Hypertension is one of the most prevalent diseases and is often called the “silent killer” because there are usually no early symptoms. Hypertension is also associated with multiple morbidities, including chronic kidney disease and cardiovascular disease. Early detection and intervention are therefore important. The current routine method for diagnosing hypertension is done using a sphygmomanometer, which can only provide intermittent blood pressure readings and can be confounded by various factors, such as white coat hypertension, time of day, exercise, or stress. Consequently, there is an increasing need for a non-invasive, cuff-less, and continuous blood pressure monitoring device. Multi-site photoplethysmography (PPG) is a promising new technology that can measure a range of features of the pulse, including the pulse transit time of the arterial pulse wave, which can be used to continuously estimate arterial blood pressure. This is achieved by detecting the pulse wave at one body site location and measuring the time it takes for it to reach a second, distal location. The purpose of this review is to analyze the current research in multi-site PPG for blood pressure assessment and provide recommendations to guide future research. In a systematic search of the literature from January 2010 to January 2019, we found 13 papers that proposed novel methods using various two-channel PPG systems and signal processing techniques to acquire blood pressure using multi-site PPG that offered promising results. However, we also found a general lack of validation in terms of sample size and diversity of populations. Hypertension (HTN), defined as a blood pressure (BP) above 140/90 mmHg on two or more occasions, is one of the most prevalent diseases, affecting over 1 billion people world-wide . In addiThe current industry gold standards of blood pressure assessment are the manual sphygmomanometer (BP cuff) and invasive blood pressure (IBP) measurements . The invPhotoplethysmography (PPG) is a technology that illuminates perfused tissue and then measures light that is either reflected or transmitted back to a photosensor, giving a measurement of blood volume . TraditiOne of the difficulties of understanding research in PPG is the interchangeability of several key terms. This inconsistency in terminology can create confusion between research questions, protocol development, and findings, so we wanted to address it early in the review. In many publications, PAT is used interchangeably with pulse transit time (PTT). These two are not the same; the key difference is when this time interval starts. The beginning of the PAT is measured from the R wave of the ECG; therefore, it includes the pre-ejection period (PEP), which is the time it takes for blood to leave the heart after the heart’s electrical impulse . PTT on p < 0.001) with systolic BP (SBP). Note that each data point in One of the limitations of the ECG–PPG method is that the PAT includes the PEP. This artifact reduces the accuracy of the BP estimation . One of We performed a literature search for all research from January 2010 to January 2019 regarding multi-site PPG as a method for BP measurement. The purpose of this review is to gain an understanding of multi-site PPG as one of the potential technologies amongst several  to assesWe conducted a thorough literature search using PubMed for articles exploring the use of multi-site PPG for BP measurement from 1/1/2010 to 1/1/2019. PubMed was used because it gave access to journal articles as well as conference proceedings. We decided to limit our search to the most recent decade, i.e., January 2010 to January 2019, because we were interested in the trend of the development of this technology in the recent decadal time frame. Note that the typical time frame is the most recent decade ,22. We uWe included any publication that examined multi-site PPG as a method for BP measurement. Our exclusion criteria included animal studies, review articles, articles that are not accessible in the English language see the , articleN = 10) and normotensive (N = 10) subjects, as well as patients undergoing coronary angiography (N = 4). Another publication collected data from patients undergoing general anesthesia (N = 35) [Our PubMed search found 13 publications that studied multi-site PPG technology; all but one paper looked at using two measurement sites with a two-probe system . The two(N = 35) .Several of the papers did not use the gold standard BP measurement methods  for their comparisons. Instead, they used less validated methods for obtaining the reference BP, such as devices that employ the volume-clamp method, other PPG methods, applanation tonometry, or no reported reference at all. The volume-clamp method, also known as finger arterial non-invasive BP, uses a PPG probe to measure the volume of blood in the fingertip and a beat-to-beat adjusting cuff that exerts pressure to maintain the volume detected by the PPG. In theory, this cuff pressure is equal to the systolic BP. Some publications have verified its validity in different applications , but othMany studies included a protocol that induced changes in BP during measurement, as shown in r). Three publications did not analyze their method in comparison to a reference BP. One publication expressed reliability in their measurements by showing that beat-to-beat variations of the measured PWV was less than 15% [r value of 0.86 ± 0.06 [The accuracy of BP estimations of each analyzed publication is summarized in than 15% . Anotherthan 15% . The thi6 ± 0.06 . Only on6 ± 0.06 .N = 20) to be poorly correlated with BP. This poor correlation is suggested to be due to the fact that the devices used had a low accuracy and sensitivity in comparison to the small changes in PTT associated with the changes in BP. In their study, the PTT varied with a massive uncertainty of ±11 ms, which was associated with a variation in the BP readings of approximately ±20 mmHg (r = 0.22). Of the 13 publications, only one study, Chen et al. , found tN = 5). This study had neither a gold standard for measuring the reference blood pressure, nor did they have any reports of accuracy of their measurements. However, they were able to show an excellent agreement within multiple measurements using different fingers (r = 0.95). One of the studies, Beckmann et al. , only loN = 15). Using machine learning, they were able to create an algorithm to predict the SBP with an accuracy of r = 0.85, which is the most accurate SBP prediction amongst the studies included here. Another study, Viejo et al.  used mulN = 35), they separated their experimental validation into two separate experiments: validation of PWV (N = 9) and validation of BP measurement (N = 26). Furthermore, they had two different age groups; therefore, it would be ideal to have a larger sample size. Another limitation of the study is that there is insufficient information about the subjects. Chen et al. notes that data acquisition was done in the operating room before, during, and after anesthesia. However, how the nature of the surgeries, health of the subjects, and the use of any medication could affect the BP is unknown. Yet another study, Chen et al. , used eaOne of the main motivators for studying multi-site PPG is its potential for incorporation into a wearable device to continuously monitor BP in an ambulatory setting. This would be designed to allow for the early detection of HTN and provide an accurate and complete profile of BP control in hypertensive patients. Based on the research found, there seem to be two major barriers to creating a wearable device. First, the device must be compact enough to be convenient to wear on a daily basis, and second, PPG measurements are usually highly sensitive to body movements, where the signal collected contains motion artifacts that can decrease the accuracy of the BP estimation.A second study, Liu et al. , proposeOne of the earliest published multi-site PPG studies, McCombie et al. , demonstSimilarly, Nabeel et al. ,29,34,36Another two studies, Liu et al. ,35, explMore recently, it has been demonstrated that the optical pulse perfusion in the human skin can be measured with a camera . This teHybrid techniques have also been developed. Zhang et al.  proposedOnly one study, Wang et al. , addressAs mentioned in the introduction, a vast majority of the publications use the term “multi-site PPG” to describe a dual PPG system. In our literature search, there was only one study that used more than two PPGs in the analysis of BP. This study used PPG analysis of three facial regions in combination with machine learning in order to develop an algorithm for BP prediction . In addi(1) to apply all recommendations in Elgendi  but in m(2) to compare the performance features extracted from signal PPG signals and multiple PPG signals for assessing BP,(3) to optimize the locations of measurement for extracting PTT that are correlated with BP,(4) to include more unhealthy subjects with co-morbidities,(5) to include subjects from different age ranges,(6) to explore different noisy environments, and(7) to investigate the integration of multiple PPG sensors (contact and non-contact) in intelligent clothing or wearable devices.A thorough literature search using PubMed revealed only 13 publications from January 2010 to January 2019 regarding the use of multi-site PPG for BP estimation. The research suggests that PPG simultaneously from a range of body sites is a promising approach for the continuous tracking of BP. The current research landscape is lacking given the increasing need for a technology to continuously and non-invasively monitor BP. The main shortcoming is that a majority of research studies do not have large enough sample sizes, nor do they include participants with co-morbidities. That being said, a surprising number of studies used a method of BP provocation, showing that their approaches are sensitive to changes in BP. There were also many different approaches to creating a device that would be convenient to wear on a daily basis. The direction of future research should be toward validating the proposed technologies using larger sample sizes and broader populations.A systematic review of multi-site PPG-based blood pressure measurement studies from January 2010 to January 2019 was conducted, revealing that there were many studies with promising PPG technologies, but were not mature enough to make a powerful and statistically significant contribution toward the reliable non-invasive measurement of arterial blood pressure. This review creates the necessary motivation for research groups around the globe to intensify their efforts toward the creation of a much needed disruptive technology offering non-invasive, cuff-less, continuous, and calibration-free measurement of blood pressure.


