry, and its parameters were acquired from all patients.P = .008 and.0003, respectively). The mean peripheral systolic blood pressure and pulse pressure were also significantly higher in the diabetic group . The average pulse wave velocity  was 9.4 ± 1.6 and 8.8 ± 1.6 in the diabetic and nondiabetic groups, respectively (P = .003).Of the 276 patients, 99 were diabetic and 177 nondiabetic. The mean systolic and pulse central blood pressure were significantly higher in diabetic patients than in nondiabetic patients (The group of hypertensive diabetic patients had significantly higher central blood pressure, peripheral blood pressure, and PWV than the hypertensive nondiabetic patients. The patients with overlapping established CV risk factors presented values of the pulse wave analysis parameters consistent with higher central pressure and greater arterial stiffness. Increasing importance has been attributed to arterial components, namely, endothelial dysfunction and arterial stiffness.,8The most reliable parameter in determining arterial stiffness is pulse wave velocity (PWV), which is the major component of pulse wave analysis. A pulse wave is generated at each systole, and its components depend on left ventricular function and arterial characteristics. Thus, the analysis of the pulse wave by a reliable, noninvasive, reproducible, and inexpensive technique constitutes a useful method in predicting CV risk and mitigating CV events. This new approach to access pulse wave parameters is feasible for outpatient use and therefore is a simple and useful equipment for primary care units. Validation studies suggest a strong correlation with invasive intra-aortic catheter measurements, which is the gold standard.–12Recently, a new oscillometric method that is noninvasive and reproducible has been developed. This method allows the measurement of peripheral and central blood pressure and pulse wave analysis parameters, notably the PWV, through the transformation of the brachial pulse waveform.Currently, the aortic PWV is a known strong predictor of CV events and all cause mortality, especially in patients who already have high CV risk based on established risk factors.Hypertension is a pathologic condition with concerning prevalence worldwide.The increase in blood pressure levels has a wide range of effects in the body, and the arteries are frequently involved. Hypertension is already considered an important cause of arterial stiffness and is related to high PWV values and other components of pulse wave analysis such as the augmentation index (AugI) and augmentation pressure (AugP).,15Diabetes is also a morbid condition regarding its effects to the arteries. Individuals with diabetes have greater PWV compared with the general population even when adjusted for age, blood pressure, and heart rate. Diabetes is also described as an independent marker of CV disease in patients with diabetes.,16,17 by comparing hypertensive patients with and without diabetes.Hypertension is an important and powerful modifiable risk factor of CV diseases in patients with type 2 diabetes. The rate of hypertension among diabetic individuals is up to 75%, which points to an important association regarding CV physiopathology alterations in these 2 conditions. This suggests an additive effect in the arterial stiffness process, evaluated mainly by the PWV. The purpose of this study was to evaluate the importance of diabetes and hypertension, as well as its coexistence, in the behavior of arterial pulse wave analysis parameters22.1This prospective cross-sectional study included 276 hypertensive individuals undergoing regular follow-up at the academic hypertension outpatient care unit of the Federal University of Triângulo Mineiro (FUTM) and Diagnostic Cardiac Center (DCC), located in Uberaba, Minas Gerais, Brazil. Informed consent was obtained and documented. The patients were examined from January to December 2016. Patients with hypertension and diabetes who were attending regular follow-up at our outpatient care were included. Patients younger than 18 years, with underlying condition(s) that required recent hospitalization, heart failure functional class 3 or 4 of the New York Heat Association, and end-stage kidney disease were excluded. The protocol of the study followed the principles of the Declaration of Helsinki and was approved by the institutional ethics committee. Written informed consent was obtained from all participants.2.2,18,19 and also registers the peripheral and central blood pressure levels. A common cuff was centered to the left upper arm. Cuff size was chosen according to the circumference of the mid upper arm. Generation of central aortic blood pressure curves based on brachial pulse waves is based on a previously published algorithm that integrates arterial impedance and aortic hemodynamics into a mathematical model. After digitalization, the signal processing is performed using a 3-level algorithm. In a first step, the single pressure waves are verified for their plausibility by testing the position of minima and the corresponding wavelengths. During the second stage, all single pressure waves are compared with each other to recognize artifacts. Thereafter, an aortic pulse wave is generated by the means of a generalized transfer function. The idea behind a transfer function is the modification of a certain frequency range within the acquired pulse signal to get the aortic pressure wave.,20After a rest period of at least 30 minutes, the oscillometric method of pulse wave analysis was performed in an appropriate room, between 9:00 AM and 3:00 PM, during peak period of antihypertensive medication. The device used, Mobil-O-Graph , is a commercially available brachial oscillometric ambulatory blood pressure monitor that has been validated according to the recommendations of the European Society of HypertensionFour consecutive measurements were taken every 2 minutes according to general recommendations for clinical studies.All individuals answered questions about personal and clinical data . Patients also underwent conventional sphygmomanometer arterial blood pressure measurement according to the guidelines of the Brazilian Hypertension Society. The diagnosis of hypertension was based on the same guidelines. and were attending regular follow-up at the diabetes outpatient care of the FUTM. We did not select any specific subgroup of diabetic patients.The hypertensive diabetic patients were diagnosed according to the American Diabetes Association recommendations2.3t test signed rank test continuous variables. Given unequal sample sizes of our data, multivariate analyses (ANOVA) with D’Agostino–Pearson test was used to compare pulse wave analysis parameters and performed adjustment for age, smoking, mean antihypertensive agents per patient, mean body mass index, mean abdominal circumference (cm). For PWV adjustment we add peripheral systolic blood pressure (pSBP), peripheral mean blood pressure (pMBP), peripheral pulse pressure (pPP), central systolic blood pressure (cSBP), central pulse pressure (cPP), and AugP.We built a database using the Excel program and performed the statistical analysis using the GraphPad Prism  and MedCalc software. All results are expressed as mean ± standard deviation. Chi-squared test was applied to compare proportions and Student 33.1± 11.9 and 59 ± 11 years, respectively. A total of 272 (98.5%) patients were taking antihypertensive medication. The population characteristics are given in Table Among the 276 hypertensive patients, 177 were nondiabetic and 99 were diabetic. There were 173 women (62.6%), and the average age of the patients in the nondiabetic and diabetic groups was 58 3.2P = .008 and .0003, respectively) . The average PWV was 9.4 ± 1.6 and 8.8 ± 1.6 in the diabetic and nondiabetic groups, respectively (P = .003).The mean cSBP and pulse pressure were significantly higher in the diabetic group in comparison with the nondiabetic group (y) Table . We obtaP = .002), pSBP 143 ± 1.9 vs 138 ± 1.4 (P = .0003), pPP 56 ± 1.3 vs 50 ± 0.9 (P = .0001), cPP 42 ± 1.1 vs 39 ± 0.9 (P = .01). And missed significant values for: pMBP 112 ± 1.6 vs 110 ± 1.2 (P = .38), cSBP 131 ± 1.8 vs 128 ± 1.3 (P = .18), and AugP 14 ± 0.8 vs 12 ± 0.6 (P = .10).After adjustment for diabetic vs nondiabetic comparisons, these differences remained significant for the following variables: PWV 9.1 ± 0.05 vs 8.9 ± 0.03 . There was no statistical difference between the average AugI and the diastolic central or peripheral blood pressure .The AugP was also higher in patients with diabetes  than nondiabetic patients (n = 203). Compared with our study, the patients on DESIR had higher systolic blood pressure values (158 ± 29 vs 160 ± 28) than our study (137 ± 17.6 vs 145 ± 22.5). Surely the differences in PWV between the studies at least in part could be explained by a difference in blood pressure. Using a new method to measure arterial stiffness, equally, we found significant differences in arterial stiffness between diabetic and nondiabetic patients, which reinforce our results.In a review based on data obtained from the French cohort, DESIR (Data from an Epidemiologic Study), diabetic patients (n = 126) showed significant higher PWV values (11. 