HYCH was defined by: (1) Symptoms of OI; (2) Orthostatic hypocapnia (low ET-CO2); (3) Abnormal decline in orthostatic CBFv due to hypocapnia; 4) Absence of tachycardia, orthostatic hypotension, or other causes of low CBFv or hypocapnia. Sixteen subjects met HYCH criteria  and were matched by age and gender to postural tachycardia patients  and healthy controls (n = 16). During the tilt, CBFv decreased more in HYCH  and POTS  compared to controls (-3.0±5.0%). Orthostatic ET-CO2 was lower in HYCH (26.4±4.2 (mmHg), p<0.0001) and POTS  compared to controls (36.9 ± 2.1 mmHg). Orthostatic heart rate was normal in HYCH (89.0±10.9 (BPM), p<0.08) and controls (80.8 ±11.2), but was higher in POTS . Blood pressure was normal and similar in all groups. It is concluded that both HYCH and POTS patients have comparable decrease in CBFv which is due to vasoconstrictive effect of hypocapnia. Blood flow velocity monitoring can provide an objective biomarker for HYCH in OI patients without tachycardia.The objective of the study was to identify markers of hypocapnic cerebral hypoperfusion (HYCH) in patients with orthostatic intolerance (OI) without tachycardia and without orthostatic hypotension. This single center, retrospective study included OI patients referred for autonomic evaluation with the 10 min tilt test. Heart rate, end-tidal CO Orthostatic intolerance (OI) represents important health problem and remains poorly understood. Typical symptoms include lightheadedness, dizziness, headache, nausea, fatigue and shortness of breath which are exacerbated during upright position and relieved by recumbency . OI sympPostural tachycardia syndrome (POTS), one of the common form of orthostatic intolerance (OI) is characterized by combination of OI symptoms and postural tachycardia. POTS is the leading cause of disability among women of childbearing age . OI pati2 and cerebral blood flow monitoring.Cerebral hypoperfusion in OI can be related to hypocapnia which causes cerebral vasoconstriction and reduces cerebral blood flow. This mechanism, e.g. hypocapnic cerebral hypoperfusion, was described in POTS . The maiThis retrospective, single-center study included consecutive patients who underwent autonomic testing between 2016 and 2017 at the Brigham and Women’s Faulkner Hospital Autonomic laboratory for evaluation of orthostatic intolerance. Patient’s electronic records were reviewed for details about past medical history, laboratory evaluations, including blood work and, as well as the use of medication. Healthy controls data were referenced from our Autonomic database at the University of Massachusetts [The study was approved by the Institutional Review Board of the Brigham and Women’s Hospital, Harvard University, as a minimal risk study and the consent form signature was waived.2.OI was defined as the presence of symptoms of cerebral hypoperfusion with standing and relieve of symptoms by recumbency . The cha2 < 30 mmHg; 2) reduced orthostatic cerebral blood flow velocity (CBFv) that can be explained by a decline in end tidal CO2. Functioning cerebral autoregulation keeps the orthostatic cerebral blood flow stable within the autoregulatory range which is 60–150 mmHg of mean blood pressure[. With normal cerebral autoregulation and in the absence of orthostatic hypotension, changes in CBFv can be attributed to variations in end tidal CO2. Hypocapnia induces cerebral vasoconstriction and reduces CBFv. A decrease in end tidal CO2 by 1 mmHg decreases CBFv by about 3% [2 would be consistent with orthostatic cerebral hypoperfusion syndrome (OCHOS) [HYCH was defined by the following inclusion criteria: 1) OI symptoms; 2) orthostatic hypocapnia during the tilt test defined as end tidal CO pressure. With noabout 3% ; POTS wa (OCHOS) .The HYCH and POTS subjects were matched with a control group by age, gender, and body mass index. All controls were asymptomatic and had normal response to tilt in heart rate, blood pressure, CBFv and respiratory variables.Exclusion criteria for both HYCH and POTS group were: (1) presence of orthostatic hypotension; (2) presence of arrhythmia, or bradycardia (HR < 50 BPM) during supine or tilt test; 3) inability to complete the 10 min tilt test for any reason including syncope; (4) any structural abnormality on brain CT or MRI imaging that could cause significant hemodynamic deficit;(5) any other cause of abnormal intracranial velocities, including cerebrovascular accident affecting large vessels, abnormal hematocrit; (6) the use of medication that affect autonomic functions, including vasoactive medication (vasodilators or vasoconstrictors) or medication that can cause orthostatic tachycardia; (7) any pulmonary, cardiovascular or systemic disorder that can cause hypocapnia including chronic fatigue syndrome and fibromyalgia [ inabilit2, CBFv in the middle cerebral artery using transcranial Doppler. Baseline supine blood pressure was obtained intermittently using an automated oscillometric blood pressure monitor Welch Allyn CVSM 6400 Monitor  from arm and continuously using Finometer®  from the third finger. During the tilt test, blood pressure was obtained every minute using the CVSM 6400 Monitor and continuously using Finometer®. Finometer-based blood pressure was calibrated with the CVSM 6400 based blood pressure. End tidal CO2 was obtained using Nonin Respsense Capnograph . A pulse oximeter (part of Welch Allyn monitor) was used to monitor the oxygen saturation usually from the left middle finger throughout the testing.All testing was performed following established standards as previously described . CardiovRight middle cerebral artery was insonated at the temporal acoustic window with a 2 Mhz probe at depth between 45 and 65 mm using a Transacranial Dopper systemMultiDop T . Transducer was attached with a head frame with a three-dimensional positioner to maintain a tight transducer fixation at the constant depth and angle. Continuous trancranial doppler monitoring was performed during the supine baseline period and during the tilt. Signals were recorded using PowerLab 16/35 data acquisition system with LabChart 8 software  and sampled at 400 Hz.Normative data for CBFv at both supine and tilt test depend on age, gender and the duration of tilt. In healthy subjects, CBFv remains either unchanged or there can be a mild drop in CBFv during the tilt. The criteria for normal drop of the mean CBFv during tilt test are equal to 90/% (1st minute), 89% (5th minute), and 85% (10th minute) of the tilt where baseline is equal to 100% .2 were analyzed at the supine baseline immediately before the tilt, and at the 1st, 5th, and 10th minute of tilt. Cerebral vascular resistance were also calculated as mean blood pressure/mean CBFv[The heart rate, blood pressure, CBFv and end tidal COAll subjects were observed for presence of orthostatic symptoms during tilt test. All subjects were assessed for the presence of sensory complaints that included self-reported the Neuropathy Total Symptom Score-6 (NTSS6) . The SurOne-way analysis of variance (ANOVA) was used to test differences between the HYCH, POTS and healthy controls at baseline, 1st minute, 5th minute, and 10th minute of tilt. The measurements at these intervals  were analyzed since previous validation studies showed clinical relevance of these intervals,15. In pIf significant, the between pairs comparisons were done using the Student’s test. The initial significance level (α) has been set to 0.05. Since there were four comparisons, the Bonferroni-corrected significance level was adjusted to 0.0125 (obtained by 0.05/4). JMP 12  statistical software was used for all statistical analyses. The measurements between the HYCH, POTS and healthy controls at supine baseline, 1st minute, 5th minute, and 10th minute of tilt were also compared using repeated measures MANOVA.Power analyses: CBFv is the main clinical variable of interest which separates HYCH from controls. Based on our data with 32 subjects (HYCH and controls) we have >99% to detect a difference 14.8 cm/s in mean CBFv at the minute 10 of the tilt with alpha 0.01.2)) 25.3 ± 5.0), satisfied criteria for HYCH. These patients were age- and gender-matched to 16 POTS patients  and 16 healthy historical controls .Sixteen patients (15/1 (women/men), (age (years)) 38.6±8.1,  and POTS  compared to controls (38.0 ± 2.1 mmHg). There was no difference between HYCH and POTS in end tidal CO2. There was no difference at the baseline in all other variables. Supine baseline CBFv (cm/sec) was 65.5 ± 7.6 (controls), 65.9 ± 9.6 (HYCH) and 66.1 ± 8.1 (POTS).Supine end tidal CO2 was not different between all groups (p = 0.32). End tidal CO2 was reduced in HYCH  and POTS  compared to controls (36.9 ± 2.1 mmHg). Heart rate was normal in HYCH (89.0 ± 10.9 (BPM), p<0.08) and controls (80.8 ± 11.2), but higher in POTS . CVR was elevated in HYCH  and POTS  compared to controls (1.45 ± 0.19). Both disease groups have higher CVR (MANOVA), p = 0.006. End tidal CO2 was higher in controls . There was no difference between HYCH and POTS in CBFv and in end tidal CO2. Blood pressure and respiratory frequency was normal and similar between the groups. In all subjects, the oxygen saturation was within normal limits throughout the testing (range 96–99%).Mean CBFv declined and the percent CBFv difference from baseline increased during the tilt (the following numbers apply to the 10 the minute of the tilt) in HYCH ((mean) 50.9 ± 6.8 cm/sec, p<0.0001 (the difference from supine baseline) -22.4 ± 7.7%, p<0.0001) and POTS  compared to controls , as compared to only 3% in controls. The study is statistically powered enough to interpret the results as being significant. Second finding is that HYCH patients had abnormally low orthostatic end tidal COMany symptoms in POTS can be attributed to cerebral hypoperfusion ,2. This 2. and elevated CVR during the tilt, similarly as POTS subjects. Since by definition patients with orthostatic hypotension were excluded, then cerebral vasoconstriction induced by hypocapnia explains the reduction in CBFv in HYCH, a mechanism already observed in POTS [2 by 1 mmHg decreases cerebral blood flow by about 3% [2 by 7 mmHg with predicted decrease in CBFv by 21%, that was equal to the CBFv obtained in this study.HYCH subjects had abnormally low end tidal COabout 3% . HYCH suHypocapnia is frequently encountered in clinical practice . The imp2 has been observed in HVS patients [The primary hypocapnia, either psychogenic or anxiety related, can be a part of hyperventilation syndrome (HSV). HSV refers to a variety of somatic symptoms which are caused by hyperventilation ,19. PatiHypocapnic hyperventilation has been previously described in other conditions such as neurally mediated syncope ,20, chroIt remains to be established if HYCH group represents a variant of hyperventilation syndrome. The concept of hyperventilation syndrome itself has been questioned because hyperventilation may be a consequence rather than a cause of the HVS disorder . Althoug2 is highly permeable to brain blood barrier and hypocapnia increases cerebral pH [In addition to reduction of cerebral blood flow, hypocapnia associated with respiratory alkalosis may have direct effect of brain functioning. COebral pH  which haebral pH 2 and CBFv are not measured. Clinical and hemodynamic variables at baseline and during tilt were similar between HYCH and POTS except of absent tachycardia in HYCH. Then the question arises whether both HYCH and POTS represent different diagnostic entities or they represent a spectrum of the same disorder of orthostatic