5 vs 13.9; ,4Our study had some limitations. The stratification of the study subjects could have been performed according to the time of diagnosis and control of the underlying disease. There was no control group of healthy individuals, which could estimate the real impact of hypertension and diabetes in the pulse wave parameters. In the diabetic group there was a higher prevalence of smoking patients; however, we could not find data about the association between the smoking habit and the PWV. An important aspect of the sample analysis was that the PWV was higher in the hypertensive diabetic patients regardless of age variation. Age is a factor that presents an independent association with increased arterial stiffness and could be a confounding variable in the analysis.The development and application of technologies that are simple, routine, cheap, and relevant are great value, especially in developing countries like Brazil where CV and metabolic diseases are important public health problems. In this context, the establishment of approaches that allow the early identification of risk factors become essential in the promotion of health in its primary level of care and prevention.Furthermore, our study raised the need for approaches that take into account the diagnostic time of diabetic patients and their glycemic control. The drug used in the treatment of hypertension is also a relevant aspect to be considered. The comparison between different glycemic levels and the CV repercussion on pulse wave parameter should motivate more studies.In conclusion, in this study, hypertensive diabetic patients had significantly higher central and peripheral systolic pressure and pulse pressure compared with hypertensive nondiabetic patients. The AugP was also statistically higher in hypertensive diabetic patients. Furthermore, diabetic patients had higher PWV regardless of age variation, and the difference was statistically significant. This study demonstrated that patients with overlapping established risk factors for CV disease had altered pulse wave analysis parameters consistent with arterial stiffness and central hemodynamic disorders.Conceptualization: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Elisabete Aparecida Mantovani Rodrigues Resende, Valdo Jose Dias Silva, Dalmo Correia.Data curation: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Formal analysis: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende.Investigation: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Methodology: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Valdo Jose Dias Silva, Dalmo Correia.Project administration: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Resources: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Software: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Supervision: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Validation: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Visualization: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Dalmo Correia.Writing - Original Draft: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Elisabete Aparecida Mantovani Rodrigues Resende, Valdo Jose Dias Silva, Dalmo Correia.Writing - Review & Editing: Luiz Antonio Pertili Rodrigues Resende, Marco Antonio Vieira Silva, Jose Augusto Mantovani Resende, Elisabete Aparecida Mantovani Rodrigues Resende, Valdo Jose Dias Silva, Dalmo Correia.


Adjusting the focal plane through the intact scalp of mice is crucial in novel angiography of cerebral vasculature using quantum dots emitting second near-infrared light at a wavelength of 1100 nm. Reagents were administered through the caudal vein. When we focused 0.4 mm below the scalp surface, based on the anatomical properties of mice reported previously, the intensity of clear fluorescence images observed transiently under a microscope became very weak within several seconds. The remaining time was extremely short to repeat adjustment of the focal plane. To investigate focus, photons exciting quantum dots at depths of 0.4, 0.8, 1.4, and 2.0 mm and emission photons were tracked in a four-layered Monte Carlo model including the scalp, skull, cerebrospinal fluid, and cortex. Based on the most near-ballistic photons emitted from quantum dots at 0.4 mm depth and specification of the microscope used, including numerical aperture and depth of field, the optimal focus plane was set.•Novel angiography for cerebrovascular structures was proposed using quantum dots with second near-infrared fluorescence.•Anatomical properties reported previously allowed focusing 0.4 mm below the surface of intact scalp before observation under fluorescence.•Clear images of cerebrovascular structures were attributed to many near-ballistic photons emitted from quantum dots at 0.4 mm depth. Specification Table2. A research MVX-10 macro-zoom fluorescence microscope  coupled with the C10633-34 InGaAs camera  was used for the imaging. A filter set comprising an excitation filter for a 785 nm laser, dichroic mirror to reflect the 785 nm laser and transmit over a wavelength of 800 nm, and a long-pass emission filter with a cut-off wavelength of 800 nm was used. Furthermore, an emission filter with a wavelength of 1100 ± 25 nm was used and placed after the filter set. Camera control and image acquisition (exposure time: 100 ms/frame) were performed using HCImage  [The surface of intact scalp from the parietal to frontal area was illuminated using the laser diode BWF1 that emitted light at a wavelength of 785 nm. The maximum excitation power on the sample stage was 25.5 mW/cmThe experimental procedures in the present study were approved by the Animal Care and Use Committee of the Maebashi Institute of Technology (AN-18-006). Data were obtained from 4-5week-old hairless male Hos:HR-1 mice . The animals were housed in cages with ad libitum access to food pellets and water. They were maintained under a 12-h light/dark cycle. The Hos:HR-1 strain loses its hair coat rapidly and completely due to an abnormal second hair cycle starting at 2 weeks of age, thereby becoming completely hairless . RecentlSeveral seconds after bolus injection of 50 μL of 1 μM QD1100 into the caudal vein of a mouse, weak fluorescence near the middle cerebral arteries appeared occasionally. Then, strong fluorescence in the venous phase masked that in the weak arterial phase. As shown with arrowheads in r. Each layer was infinitely wide, and the thickness of the scalp, skull, and cerebrospinal fluid (CSF) was set to 0.2, 0.1, and 0.1 mm, respectively, based on the anatomical properties of the mouse. Except the scattering coefficient sμ and anisotropy g of CSF, optical parameters to simulate the observation space of mouse head were found in literature. Because CSF is a clear solution, sμ and g was set to 0 and 1, respectively, according to their definitions. Scalp was regarded as skin. As shown in The four-layered Monte Carlo model was developed based on the three-layered model . The sof6 photons entered from the skin surface. The path length to a photon-tissue interaction was calculated based on random numbers and total attenuation coefficient: tμ = sμ + aμ. Each photon had an initial weight corresponding to 1. As photons reached an interaction site, they lost aμ/tμ of their weight. Toward the next interaction site, an isotropic azimuth and a zenith angle with highly forward scattering property were generated using different random numbers. When photons crossed the boundary between two layers, they were reflected or refracted according to the Fresnel reflectance. When light of an intensity I0  crosses the boundary from a medium with refractive index n1 to that with refractive index n2, intensity decreases to I,An infinitely narrow beam comprising 10α is larger than critical angle cθ, light is reflected at emergent angle α.When incident angle α is smaller than critical angle cθ, light is refracted at refraction angle β.When