intolerance. POTS is a syndrome of multiple causes. It is possible that there is overlap between HYCH and some forms of POTS. Nevertheless, further studies are necessary to establish link between HYCH and POTS and other disorders of OI.HYCH and POTS have similar clinical presentations; in fact more than 80% of patients with HYCH were referred for evaluation of suspected POTS. Historically postural tachycardia has become a major criterion for OI. Therefore many patients without tachycardia remain undiagnosed by the tilt test if the end tidal COThis study emphasizes the importance of respiration in OI as hypocapnia underlies abnormal CBFv and likely results in cerebral hypoperfusion in OI. Future interventions may be directed to alter the respiratory dynamics perhaps in combination with reconditioning  which ma2 and of respiratory frequency [2, normal oxygen saturation as well as basic demographic characteristics .This study has number of limitations. The study was retrospective where the diagnostic criteria were applied to already acquired data. A referral bias may affect selection of subjects and, therefore, the studied population may not be representative. Furthermore, the studied population has been narrow as it did not include other disorders of OI. However, the purpose of this study was to exclude disorders with multifactorial causes such as syncope, chronic fatigue syndrome or fibromyalgia in order to demonstrate the link between hypocapnia and low CBFv in well-defined OI population. Other technical aspects may influence the results. The cerebral blood flow was assessed indirectly using CBFv. Transcranial Doppler measures flow velocity instead of blood flow. The velocity is not only proportional to flow but also depends on the diameter of the insonated vessel. The MCA diameter does not change during orthostatic stress and, therequency , therefo2, as described with HYCH, can be an objective biomarker of OI subjects without tachycardia. Therefore, continuous monitoring of cerebral hemodynamic and end tidal CO2 provides additional diagnostic value in symptomatic OI patients without tachycardia. The spectrum of orthostatic intolerance is broad and includes several overlapping presentations. A decrease in orthostatic cerebral blood flow may be one of the unifying mechanisms of OI.Decreased of cerebral blood flow and end tidal CO


This study aimed to quantify the associations of regional fat mass and fat‐free mass with systolic blood pressure.This analysis combined individual participant data from two large‐scale imaging studies: UK Biobank and Oxford BioBank. In both studies, participants were interviewed and measured, and they underwent dual‐energy x‐ray absorptiometry imaging. Linear regression was used to relate systolic blood pressure to anthropometric measures of adiposity  and dual‐energy x‐ray absorptiometry–derived measures of body composition .Among 10,260 participants , systolic blood pressure was positively associated with visceral android fat  and fat‐free mass , but there was no evidence of an association with subcutaneous android or gynoid fat. Associations of systolic blood pressure with BMI were slightly steeper than those with waist circumference or waist to hip ratio; these associations remained unchanged following adjustment for fat‐free mass, but adjustment for visceral android fat eliminated associations with waist circumference and waist to hip ratio and more than halved associations with BMI.This analysis indicates that visceral fat is the primary etiological component of excess adiposity underlying the development of adiposity‐related hypertension. Adiposity is an established risk factor for vascular disease, and this association is known to be mediated in part by raised blood pressure. Randomized controlled trials of weight loss interventions (and Mendelian randomization studies of blood pressure in relation to adiposity‐related genetic variants) support the causality of the relationship between adiposity and blood pressure 2 higher is typically associated with approximately 1‐mmHg higher systolic blood pressure Previous studies of adiposity and blood pressure have mostly used indirect measures of adiposity, such as BMI, waist circumference, or waist to hip ratio. The etiological relevance of general adiposity (as measured by BMI) and central adiposity (as measured by waist circumference or waist to hip ratio) has been inferred from these findings. Overall, BMI has been found to be more strongly related to blood pressure than measures of central adiposity Measures of body composition that directly quantify regional fat distribution  can overcome some of these limitations. However, it has only recently become feasible to conduct large‐scale studies with whole‐body imaging scans. Evidence for the associations between regional body composition and blood pressure is, therefore, limited to a small number of studies, none of which was sufficiently large to reliably estimate the independent effect of different fat deposits on blood pressure levels This study examines the cross‐sectional associations of DXA‐derived measures of regional fat mass with systolic blood pressure, known to be more strongly predictive of vascular disease than diastolic blood pressure In 2006 through 2010, 503,000 adults (aged 40‐70 years) were recruited from the general population in the United Kingdom into a prospective cohort study In 1999, the Oxford BioBank study recruited 5,425 adults (aged 29‐55 years) from the general population of Oxfordshire, UK In both studies, DXA imaging scans were used to estimate fat mass in four mutually exclusive regions of the body: visceral android fat, subcutaneous android fat, subcutaneous gynoid fat, and other fat mass. Android, gynoid, and visceral fat were provided as automated output from the device, as estimated by using proprietary algorithms n = 335), 10,260 participants from both studies were available for analysis (Supporting Information Table r). Linear regression was used to estimate the mean differences in each DXA‐derived measure of body composition  per 1‐SD difference in the levels of each anthropometric measure of adiposity.A meta‐analysis of the individual results from the UK Biobank and Oxford BioBank was performed. After excluding those with missing or extreme values of adiposity measures or systolic blood pressure (n = 657), linear regression was also used to relate systolic blood pressure to each DXA‐derived measure of body composition  and to each anthropometric measure of adiposity (with and without further adjustment for selected DXA‐derived measures of body composition); these analyses were further adjusted for height (sex‐specific fifths) and alcohol consumption (five groups) based on theoretical importance and significant contributions to the model.After excluding participants on blood pressure–lowering therapy (r) between repeat measures at imaging and resurvey (2.1 years prior to imaging) in 2,063 UK Biobank participants (Supporting Information Table r.Because of measurement error (or within‐person variability over time), regression analyses that use measurements of anthropometry on a single occasion may systematically underestimate the strength of the associations between anthropometric variables and other factors such as blood pressure (a phenomenon known as “regression dilution bias”) Sensitivity analyses were conducted to assess the effect of blood pressure–lowering medication on the findings and to assess the effect of potential confounding by other lifestyle factors, such as smoking and physical activity. Analyses were also repeated by using log‐transformed, DXA‐derived variables. Ethics approval was obtained from the Oxford Ethics Committee and the Ethics and Governance Council for the Oxford BioBank study. The UK Biobank study received ethics approval from the National Information Governance Board for Health and Social Care and the National Health Service North West Multicentre Research Ethics Committee. All participants provided written informed consent. All analyses were conducted by using SAS version 9.3  and R version 3.3.1 .After exclusions, 10,260 participants contributed to the main analyses Table . Almost On average, men had higher BMI, waist circumference, and waist to hip ratio than women. The mean difference in weight between men and women (approximately 15 kg) was almost entirely due to differences in fat‐free mass. The distribution of body fat also differed between the sexes, with men having on average less subcutaneous fat in android and gynoid regions but more than twice the amount of visceral android fat (1.4 kg in men vs. 0.6 kg in women).r = 0.87 in men and 0.86 in women), and both measures were less strongly correlated with waist to hip ratio , and there were more modest correlations of these variables with visceral android fat (r = 0.46‐0.72). Fat‐free mass was only weakly correlated with DXA‐derived measures of regional body fat mass (r = 0.28‐0.53).BMI was strongly correlated with waist circumference  have described cross‐sectional associations between blood pressure and regional fat mass or fat‐free mass The relation between systolic blood pressure and the anthropometric measures of adiposity used in the present report have been well described in previous studies The associations of systolic blood pressure with each of the anthropometric measures of adiposity were strongly attenuated when adjusting for visceral android fat, with complete attenuation of the associations with central adiposity (waist circumference and waist to hip ratio). This indicates that the relation between central adiposity and systolic blood pressure is mediated largely by visceral android fat  and suggests that there are additional mechanisms by which general adiposity (as measured by BMI) may influence blood pressure. Visceral fat is known to have distinct biological properties compared with body fat stored elsewhere, but the mechanisms by which it may influence systolic blood pressure are not clear; potential mechanisms involve abnormal renin‐angiotensin‐aldosterone regulation and sympathetic nervous system activation The key strengths of the present study include its large sample size; the high‐quality measurements of body composition, blood pressure, and potential confounders; and the repeated surveys in a subset of UK Biobank participants to allow correction for measurement error of anthropometric variables. However, the cross‐sectional study design limits the ability to infer causality. There were rigorous quality‐control procedures for the DXA machines and digital sphygmomanometers in both studies. Importantly, however, regional fat was estimated using proprietary algorithms, and although externally validated, different DXA models used by the two studies may have produced estimates of varying accuracy. Furthermore, DXA imaging technology is unable to quantify ectopic fat within internal organs (such as the heart and liver), of which others have shown stronger correlations with metabolic disease, independently of total visceral fat Some of the observed variability of anthropometric measures may reflect real temporal variation in addition to measurement error. However, similar self‐correlations were observed between baseline and imaging visits (approximately 7 years apart; Supporting Information Table Adiposity is an established risk factor for vascular disease, and this association is considered to be largely mediated by blood pressure, insulin resistance, and cholesterol  Click here for additional data file.


The autonomic nervous system interacts with the pain system. Knowledge on the effects of high velocity low amplitude spinal manipulations (SM) on autonomic activity and experimentally induced pain is limited. In particular, the effects of SM on autonomic activity and pain beyond the immediate post intervention period as well as the relationship between these two outcomes are understudied. Thus, new research is needed to provide further insight on this issue.The aim was to assess the effect of a single SM (i.e. SM vs. sham) on cardiovascular autonomic activity. Also, we assessed the relationship between cardiovascular autonomic activity and level of pain threshold after the interventions.We conducted a randomized, cross-over, sham-controlled trial on healthy first-year chiropractic students comprising two experimental sessions separated by 48 h. During each session, subjects received, in a random order, either a thoracic SM or a sham manipulation. Cardiovascular autonomic activity was assessed using heart rate and systolic blood pressure variabilities. Pain sensitivity was assessed using pressure pain threshold. Measurements were performed at baseline and repeated three times (every 12 min) during the post intervention period. Participants and outcome assessors were blinded. The effect of the SM was tested with linear mixed models. The relationship between autonomic outcomes and pressure pain threshold was tested with bivariate correlations.Fifty-one participants were included, forty-one were finally analyzed. We found no statistically significant difference between SM and sham in cardiovascular autonomic activity post intervention. Similarly, we found no post-intervention relationship between cardiovascular autonomic activity and pressure pain threshold.Our results suggest that a single SM of the thoracic spine has no specific effect on cardiovascular autonomic activity. Also, we found no relationship between cardiovascular autonomic activity and pressure pain threshold after the SM. Further experimental research should consider the use of several markers of autonomic activity and a more comprehensive pain assessment.NCT03273868. Registered September 6, 2017.N°  Spinal manipulative techniques, i.e. mobilizations or high velocity low amplitude (HVLA) manipulations, are commonly used to treat musculoskeletal pain by chiropractors, osteopaths, and physical therapists . DespiteThe autonomic nervous system is a major part of the nervous system. It is divided into three parts: the parasympathetic nervous system, the sympathetic nervous system, and the enteric nervous system. Its ultimate responsibility is to ensure the maintenance of homeostasis by regulating cells, tissues, and function of organs . The autEvidence from experimental research suggests that mobilizations and HVLA manipulations may produce acute changes in autonomic activity. Indeed, three reviews of the literature reported that spinal mobilization may have a sympato-excitatory effect reflected by an immediate, statistically significant, increase in skin conductance compared to a sham procedure –14. EvidIn addition to this possible autonomic effect, mobilizations and HVLA manipulations seem to have at least a short-term hypoalgesic effect, as shown by a decrease in sensitivity to experimentally induced pain (e.g. an increased pain threshold) , 11. PaiTo summarize, there are gaps in the current knowledge regarding the effects of spinal manipulative techniques on autonomic mediated physiology and experimentally induced pain that make additional randomized controlled trials relevant. In particular, the effects beyond the immediate post intervention period as well as the relationship between these two outcomes  are largely unknown. Additionally, the certainty of evidence on the effects of HVLA spinal manipulation on autonomic activity is low . TherefoFinally, to provide the best quality evidence on the specific effect of the joint manipulative techniques using randomized controlled trials, the untreated control group should receive a sham intervention. This allows differentiating responses caused by the specific action of the supposed effective intervention to those attributable to context information (e.g. placebo responses) .The aim was to assess, in a randomized sham-controlled trial on healthy young subjects, the specific effect of a thoracic HVLA manipulation on cardiovascular autonomic activity (i.e. heart rate and systolic blood pressure variabilities), measured repeatedly during the post intervention period. An additional aim was to assess the relationship between pressure pain threshold (PPT) and cardiovascular autonomic activity after the interventions.Please note that another report deals with the assessment of the specific effect of the spinal manipulation on pressure pain threshold .This report follows the CONSORT statement .We conducted a randomized, cross-over, sham-controlled trial comprising two experimental sessions separated by 48 h and scheduled at the same hour both days with each session lasting about one and a half hour. During each session, the study subject received, in a random order, either a thoracic HVLA manipulation or a sham manipulation. During each session we assessed sensitivity to experimentally induced pain (i.e. pressure pain threshold) and cardiovascular autonomic activity (i.e. HRV and systolic blood pressure variability). Measurements were performed at baseline and repeated three times (on average every 12 min) during the post intervention period. The study subjects rested for 10 min lying on their back to stabilize the cardiovascular system before baseline measurements. The experimental design is shown in Fig. Participants were recruited among the first-year chiropractic students at the Institut Franco-Europeen de Chiropraxie, an independent chiropractic college situated in France. We chose first-year students, since they are expected to be relatively naïve to spinal manipulation and to the various types of studies dealing with this issue. Each volunteer was screened by a licensed chiropractor for eligibility criteria at the beginning of the first experimental session. Inclusion criteria were healthy volunteers, aged at least 18 years, without contra-indication to manipulative therapies. Non-inclusion criteria were pain at the time of the study, any contra-indications to spinal manipulation , cardiovThe experiment was conducted in a laboratory room at the college from September 2017 to February 2018 and from September to October 2018. Environmental noise was kept to a minimum and the temperature was maintained at about 21 °C. The study subjects were placed on a treatment table (prone or supine position) throughout the experimental session, i.e. from the rest period to the last measurements.We used a drawing lot method for randomizing the order of the interventions, i.e. spinal manipulation-sham sequence or sham-spinal manipulation sequence. Allocation concealment was ensured by sealed opaque envelopes. The study subjects drew a sealed envelope from an opaque box. The sealed envelope was opened by the treating chiropractor immediately before the intervention during the first session, to ensure that ‘blinding’ to type of intervention remained unknown until this time. The study subjects were not informed that one of the interventions consisted of a sham procedure.spinal HVLA technique, the chiropractor first localized de C7 spinous process and then palpated the spinous processes up to the T5 vertebra. Then he applied a preload force with both hands placed over the transverse processes of the targeted vertebra (T5), followed by a firm thrust directed postero-anteriorly. We mainly chose this level (i.e. middle of the thoracic spine) because it is simple to perform both manipulation and a sham in this region. The sham procedure was applied with the subject in the same position, but the chiropractor contacted the medial border of the right scapula positioned in external rotation, applied a preload force that was followed by a thrust in the plane of the scapula-thoracic interface. This type of sham procedure has been previously used in an experimental study [The HVLA technique and the sham procedure were performed by the same licensed chiropractor during the whole trial. The study subject lay on a treatment table in a prone position for both interventions. For the al study  and repoal study . This shSBMEDIC Electronics, Sweden) with a 1 cm2 probe, with the study subject in the prone position. PPT was measured at two different localizations, on the paravertebral tissues (i) just right of the spinous process of the T5 vertebra, and (ii) just right to the spinous process of the L4 vertebra. An assessor, trained to assess PPT and blinded to the interventions, performed all measurements. The assessor increased pressure manually and perpendicularly to the skin with an application rate set at 50 kilopascal/s. The subject was instructed to press a button placed in his/her right hand to indicate when the pressure became painful (i.e. when the PPT was reached). The PPT was measured three times at each localization and at each time point . There was a 30 s rest period between each measurement. The mean of the three recordings for each time point was used in the statistical analysis, as this has been shown to be reliable in previous studies [O’Neill et al. [To assess the effect of the thoracic HVLA technique on pain sensitivity we measured the PPT. The PPT was defined as the pressure at which the subject first indicated it became painful. This was measured in kilopascal using an Algometer type 2 . The stPaced breathing, Trex LLC) on a smartphone. The ECG was recorded using three electrodes connected to the PowerLab system . These three electrodes were placed on the right clavicle (earth), on the sixth left rib (positive), and on the left clavicle (negative) of each study subject. The analogous signal of the ECG was amplified with a Dual Bio Amp , connected to a PowerLab 16/35 . Noninvasive beat-to-beat blood pressure was recorded with a Finometer  using a finger cuff placed on the right middle finger. The Finometer was also connected to the PowerLab 16/35. The ECG and finger blood pressure signals were digitized at a sampling rate of 4000 Hz with the PowerLab device. Signals were further analyzed with LabChart on a personal computer. The assessor set up the equipment and prepared the study subjects, e.g. cleaning skin with alcohol, positioning the electrodes, the finger cuff, calibrating the Finometer. Study subjects were prevented from standing up, when they changed from the prone position (PPT assessment) to the supine position (autonomic assessment) to avoid orthostatic autonomic reflexes.Electrocardiogram (ECG) and continuous finger blood pressure were recorded for 7 min immediately after the PPT assessment for each period of measurements . Subjects were placed in a supine position and were instructed to breathe at a pace of 0.25 Hz during the recordings, either with an auditory or visual guide, with the help of a metronome application (LabChart tools (e.g. HRV module and its beat classifier tool).A blinded assessor, who underwent a training in autonomic measures and data management, selected 5-min blocks from the 7-min recordings  for each time point  unaware of whether data pertained to spinal HVLA technique or sham . He performed (i) an automated and visual inspection of the ECG signal and (ii) a visual inspection of the blood pressure signal to detect abnormal beats, and other measurement issues (e.g. artifacts). Finally, he edited the recording using LabChart. This was performed in both (i) the time domain , and the standard deviation of the inter beat interval of normal sinus beats (SDNN)) and (ii) the low frequency (LF) and high frequency (HF) domains  according to Task Force of the European Society of Cardiology and The American Society of Pacing and Electrophysiology [LabChart’s HRV module is performed with a Lomb-Scargle Periodogram, “… the Lomb method also allows for the exclusion of ectopic beats without requiring an approximated beat to be put in its place as it is perfectly capable of dealing with gaps in the data set, giving you a more accurate analysis that is less affected by ectopic or missing beats.” [LabChart, and if important deviations were noted, data were excluded (because this meant that the subject had not followed the paced breathing). Reliability of short term measurements of HRV at rest in healthy subjects is reported as moderate to good [HRV  was further analyzed using the HRV module in ysiology . Please  beats.”  . In sho beats.” , althoug to good .HRV is dependent of heart rate for both mathematical  and physiological (i.e. autonomic control) reasons –45. ThusLabChart. The low frequency oscillations in systolic blood pressure (LF-SBP) are proposed as a marker of the sympathetic activity to the alpha-adrenergic receptor of vasculature [The beat-to-beat variation in systolic blood pressure was resampled to obtain a smoother trace and to permit further spectral analysis (using fast Fourier transformation) of systolic blood pressure variability in the low frequency band (0.04–0.15 Hz) in culature  and was culature .The means of heart rate, systolic blood pressure, diastolic blood pressure, and blood pressure were also calculated from each selected 5-min block.The study subjects did not have access to the content of the envelope used for the randomization at any time during the whole session and were not informed of the ‘treatment’ that they would receive. At the time of the information they had been told that that the aim of the study was to assess the effect of different techniques used in manual therapies on physiological outcomes and that they would receive the same type of intervention during both sessions. Thus, we attempted to keep them naïve to the purpose of the study. Further, they were informed that the different researchers participating in this trial would not answer questions dealing with the interventions until the end of the study. They were also blinded to the recordings during the whole trial .Finally, we assessed if our sham procedure had been successful to blind the study subjects. This was done using a post session questionnaire about their beliefs on the effectiveness of each intervention (HVLA manipulation and sham), to see if these were similar or if study subjects could differentiate ‘treatment’ from sham . Thus, the assessor was blinded to the intervention delivered. During experimental sessions, ECG and continuous blood pressure signals were directly recorded on a computer, and the research team had no interaction with the study subjects during these recordings.During the treatment of the raw data, i.e. the selection of 5-min blocks of ECG and continuous blood pressure recordings and the data cleaning process , the assessor was blinded to the link between the type of intervention and data. The blinding of this procedure was ensured by using transformed data file names.Finally, the main statistical analysis (except for bivariate correlations) was also performed in a blinded manner by transforming names of the sets of data. The study groups were uncovered only at the time of data interpretation.The present study assessed the effect of a thoracic HVLA manipulation on several outcomes. Therefore, it would be difficult to justify a power calculation on one particular outcome over another, as all variables had the same importance (i.e. there was no primary outcome). Instead, we determined our sample size on ‘the rule of thumb’, guided by advice of a statistician and previous literature . Thus, amean with standard deviation (SD) for continuous variables. Also, mean (SD) was calculated for each dependent variable for both sessions and all time points. We assessed the distribution of data with histograms and QQ plots. Dependent variables with a skewed distribution were transformed using a logarithm function (Log10) to achieve normality. Log transformation is usual for HRV parameters [SPSS Statistics for Windows, version 25  was used for all analyses. Descriptive data are presented as frequencies for categorical variables and rameters . Log trat-tests or Wilcoxon signed rank tests, when data were skewed. In addition, for each outcome variable, we assessed the risk of carry over effect by comparing baseline values of subjects allocated to the spinal manipulation-sham sequence to those allocated to the sham-spinal manipulation sequence using independent t-tests or Mann Whitney U tests for skewed data.Differences at baseline between spinal manipulation and sham were determined for each outcome variable using paired Intervention , Time , and the interaction between intervention and time (Intervention × Time). Time was treated as a continuous, linear variable in all models. Quadratic effect of Time and its interaction with Intervention were added only if they improved the fit of the model . Random intercepts were included to account for individual differences. Generalized Linear Mixed Model with a gamma distribution and a log link function was used for LF-SBP due to a skewed distribution. Within-subject correlations arising from the crossover design were taken into account in all models. Sex was found not to be a confounder and therefore excluded from the analyses. The range of age in our study subjects was too narrow to be of any importance. A statistically significant Intervention × Time interaction was interpreted as an effect of the spinal manipulation.To assess the effect of the thoracic HVLA technique for each outcome variable  we used Generalized Linear Mixed Models. Fixed effects of the models were In a sensitivity analysis, we also analyzed corrected HRV parameters. However, results were not reported if they yielded similar conclusion as with non-corrected HRV parameters.We visually inspected the presence of a relationship between cardiovascular autonomic outcomes and PPT  using scatter plots. Also, we analyzed bivariate correlations  between cardiovascular autonomic outcomes and PPT. Distribution of change scores were assessed with histograms and QQ plots. We used (i) Pearson’s (parametric) or (ii) Spearman’s (non-parametric) correlation coefficient, respectively (i) if the two variables (i.e. autonomic outcomes and PPT) followed a normal distribution or (ii) if at least one of the outcome variables did not follow a normal distribution , 49. CorThe statistical level of significance was set at 0.05. Bonferroni corrections  were applied for bivariate correlations to compensate for the risk of obtaining a significant finding by chance when performing multiple tests (i.e. Type I error).Fifty-four volunteers were screened for eligibility criteria, 51 were included and 41 were finally analyzed. Figure The blinding was interpreted in two different ways.Our results suggest that the sham procedure was successful in blinding subjects in 85% of cases Table . row A oIt is also possible to consider that subjects who thought that both interventions were effective but with a stronger certainty for the spinal manipulation Table . row C wIn any case, since 80% or 85% of the subjects were probably blinded and that, among them, the large majority had exactly the same beliefs regarding the effectiveness of both interventions, we can reasonably conclude that the sham procedure used in this trial was generally effective to control the brain-body responses to context information (e.g. placebo responses).Descriptive data for each outcome variable are shown in Table There were no statistically significant differences at baseline between the spinal manipulation and the sham sessions for any of the cardiovascular autonomic outcome variables. In addition, there were no statistically significant differences at each baseline  between subjects randomized to the spinal manipulation-sham sequence and those to the sham-spinal manipulation sequence for any of the cardiovascular autonomic outcome variables except systolic blood pressure and mean blood pressure (see below). For instance, for the sham session at baseline, there was no significant difference in outcome variables between subjects who already underwent the supposed effective treatment during the first session  and those who started the experimentation . Regarding, systolic blood pressure and mean blood pressure, the difference occurred only at baseline for the spinal manipulation session, i.e. between subjects who already underwent the sham (i.e. ineffective intervention) compared to those starting the study. Thus, we can conclude that the ‘effects’ of the spinal manipulation in the first intervention period did not carry on into the next one . The results of these different analyses are available in Additional file Intervention × Time interactions) for any of the cardiovascular autonomic outcomes . Also, there were small  increases in systolic, diastolic and mean blood pressure over time. However, there were no statistically significant changes over time in LF-SBP. Please see Table There were statistically significant increases in RR intervals (i.e. decrease in heart rate), log HF-HRV, log LF-HRV, log LF/HF, log RMSSD, and log SDNN over time .Visual analysis of scatter plots with PPT plotted against cardiovascular autonomic outcomes suggests neither monotonic (linear or non-linear) nor other types of relationships between the two variables.p <  0.006) positive associations between changes in distal PPT and changes in both Log LF-HRV and systolic blood pressure during the sham session  correlations for changes from baseline to post intervention measures between cardiovascular autonomic outcomes and PPT  after both spinal manipulation and sham interventions Table . It is won Table .Table 5In addition, negligible or weak  correlations were found between PPT and cardiovascular autonomic outcomes at each time point and for each type of intervention  after the intervention, in healthy young subjects. We found no statistically significant effect of the thoracic HVLA technique on the cardiovascular autonomic activity. In other words, there was no difference on the outcomes between the thoracic HVLA technique and a valid sham procedure. In addition, we found neither monotonic (linear or non-linear) associations nor evidence of other types of relationship between cardiovascular autonomic activity and PPT after the spinal manipulation.We noticed a decrease in heart rate over time during sessions. This was probably caused by an increase in cardiac vagal activity, as shown by the increase in log HF-HRV, log RMSSD, log SDNN. The increase in log LF-HRV over time might also indicate an increase in vagal activity. These observations may be explained by a decrease in stress after the interventions and an increase of the time spent in a recumbent position.Concerning cardiovascular autonomic control, our results are in agreement with the conclusions of a recent review of the literature on randomized sham-controlled trials, suggesting that spinal HVLA techniques may have no effect on frequency domain indices of HRV immediately after the intervention . This waHowever, it should be acknowledged that in the current trial, the cardiovascular autonomic activity was assessed 5 min after the interventions (i.e. not during the very immediate period after the interventions), since we first measured the sensitivity to experimentally induced pain. This is different from the sham-controlled trials included in the previous review which measured HRV within the 5 min , 50–52 aHowever, a recent sham-controlled trial  reportedIt is also worth noting that a recent good quality sham-controlled study testing the effect of spinal mobilization reported also no effect on HRV and PPT .Further, we found no relationship between autonomic activity and pain sensitivity after the spinal HVLA technique, which is in contrast with a previous study on chronic pain patients dealing with spinal mobilization . That stWe found, however, a moderate  positive correlation between changes in systolic blood pressure and distal PPT during the sham session, which might