incident angle Quantum dots were excited and emitted when photons entering from the skin reached them. Subsequently, they became a new light source that emitted isotropic fluorescence depending on the sum of excitation photon weights at a specific depth. Near-axis coaxial fluorescence photons were captured by an image sensor using an optical system of our epifluorescence macro zoom microscope .r, z). Here, fluence is a quantity converted per photon and per cm2. In the skin, the absorption coefficient at 785 nm in NIR-I was 4-fold higher than that at 1100 nm in NIR-II. In addition, the scattering coefficient at 785 nm was 1.4-fold higher than that at 1100 nm. Thus, the spatial distribution of photons in NIR-I in the skin spread wider than that in NIR-II and the strong absorption made the area with high photon fluence wider than NIR-II. Near-axis NIR-II photons transmitted though the skin were considered more in number than NIR-I photons and to have more weight than NIR-I photons. On the other hand, in the skull, the absorption coefficient at 1100 nm in NIR-II was 4-fold higher than that at 785 nm in NIR-I although the scattering coefficient in NIR-II was 83% of that in NIR-I. This resulted in a high photon fluence area in NIR-II wider than that in NIR-I. The results for transverse propagation within CSF were similar to those of goniometric measurement for estimating anisotropy g [z = 0.4 mm and r =0 mm. Excitation photons propagated deeper when the excitation light became stronger, i.e., photon weight increased. In contrast, emission photons propagated more wide because of isotropic emission from quantum dots.First, we compared the differences in photon propagation between NIR-I and NIR-II using the four-layered Monte Carlo model, which simulated a hairless mouse head including the scalp, skull, CSF, and cortex. When photons entered the model with a strong forward scattering property, most photons propagated along and from the light axis, and partially lost weight due to their interaction with tissue of scattering and absorption. −2 and was the strongest among all depths. Deeper quantum dots got, the weaker the intensity became. The intensity at 0.8 mm depth equivalent to a 0.4 mm depth from the cortical surface was 33 cm−2 which was 1.1% of the intensity at 0.4 mm depth. The intensity at 1.4 mm and 2.0 mm depths was 0.15% and 0.054%, respectively. Thus, strong light scattering due to tissues resulted in a decrease in intensity in deeper tissues. Furthermore, it would affect the distribution of photons apart from the light axis.Next, we examined the excitation efficiency of quantum dots at different depths from the skull surface. 6 photons with an initial weight of 1 were used as a point source in simulation at 0.4 mm depth, and compared them with those with weights of 0.011 at 0.8 mm depth, 0.0015 at 1.4 mm depth, and 0.0005 in 2.0 mm depth depending on the excitation efficiency shown in 5 fluorescence photons were emitted from 0 to 0.4 mm depth. As the photons emitted from deeper points were counted, the number of photons decreased remarkably; 3.1 × 103 at 0.8 mm depth, 40 at 1.4 mm, and 2 at 2.0 mm. Furthermore, similar to excitation, the deeper the quantum dots got, the more widely fluorescence photons spread (data not shown).Finally, we examined emission properties when 10The use of this optical system facilitated preferential collection of these near-ballistic fluorescence photons relative to the diffusely scattered photons. Therefore, the fluorescence photons at 0.4 mm depth permitted brighter and clearer imaging of the vascular system near the cortical surface using this optical system, unlike photons emitted at 0.8, 1.4, and 2.0 mm depth. In contrast to the photons emitted in the visible region, the near-ballistic photons emitted in NIR-II tended to survive because photons that traveled deeper due to tissue scattering were absorbed by water, which can improve image quality. The depth of field of the microscope used in the present study was 0.3 mm when it was calculated using wavelength, numerical aperture, and magnification, according to the Berek equation . This vaSo far, in a simple approach for nTOVI, the standard near-infrared fluorescent dye used was excited at a wavelength of 720 nm in NIR-I and had fluorescence spectra with the emission maximum at 780 nm in NIR-I. The preliminary treatment such as scalp shaving just before imaging facilitated non-invasive cerebrovascular observation. The quantum dots used in the present study were excited at a wavelength of 785 nm in NIR-I and emitted fluorescence at a wavelength of 1100 nm in NIR-II. For noninvasive imaging of a cerebrovascular structure, the use of hairless mice was feasible by injecting QD1100 under general anesthesia. As suggested by Monte Carlo simulation, the cerebrovascular structure was observed by setting focal plane 0.4 mm below the scalp surface.　We have no conflict of interest with any company or commercial organization.


Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4. We demonstrate that in aqueous systems fluorescent dyes form a self-stabilized (pseudolyophilic) emulsion of auto-fluorescing colloid particles. Sample shaking and addition of surfactants enhance auto-fluorescence due to increased dispersion and, in the presence of surfactants, stabilization of the dye emulsion. Bacteriophages with genome sizes <100 kbp (i.e. λ & P1) did not generate a distinct population signal to be detected by one of the most sensitive FCM instruments available (BD LSR Fortessa™ X-20), whereas the larger T4 bacteriophage was resolved as a distinct population of events. These results indicate that the use of fluorescent dyes for bacteriophage enumeration by flow cytometry can produce false positive signals and lead to wrong estimation of total virus counts by misreporting colloid particles as virions, depending on instrument sensitivity.  •Fluorescent dyes form auto-fluorescent colloidal particles in aqueous solutions.•Presence of surfactants enhances non-specific dye fluorescence.•Successful virus enumeration relies on correctly identified target population.•Stained virus samples demand gentle handling. They inTo investigate viruses in environmental waters, transmission electron microscopy (TEM) was one of the first methods to be utilized, which demonstrated much higher abundances of viruses in marine waters compared to plaque forming unit enumeration . With th® Green I is preferred for virus staining since this fluorescent dye is affordable and results in higher virus counts when compared to other dyes , P1, and T4 bacteriophage enumeration compared to PFU estimations.The aims of this study were to illustrate likely artifacts and understand their mechanisms when staining bacteriophages with SYBR22.1E. coli hosts TG1 (Lucigen), MG1655 (ATCC 47076), and BL21DE3 (Sigma-Aldrich) respectively. The E. coli cultures were grown in LB broth  at 37 °C and 250 rpm to optical densities of 0.6–0.7, then infected with appropriate bacteriophage and the incubation was continued overnight at 37 °C with no shaking.Bacteriophages of three genome sizes: 48,502 bp dsDNA lambda ; 93,601 g for 30 min to precipitate bacterial cell debris, supernatant was filtered through 0.22 μm syringe filter  into a sterile Amicon Ultra 100 K centrifugal filter device , and centrifuged again at 4,000 g for 20 min to eliminate any influence of growth media on flow cytometry analysis. Bacteriophage remaining on the filter part of the device (in about 250 μL), was treated with DNAse I  to remove residual host DNA by adding: 25 μL of 10x DNAse I buffer  and 1 μL of 2.5 mg/mL DNAse I, dissolved in storage buffer (20% glycerol in 75 mM NaCl) to the bacteriophage suspensions and incubated for 45 min  at 37 °C. All chemicals were purchased from Sigma, unless stated otherwise.Overnight cultures were centrifuged at 4,000 After the incubation, bacteriophage samples were rinsed with 10 mL of 1x HyClone PBS  that was filtered through 1 kDa Macrosep Advance Centrifugal device , resuspended in PBS to the initial volume and analysed.2.2Solid and soft Trypticase Soy Agar was prepared from BBL Trypticase Soy Broth  with addition of 1.5 and 0.6% agar respectively. Triplicate decimal dilutions of bacteriophage  samples were prepared in 900 μL of 1x HyClone PBS and the double-layer agar assay was carried out as described previously . Standar2.3® Green I ) implies a hydrophobic compound, which is not fully soluble in aqueous solvents. Hence, to estimate fluorescence of colloidal SYBR particles, we prepared stabilized emulsions of SYBR with each of the following surfactants: Triton-X100, IgePal-630, Tween 20, NP 40, Brije 35, and Sodium Dodecyl Sulfate (SDS). SYBR Green I  was added to 1% solution of a surfactant in 1 kDa – filtered Tris-EDTA (TE) buffer pH 8.0 to final concentration of 50 x. All samples and SYBR Green I stock in this study were diluted, stained, and stored in black microcentrifuge tubes .The molecular structure of SYBR® (ThermoFisher Scientific #F8806) were added to a final concentration of 3.4 × 107 beads.mL−1 for quality control.Duplicate dilutions of SYBR in TE were prepared at 0.5x, 1x, 5x, and 50x concentrations; one set was heated at 80 °C for 10 min, and the other was analysed unheated. All TE buffer was 1 kDa – filtered before use. Crimson fluorescent 0.2 μm FluoroSpheresThe working stock of SYBR Green I should not be filtered due to interactions that remove this hydrophobic dye from solution . This efFluorescence was observed with a conventional benchtop UV transilluminator  as well as an EVOS FL fluorescent cell imaging system (ThermoFisher Scientific). For the wet mount, 25 μL of fresh samples were placed on new pre-cleaned microscope slides (ThermoFisher Scientific #12-550-A3) and glass coverslips (ThermoFisher Scientific #12-540B). The EVOS images were captured in TxRed (585/29 Ex 624/40 Em), GFP (470/22 Ex 510/42 Em), and TRANS channels and image overlays were created.2.4® Green I samples were diluted in TE buffer to final concentrations 0.1x, 0.2x, 0.5x, 1x, and 2x, with one set heat treated and the other not, as described above.SYBR® Green I. TE buffer was also prepared with the SYBR dye as negative control.Bacteriophage decimal dilutions were prepared in triplicate in TE buffer and stained as described  with 0.5® . The beads were first briefly vortexed and then bath-sonicated for 1 min as recommended by the manufacturer; noting that vortexing only gave inconsistent results (data not shown). Triplicate 100-fold serial dilutions were prepared to 10−4, and then decimally to 10−6, immediately after the sonication step. It is important to pay attention that no droplet was left on the outer side of the pipette tip. Dilutions, used for analysis, were briefly vortexed and sonicated again right before being analysed. As each batch of beads has a Certificate of Analysis with the number of beads per mL indicated, it was possible to calculate the number of beads per mL of the working dilution. To calculate the flow rate, the number of events in the bead population was divided by bead concentration in the working dilution. Flow rate was calculated each time samples were analysed.Flow rate was estimated with 1 μm latex bead FluoroSpheresFlow cytometric analysis was carried out with BD LSRFortessa™ X-20  cell analyzer  equipped with 488 nm excitation laser with standard filter setup. The trigger was set on green fluorescence (FITC channel). Data was collected using FITC-W/SSC-W dot plots. Events were gated based on SYBR in TE samples with no virus and T4 SYBR-stained decimal dilutions.Also, an older model of flow cytometer, Gallios™ (Beckman Coulter), also equipped with 488 nm excitation laser, was used to compare sensitivity of the two instruments. Data were collected as FL1 INT/FL2 INT and/or FL1 TOF/SSC TOF plot, with the same no virus and T4 SYBR-treated samples used on the BD LSRFortessa™.33.1® Green I partly dissolved in TE buffer revealed the presence of fluorescent particles in all dilutions, in both heated and unheated samples. Critical to the presence of possible artifacts analysed by FCM, this dye produces small crystals or amorphous mass, which may also lead to uneven distribution of the SYBR fluorophore among the aliquots used for sample staining  resulte Green I  even witcroscopy , and FCM® Green I in TE demonstrated a distinct population of fluorescent particles. Event counts in some random sample tubes were much higher than in other replicate tubes with supposedly the same concentration of SYBR (data not shown). Most likely, this variability was the effect of non-uniform dispersion of SYBR® Green I in the stock solution. Moreover, the event counts noticeably increased after bath-sonication, pipetting, or just hand shaking of the samples and decreased in the samples subsequently kept undisturbed, as illustrated in Flow cytometric analysis of various concentrations of SYBR3.2−1 of T4, 10.36 ± 0.25 log PFU.mL−1 of P1, and 9.3 ± 0.15 log PFU.mL−1 of λ bacteriophages. However, both Fortessa™ X-20 and Gallios™ instruments failed to detect Lambda (data not shown) and P1  bacteriophages. On the other hand, bacteriophage T4 was resolved as a distinct population of events when analysed on the Fortessa™ X-20 ) there are inherent problems in using such fluorophores when targeting small particles like viruses by FCM. Though not widely discussed in the microbiological literature, SYBR® Green I forms a disperse colloid rather than a homogenous molecular solution. Disperse systems can be formed via two main routes: mechanical dispersion or condensation from oversaturated solutions  was also shown to self-stabilize in solution in the same manner, which we confirmed by flow cytometry (S4). Furthermore, the addition of surfactants to a panel of SYBR® Green I solutions generated and stabilized artifact particles into emulsions  could be identified by gating it/them from the total stained suspension signal. As illustrated in the current work, serial dilutions of the sample need to be correlated with the decline in target signal, which should be independent of dye concentration and should appear as a defined target population e.g. . Once thIn addition, staining of virus particles with nucleic acid stains may require heating of the samples to 80 °C, in order to expose viral nucleic acid . Success5Commonly used fluorescent dyes create pseudolyophilic colloid systems, which auto-fluoresce as stained virus-like particles even in the absence of DNA. The presence of surfactants further enhances non-specific fluorescence of such dye colloids and, therefore, use of surfactants for sample preparation should be avoided. Altogether, these interfere with small-particle enumeration by fluorescence-based assays, such as flow cytometry.Successful enumeration relies on correct identification of the target population by the careful use of negative virus control samples. The instrument sensitivity should be assessed by comparison with established culture-based methods.Given the pseudolyophilic colloidal nature of fluorophores used in FCM, sample handling can additionally affect the accuracy of virus enumeration. Overall, further research is needed to optimize the use of fluorescent dyes for virus quantification from environmental matrices by sensitive assays, such as flow cytometry.The authors have no conflict of interest to declare.


Work-related musculoskeletal pain is a major cause of work disability and sickness absence. While pain is a multifactorial phenomenon being influenced by work as well as lifestyle, less is known about the association between specific lifestyle factors and the type of musculoskeletal pain. The aim of the study was to investigate if a dose-response association existed between lifestyle factors and musculoskeletal pain intensity in the low back and neck-shoulder.N = 10,427) replied in 2010 to questions about work environment, lifestyle and health. Logistic regression analyses adjusted for various confounders tested the association of alcohol intake, physical activity, fruit and vegetable intake, and smoking (explanatory variables) with low back pain and neck-shoulder pain intensity .Currently employed wage earners  of 0.95 (95% CI 0.90–1.00) and 0.90 (95% CI 0.82–0.99), respectively. No association was found between alcohol intake and pain.Being physically active associated with lower risk of having musculoskeletal pain, while smoking habits and healthy eating were associated with higher pain when adjusting for age and gender. Considering the continuously increasing retirement age in many societies, initiatives to promote healthy habits should still be a political priority to help the workers to stay healthy and cope to their work. Low back pain and neck-shoulder pain affect between 51 and 90% and 14–71% of people at some point during their lifetime, respectively , and areLow back pain and neck-shoulder pain are multifactorial disorders related to both individual, physical and psychosocial work environmental factors . In the Physical activity and exercise programs are increasingly being promoted and offered in various healthcare systems. A Cochrane review from 2017  indicateConsidering