be supported by previous literature showing an association between elevated blood pressure and a decrease in pain sensitivity . HoweverAs our study subjects were healthy and young, the findings might not be applicable to other populations, such as people in pain or with chronic disorders.selection bias was low. There was a roughly equal proportion of subjects allocated to the two sequences of interventions limiting the risk of period effects. The risk of carry-over effect was also low, as we used a wash-out period, and there were no results suggesting the presence of such risk.We used a drawing lots method to generate the randomization as well as sealed opaque envelope for allocation concealment. Thus, the risk of (performance bias). However, we found with the post-session questionnaires that (i) the sham procedure was successful in blinding subjects and that (ii) beliefs in the effectiveness of each intervention to change the outcomes were generally similar. Hence, brain-body responses caused by the intervention context (e.g. placebo responses) [performance bias.It is difficult to blind study subjects to interventions in controlled trials dealing with spinal HVLA manipulations, since these techniques are generally well-known and easy to recognize by the general population and, in particular, by chiropractic students. Thus, including only chiropractic students might be viewed as a limitation, as they are likely to discover the true nature of both interventions . Theoretically, this could increase the ‘effect’ sponses)  were prodetection bias was low. In addition, we performed most of the statistical analysis in a blinded way (except for the correlations).During each session, physiological signals were directly recorded on a computer and further extracted and processed by a blinded assessor. Data collection for PPT was also performed by a blinded assessor. Thus, the risk of Some subjects were excluded from the final analyses because of technical issues during the experiments. In particular, blood pressure was difficult to record in these conditions (long period in a recumbent position), especially in women . These exclusions reduced the statistical power, but they did not lead to any attrition bias as data from both sessions, for the remaining subjects, were analyzed. The larger number of subjects excluded for issues on blood pressure recording is briefly discussed below.The study was limited to the assessment of the effect of a spinal HVLA technique applied on the middle part of the thoracic spine. Thus, the results may not be applicable to manipulation in other parts of the spine.We used a sham procedure adopting the same physical cues as the spinal HVLA technique (i.e. preload and thrust) to improve its credibility as well as to produce similar levels of mechanical stress. This was done to control for non-specific autonomic reactions that might be caused by mechanical stress. The sham procedure was performed outside of the spinal joints complex to avoid the stimulation of the supposed ‘active ingredients’ of spinal manipulation . Our observations suggest, at least in part, that the sham did not produce ‘spinal’ stimulation since there was, generally, no cracking sound  during its execution contrary to the spinal technique.The mechanical parameters of the interventions  and thus the resulting rate of force application during the thrust might have an impact on some outcomes, as shown on the immediate neuromuscular response following HVLA manipulation –9. We diWe assessed only cardiovascular autonomic activity, meaning that other autonomic sub-systems have not been assessed, e.g. skin sympathetic nerve activity, which was previously found to increase following mobilization with oscillatory movements as compared to a sham –14. ThusN = 30), our results should be interpreted with caution and thus, replication of the results is needed.In addition, considering that the use of systolic blood pressure variability in this research context is still limited and that we based our conclusions on a smaller number of subjects for this particular outcome  no effect on local and distal PPT  and (ii)Our assessment of the autonomic activation following a spinal manipulation might also allow some clinical considerations. Clinical evidence suggests that in some chronic pain condition, an increase in sympathetic activity may lead to an increase in pain –57 and tWe are of the opinion that it is still reasonable to conduct experimental research on this issue because our study assessed only a limited part of the autonomic and pain systems. Further studies should then consider assessing several markers of autonomic nervous system activity (i.e. assessing various autonomic sub-systems) such as HRV and skin conductance and, very importantly, assessing several pain dimensions. Also, the potential effect of HVLA techniques applied in other parts of the spine should be considered in further studies. Sham-controlled trials should be used to control for non-specific responses and an assessment made to establish if this control procedure was effective, e.g. with the use of post-trial questionnaire. It would also be relevant to conduct such studies in a clinical context, especially on chronic pain patients who may have a disturbed cardiac autonomic  control. This would make it possible to explore if autonomic modulations after spinal manipulations are linked to health outcomes (e.g. self-reported pain) and if a course of treatments would permit to improve the cardiac autonomic  control.Our results suggest that a single HVLA manipulation of the thoracic spine has no specific effect on cardiovascular autonomic activity. Also, we found no relationship between cardiovascular autonomic activity and pressure pain threshold after the spinal manipulation. It is reasonable to conduct new experimental studies on this topic using several markers of autonomic activity with a more comprehensive pain assessment not limited to the immediate post intervention period. Even more relevant is, perhaps, to perform clinical research on people with chronic pain.Additional file 1. Post session questionnaireAdditional file 2. Additional analyses and data


Cardiovascular disease (CVD) is common in chronic kidney disease (CKD), while major CV events are rare in young CKD patients. In addition to nitric oxide (NO)-related biomarkers, several surrogate markers have been assessed to stratify CV risk in youth with CKD, including 24-h ambulatory blood pressure monitoring (ABPM), carotid artery intima-media thickness (cIMT), pulse wave velocity (PWV), ABPM-derived arterial stiffness index (AASI), flow-mediated dilatation (FMD), and left ventricular mass index (LVMI). The aim of this study was to identify subclinical CVD through the analysis of indices of CV risk in children and adolescents with CKD. Between 2016 and 2018, the prospective observational study enrolled 125 patients aged 3 to 18 years with G1–G4 CKD stages. Close to two-thirds of young patients with CKD exhibited blood pressure (BP) abnormalities on ABPM. CKD children with abnormal office BP showed lower plasma arginine levels and arginine-to-asymmetric dimethylarginine (ADMA) ratio, but higher ratios of ADMA-to-symmetric dimethylarginine (SDMA) and citrulline-to-arginine. High PWV and AASI, indices of arterial stiffness, both strongly correlated with high BP load. Additionally, LV mass and LVMI exhibited strong correlations with high BP load. Using an adjusted regression model, we observed the citrulline-to-arginine ratio was associated with 24-h systolic and diastolic BP, systolic blood pressure (SBP) load, and diastolic blood pressure (DBP) load. Early assessments of NO-related parameters, BP load abnormalities, arterial stiffness indices, and LV mass will aid in early preventative care toward decreasing CV risk later in life for children and adolescents with CKD. Non-communicable diseases (NCDs) present the greatest public health challenges of the 21st century and account for two-thirds of all global deaths . By the Surrogate markers have been required to stratify risk for CVD in children. Some biomarkers could evolve to act as surrogates including biomarkers, structural markers, or functional markers. Nitric oxide (NO), a potent vasodilator, plays a relevant role in the regulation of blood pressure (BP) and renal physiology . EmerginSeveral structural and functional markers have been proposed to stratify the cardiovascular risk in CKD patients, including left ventricular mass , 24-h am2, which indicates that most participants had CKD in the early stage. Congenital anomalies of the kidney and urinary tract (CAKUT) were the underlying kidney disease in 64% of patients. The diagnosis of office BP depends on age, gender, and height. Systolic BP (SBP) and/or diastolic BP (DBP) in the 95th percentile or greater was considered abnormal . The adult cut-off was applied at persons aged 16–18 years [We enrolled a total of 125 children and adolescents with CKD stage G1–G4 in this study. The clinical characteristics of the study participants are presented in 18 years . Of the p = 0.021) while a lower ADMA-to-SDMA ratio (1.37 (IQR 1.03–1.83) vs. 1.74 (IQR 1.55–2.05), p = 0.042) than those with CKD stage G1. No significant differences in arginine, ADMA, SDMA, arginine-to-ADMA ratio, and citrulline-to-arginine ratio were observed between CKD stage G2–G4 and the CKD stage 1 group.The plasma levels of NO-related parameters and their combined ratios stratified by the office BP are illustrated in p < 0.001), 24-h DBP (p = 0.043), SBP load (p = 0.004), and DBP load (p = 0.039) than those with CKD stage G1. The rate of patients with abnormal 24-h, daytime BP, nighttime BP, and BP load was higher among patients with CKD stage G2–G4 than those with CKD stage 1. The PWV, an arterial stiffness index, and LV mass were higher in children with CKD stage G2–G4 compared to those with G1. However, cIMT, FMD, AASI, and left ventricular mass index (LVMI) were not different between CKD children with stage G1 and G2–G4. Also, we analyzed the differences in ABPM profile and cardiovascular indices between CKD patients with CAKUT and with non-CAKUT. Nevertheless, we found all cardiovascular indices were comparable between these two groups (All p > 0.05).A complete analysis of 24-h ABPM study was feasible in 76 patients aged 6–18 years with CKD . After AAs shown in r = 0.613, p < 0.001), the citrulline-to-arginine ratio was significantly associated with 24-h systolic BP (p = 0.006) controlling for age and creatinine. Also, the citrulline-to-arginine ratio was associated with 24-h diastolic BP (p < 0.001) controlling for creatinine. We also found that both citrulline-to-arginine ratio and citrulline had significant associations with SBP load  controlling for creatinine in a predictive model applied for the above-mentioned factors . Additionally, a negative association was found for citrulline-to-arginine ratio and DBP load in the adjusted regression model .Associations between NO-related biomarkers and BP load were further examined in a multivariate linear regression model adjusted for age, sex, and creatinine . To specr = 0.346, p = 0.003) and PWV . Furthermore, citrulline-to-arginine ratio had a negatively correlation with cIMT , LV mass , and LVMI . However, citrulline, ADMA, SDMA, arginine-to-ADMA-ratio, and ADMA-to-SDMA ratio were not correlated with any of the cardiovascular surrogate markers. A multivariate linear regression model was further analyzed to control for the effects of covariates  on cardiovascular surrogate markers. When the covariates were taken into account, only arginine had a significant association with cIMT  in a multivariable regression model applied for age, sex, creatinine, and NO-related biomarkers . However, other relationships were no longer significant.Correlation analysis of NO-related biomarkers and cardiovascular surrogate markers is detailed in This study provides insight into a link between NO-related biomarkers, structural and functional markers, and cardiovascular risk in children and