that cigarette smoking is one of the leading causes of premature morbidity and mortality , systemaAlcohol intake is another risk factor that needs to be considered. Few reviews have been carried out to investigate the association of alcohol intake with low back pain. In 2000, Leboeuf-Yde  performeMoreover, healthy lifestyle habits such as consumption of fruit and vegetables have also been investigated, however the number of studies is limited. This habit is considered as a part of healthy lifestyle behavior  or optimAltogether, more data is necessary to better understand the association between healthy lifestyle behavior and musculoskeletal disorders in workers. Because the working population is aging in many societies, a healthy lifestyle behavior may be important for staying healthy and cope to the work until high age. Thus, the present study aims to examine the joint association between four lifestyle behavior factors  and musculoskeletal pain in the low back and the neck-shoulder among more than 10,000 adults from the general working population in Denmark.n = 10,427), i.e. excluding self-employed people and people not affiliated with the labour market [The present study is a cross-sectional study consisting a questionnaire survey about work environment and health in the general working population in Denmark, the Danish Work Environment Cohort Study (DWECS) . The quer market . BecauseThe present study has been notified to and registered by The Danish Data Protection Agency . According to the Danish law, questionnaire- and register-based studies do not need approval by ethical and scientific committees, nor informed consent . All data were de-identified and analyzed anonymously.Low back pain and neck-shoulder pain intensity were assessed by replying to the question “Have you experienced any trouble (pain or discomfort) in your (body region) during the last 12 months?” on a scale ranging from 0 to 9, where 0 is no pain and 9 is the worst imaginable pain. For further analyses, back pain was dichotomized into “High pain” (pain intensity ≥4), “No or little pain” (pain intensity 0–3).Physical activity was assessed by the question: “How much time did you on average spend on each of the following leisure-time activities during the last year (including commuting to and from work)?”It had the following three sub-questions of 1) low, 2) moderate and 3) high-intensity exercise: 1) “Walking, biking or other low-intensity exercise, where you do not get out of breath or sweaty ?” 2) “Exercise training, heavy gardening or fast walk/cycling, where you get out of breath and sweaty?” and 3) “Vigorous exercise or competitive sports?”. The response options were: Do not perform this activity; < 2 h per week; 2–4 h per week; or > 4 h per week . These fAlcohol intake was assessed by two questions: (1) “How much alcohol do you drink on average on weekdays (Monday to Thursday)?”, and (2) “How much alcohol do you drink on average Friday, Saturday and Sunday?” The amount of alcohol was considered as number of units per day. One unit = 1 bottle of beer, 1 glass of wine or 2 cl. liquor.For further analyses, the number of units from the two questions were summed and further grouped into: “0-7 units per week”; “8-14 units per week”; “15-21 units per week”; “>21 units per week”.The smoking status was assessed by the following question: “Do you smoke?”. The response categories were: (1) “Yes, daily”; (2) “Yes, sometimes”; (3) “Used to smoke but not anymore”; (4) “Have never smoked”. For subsequent analyses, the answers were allocated to the following categories: “No, never,” “Ex-smoker” or “Yes”.Furthermore, the quantity of smoking was assessed by the following question: “If you smoke, how much do you smoke per day on average?” The answers were allocated in the following groups: “0 cigarettes per day”; “1-9 cigarettes per day”; “10-20 cigarettes per day”; “≥ 20 cigarettes per day”.The consumption of fruits and vegetables were assessed by the following question: “How often do you eat fruit, salad / raw food, cooked vegetables - apart from potatoes?”. The response options were: (1) “At least 3 times per day”; (2) “Twice per day”; (3) “1 time per day”; (4) “3-6 times per week”; (5) “1-2 times per week”; (6) “Rarer”. For subsequent analyses, the response categories were divided into: “≤ 2 days per week”; “3-6 days per week”; “Daily”.2), physical activity at work  [Control variables for the analyses in the present study included age  , gender us work) , psychosus work) , job grous work) , and chrus work) . ChronicUsing logistic regression analyses (PROC GENMOD of SAS version 9.2), the risk for high pain  in the low back and neck-shoulder, respectively, was estimated. Risk ratios (RR) and 95% confidence intervals (95% CI) were calculated for high pain as outcome variable. Explanatory mutually adjusted factors were the four lifestyle factors. Model 1 was, besides the explanatory variables , additionally adjusted for age and gender. Model 2 was, besides age and gender, additionally adjusted for BMI, physical activity at work, psychosocial work factors, job group, and chronic disease.Table Table In the fully adjusted model (model 2), only a high level of physical activity (i.e. 5 h per week) was associated with lower risk of low back pain and neck-shoulder pain, with RRs of 0.95 (0.90–1.00) and 0.90 (0.82–0.99), respectively.Estimates for the psychosocial factors that were used as continuous control variables  are provided here (not discussed): For emotional demands, the OR’s were 1.001 (95% CI 1.001–1.002) for low back pain and 1.003 (95% CI 1.002–1.005) for neck-shoulder pain, i.e. higher emotional demands is associated with higher pain. For influence at work, the OR’s were 0.998 (95% CI 0.998–0.999) for low back pain and 0.997 (95% CI 0.996–0.998) for neck-shoulder pain, i.e. higher influence at work is associated with lower pain.The present study found that physical activity during leisure was associated with lower risk of having low back pain and neck-shoulder pain when adjusted for age and gender (model 1). Furthermore, when adjusting for age and gender, the level of smoking and physical activity were associated with higher and lower risk of low back pain and neck-shoulder pain, respectively. Daily fruit and vegetable intake was associated with low back pain when adjusted for age and gender, while no association was observed between alcohol intake and pain.Physical activity is one of the most important characteristics for retaining health , 42, andLunde and co-workers  performeSitthipornvorakul and co-workers  publisheAnother life-style habit that showed assotiations with low back pain and neck-shoulder pain was smoking status, however with adjustment for further covariates no associations were observed in the present study. Smoking could theoretically be a risk factor for developing chronic pain due to the nicotine which leads to an activation of the immune system . The nicBoth studies , 18 are To our knowledge, the existing literature on neck-shoulder pain seems to be in accordance with our study results. A study among software engineers observed an association between smoking and current neck pain . FurtherConsidering the other lifestyle factors examined in the present study, neither alcohol nor vegetable and fruit intake showed a consistent association with low back pain and neck-shoulder pain in both the mutually adjusted models. A previous systematic review observed that an association between alcohol consumption and low back pain only appears in complex cases of low back pain and only in people with alcohol consumption addiction . RegardiFinally, it is important to note that the wide variation in prevalence of pain could be partially explained by methodological, racial/ethnical, or cultural differences . Still, The present study has both limitations and strengths. A limitation of the study is the use of self-reported questionnaires, which may have led to reporting bias. For instance, there could be different understandings and perceptions in people with chronic pain, who often report problems with cognitive abilities, such as memory or attention . HoweverBeing physically active during leisure associated with lower risk of having musculoskeletal pain, while smoking habits and healthy eating were associated with higher pain when adjusting for age and gender. Considering the relatively high prevalence of unhealthy habits in the general working population, initiatives to promote healthy habits should still be a political priority to help the workers to stay healthy and cope to their work.


Telfairia occidentalis) and amaranth (Amaranthus spp.) are common leafy vegetables produced and consumed in southwestern Nigeria. These vegetables attract insect pests which are controlled by pesticides that may have adverse impacts on human health.Fluted pumpkin (To determine the levels of dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexane (HCH) pesticide residues in the two vegetables and evaluate the potential health risks associated with their consumption.The pesticide residue levels were quantitatively and qualitatively determined using a gas chromatograph coupled with electron capture detector. Health risk assessment were performed using estimated average daily intake and hazard indices for two weight categories: children (16.7 kg) and adults (60 kg).delta-HCH, DDT and methoxychlor were predominantly detected in the two vegetables from both farms and markets. In amaranth, the mean concentration of methoxychlor, DDT and delta-HCH were 4.590 ± 2.774 mg/kg (dry weight (dw)), 0.757 ± 0.457 mg/kg (dw) and 0.577 ± 0.390 mg/kg (dw), respectively, while fluted pumpkin levels were 6.223 ± 2.489 mg/kg dw (methoxychlor), 0.504 ± 0.056 mg/kg dw (delta-HCH) and 0.486 ± 0.123 mg/kg dw (DDT). The levels of HCH and DDT residues were generally above the United Kingdom/European Commission maximum residue limit. The analysis of health risk estimates for non-carcinogenic risk revealed that for both vegetables, the hazard quotient for p, p′ DDT and methoxychlor was >1 for both children and adults. The health risk estimates for carcinogenic risk revealed that hazard indices values were >1 for children for both vegetables and alpha-HCH had a hazard index >1 for adults for amaranth. This means the residue exceeds acceptable standard and present potential risk to consumers of these vegetables.The results showed that alpha-HCH could pose a carcinogenic risk for adult consumers. The consumption of the two vegetables could pose both non-carcinogenic and carcinogenic health risks to children and adults. Therefore, there is need for strict enforcement of regulations on pesticide usage to minimize human health risks.The results obtained from the present study indicate that consumption of amaranth contaminated with The authors declare no competing financial interests. Telfairia occidentalis Hook F.) and amaranth (Amaranthus spp.) are leafy vegetables which are widely cultivated and commonly consumed in southwestern Nigeria. Nigeria is the fourth highest producer of vegetables in the world and highest producer in Africa.4Fluted pumpkin (Dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexane (HCH) are organochlorine pesticides (OCPs) and also regarded as persistent organic pollutants.10et al. and fluted pumpkin by Ibrahim et al.et al., and cocoa beans by Oyekunle et al.et al. assessed the potential health risk of contaminated vegetables in Lagos such as cabbage, carrot, pepper, tomatoes and lettuce, and a study by Sosan and Oyekunle assessed the potential health risk from kolanut consumption.Amaranthus spp.) and fluted pumpkin . In order to guarantee that vegetables produced and marketed are safe for consumption, the present study was undertaken to evaluate the levels of pesticide residues and associated health risks in two leafy vegetables collected from selected farms and markets in Osun and Ekiti States in southwestern Nigeria.Pesticide residues such as HCHs and DDT have been reported in amaranth, cabbage, lettuce, pumpkin and spinach in China; and in cabbage, lady's finger, tomato and brinjal in India.DCMDichloromethaneDDDDichlorodiphenyldichloroethaneDDEDichlorodiphenyldichloroethyleneDDTDichlorodiphenyltrichloroethanedwDry weightEADIEstimated average daily intakeHCHHexachlorocyclohexaneHQHazard quotientMRLsMaximum residue limitsOCPsOrganochlorine pesticides. The sampling sites in selected locations from Ekiti State were Ogbese, Erinfun, Odo-Ado, Akuro Alapo, Igboata farms, and Awedele, Oja-oba, Oja Bisi, Olojudo and Ido daily markets. In Osun State, selected locations were Ede road, Oju-ako, Oke-osun and Osun Agricultural farms, and Oluode, Alekuwodo, Obafemi Awolowo University Central and Ile-Ife Central markets. The two states were selected based on their agrarian nature and the locations or towns were selected based on populations of vegetable farmers, their production levels and size of markets. Each of the vegetable types were purchased from three randomly selected market stalls and farms within sampling sites (November 2017–January 2018). Vegetable samples collected from each location were wrapped in aluminum foil to avoid cross contamination. The foil was wiped clean with cotton wool soaked in acetone. A total of 96 samples (48 for each of the vegetables) were later bulked into 32 composite samples and labelled before being transferred to the laboratory.The leafy vegetable samples (amaranth and fluted pumpkin) used for the present study were obtained from selected farms and open markets in Ado-Ekiti (7° 37′ N and 5° 13′ E) and Ido-Ekiti (7° 50′ N and 5° 10′ E), Ile-Ife (70° 50′ N and 4° 69′ E) and Osogbo (7° 46′ N and 4° 34′ E) in southwestern Nigeria Each vegetable sample was rinsed with distilled water, allow to drain properly and chopped with a clean sharp knife on a chopping board and oven dried at 45°C for three days until a constant weight was attained. To obtain a homogenous representative sample, each composite sample was macerated and pulverized to a homogenous powdered form using a Nakai blender (Japan). The knife, chopping board and blender were washed thoroughly with water and rinsed with acetone to avoid cross contamination. Each sample was then placed in Ziploc bags, well labelled and stored in a cool place prior to further analysis.et al.; the sample was Soxhlet extracted for 4 hours using dichloromethane (DCM) as the extraction solvent.For each sample, 20 g of homogenized vegetable was weighed into a pre-extracted Whatman thimble. Following the method of Oyekunle A clean-up experiment was carried out to separate analytes from interfering compounds of a different polarity or removal of impurity (chlorophyll) from extracts, a column of about 15 cm (length) × 1 cm  was packed first with glass wool and then 5 g activated silica gel prepared in a slurry form in DCM. About 5 g of anhydrous sodium sulfate was placed on top of the column to absorb any water in the sample or the solvent. The pre-elution was done with 15 mL of DCM without exposing the sodium sulfate layer to air so as to prevent the cracking of packed silica gel adsorbent. The reduced extracts were run through the column and allowed to sink below the sodium sulfate layer. Elution was done with 3 × 10 mL portions of DCM.The eluents were collected and the accompanying solvent was then evaporated to dryness under a stream of pure nitrogen (99.99%).The instrumental analysis was carried out at the Nigeria Institute of Oceanography and Marine Research Laboratory, Lagos, Nigeria. Detection and determination of the pesticide residues were performed by reconstituting the dried sample eluents with 2 mL n-hexane before injecting 1 μL of the cleaned-up eluents into injection port of an Agilent 7890A gas chromatograph system equipped with an electron capture detector. The separation was performed on a fused silica capillary column . The temperatures of the injector and detector were 250°C and 290°C, respectively. Oven temperature started at 150°C and increased to 280°C at 6°C per minute. The injection was through a splitless injector, using helium as a carrier gas at a flow rate of 2 mL/min and nitrogen as a make-up gas. The run time was 21.67 minutes. Quantification of the OCPs was based on external calibration curves prepared from the standard solutions of alpha (α)-HCH, beta (β)-HCH), delta (δ)-HCH, gamma (γ)-HCH, dichlorodiphenyldichloroethane (DDD), dichlorodiphenyldichloroethylene DDE), DDT, and methoxychlor.All analytical procedures were monitored using strict quality assurance and control measures. Materials used for preparation of samples were well washed and rinsed with acetone before re-use. Chemicals used in sample preparation and analyses were of analytical grade. Blank determination, percentage recovery determination, response factor, and limit of detection determination were also carried out for quality control and assurance.The background value of OCPs in the analytical grade DCM and eluent of the pre-extracted thimble was determined by injecting 1 μL of the cleaned-up eluents and solvent into the gas chromatograph coupled with electron capture detector. No organochlorine pesticide residue was detected on blank samples.