adolescents with early-stage CKD. Our major findings are as follows: (1) approximately two-thirds of children and adolescents with CKD stage G1–G4 had BP abnormalities on ABPM; (2) plasma arginine level and arginine-to-ADMA ratio were lower, whereas ADMA-to-SDMA and citrulline-to-arginine ratios were higher in CKD children with abnormal office BP; (3) PWV and LV mass were increased in the case of CKD stage G2–G4 compared to those with stage G1; (4) PWV and LVMI was higher in CKD children with abnormal vs. normal ABPM profile; (5) PWV, LV mass, and LVMI exhibited stronger correlations with elevated BP load than other cardiovascular surrogate markers; and (6) the citrulline-to-arginine ratio was associated with 24-h SBP, 24-h DBP, SBP load, and DBP load in the adjusted regression model.In line with previous studies in pediatric CKD ,16,23,24Although most evidence indicates NO deficiency in CKD and hypertension ,7,8, somAs expected, the severity of CKD is associated with certain markers of adverse CV health, like PWV and LV mass in the current study. Although PWV has been used in children, age-specific reference ranges are still unavailable . In the Our study was not without its limitations. First, additional assessments of the above-mentioned CV markers with longer follow-ups are essential in view of the long-term nature of childhood CKD. Second, there are no reference values established for PWV, AASI, cIMT, and LVMI to define a cut-off point between healthy and CKD children . Third, 2, or were on dialysis maintenance or had ever received a renal transplantation; or (4) were unable to follow-up protocol or cooperate with the assessment. All participants were categorized to eGFR categories: G1 (eGFR ≥ 90 mL/min/1.73 m2), G2 (eGFR 60–89 mL/min/1.73 m2), G3 (eGFR 30–59 mL/min/1.73 m2), or G4 (eGFR 15–30 mL/min/1.73 m2). The analysis was restricted to children with a baseline eGFR > 15ml/min/1.73m2, who measured renal and cardiovascular parameters described in the following section. The causes of kidney disease were divided into two categories: Congenital anomalies of the kidney and urinary tract (CAKUT), or non-CAKUT. CAKUT structural anomalies range from renal agenesis, kidney hypoplasia, kidney dysplasia, multi-cystic kidney dysplasia, horseshoe kidney, duplex collecting system, posterior urethral valves, and ureter abnormalities [The prospective cohort study was approved by the Institution Review Board and Ethics Committee of Chang Gung Medical Foundation, Taoyuan, Taiwan  and adherent to the principle of the 1964 Declaration of Helsinki and its later amendments. All participants gave written informed consent. The present study includes 125 participants who contributed at least one visit to a pediatric nephrology clinic in a Taiwan medical center between December 2016 and October 2018, while they were between 3 to 18 years of age with CKD. CKD is defined as kidney damage or decreased kidney function over at least three months . Kidney malities .Fasting plasma specimens, spot urine, and fecal samples were aliquoted and stored at –80 °C until analysis. Blood urea nitrogen, creatinine, uric acid, glucose, total cholesterol, LDL, triglyceride, sodium, potassium, calcium, phosphate, hemoglobin, and urine total protein-to-creatinine ratio were measured by the hospital central laboratory. We directed the families to prevent their children from having an excessive intake of foods rich with arginine and citrulline  for 1 week before blood and urine sampling.BP measurements were taken at the clinic visit. The participant rested quietly for 5 min in a seated position. Three BP recordings were obtained 30 s apart by experienced specialist nurses using a standard mercury sphygmomanometer . A cuff with a bladder sized by a 1:2 width to length ratio according to the size of the arm was chosen. The mean value was used as the participant’s office BP for analysis. The 24-h ABPM data were collected for subjects aged 6–18 years using an Oscar II monitoring device  handled by a trained specialist nurse as previously reported . BrieflyAn abnormal ABPM profile was determined based on: (1) awake, asleep, systolic, or diastolic BP loads exceeding the 95th percentile based on gender and height using ABPM reference data ; (2) awa2.7 [The ultrasound studies were performed by two experienced pediatricians (P.-C.L. and I.-C.L.) as previously reported . Patient2.7 .Plasma levels of citrulline, arginine, ADMA, and SDMA were determined using high-performance liquid chromatography . O-phthaldialdehyde 3-mercaptopropionic acid (OPA-3MPA) was used for the fluorescent derivatization . The stan (%), as appropriate. Group differences were analyzed by Mann–Whitney U-test or Chi-square test, as appropriate. The associations between variables were assessed using Spearman’s rank correlations. A linear regression model was performed followed by the stepwise multivariable analyses integrating relevant parameters to explain BP load. A value of p < 0.05 was considered statistically significant. Statistical Package for the Social Sciences (SPSS) software 14.0  was used. Data were expressed as median (25th percentile–75th percentile) or In conclusion, a significant proportion of children with CKD, even in early-stage, exhibit BP abnormalities and an adverse CV risk profile. Although the mechanism underlying the NO pathway and CVD in CKD children has not yet been completely clarified, our results cast a new light on the link between NO pathway, BP load, and arterial stiffness in children with CKD. Early detection of NO-related biomarkers and structural and functional surrogate markers may assist in advancing knowledge of CVD and initiating treatment at an early stage with respect to improving CV outcome in at-risk children with CKD.


Fascioliasis is a neglected zoonosis with major public health implications in humans. Although triclabendazole (TCBZ) is the drug of choice, there are records of TCBZ failure worldwide. TCBZ-resistant fascioliasis is treated with alternative approved drugs including nitazoxanide (NTZ), with varying levels of efficacy. Data on NTZ efficacy after TCBZ failure in Egypt is scarce. This study evaluated the efficacy of NTZ in cases of TCBZ failure during an outbreak of fascioliasis in Assiut governorate of Upper Egypt.Fasciola antibodies, and hepatic focal lesions (HFL) or ascites on abdominal ultrasound or computed tomography. All patients initially received TCBZ at recommended doses. Patients were followed up after 1 month to assess response. According to the responses, patients were categorized as non-responders and responders. The non-responders received a trial of NTZ and were re-assessed for response based on clinical manifestations, eosinophil count, and abdominal ultrasound. Patients not responding to NTZ received additional doses of TCBZ.This prospective study included 67 patients from the outpatient clinic in Manfalout locality of Assiut governorate with clinical manifestations of acute fascioliasis. These included high eosinophilia , positive anti-One month after initial TCBZ treatment, 37 patients responded well to TCBZ, while 30 patients failed to respond with persistence of fever, abdominal pain, high eosinophilia, and HFL. Most non-responders were male (56.7%); females predominated among TCBZ responders (62.2%). The mean age of the non-responders was relatively lower, at 20.57 ± 14.47 years (p = 0.004). Following NTZ therapy, HFL disappeared in 9/30 (30%) patients and eosinophil counts normalized in only 2 (6.7%) patients, indicating an overall efficacy of 36.6%. The remaining cases received additional doses of TCBZ with complete clinical, pathological, and radiological resolution.Nitazoxanide was partially effective in TCBZ failure in acute human fascioliasis in Upper Egypt. Further studies with larger samples are highly encouraged and further research is urgently needed to find new therapeutic alternatives to TCBZ. Fascioliasis is a neglected zoonosis with major public health implications in humans. Triclabendazole (TCBZ) is the drug of choice, but alternative approved drugs are necessary in cases of TCBZ failure. Nitazoxanide (NTZ) is an alternative used in such cases. However, the efficacy of NTZ in TCBZ-failure cases among patients in Egypt remains unclear. In this study, the efficacy of NTZ was evaluated in cases of TCBZ failure during an outbreak of human fascioliasis in Assiut governorate of Upper Egypt. This study enrolled 67 patients diagnosed with fascioliasis based on clinical, laboratory, and radiological findings. These patients were referred from the outpatient clinic in Manfalout locality of Assiut governorate in Egypt. All patients received TCBZ at recommended doses as initial treatment. Those failing to respond were treated with NTZ at standard doses; following therapy, lesions in the liver and high eosinophil counts were resolved in 30% and 6.7% patients, respectively, indicating an overall efficacy of 36.6%. Therefore, in this outbreak of human fascioliasis in Upper Egypt, NTZ was found to be partially effective in cases with TCBZ failure. Fasciola (F. hepatica and F. gigantica). In the past few decades, the incidence of human fascioliasis has considerably risen in different parts of the world. The rise has been particularly remarkable in South America, Asia, and Africa including Egypt, where the two common species of Fasciola coexist [Fascioliasis has emerged as a notable zoonotic disease with considerable impact on veterinary and public health. This prompted the World Health Organization (WHO) to include human fascioliasis among the important neglected tropical diseases (NTDs) . It is a coexist . Recent  coexist .Fasciola worms in the bile ducts and may last for months or even years [Fasciolids are parasites of the hepato-biliary ducts, and the disease is mostly confined to the liver. Therefore, the main pathogenic sequelae are hepatic lesions, fibrosis, and chronic inflammation of the biliary passages. The pre-patent period together with the time to onset of signs/symptoms of the disease may range from a few days to 2–3 months or longer. There are 2 main clinical stages in fascioliasis. The acute stage coincides with larval migration and mechanical destruction of the liver tissue. This stage extends till worm maturation in the hepatic tissues, and lasts for 2–4 months. The chronic stage coincides with the persistence of adult In Egypt, fascioliasis has probably been prevalent for a very long period, since the times of the pharaohs , 6. HighThe mainstay of treatment in fascioliasis affecting animals and humans is triclabendazole (TCBZ), which targets both the immature stages and mature adult worms . Older dThe widespread use of TCBZ in the livestock industry led to the emergence of resistance in fluke populations affecting ruminants in both, developed and developing countries including Ireland, Spain, Australia, Peru, and Argentina . The zooReliance on monotherapy poses a risk for the treatment of fascioliasis, particularly in the absence of a vaccine for the prevention of the disease . As caseSeveral trials were conducted in the search for effective alternative drugs for fascioliasis. Nitazoxanide (NTZ), which is a broad spectrum antiparasitic agent, has been found to be well tolerated by humans, with adverse effects similar to that of placebo . Across The aim of the present study was to investigate the efficacy of nitazoxanide as a treatment for fascioliasis in the face of incomplete response to triclabendazole in Upper Egypt.This prospective study was conducted between August and November 2018.The study protocol was approved by the Institutional Review Board of the Faculty of Medicine of Assiut University, Egypt. Written informed consent was obtained from all patients prior to participation in this study.A total of 74 patients with diagnosed or suspected fascioliasis were recruited in the study. All these patients were referred to the outpatient clinic in the Tropical Medicine and Gastroenterology Department at the Al-Rajhi Liver University Hospital during an outbreak of fascioliasis in Manfalout locality of Assiut Governorate in Upper Egypt.The included patients had symptoms and signs suggestive of fascioliasis such as fever, abdominal pain, jaundice, and hepatomegaly. The complete blood count (CBC), including eosinophil percentage and absolute eosinophil count was individually assessed using the ADVIA 2120i Hematology System . Stool examination was also performed for all cases for the qualitative diagnosis of fascioliasis using the native lugol and formalin ethyl acetate sedimentation method . Stool sF. hepatica IgG Enzyme-linked immune sorbent assay (ELISA) kits  for qualitative diagnosis of fascioliasis. The result was read photometrically at 450 nm (TECAN Sunrise Absorbance Reader).  