−1 standard mixture consisting of some of the available organochlorine pesticides of interest. The mixture was thoroughly mixed together to ensure maximum homogenization. The other two vegetable samples were left un-spiked. The samples (amaranth and fluted pumpkin) were extracted and cleaned up following procedures of Oyekunle et al. and followed by gas chromatograph coupled with electron capture detector analysis.Four samples of homogenized vegetable, each weighing 20 g, were chosen. For each vegetable, one sample was spiked with 10 mg kgThe percent recovery obtained was within the 70–120% range for acceptable recovery values stipulated by the European Union's guidelines for evaluating the accuracy and precision of a method.The response factor of the standard OCPs was obtained by the method of Sosan and Oyekunle.The limit of detection of each of the OCPs was evaluated using the relationship: limit of detection = 3.3 S/b, where S is the residual standard deviation of the calibration function and b is the slope of the calibration curve.22The results obtained from the chromatographic analysis were summarized using descriptive statistics  and charts on a log (x+1) scale and normal scale. The Wilcoxon (Mann-Whitney U test) test was conducted to examine differences between market and farm samples of amaranths and fluted pumpkin using Statistical Analysis System (SAS) 9.0 version.Data on pesticide residues levels were compared with maximum residue limits (MRLs) recommended by the United Kingdom/European Commission (UK/EC) for leafy vegetables.−1) in the food of interest and the food consumption rate (kg d−1) and dividing by body weight.The EADIs of a pesticide residues and the food consumption assumption were used to determine long term health risks to consumers. The EADI was obtained by multiplying the mean residual pesticide concentration . The cancer benchmark concentration for carcinogenic effect is derived by setting the risk to one in one million due to lifetime exposure. The oral slope factors for the pesticide were obtained from the United States Environmental Protection Agency (USEPA).30Risk is the maximum acceptable risk level (1 × 10−1. The blank determination recorded no peak.The levels of HCHs and DDTs in amaranth and fluted pumpkin from selected markets and farms in southwestern Nigeria are shown in −1 (dry weight (dw)), 1.251 ± 0.737 mg kg−1 (dw), and 0.986 ± 0.448 mg kg−1 (dw), respectively, were detected in amaranth from farms and 0.643 ± 0.365 mg kg−1 (dw), 0.263 ± 0.263 mg kg−1 (dw) and 0.168 ± 0.068 mg kg−1 (dw), respectively, in market samples Supplemental Material, Table 1), 0.542 ± 0.080 mg kg−1 (dw) and 0.254 ± 0.174 mg kg−1 (dw), respectively, for farm samples and 5.988 ± 2.533 mg kg−1 (dw), 0.467 ± 0.147 mg kg−1 (dw) and 0.718 ± 0.203 mg kg−1 (dw), respectively, for market samples Supplemental Material, Table 1).The mean concentration (log x+1 transformed) of DDTs and HCHs are presented in Supplemental Material, Table 2)/DDT detected in amaranths and fluted pumpkin was 0.4 and 0.2, respectively The mean concentration of pesticide residues detected in amaranth and fluted pumpkin in comparison with respective MRLs is presented in p, p′ DDT and methoxychlor, respectively, and γ-HCH (2.00), p, p′ DDT (8.20) and methoxychlor (10.40) detected in fluted pumpkin had a hazard quotient (HQ) > 1, indicating a non-carcinogenic (systemic) health risk to children who consume these vegetables. For adults, the hazard quotient for methoxychlor (1.37) and DDT (2.25) were detected in amaranths while the HQs obtained for methoxychlor and DDT in fluted pumpkin were 1.86 and 1.40 respectively, which indicated a non-carcinogenic (systemic) health risk to adult consumers of these vegetables.The non-carcinogenic health risk analyses for amaranth and fluted pumpkin samples are presented in p, p′ DDT (11.454), β-HCH (9.625), γ-HCH (5.561) and p, p′ DDD (2.567) detected in amaranth. All DDTs and HCHs detected in fluted pumpkin for adults had a hazard index < 1, indicating no carcinogenic risk to consumers or a risk below the maximum level of 1 in 1 million. Hazard ratio values of 7.454, 2.888 and 4.171 were obtained for p, p′ DDT, β-HCH, and γ-HCH, respectively, indicating a carcinogenic health risk to children consuming fluted pumpkin.The carcinogenic health risk analysis for DDTs and HCHs detected in amaranth and fluted pumpkin is shown in The levels of organochlorine pesticide residues in amaranth and fluted pumpkin sampled from markets and farms followed similar trends of ∑HCH < ∑DDT, suggesting that similar pesticides were used on both vegetables for protection against insect pests. The δ-HCH was the most prominent HCH in amaranth samples, while γ-HCH and δ-HCH were the most predominant in fluted pumpkin. The high levels of δ-HCH from the two vegetables suggest the use of technical HCH, a commercial mixture of both γ and other isomers in which δ-HCH probably had the highest concentrations or as a result of degradation of γ-HCH and other isomers, while the predominance of γ-HCH in fluted pumpkin could be as a result of recent usage as it had not degraded to its metabolites of α-HCH and β-HCH.16The ratio of α-HCH to γ-HCH can be used to monitor sources of HCHs. A ratio between 4–7 indicates technical HCH and a value of nearly zero indicates technical lindane.®, Gammalin super 20 EC® and Capsitox 20 EC® is already banned in Nigeria and worldwide. These pesticides and other organochlorine pesticides have been reported in vegetables in Nigeria and other countries of the world as shown in et al. previously reported lower concentrations of DDTs and HCH isomers in vegetables from some locations in southwestern Nigeria and the levels reported by Ibrahim et al. were found to be comparable with the results obtained in the present study.et al. reported that the level of γ-HCH pesticide residues in cowpea grains in Ile-Ife market was 0.085 mg kg−1 and this value is within the range of γ-HCH detected in amaranth and fluted pumpkin from farms and markets in the study area, indicating that the pesticides were still being used as a general purpose pesticide for insect pest control.et al. previously reported the presence of 15 OCP residues, including α-HCH, β- HCH, lindane, p, p′-DDD and p, p′-DDT in water from the Ogbese river in Ekiti State.γ-HCH (lindane) commercially available as Gammalin 20 ECThe prominence of methoxychlor and DDT in amaranth in the present study could be from the soil on which the vegetables were planted. For example, the presence of organochlorine pesticides was reported in soils from Oke-Osun farm settlement, which lies within one of the sample locations in Osun State.et al. reported that the ratio of (DDE+DDD)/DDT >1 shows no recent inputs of DDT or degradation from previous residues.DDT is biodegradable into DDD through reductive de-chlorination under anaerobic conditions and to DDE under aerobic conditions through de-hydrochlorination (an oxidative process).et al. in northern Nigeria reported that concentrations of pesticides detected in five freshly harvested vegetables were observed to be significantly higher than MRLs set for vegetables; Sosan and Oyekunle reported organochlorine pesticide residues concentration detected in kolanut samples from selected markets in Osun State, southwestern Nigeria to be 44%–92% above EU-MRLs for DDTs and 76%–100% above EU-MRLs for HCH isomers.et al. reported that levels of lindane (γ-HCH) detected in all cowpea samples and 90% of dried yam chips samples were above the recommended EU-MRL of 0.01 mg kg−1, which was higher than the present study.All the HCHs and DDTs detected in the vegetables were above their respective maximum residue limits. The results of the present study corroborated those of earlier studies. For example, Akan , the hazard risk values in the present study were higher than those of vegetables  collected from selected markets in Lagos, Nigeria which posed no risk to adults or children.et al. was lower compared to the current study.et al.et al. and Wang et al. in Pakistan and Cambodia, respectively, which reported hazard ratios greater than 1 for adult consumers of vegetables.34Methoxychlor, γ-HCH and DDT posed a non-carcinogenic threat or risk to children consumers of the selected vegetables, while only methoxychlor and DDT posed a non-carcinogenic threat or risk to adult consumers of contaminated amaranth and fluted pumpkin. In comparison with earlier studies Consumption of the γ-HCH-contaminated amaranth and fluted pumpkin could cause liver and kidney toxicity for child consumers of these vegetables. Consumption of both vegetables contaminated with DDT and methoxychlor by children and adult consumers could cause liver lesions as well as disrupt reproductive and developmental functions.The present study revealed a high occurrence of DDTs and HCHs in amaranth and fluted pumpkin from the selected locations in southwestern Nigeria with contamination levels greater than their respective MRLs. The hazard ratio values for adults revealed that exposure through consumption of amaranth contaminated with α-HCH poses carcinogenic health risks. The estimate suggested a potential non-carcinogenic and carcinogenic health risk for children and adult consumers of the two vegetables. If pesticides used in production of vegetables in Nigeria are not regulated, there is a possibility of an upsurge in potential risks with resultant health hazards associated with their usage. Strict enforcement of regulations on pesticide usage and regular monitoring of pesticide residues in food products in Nigeria is needed to minimize health risks.Click here for additional data file.


The effectiveness of alcohol-based hand rubs (ABHRs) depends substantially on their acceptability and tolerability. In this study, we assessed the acceptability and tolerability of a new ABHR (product EU 100.2018.02).Among physicians, nurses, and cosmetologists who used the ABHR for 30 days, we assessed the product’s acceptability and tolerability according to a WHO protocol. Additionally, we used instrumental skin tests. Participants assessed the product’s color, smell, texture, irritation, drying effect, ease of use, speed of drying, and application, and they gave an overall evaluation. Moreover, they rated the tolerability, i.e. their skin condition, on the following dimensions: intactness, moisture content, sensation, and integrity of the skin. The tolerability was also assessed by an observer as follows: redness, scaliness, fissures, and overall score for the skin condition. Instrumental skin tests included transepidermal water loss, skin hydration, sebum secretion, and percentage of skin affected by discolorations. All assessments were made at baseline (visit 1), and 3–5 days (visit 2) and 30 days (visit 3) later.p < 0.001), whereas skin hydration, sebum secretion, and the percentage of skin affected by discolorations did not change significantly during the study (p ≥ 130).We enrolled 126 participants (110 [87%] women) with a mean age of 34.3 ± 11.65 years. Sixty-five participants (52%) were healthcare professionals , and 61 (48%) were cosmetologists. During visit 2 and visit 3, about 90% of participants gave responses complying with the WHO’s benchmark for acceptability and tolerability. Similarly, the ABHR met the WHO criteria for observer-assessed tolerability: on all visits, in more than 95% of participants, the observer gave scores complying with the WHO benchmark. Transepidermal water loss decreased from baseline to visit 3 (The EU 100.2018.02 had both high acceptability and tolerability, meeting the WHO criteria. The WHO protocol proved useful in the analysis of acceptability and tolerability of ABHRs. Hand hygiene plays a central role in the prevention of infections, including those with multidrug-resistant pathogens , 2. HoweAlthough consistent use of alcohol-based hand rubs (ABHR) improves hand hygiene substantially, poor acceptability and tolerability of ABHRs in the workplace is one of the most common causes of ineffective hand hygiene , 6. ThusIn this study, we evaluated the acceptability and tolerability of a new ABHR (product EU 100.2018.02) among healthcare professionals and cosmetologists. We followed the WHO protocol and used additional instrumental skin tests.We enrolled adult participants (> 18 years) among physicians and nurses of a gynecological ward and among cosmetologists of a private clinic. We excluded people with skin diseases or known hypersensitivities to the ingredients of the investigational product. The study was approved by the Bioethics Committee of the Medical University of Warsaw (KB213/2018). All participants signed informed consent before enrollment. Basic characteristics of participants were gathered in accordance with the WHO protocol .The investigational product, an ABHR , complied with the EN 1500:2013–07 and EN 12791 + A1:2017–12 standards. The ABHR contained ethanol CAS 64–17-5 (80 g/100 g), vegetable glycerin, vitamin E, bisabolol, and flavonoids.The study assessed the tolerability and acceptability of the investigational product according to the protocol proposed by the WHO . Brieflycolor (“unpleasant”-“pleasant”),smell (“unpleasant”-“pleasant”),texture (“sticky”-“non-sticky),irritation (“very irritating”-“not irritating”),drying effect ,ease of use (“very difficult”-“very easy”),speed of drying (“very slow”-“very fast”),application (“unpleasant”-“pleasant”), and.overall evaluation (“dissatisfied”-“satisfied”).appearance ;intactness ;moisture content ;sensation ;overall integrity of the skin .Similarly, on a seven-point Likert scale, participants assessed the skin condition of their hands:redness ,scaliness ,fissures , overalloverall score for the skin condition .Skin condition was also rated by an observer, as follows:All evaluations were carried out at baseline and 3–5 and 30 days after using the ABHR. All participants used 30 ml or more of the ABHR daily. Each participant received a personal container of the ABHR. Participants were allowed to use hand lotion or hand cream throughout the study.2/h, with higher values indicating worse skin hydration. The Derma Skin Visualizer uses parallel and crossed polarized light to measure pigment discoloration on the skin; the result is given as the percentage of the studied area with discolorations.During all three visits, the skin on hands was assessed with an MPA-5 Corneometer to evaluate skin hydration; an MPA-5 Sebumeter to evaluate sebum secretion; a TM300 Tewameter with a cylindrical probe to evaluate transepidermal water loss; and a Derma Visualizer camera to assess the percentage of skin affected by discolorations . The Corneometer measures hydration in the stratum corneum of the epidermis based on the electrical properties of the skin; skin hydration is reported in units from 0 to 130 (each unit indicates 0.02 mg of water per square centimeter of the stratum corneum). The Sebumeter measures sebum content in a foil attached previously to the skin for 30 s; the results range from 0 to 350 micrograms of sebum per square centimeter of skin. The Tewameter estimates transepithelial water loss based on the skin’s wetness and temperature, which are used to calculate water vapor pressure. Transepithelial water loss is reported in g/m≥ 50% of participants responding above 4 for “Color” and “Smell”, and ≥ 75% of participants responding above 4 for “Texture”, “Irritation”, “Drying effect”, “Ease of use”, “Speed of drying”, “Application”, and “Overall evaluation”.The WHO criteria for product acceptability were as follows:≥ 75% of participants responding above 4 for the skin’s “Appearance”, “Intactness”, “Moisture content”, “Sensation”, and “Overall integrity”≥ 75% of participants with scores below 2 on the skin evaluation by the observer.The WHO criteria for skin tolerability were as follows:P < 0.05 was considered statistically significant. All calculations were carried out with SPSS software (version 2018).Variables are presented as counts and percentages or as means±standard deviations (SD). Comparisons were carried out with repeated-measures analysis of variance (ANOVA) and post-hoc Scheffe contrasts. We enrolled 126 participants (110 [87%] women) with a mean age of 34.3 ± 11.65 years. Sixty-five participants (52%) were healthcare professionals  and 61 (48%) were cosmetologists. Their mean professional experience was 9 ± 10 years; 88 (67%) participants worked full-time. Thirty-three (26%) participants had non-occupational activates that could damage skin, 10 (15%) participants declared frequent skin irritation, and 88 (70%) participants declared non-frequent skin irritation. Fifty-nine (48%) participants used ABHRs for five years or longer. Forty-five (36%) participants had Fitzpatrick skin type 1, 58 (46%) had Fitzpatrick skin type 2, and 23 (18%) had Fitzpatrick skin type 3. Table During visit 2 and visit 3, the ABHR met the WHO criteria for self-assessed acceptability. More than 90% of participants gave responses of 4 or more to all questions about the product’s acceptability Fig. . SimilarThe ABHR met the WHO criteria for observer-assessed tolerability. On all visits, in more than 95% of participants, the observer gave scores of ≤1 to all items that assessed skin tolerability Table .Table 2p < 0.001); only the difference between visit 1 and visit 3 was significant on post-hoc comparisons  had good acceptability and tolerability among healthcare professionals and cosmetologists. The ABHR met all WHO criteria for both acceptability and tolerability. Moreover, participants declared that the product could improve hand hygiene in their workplace.Hand hygiene is insufficient among healthcare professionals and other staff . For exaAcceptability and tolerability of hand sanitizer products are also very important for consistent hand hygiene. Therefore, improvements in acceptability and tolerability can translate into fewer infections. Hand products, particularly those containing sodium laureth sulfates, irritate the skin and deplete skin lipids –16. ABHRBecause ABHRs are important for hygiene, the WHO put forward both a protocol and criteria for assessing the acceptability and tolerability of these products. To our knowledge, only one published study followed this protocol. In that study, in contrast to our study, the product investigated did not meet all the WHO criteria for acceptability . In the We found that the ABHR significantly reduced transepidermal water loss over a month of use, although previous studies on ABHRs found no such effect , 30. MorIn our study, each participant received a personal container of the tested ABHR, which could have had an effect of the perceived acceptability and tolerability. Wolsfensbergeret et al. found that hand sanitizers available from personal containers might improve self-reported assessments . ParticiIn conclusion, we found that the investigational ABHR (EU 100.2018.02) had high acceptability and tolerability. Thus, it could improve hand hygiene. The WHO protocol proved to be a useful tool in the analysis of acceptability and tolerability of ABHRs.