Indirect hemagglutination assay (IHA) using Distomatose Fumouze  was also done to compare antibody titers (a titer ≥ 1/320 was considered to be positive). Abdominal ultrasound (US), and abdominal computerized tomography (CT) were also done. Endoscopic retrograde cholangiopancreatography (ERCP) was performed in cases presenting with obstructive jaundice and a dilated common bile duct (CBD) on abdominal US and/or CT.Further investigations included; serological analysis was done by All patients received a double dose of triclabendazole  at a dose of 10 mg/Kg/dose, at 12-hour interval in a joint venture with the WHO. Patients were advised to avoid vegetables that posed a risk for re-infection.The endpoints for treatment response were evaluated on follow up after 1 month. Evaluation was based on 3 parameters, namely, resolution of clinical symptoms and signs, normalization of eosinophil counts, and improvement of hepatic lesions on US. According to the WHO criteria, persistence of symptoms or signs with either eosinophilia  or hepatic focal lesions, was considered to be a probable indicator of treatment failure with TCBZ [The non-responders received NTZ at a dose of 500 mg orally every 12 hours for 7 days. Patients were clinically assessed for response after 1 month. Resolution of both, eosinophilia in the CBC and/or hepatic focal lesions on US were indicative of response. Patients who failed to respond to NTZ were re-treated with TCBZ at doses similar to the initial dose and were followed up for response.Patients who received any other anthelminthic drugs within 1 month before TCBZ or NTZ therapy including bithionol, praziquantel, albendazole, dihydroemetine, or emetine hydrochloride, and patients who showed hypersensitivity to nitazoxanide were excluded from this study.Data entry and analysis were performed using the IBM SPSS Statistics for Windows, Version 20.0.  software package. Data were presented as numbers, percentages, means, and standard deviations. The Chi-square and Fisher’s exact tests were used to compare qualitative variables. The Mann-Whitney test and the Wilcoxon signed rank test were used to compare variables between independent and dependent groups, respectively. In case of non-parametric data, the Wilcoxon signed rank test was used to compare the quantitative variables before and after treatment. A P-value < 0.05 was considered statistically significant.In this prospective study, 74 patients were initially recruited. Among them, 67 patients with symptoms and signs suggestive of acute fascioliasis were included for the NTZ trial; 7 patients were excluded as they presented with obstructive jaundice and a dilated CBD on ultrasound (suggesting chronic fascioliasis). These 7 patients underwent endoscopic sphincterotomy and extraction of the adult worm by ERCP followed by TCBZ therapy. The included patients received initial treatment with a double-dose of TCBZ.Fasciola eggs in 7 of 67 patients (10.4%) with absence of other co-existing parasitic infections that could, potentially, construct immunological cross-reactions or clinical symptoms overlapping with fascioliasis.The pretreatment demographic, clinical, and laboratory data of the studied patients are shown in Tables The studied patients were followed up after 1 month to evaluate the response to first line TCBZ. A total of 37 cases (55.2%) showed good response to TCBZ (the responder group) as evidenced by disappearance of signs and symptoms, normalization of peripheral eosinophil counts, and resolution of HFL. The remaining 30 cases (44.8%) (the non-responder group) showed persistence of infection, as evidenced by persistence of clinical manifestations, high eosinophilia, and HFL. This group received nitazoxanide and were followed up after 1 month .The demographic, clinical, and sonographic characteristics of both groups, as summarized in As shown in Furthermore, the assessment of response to first line TCBZ showed complete resolution of the clinical manifestations in all patients in the responder group; patients in the non-responder group had persistent fever and abdominal pain. The pre-treatment eosinophil counts were not significantly different between the groups (p = 0.081). After treatment, limited improvement in eosinophil counts was observed in the non-responder group, with a reduction from 26.72% ± 13.21 to 20.00% ± 11.28. In the responder group, the counts reduced from 30.47% ± 15.18 to 3.6% ±1.7, showing statistically significant difference between the groups (p = 0.000).After NTZ treatment in the non-responder group, HFL disappeared in 9/30 patients (30%) as opposed to all patients in the TCBZ responder group; this difference was statistically significant (p = 0.015) . In addiF. hepatica and F. gigantica infections in humans [Owing to its activity against juvenile and adult forms of the parasite, TCBZ is the drug of choice in the treatment of n humans . Mass con humans .Several previous studies have documented the clinical efficacy of TCBZ with various treatment regimens in different regions including Egypt –23. The Indeed, TCBZ is the only first-line medication with reports of high efficacy in humans. Therefore, the effective management of resistance to this drug is of utmost importance . ClinicaFasciola antibodies. However, stool examination was positive in only 7 cases (10.4%) with a low egg burden. In the present study we could not rely only on coprological examination for the diagnosis and follow up of cases. This is attributed to many factors, including prepatent or acute infections (where the patients were symptomatic prior to the appearance of eggs in the stool) [Fasciola worms to produce eggs (due to its lack of adaptation to the human host), encapsulation of eggs in granulomas or abscesses in the liver, and low egg shedding related to low infection burdens [In the current study, all cases of acute fascioliasis were defined based on clinical manifestations, high eosinophilia, and radiological signs with positive anti-e stool) , the ina burdens . Coprolo burdens .Fasciola eggs were detected in only 2 cases (8.6%) as the patients were diagnosed in the hepatic phase [In a study previously conducted in Egypt including 23 cases, ic phase . An immaic phase .Fasciola antibodies by the ELISA test is a reliable and sensitive test for diagnosis of fascioliasis compared with stool examination. The main advantage is that results are positive as early as 2 weeks post infection. However, since serum antibodies may persist for 4–5 months after successful treatment, it is not a reliable test in the evaluation of response during follow up [The detection of anti-ollow up . Eosinopollow up .As described by Marcos et al., the primary outcome measures for clinical cure after treatment are defined by resolution of the clinical picture and eosinophilia during follow up . TherefoIn the present study, as evidenced by the disappearance of signs and symptoms, normalization of peripheral eosinophil counts, and resolution of HFL, 37 patients (55.2%) showed good response to TCBZ. The remaining 30 cases (44.8%) were suspected to have TCBZ failure and were treated with NTZ. The mean age of the non-responder group was lower than that of the TCBZ responders; this may have had an impact on the treatment response. A double blinded placebo-controlled trial in Peru, which employed NTZ for the treatment of chronic fascioliasis, has shown a low cure rate in children (40%) and a slightly higher efficacy in adults (60%) .F. hepatica [In our cohort, most non-responders were male (56.7%); females were predominant among the responders (62.2%). The gender of the studied patients did not significantly differ between groups. However, previous studies have indicated differences in sensitivity to flukicides depending on the sex of the host animals infected with hepatica .Fasciola infection. However, parasitological cure (the absence of eggs in the stools) was not reported during follow up.Notably, in the current study, patients who did not initially respond to TCBZ in the acute stage, responded to the subsequent trial of TCBZ administered 2 months after the initial dose, in the chronic stage. This relationship between response to TCBZ and the stage of the disease has also been previously mentioned by Marcos et al. , who repFasciola hepatica infection [The difference in TCBZ susceptibility between juvenile and adult parasites has been previously described in an in vitro study with nfection . Howevernfection , 33.F. hepatica infection, which is considered a large number. They received a trial with NTZ at a dose of 500 mg twice daily for 7 days, that showed an overall efficacy of 36.6% (11/30 patients), based on the improvement of eosinophil counts and HFLs.According to our results, 30 patients showed clinical evidence of the presence of TCBZ-resistant Fasciola has been studied in rabbits experimentally infected with F. gigantica. NTZ was found to be partially effective (47%) against the juvenile stages of the parasite, but completely effective (100%) against the adult stage [NTZ has been widely used in the management of different parasitic infections with reportedly high efficacy and tolerability. The efficacy of NTZ against lt stage .F. hepatica eggs in the stool on day 30 after treatment with NTZ; the serological and eosinophilic patterns had also improved [A few clinical trials have been conducted on the efficacy of NTZ in the treatment of human fascioliasis with considerably variable efficacy. In Egypt, an open-label clinical study including 125 Egyptian patients with chronic fascioliasis demonstrated 97% clearance of improved . A seconimproved . Similarimproved .F. hepatica received a 7-day course of NTZ. Compared to the placebo group, 60% adults and 40% children were cured [A much lower efficacy rate was observed in a double-blinded placebo-controlled study in northern Peru, where 50 adults and 50 children infected with re cured . These rFasciola in various regions [Conversely, some studies have revealed a lack of efficacy of NTZ in 24 cases of liver fluke infection in Cuba  and in a regions . This inAlthough TCBZ resistant fascioliasis has been widely described in livestock, the understanding of the mechanism of resistance to TCBZ remains incomplete, with a knowledge gap in terms of its capacity to spread and strategies for control . It has Fasciola exist, there is no documented strategy for the management of TCBZ treatment failure in humans. To minimize the development of drug resistance, the use of synergistic drug combinations has been suggested [In contrast to veterinary medicine where other treatment options for uggested . Howeveruggested .Although the small sample size has limited the scope of this study, to the best of our knowledge, this is the first report of TCBZ failure in humans with acute fascioliasis in Egypt. Further multicenter randomized studies including larger sample sizes are required to evaluate predictors of TCBZ failure. This will help to determine the optimum timing for repeating TCBZ after failure of the initial dose. Also, further research is urgently needed to find new therapeutic alternatives to TCBZ for controlling fascioliasis.In this first report of TCBZ failure in acute human fascioliasis in Upper Egypt, NTZ proved to be partially effective.S1 Checklist(DOCX)Click here for additional data file.